Pseudotyping systems and their use in
coronavirus entry research
T.P. Noorbergen, 3384578, Biomedical Sciences, Utrecht University
Department of Infectious disease and Immunology, division Virology,Faculty of Veterinary Medicine,
Utrecht University
The research of virus host cell entry is essential for anti-viral drug development. However, for
coronaviruses, such as the highly pathogenic SARS virus, the entry mechanisms are not yet fully
discovered. Therefore, many research groups are currently studying the activation of the
coronavirus spike protein, which mediates fusion between the viral envelope and plasma
membrane. In many fundamental studies, artificial assays are used instead of genuine virus to
circumvent the requirement for a high Biosafety level. In this thesis, we discuss systems as
pseudotyping assays, cell-cell fusion assays, virus-like particles and some reconstituted systems as
well. Some of these systems also have purposes outside viral entry research, for example in gene
therapy, targeted drug-delivery and anti-cancer treatment by specifically altering the tropism of
the vector to the target cells.
Pseudotyping and other artificial assays have the advantages of being allowed to be performed
less stringent biosafety conditions and making reverse genetics easier to perform. However, all
experimental systems have limitations which we evaluated. While VSV∆G and retrovirus based
pseudotyping assays gave comparable results as authentic virus, real virus has still to be taken
along as well.
Cell-cell fusion assays are simplistic systems to study the basic functionality of a fusion protein.
However, these assays gave contradictory results in comparison to virus entry in some studies,
which can be explained by the difference in the interaction context. Therefore, cell-cell fusion
assays are not suitable to study entry mediation by fusion proteins.
The experimenter has to choose the systems carefully to approach his questions, because of the
limitations of the experimental set ups.
Finally, we try to experimentally set up a new pseudotyping system based on the coronavirus
Murine Hepatitis Virus (MHV). We also make a stable cell line expressing recombinant MHV spike
protein which could be used for both retrovirus and this new pseudotyping assay.
Introduction
Viruses are responsible for many diseases, causing high numbers of victims over the world. Unlike
bacteria, viruses are non-cellular pathogens basically composed of a protein shell which contains its
genomic material. For their replication, viruses are entirely depended on host cell’s transcription and
replication machinery. Because of this dependency, viruses are continuously and successfully
co-evolving and adapting to its hosts. Some viruses are enveloped, thus containing a lipid membrane
around the nucleocapsid, while others like the poliovirus are non-enveloped. The first step of the lifecycle of a virus is entry of a host cell, in which the genomic information is introduced into the cell for
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replication and transcription. Virus entry begins with binding to the receptor on susceptible host
cells. Entry of enveloped viruses involves fusion between the viral envelope and a host cell
membrane. In this thesis, we mainly focus on research of this fusion process.
Fusion of the viral envelope with either the plasma membrane or intracellular membrane releases
the content of a virus, some viral proteins and genetic material, into the cell. The envelope of the
virus contains proteins which mediate membrane fusion, the fusion proteins. There are three classes
of fusion proteins: Class I which has a central α-helical coil (e.g. Influenza hemagglutinin (HA), HIV
Env, coronavirus Spike (S)), class II which mainly consists of β-sheets (Dengue virus E and Semliki
Forest virus E1 proteins) and class III with a combined structure of an α-helix and β-sheets (VSV-G,
baculovirus gp64)46. Class I fusion proteins are proteolytically cleaved by host proteases to become
active. This cleavage can occur during assembly, such as in the case of HIV Env, before binding to the
host cell (influenza A) or in the case of coronaviruses after endocytosis.
The fusion process is schematically presented in Figure 1. The fusion peptide of fusion proteins is
hydrophobic and thus shielded in the virus when it is inactive. Activation of the fusion protein results
in the exposure of the fusion peptide and subsequent insertion into the target membrane.
Subsequent folding of the protein brings the viral envelop close to the target membrane. First, the
outer lipid layers of the viral envelope and target membrane merge while the inner layers remain
intact, forming a fusion stalk which expands into a hemifusion. Subsequently an expanding fusion
Figure 1 – Schematic overview of Class I fusion protein function{{68 Jardetzky,T.S. 2004;}}: (a) Inactive class I fusion
protein in a trimeric arrangement with fusion domain (yellow), helical domain (pink) and transmembrane domain (purple).
(b) The fusion peptide (red) is inserted into the target membrane after conformation change of the fusion domain. (c)
Multiple fusion protein trimers are believed to be involved in fusion. (d) Folding of the fusion proteins brings the target
membrane in proximity of the viral envelope. (e) First, the outer lipid layers of both membranes merge, forming a fusion
stalk. (f) A fusion pore is formed when the inner layers fuse, releasing the content of the virus into the cytoplasm of the cell.
pore originate when the inner lipid layers fuse, releasing the content of the virus into the cytoplasm.
There are different triggers which can activate fusion proteins.
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Some fusion proteins are activated by binding to the host receptor alone. For example, the HIV Env
protein (or gp160), which is arranged in trimers, undergoes conformational changes after sequential
interaction with its receptor CD4 and a co-receptor such as CXCR4 or CCR51. HIV Env is cleaved by
cellular proteases into two subunits, gp41 and gp120 (glycoprotein) during assembly. The gp120 is
the receptor-binding part while gp41 contains the fusion peptide. The gp41 subunit is initially
shielded by gp120, thus kept in its non-fusogenic state. Binding of gp120 to CD4 and co-receptor
results in a conformational change of Env to an extended pre-hairpin form. Gp41 is released,
anchoring the fusion peptide into the plasma membrane and membrane fusion occurs as described
above1, 43.
A pH trigger could also elicit a conformational change in the protein that renders the fusion domain
active. The influenza hemagglutinin (HA) protein is an example of a fusion protein activated by
acidification of endosomes after binding to its receptor, usually a sialic acid, followed by endocytosis
of the virus. The pH decrease triggers a loop-to-helix transition of an interhelical loop in the fusion
domain by protonation of amino acid residues in this loop, resulting in a conformational change of
the HA protein and subsequent release of the fusion peptide into the target membrane46.
Other fusion proteins such as those of coronaviruses, are believed to be activated by the
proteolytically cleavage by host proteases, although the molecular mechanism of Spike protein
activation remains unclear. SARS-CoV S is cleaved on two distinct sides by proteases like cathepsin L
in endosomes of the host cell. The first cleavage in the S1-S2 junction promotes second cleavage at a
S2’ site, which result in a release of the fusion peptide3.
Virus entry is a well studied target for antiviral drugs to reduce symptoms of disease after infection.
To develop such drugs it is crucial to understand the fundamental mechanisms of entry of the virus.
Therefore, many studies are focused on this intriguing aspect of the viral life cycle. Various
approaches to study viral entry are developed by researchers. Unmodified, live viruses can be used
on cultured cell lines and animals. In many fundamental studies pseudotyping systems are used
beside or instead of life virus. Well known are vesicular stomatitis virus delta G (VSV∆G) and
retrovirus based pseudotyping. Pseudotyping is basically replacing a fusion protein of a virus with an
exogenous fusion protein, thereby changing the host cell tropism and mechanism of membrane
fusion. Other experimental systems used in virus entry research are the cell-cell fusion, virus-virus
fusion, virus-like particle (VLP) and virosome based assays. These systems are used as artificial
models mimicking entry of real, possibly highly pathogenic viruses, making the experimental work
easier and exposing the experimenter to lower risk. There are limitations to the different systems
and also it is questionable how strong they represent genuine virus. In this thesis, we evaluate these
limitations and representation of virus entry based on literature examples of studies on coronavirus
entry.
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1 Experimental systems in viral entry research
As mentioned previously, different experimental systems are used to study virus entry and fusion
protein activation. The basic principles and experimental procedures of the different systems are
described in this chapter and examples of their use in coronavirus entry research are given as well.
1.1 Vesicular stomatitis virus ∆G
The vesicular stomatitis virus (VSV) is a virus in the genus of vesiculovirus of the Rhabdoviridae
family, the same as the Rabies virus. Virions of VSV and most Rhabdoviridae are bullet-shaped and
enveloped. Its genome consists of a single-stranded negative RNA molecule of 11-15 kb bound to the
nucleoprotein (N). The N protein forms together with viral polymerase (L) and phosphoprotein (P)
the (ribo) nucleocapsid. Matrix proteins (M) condense the nucleocapsid into a helix within the capsid,
while the glycoprotein (G) is located in the envelope. Entry of VSV into host cells is mediated by the
VSV-G protein, the only fusion protein of VSV35. Since VSV-G binds to many different receptors,
probably even phospholipids, VSV is able to infect various types of insect and mammalian cells. VSV is
investigated for many years and its entry is very well understood, but up until today new details are
found35.
VSV is also used by various researchers for studying virus fusion protein behaviour of other viruses by
pseudotyping.
When the coding sequence of the G protein is deleted from the viral genome, VSV lacking G will be
formed, called VSV∆G. The infectivity of these particles can be rescued by transfecting producer cells
with an expression vector coding a viral glycoprotein as described by Fukushi et al., 200613. This
process is called transcomplementation. It allows the researcher to incorporate exogenous fusion
proteins, for example from foreign viruses, into VSV, thereby generating pseudotyped VSV∆G.
Pseudotyped VSV particles have the host cell tropism of the donor of the fusion protein and cell
entry occurs characteristic of the fusion protein. Infection of cells by the pseudotyped virus can be
detected by replacing the G gene for a reporter gene, such as green fluorescent protein (GFP). The
number of infected cells can be quantified by fluorescence microscopy. This makes VSV∆G a model
for studying viral entry behaviour without working with genuine virus itself, thereby circumventing
safety concerns. In a typical experiment, producer cells are transfected to produce a fusion protein of
interest. After 1-4 days the expression can be checked by protein biochemistry. Next, the cells are
transfected with VSV∆G. A simplified schematic drawing of this procedure is shown in Figure 2. It
takes about 48 hours to generate pseudotyped VSV∆G and infectivity of the VSV∆G pseudotype can
be measured in about 7 to 16 hours after inoculation of the cells with the pseudovirus13.
Fukushi et al. 2006 used the VSV∆G pseudotyping assay to study inhibition of SARS-CoV S mediated
infection by specific inhibitors and neutralization by antibodies. They showed that infection by
VSV∆G pseudotyped with SARS-CoV Spike protein is solely dependent on functional Spike protein,
since both neutralizing anti-SARS-CoV antibody and the ACE2-specific peptide inhibitor DX600
inhibits infection by VSV∆G-SARS S but not by VSV∆G-G13.
The VSV∆G pseudotype was also used in 2008 by Glende et al. to study the cholesterol dependency
for SARS S mediated infection15. Cells were first treated with methyl-β-cyclodextrin, a drug that
sequesters cholesterol from the plasma membrane. Next, the cells were infected by either VSV∆G
pseudotyped with SARS S or VSV-G. The infectivity of the S pseudotyped VSV∆G was reduced while
that of VSV∆G transcomplemented with G was not.
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In another study by Schwegmann-Weßels in 2009 the spikes of two coronaviruses, SARS-CoV and
porcine transmissible gastroenteritis virus (TGEV), are compared in their ability to mediate infection
by pseudotyped VSV. It was observed that the pseudotypes have the same host cell tropism as the
donor virus36.
Figure 2 – Simplified overview of VSV∆G-SARS S generation. To generate a VSV∆G pseudotyped with the spike of SARSCoV, a replication incompetent VSV∆G pseudotyped with G is created (normally bullet-shaped but simplified in this
drawing). Infection of producer cells with this virus and co-transfection of a vector expressing SARS-CoV-S results in VSV∆GSARS-S pseudotype production.
1.2 Retrovirus / Lentivirus based pseudoparticles
Retroviruses and the subgroup of lentiviruses are enveloped single-stranded RNA viruses and have a
diploid genome with a length of 8-11 kb. The host species of all retroviruses belong to the
vertebrates. One of the most known lentiviruses is the human immunodeficiency virus (HIV), which
causes acquired immunodeficiency syndrome (AIDS) and is responsible for many victims over the
world. Retroviruses reverse transcribe their RNA genome into double stranded DNA and integrate it
into the host cells genome by the integrase enzyme. Retroviruses are therefore investigated as a
gene delivery tool.
For virus entry research, pseudotyping assays based on retroviruses such as HIV and MLV are
commonly used4, 14, 17, 25, 37. Creating single-round infectious pseudotyped retrovirus pseudoparticles
is performed by transfecting producer cells with a plasmid containing retroviral Gag-Pol (HIV or MLV),
a second vector that allows expression of the fusion protein of interest and a third plasmid
containing a packaging construct (see Figure 3). According to Belouzard et al. 2009, it takes 72 hours
to incubate cells with the three plasmids to produce pseudotyped retroviral particles before
infectivity can be measured.
Gag (group-specific antigen) codes all structural and core proteins, the matrix (MA), capsid (CA) and
nucleocapsid (NC) proteins. Pol (polymerase) codes the viral reverse transcriptase, protease and
integrase. The Gag-Pol poly-protein is able to mediate membrane-budding by itself when expressed,
thereby forming virus-like particles20. The packaging construct contains a retroviral Psi packaging
element, an element in the retroviral genome which regulates the packaging of the genetic material
into the capsid of a retrovirus during assembly. It can be constructed to contain a reporter gene like
GFP to monitor transduction of cells by the retroviral pseudoparticles. When the producer cells
express the fusion protein of interest on the plasma membrane, the budding pseudoparticles takes
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some of them in their envelope as well. The efficiency of their incorporation differs between fusion
proteins. The reporter gene is integrated into the genome of transduced cells and enables detection
of infection by the formed retroviral pseudoparticles. This way it is possible to compare different
variants of a fusion protein, like wild type and mutants or multiple serotypes for their capability to
mediate entry into cells.
Pseudotyping of retroviruses with the spike protein of the SARS-CoV was first studied by Giroglou et
al. in 200414. It was found that MLV pseudotyped with SARS-CoV S has the same tropism of host
species and cell types as genuine SARS-CoV. These results indicate that the SARS-CoV is able to
mediate virus entry of retroviral pseudoparticles, suggesting retroviral pseudotypes as a valuable
system for investigating entry mediation by the fusion protein.
In studies by Belouzard et al. in 2009 and 2010, SARS-CoV S pseudotyped MLV assays were used to
investigate the cleavage sites in the spike protein. In the 2009 study, it was revealed that cleavage by
trypsin in the S1-S2 and S2’ sites of S is required for infection when the endosomal entry route is
blocked by NH4Cl. Based on the observations with the pseudotype assay, it was concluded that for
SARS-CoV entry, sequential cleavage at both S1-S2 and S2’ occurs, with the first cleavage promoting
the second, before membrane fusion3.
The 2010 study utilized MLV-SARS S pseudotypes to study spike cleavage by elastase, a serine
protease. It was observed that elastase mediated transduction by the pseudotyped virions was
decreased by an amino acid substitution in the S2’ site, indicating that this amino acid is important
for cleavage by elastase4.
Shulla et al. used MLV pseudoparticles to investigate the importance of transmembrane
protease/serine subfamily member 2 (TMPRSS2) in binding of SARS S to ACE2. Observed was that
TMPRSS2 co-localizes with ACE2 on the plasma membrane and that it promotes entry of both
genuine SARS-CoV as SARS S pseudotyped HIV particles37. It was earlier reported by Matsuyama et al.
in 2010 that SARS-CoV cell tropism correlates with TMPRSS2 rather than ACE227, confirming the
importance of TMPRSS2 in SARS-CoV infection as found by Shulla et al..
Figure 3 – Simplified overview of retroviral pseudotyping. To generate retroviral pseudoparticles,
producer cells have to be co-transfected with three plasmids: first containing retrovirus gag-pol gene,
second containing a packaging construct with a reporter gene and third expressing a viral fusion
protein of interest (SARS-S in this figure).
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1.3 Cell-Cell fusion assay
The cell-cell fusion assay is a widely used model to study membrane fusion induced by viral fusion
proteins. This assay is based on the principle that two batches of cells are mixed together: one
transfected with plasmids expressing the viral fusion proteins, and the other with plasmids
expressing the host virus receptor. If active fusion protein and matching receptor are present, the
plasma membranes of both cells fuse together. A reporter gene, like luciferase, that is only
expressed upon fusion of both cells is usually used. By performing a cell-cell fusion assay, the
function of fusion proteins and the mechanism of their activation as well as their receptor and its
specificity can be studied without any viral particle.
In a study by Madu et al. 2009, mutated forms of the SARS-CoV S protein were assessed for their
fusion activity. They used BHK-21 cells co-transfected with mutant or wild type spike protein and a T7
polymerase-driven luciferase gene and overlaying with susceptible Vero E6 cells transfected with the
T7 polymerase gene 25. They used alanine scanning in the S2 domain to find a region that is essential
for activity of the fusion protein 25.
Besides the MLV retroviral pseudotyping assay as mentioned previously, Belouzard et al. 2010 also
uses a cell-cell fusion to investigate the importance of the T795 amino acid residue for elastase
mediated activation of the SARS S protein. By comparing the luciferase activity after fusion of HEK
293T cells transfected with either wild type SARS-CoV S or T795D S and Vero E6 cells, it was found
that T795 is at least in vitro important for elastase-mediated fusion activity of the SARS spike protein,
confirming the observations of the pseudotyping assay4.
A cell-cell fusion assay was also used by Simmons et al. 2011 to study SARS S cleavage by multiple
host proteases. A twofold higher luciferase activity was measured for the T760R variant of the spike
protein in comparison to wild type SARS-CoV S, suggesting that this amino acid substitution
augments the activation of S. It was also found that overexpression of furin enhances the fusion
activity of both wild type and T760R S. The same researchers used a similar assay in 2011, to
determine the role of amino acid residue R667 in the proteolytic cleavage of SARS-CoV S by both
trypsin and cathepsin L39.
1.4 Virus-virus fusion assay
Another method to study virus fusion is the virus-virus fusion assay, also called an intervirion fusion
assay (see Figure 4). In this system, two types of viral particles are generated, one expressing viral
fusion proteins and the other expressing a target receptor. When mixed together, fusion of the viral
envelopes occurs after binding and activation of the fusion protein.
One particle usually harbours a studied fusion protein, and contains a reporter gene. The second
particle expresses the receptor for the fusion protein and a reporter fusion protein of a different
virus. To monitor the fusion between the two viruses, the fused virus is allowed to infect cells that
are susceptible only to the particle with the reporter fusion protein, but not by the particles with the
reporter gene and glycoprotein of choice. After infection, the reporter gene is expressed in the target
cells. Only fused particles are able to infect cells and transduce the reporter. The initial particles are
either missing the fusion protein or the reporter gene.
A lipid mixing assay, with one of the particles containing labelled lipids like R18 in the envelope, could
also be used to monitor fusion. R18 is a self-quenching molecule that inhibits emission of light at a
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high density. After fusion of the envelop of both viral particles, the R18 disperses over the membrane
of the fused virions and starts to emit light, which can be detected.
In 2005 Simmons et al. reported the use of a virus-virus assay to determine the impact of cathepsin L
inhibitors on the infectivity of SARS-CoV. HIV-luc(ACE2) particles (HIV particles covered with ACE2,
the SARS-CoV receptor, and expressing luciferase) were allowed to bind to HIV-gfp(SARS S/ASLV-A)
particles (HIV particles bearing S and Avian Sarcoma Leukosis Virus Envelope-A (ASLV-A) and
encoding GFP). By infecting HeLa cells, which express Tva, the cellular receptor for ASLV-A, with the
mixed virions, the genome encoding luciferase is transferred, indicating that intervirion fusion
between HIV-luc(ACE2) and HIV-gfp(SARS S/ASLV-A) has occurred.
The same researchers used a similar assay in 2011, to determine the role of amino acid residue R667
in the proteolytic cleavage of SARS-CoV S by both trypsin and cathepsin L39.
Figure 4 – Virus-virus fusion assay with SARS-S pseudotypes. SARS-S/Avian Sarcoma leukosis virus (ASLV) A pseudotyped
retrovirus are allowed to fuse with ACE2 pseudotyped GFP encoding virus. ASLV susceptible ACE2 - cells are infected with
the fused virus. Infection results in expression of GFP. Since the ACE2 pseudotyped virus carries the GFP gene, only fused
virus results in GFP expression.
1.5 Virus-like particles
Virus-like particles, or VLPs, are essentially empty virions composed of some structural proteins, not
containing a viral genome. To produce VLPs, multiple structural proteins need to be produced in
sufficient amounts and have to be assembled correctly into a particle resembling the capsid of a life
infectious virus. A good expression system is necessary to meet the first requirement. The expression
of viral structural proteins of some viruses is sufficient for membrane budding to occur and thereby
for the assembly of VLPs. The VLPs are believed to behave similar to real virus particles, attaching to
cells and have their membrane fusing with the plasma membrane of the target cells.
In 1997, Bos et al. used VLPs to study the infection mediated by the fusion protein of MHV-A597. It
was found that cleavage of the spike protein is not necessary for infection. However, the specific site
of the cleavage was not mentioned in the publication.
Mortola et Roy reported in 2004 that they successfully produced SARS VLPs by expressing the S, E
and M proteins using a recombinant baculovirus. While co-infection with two viruses expressing E
and M results in assembly of VLPs, expression of E, M and S by a single recombinant virus resulted in
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both assembly and release of VLPs. By using electron microscopy (EM), it was observed that the VLPs
resemble life SARS virions with spike protein on the surface (Figure 5)29. These VLPs were not
assessed for their capability to fuse with target cells, so it remains uncertain whether these particles
were infectious.
VLPs can also be reconstituted for non-enveloped, or naked, viruses like poliovirus. Bräutigum et al.
generated in 1993 poliovirus-like particles by using a recombinant baculovirus expressing the VP0,
VP1 and VP3 structural proteins of poliovirus. The VLPs generated this way resembles poliovirus
virions in size, antigenicity and form. However, the amount of VLPs isolated was rather low8.
Figure 5 – SARS-VLPs (adopted from Mortola et Roy, 2004)29. (Left) EM-photograph of SARS-VLPs. The VLPs resemble SARS
virions. (Right) Immunogold labeling of SARS-Spike protein.
1.6 Lipoparticle/Virosome
Giant unilamellar vesicles (GUV) and giant plasma membrane vesicles (GPMV) are used as model
systems for (intra-)cellular membrane processes. GUVs are commonly produced by sonication of an
aqueous solution with lipids, thereby forming bilayered vesicles of different sizes. GUVs have a size of
approximately 50 µm. Membrane proteins, such as virus fusion proteins, can be reconstituted into
membrane of the GUVs by solubilising the proteins with a detergent. The detergent allows
spontaneous insertion of the proteins into the membrane of the vesicles.
In contrast to the in vitro reconstituted GUVs, GPMVs are obtained from cells. GPMVs can be
produced by incubating cells with special buffer containing formaldehyde and dithiothreitol for 1
hours. Constant shaking at 37°C will cause formation of membrane blebs which eventually pinch of
from the cell surface, thus forming plasma membrane derived vesicles. These vesicles contain
proteins expressed on the surface of the cells they derived from and cytoplasmic factors as well. By
transfecting the cells from which the GPMV originates first with vectors expressing a viral fusion
protein, a fusogenic GPMV can be produced.
Both GUVs and GPMVs can be used to study activation of fusion proteins by different factors.
Nikolaus et al. (2010) utilized GUVs and GPMVs to study the localization of by influenza
hemagglutinin (HA) into lipid rafts32. They chose to use GUVs and GPMVs because in cellular plasma
membranes the rafts they want to study are usually of submicroscopic size, making it difficult to
study raft formation by HA with light microscopy. It was found that HA preferentially localizes to
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specific lipid domains in the membranes in both GUVs and GPMVs by using both fluorescent labelled
lipids as labelled HA. These domains (called liquid-disordered or ld) resemble the rafts of a plasma
membrane. Based on these observations, it was proposed that influenza virus assembly occurs in
lipid rafts.
Liposomes can also be used in a liposome binding assay for studying fusion protein activation. By
incubating a mix of liposomes with soluble virus receptor and virus, virus will bind to the liposomes
when the fusion peptide is released after binding to the receptor. After centrifuging the mix through
a sucrose gradient in a tube, virus bound to the liposome will co-localize with a fraction of liposomes
within the gradient. Different fractions of the gradient can be isolated and checked for the presence
of virus by either immunoblotting (viral proteins) or real-time PCR (viral RNA).
Matsuyama used this assay in 2009 for studying MHV-2 Spike protein activation. By mixing virus with
liposomes in the absence or presence of soluble MHV receptor (soMHVR), it was found that
approximately 50% of the virus was bound to the liposomes in the presence of soMHVR. Based on
these observations it was concluded that receptor binding of MHV induces the release of the fusion
peptide and interaction with the target membrane26.
Devesa et al. reconstituted CD4 and CCR5, the receptor and co-receptor of HIV respectively, into
liposomes11. They did this by adding special tags to the receptors and expressing them in mammalian
cells. The proteins were then purified with a detergent and reconstituted into the liposomes with the
same detergent. Both soluble gp120, the HIV fusion protein, as gp120 expressing cells were able to
bind to liposomes, indicating that the receptors are oriented rightly. Although the experimenters of
this study mainly focused on setting up a new system to reconstitute proteins into liposomes, this
method can also be used to study interactions of fusion proteins with receptors and co-receptors11.
1.7 Coronavirus based pseudotyping
Yount et al., 2002, reported about the successful assembly of the coronavirus Mouse Hepatitis Virus
strain A59 (MHV-A59) from a full-length infectious cDNA clone47. This method can be used for easy
genetic modification of the viral genome.
It could also be used to set up a new pseudotype system based on coronaviruses. However, this
approach is not yet successful and is still under development. Easy genetic modification of cDNA
clones makes it possible to produce spike lacking MHV-A59 without the need for selection. We are
currently reproducing the generation of icMHV-A59 as described in 2002 by Yount et al.. In multiple
steps a sufficient yield has to be gained for a successful production of a MHV-A59 clone. Seven
plasmids, containing subsequent fragments of the MHV-A59 genome, have to be prepared. The
fragments have to be excised from the plasmid preparation by restriction digest. Ligation of all
fragments is performed to make a full-length cDNA clone. The cDNA is then transcribed to make
infectious RNA. The genome size of MHV-A59 is approximately 31.5 kb. Since transcription has a
lower fidelity than DNA replication, RNA transcripts will contain errors or the transcription is even
aborted before completion. Therefore, the yield of full-length infectious RNA from the in vitro
transcription is low. Nevertheless, a single, complete RNA molecule could be enough to generate
icMHV-A59 de novo.
Another positive single-stranded (+ssRNA) RNA virus of which infectious transcripts from full-length
cDNA clones was created, is the Sindbis virus, as reported by Rice et al. in 198734. The produced virus
was identical to authentic Sindbis virus. In the publication, multiple applications for full-length cDNA
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clones of viruses are reported. The generation of cDNA makes side directed mutagenesis by
homologous recombination easier and as the cDNA fragments are usually ligated into vector
plasmids, it can be easily amplified in bacteria.
If the generation of MHV-A59 from a cDNA clone is optimized, the same applications mentioned by
Rice et al. are possible for MHV. This makes it a very promising method to create a coronavirus based
pseudotyping system, as by homologous recombination a MHV∆S cDNA clone can be produced.
MHV∆S allows the transcomplementation of MHV with a fusion protein of interest.
In the experimental section of this thesis, the in vitro generation of icMHV-A59 is described.
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2 Uses of pseudotyping outside virus entry research
As described in the previous chapter, pseudotyping and other experimental systems are useful for
virus entry research. However, there are multiple other purposes, worth mentioning. Pseudotyping
can be applied in gene therapy, anti-cancer therapy, targeted drug delivery and to study virus
spreading in a host. In this chapter, some of these uses are presented.
2.1 Gene therapy
Gene therapy is based on altering the genome in cells of some tissues to hopefully cure a disease,
either genetic, like cystic fibrosis (CF)40, or non-genetic such as diabetes33. To introduce the genes of
interest into the host cells, viral vectors can serve as vehicles. Retroviruses are well studied examples.
A characteristic of retroviruses is the permanent integration of their genome into the host cells
genome. Thereby, the introduced genetic information will be reproduced during each cell division,
passing it on to each daughter cell along with the host’s genome. This results in having to admit few
doses of the retrovirus vector for prolonged therapeutic effect, although the retrovirus vectors are
only infectious for a single round.
One of the safety concerns of retroviral vectors is the risk of insertional mutagenesis and oncogenesis
if a gene of cell-cycle regulation or an oncogene is affected. A possible approach to overcome this
concern is reported by Lim et al. in 2010. Inserting Zink-finger domains at different sites into the GagPol gene of MLV directed the integration inside the host genome24. With the results of this study,
retroviral vectors could be further developed to integrate their genetic information safely.
Given that retroviruses have a limited range of cells they can infect, pseudotyping is a tool to retarget
the vector only to the cell types in which the therapeutic genes need to be inserted. This way, a
lower dose of vector is sufficient and the risk of adverse events is possibly reduced.
2.2 Anti-cancer therapy
Tumour cells are normally killed by the immune system through cytotoxic T cells and natural killer
cells. The immune cells recognize the abnormal cells because they express either abnormal proteins
at their surface or normal proteins in an increased number, so called tumour markers. However, if
the immune system fails to clear these cells, the tumour can further grow into cancer. Viruses are
developed which specifically target the tumour markers and can be used as therapeutic agents
against cancer.
VSV is studied for its proposed oncolytic capabilities. Stojdl et al. 2000 found that VSV is very
sensitive for the cellular antiviral responses of the host and therefore VSV infections rarely result in
disease in healthy persons. Since this antiviral defence is often defective in tumour cells, these cells
are particularly sensitive to VSV infection while healthy cells are mostly remained intact41. This was
revealed in an experiment in which both tumour cells and normal cells were infected with VSV after
exposure to high concentrations of interferons, a class of signalling molecules which triggers
uninfected cells to go into an antiviral state, making them less susceptible to viral infection.
To even more specifically target cancer cells, pseudotyped vectors can be used. Muik et al. 2011
described VSV pseudotyped with the glycoprotein of lymphocytic choriomeningitis virus (LCMV)
which was able to destroy brain tumour cells but spares normal neuronal cells, as LCMV is a nonneurotropic virus31. While VSV has oncolytic activity, as described before, it also has a very broad cell
tropism, infecting nearly any type of cells and especially neurons. Therefore, pseudotyping might be
necessary to target this virus specifically to tumour cells while sparing healthy tissue, reducing the
risk of adverse events, just as with gene therapy.
12
2.3 Targeted drug delivery
VLPs and liposomes can be constructed to contain drugs. By reconstituting these particles with the
envelope glycoproteins of a virus, they can be used to deliver the drugs specifically at the targeted
cells.
Brown et al. described in 2002 a method of creating bacteriophage VLPs which contain
macromolecular drugs9. They reported that the use of drugs brings multiple obstacles to overcome,
such as negative side effects, immune responses and accessibility of targets and that VLPs could be a
useful tool to beat these challenges.
2.4 Vaccines
Both VLPs and pseudotyped viruses are can be used as a vaccination strategy. VLPs are already in use
as a subunit vaccine against human papilloma virus (HPV) and are studied for use against influenza
virus as well. In a study by Bai et al. 20082, VLP’s were created by co-infecting cells with two
recombinant Baculoviruses, one expressing the S protein of SARS-like Coronavirus(SL-CoV) from bats
and one expressing the E and M proteins of human SARS-CoV. The resulting BVLPs were shown to
induce cytokine production by dendritic cells and activation of T cells. This suggests that VLPs with S
protein might be able to induce an immune response, thereby possibly a strategy for vaccination
against SARS-CoV2.
Kapadia et al. reported in 2008 about VSV∆G pseudotyped with SARS-S as a vaccine strategy against
SARS-CoV23.. The Food and Drug Administration usually hardly approve vaccines based on life virus,
because of the risk that mutations or recombination with wild-type viruses could render the vaccine
virus pathogenic again. The VSV∆G-S pseudotype generated by Kapadia et al. was shown to be
infectious for one round only, while still eliciting a neutralizing antibody response in mice. Therefore,
VSV∆G-S vaccine could be a safe alternative for life attenuated virus as a vaccine23. A possible
explanation of S not being able to mediate infection of VSV∆G-S, is that here full-length S is used,
which according to Fukushi et al. and Giroglou et al. is not efficiently incorporated into heterologous
viruses 13, 14. Kapadia et al. reported that the amount of S on VSV∆G-S was small, confirming the
observations of Fukushi et al.13, 23.
2.5 Testing anti-viral drugs and monoclonal antibodies
Pseudotyping systems can also be used to study the effect of anti-viral drugs and monoclonal
antibodies on the infection. Bian et al. (2009)5 used both HIV-based pseudovirus and cell-cell fusion
assays to test the effect of monoclonal antibodies against two amino acid epitopes in the SARS spike
protein. From information from both assays it could be concluded that the monoclonal antibodies
were able to neutralize SARS CoV, as was confirmed in an experiment using life virus 5. Because of the
low biosafety, pseudotyping can be used in high throughput facilities for drug assays.
2.6 Study mechanisms of viral spreading in the host
Besides viral entry into host cells, the transport of the virus through the body of the host can also be
studied by using pseudotyping systems. Mazarakis et al. (2001) 28 pseudotyped retroviral vectors with
the G protein of Rabies virus. While VSV-G pseudotyped retroviruses were able to transduce neurons
anterograde after injection in the central nervous system (CNS), retroviral particles pseudotyped with
Rabies G were able to transducer neurons retrograde after intramuscular injection and even reaches
the CNS. Based on these conclusions, it can be concluded that retrograde axonal transport is solely
mediated by the G protein of Rabies virus. This may be valuable information for producing a noninvasive therapy against neurological disease in the CNS based on a pseudotyping system28.
13
3 Advantages and disadvantages of pseudotyping for experimenter
Pseudotyping systems do have advantages and disadvantages for the experimenter.
As many viruses are hazardous pathogens for men and animals, biosafety has to be ensured to be
allowed to work with them. Therefore, the work with genuine viruses is restricted to specialized and
strictly controlled laboratories. Although this increases safety, it also restricts the experiments. By
using pseudotyping systems these restrictions can be bypassed.
Psuedotyping systems allow the experiments to be performed in a Biosafety Level 2 (BSL-2)
laboratory6, 30. There are five Biosafety Levels, from BSL-0 to BSL-4. Higher Biosafety Levels means
more restriction, as the biohazard of the materials worked with is also higher. This makes it possible
for the experimenter to study entry of highly pathogenic viruses, such as SARS or Ebola, without
exposing himself to biohazards and work under less stringent regulations, making the research more
convenient30. In a Biosafety Level 3 or 4 laboratory (BSL-3 or 4) all personal are required to wear
special protective cloths and work in specific containment devices like a safety cabinet or hood.
Because everything in these labs needs to be disposed after use and maintenance is complicated in
Biosafety labs, it is also expensive to maintain such a facility. Therefore, research institutes rarely
have BSL-3 or 4 labs. To be allowed to work in a high level safety lab, an experimenter has to be
trained thoroughly in handling biohazardous material and has to be supervised by a qualified
scientist. Every experimental step has to be precisely documented and prepared beforehand.
Some viruses cannot be propagated in cultured cell lines or there are only few, often adapted strains
of the virus which do so. Viruses need both susceptible and permissive cells to grow. Susceptibility
means that a virus is able to bind to and enter host cells, while permissibility stands for the ability of
a virus to establish productive replication. VSV and retroviruses, both widely used as pseudotyping
vectors, are able to grow on a many types of cells with high titres; they are easy to culture.
Therefore, using pseudotyping systems can make studying entry of viruses with a narrow tropism
easier. HEK293T cells, which are often used in virus entry research, also have the advantage of being
easy to transfect with foreign genetic material.
Reverse genetics is a popular tool in virology and molecular biology to study the function of a protein
by introducing mutations and analyzing the effects of the mutation on the phenotype of the protein.
For example, site directed mutagenesis helps to study the activation mechanisms of fusion proteins
by altering the amino acids of important parts of the protein, such as in proximity or in protease
cleavage sites. Since most pseudotyped systems exploit plasmid vectors to transcomplement the
pseudovirus with a foreign fusion protein, the gene for this protein is more accessible for targeted
mutagenesis. To introduce mutations into a life virus more complex procedures like homologous
recombination or full-length cDNA clones are necessary. By homologous recombination, genomes of
RNA viruses can be altered. This is usually performed by infecting cells with life virus and co-transfect
them with a cDNA or RNA molecule which contains the mutated gene. The viral polymerase can
switch from the viral gene to the introduced, modified template, creating recombinant genomes.
Since this does not occur efficiently, a mix of parental and recombinant virus is produced, which
requires subsequent negative selection. De novo generation of virus from full length cDNA is
described above for RNA viruses (Section 1.7). The experimental procedure is difficult for viruses with
large genomes, but commonly used for viruses with a small genome. For some viruses the expression
14
of helper proteins is essential to start infection. The advantage of the virus generation by cDNA is the
reduced need for selection of the correct clone. Moreover the method can also be applied for DNA
viruses.
Generating mutant variants of life viruses by targeted mutagenesis usually takes multiple steps to be
performed precisely. Because the release of genetically altered organisms into the environment is
undesired, experiments with them need to be performed in an enclosed environment. Under high
biosafety conditions, as required when working with highly pathogenic viruses, there may not be a
protocol available to perform such experiments and establishing experimental procedures and
protocols is very inconvenient. For pseudotyping systems and different fusion assays, many
descriptions of the procedures are widely available and can often be transferred to analogous
research questions. Pseudotyped viruses can also easily be constructed with a reporter gene
packaged in the particles. Examples of these genes are β-galactosidase, (enhanced) GFP and
luciferase. These genes are introduced into the target cells in case of infection. Their expression is
visualized by staining with X-gal (β-galactosidase) or by detecting light emission in the cases of GFP
and luciferase. By using reporter genes, infection by the pseudovirus can be measured and compared
between different subtypes. For that reason, many researchers choose to use pseudotyping systems
instead of studying life viruses.
Maintaining a biosafety level laboratory requires parallel facilities tools with the normal laboratory.
The tools used in a high biosafety laboratory for experiments are not allowed to be taken out unless
decontaminated. This includes pipettes, centrifuges and incubators. Apparatuses, like enzyme linked
immune sorbent assay (ELISA) scanners, fluorescence-activated cell sorting (FACS) systems and
modern microscopes are rather expensive. Another advantage of pseudotyping is that it allows the
experiments to be less expensive since most work can be performed in the more common
laboratory. Also, the maintenance in high Biosafety laboratories is an obstacle, as the maintenance
personal is not allowed to visit the facility or need special training. These costs of performing
experiments in biosafety facilities are another reason to prefer working with pseudotyping systems
instead of genuine virus.
However, as described further in this thesis, the representation of real virus behaviour by
pseudotyped viruses is questionable. When the experimenter defends his experimental approach, he
has to convince the peers that the pseudotyping system is comparable to life virus entry. The
experimenter has either to deliver evidence from other studies that the experimental methods
deliver plausible results or compare entry of pseudotyped viruses with life virus itself, the latter still
need to be performed in a BSL-4 lab. Nevertheless, all experiments to test a hypothesis can be
performed with pseudotyping assays, limiting the number of experiments to confirm the
observations. Therefore, the use of pseudotyping systems significantly reduces the need to work in a
high biosafety laboratory, thereby making studying virus entry behaviour more convenient and costbeneficial to perform.
15
4 Technical limitations of pseudotyping systems
The assembly of viruses requires multiple structural proteins to be correctly incorporated in a
concerted fashion. Structural proteins have a specific binding adaptors or binding partners and often
require modifications that allow them to form complex structures to assemble the virus particle.
Pseudotyping is the replacement of a fusion protein with an exogenous protein. It is essential that
the pseudotyped virus allows sufficient incorporation of the fusion protein of interest. An important
aspect under consideration is whether the fusion protein is efficiently located to the same cellular
location as where assembly of the pseudoparticles takes place. Nevertheless, not every fusion
protein is suitable for efficient pseudotyping.
An example of problematic incorporation into pseudotyped virus particles is the Human
Parainfluenza type 3 (HPIV3). Jung et al. pseudotyped lentivirus particles with HPIV3 envelope
proteins hemagglutinin-neuramidase (HN) and fusion protein (F), but the particles had a low
infectious titre21. In another study, also by Jung et al., in 2007, the efficiency of the incorporation of
the HPIV3 envelope glycoproteins was low22. They compared the expression of HN and F by HPIV3
infected cells with cells transiently transfected with plasmids encoding these glycoproteins. The
amount of cytosolic mRNA of HN and F were similar between both, but there were fewer
glycoproteins on the cell surface of transfected cells than on infected cells. This might implicate that
other viral proteins expressed during infection mediate transport to the plasma membrane, such as
accessory proteins. To improve the quantity of surface expressed HN and F, codon optimized
plasmids were used. The amount of HN and F on the cell surface increased, as well as the titre of
lentiviral pseudoparticles. However, the quantity of glycoproteins on these particles was much lower
than the amount of genuine lentiviral envelope protein on wild type virus. The study suggests that
retroviruses are a suitable pseudotyping vector, but it has to be evaluated if the used glycoprotein
for pseudotyping is incorporated efficiently enough for proper research. It also indicates that
pseudotyping does not resemble natural incorporation of fusion proteins.
The spike protein of SARS-CoV and maybe that of other coronavirus as well, is another example of a
viral fusion protein which is not efficiently incorporated into pseudoparticles.
Giroglou,T et al. 2004, reported about a study in which the infectivity of MLV pseudotyped with
different SARS S constructs with C-terminal truncations or even transmembrane domain deletions
was compared with wild type S. MLV pseudotyped particles with SARS S expression constructs with a
cytoplasmic truncation were more infectious than with full-length SARS S14.
A similar observation was seen in a study by Fukushi et al., 2006 with a VSV∆G based assay. They
compare the infectivity of VSV∆G* (VSV∆G with G replaced by a GFP gene) pseudotyped with either
wild type SARS S or SARS S in which 19 C-terminal amino acid residues were truncated (SARS St19).
They found that VSV∆G* pseudotyped with SARS St19 was significantly more infectious than VSV∆G*
pseudotyped with wild type SARS. This indicates that wild type SARS S is less efficiently incorporated
into VSV particles and that C-terminal truncation is necessary to efficiently produce infectious SARS S
pseudotyped VSV particles13.
The study by Schwegman-Weßels et al. revealed that VSV∆G-SARS S pseudotypes where more
infectious than VSV pseudotyped with TGEV S. They also confirmed the observation of Fukushi et al.
that cytoplasmic truncation of SARS S results in higher infectivity of VSV pseudotypes. For TGEV,
deletion of the retention signal, which normally keeps the S protein in the endoplasmic reticulum
16
(ER) or Golgi, is even required to produce infectious pseudotyped particles. Exchange of SARS-S and
TGEV S cytoplasmic tails was detrimental for infectivity36.
The similarity between the observations of Giroglou et al. with MLV pseudoparticles and both
Fukushi et al. and Schwegmann-Weβels et al. with VSV∆G suggests that the cytoplasmic part of wild
type SARS spike protein hinder the incorporation into pseudoparticles. Giroglou et al. mentioned two
possible explanations for these observations. One was that the cytoplasmic part of the spike protein
contains endoplasmic reticulum (ER) retention signals, keeping the protein in the ER, while both MLV
and VSV are assembled at and bud from the plasma membrane. Deleting these retention signals
should allow more efficient transport of SARS spike protein to the plasma membrane. As none of the
truncated S constructs showed an increased surface expression compared to wt Spike protein, this
hypothesis can be rejected. The second was that the size of the cytoplasmic domain could interfere
with formation of viral particles. Attempts to create MLV pseudotyped with a chimeric construct with
the ectodomain of SARS spike protein and the cytoplasmic domain of MLV envelope glycoprotein did
not result in efficient expression on the cell surface14.
Deletion of highly conserved cysteine’s within the cytoplasmic domain results in a drastically reduced
infectivity of MLV pseudotyped with SARS S, suggesting that some parts of the cytoplasmic domain
are essential for the function of the protein14. The cytoplasmic tail can possibly be involved in the
folding of the transmembrane and extracellular domains, thereby being responsible for the function
and structure of the whole protein. It could be paramount for proper receptor binding, fusion
mediation and incorporation into the virus particles. In case of pseudotyping assays in which
truncation is necessary for efficient generation of pseudoparticles, it is an important question
whether the fusion protein is still behaving in the same way as the wild type form. Similar receptorspecificity (viral tropism) or inhibition by drugs between wild-type and pseudotyped virus indicate a
normal function of the fusion protein, so these are performed as control assays. In the studies by
Giroglou et al., the pseudotyped MLV pseudoparticles with SARS S protein truncated at different
length show similar host cell tropism as SARS-CoV, indicating that the protein was still functional.
One of the difficulties of having glycoproteins incorporated into vector pseudoparticles with less
efficiency is that the generated pseudotype also has a lower infectivity then an authentic virus.
Because of this issue, the infectivity of a pseudotyping system can only be qualitatively compared to
that of a life virus and not quantitatively.
17
5 Comparison between different pseudotyping systems and life virus
There are various pseudotyping systems to study virus entry. Therefore it is of importance to
compare and understand each system and evaluate if they represent real virus entry behaviour.
5.1 Cell-cell fusion compared to virus-cell fusion
The cell-cell fusion assay is used to study membrane fusion by viral fusion proteins. No viruses are
directly involved, because this assay is based on the fusion between the plasma membranes of two
cells. Therefore, it is necessary to evaluate if the fusion between two cells resemble natural fusion
between a virus envelope and a target membrane.
In 2006 Follis et al. studied the influence of cleavage on the fusion capacity of SARS-CoV S12. To allow
Spike cleavage by furin, a furin cleavage site was introduced at the S1-S2 domain junction. Furin is a
proprotein convertase enzyme which converts a proprotein to its active form. The effect of furin on
the membrane fusion was assessed by both a cell-cell fusion and retrovirus based pseudotyping
assay. In the cell-cell fusion the addition of purified furin protease to the cells enhances the fusion
capacity of the spike protein. In contrast, the infectivity of the retroviral pseudotypes was not
increased by the presence of furin during infection. A possible explanation for these contradictory
observations is that SARS-CoV entry requires two cleavages in the spike protein, one in the S1-S2
junction and the second at the S2’ site, as reported by Belouzard et al. 20093. While furin might
mediate the first cleavage, which could be sufficient for membrane fusion, a second cleavage is still
required for entry of virions.
Similar observations were done earlier by de Haan et al. in 2004 for the coronavirus MHV-A5918. Cellcell fusion but not entry of virus was affected by a furin inhibitor peptide (Figure 6). This indicates
that cell-cell fusion is dependent on furin cleavage and that for virus entry other processes are
involved which have different requirements. Possible explanations for the contradictories between
cell-cell fusion and virus-cell fusion were given by the authors. The difference in composition of a cell
membrane and virus envelope can be of great importance to how the fusion protein acts. The plasma
membrane and virus envelope differ in lipid composition, fusion protein density and other
membrane proteins18. The plasma membrane contains more cholesterol which makes it more
flexible. The plasma membrane contains more cholesterol which makes it more flexible. Therefore,
fusion between two plasma membranes might occur easier than fusion between a viral envelope and
a cellular membrane. Also, the possible contact area between the plasma membranes of two
neighbouring cells is larger than that between a viral envelope and cell membrane, allowing more
spike protein to receptor complexes to be formed which are involved in membrane fusion.
Simmons et al. 2011 also provided inconsistent results between cell-cell fusion and virus-cell fusion
when studying the dependency of Cathepsin L for spike activation. They showed that cell-cell fusion
happens independently of cathepsin L, while virus-cell fusion is dependent39. The explanation could
be that cathepsin L is a lysosomal protease and is activated by low pH. The lysosome is the natural
location of virus-cell fusion by SARS spike protein occurs after endocytosis of the virus particle. Since
in a cell-cell fusion assay fusion only occurs at the surface of the cells, cathepsin L is likely not
involved. Therefore, other proteases must be responsible for coronavirus S activation on the plasma
membrane. They could have provide endogenous cathepsin L and decrease the pH to activate it in a
similar way as they did with the virus-virus fusion assay.
18
However, Belouzard et al. (2010) find correlating results in both a cell-cell fusion and life SARS-CoV
assays when studying the role of T795 in elastase-mediated cleavage of Spike. Mutation of T795
resulted in both a reduction of cell-cell fusion and infectivity of life SARS-CoV after treatment with
elastase4.
The contradictory observations between cell-cell fusion and virus entry as described by De Haan et
al. (2004) and Follis et al. (2006) but comparable results by Belouzard et al. (2010) indicate that cellcell fusion assays are limited experimental systems. The viability of the cell-cell fusion assay depends
on the research question. In the first two studies, the cleavage at the S1-S2 junction was
investigated, while Belouzard et al. studied the S2’ cleavage by elastase. In a previous study by
Belouzard et al. 2009 it was stated that possible cleavage in S1-S2 is more important for virus
infection than cell-cell fusion3. Until today, the precise molecular role of the two distinct cleavage
sites in virus entry is unclear.
Although cell-cell fusion assays could provide information about fusion protein activity and have the
advantages of easy establishment and manipulation, the results of studies by Follis et al. and de Haan
et al.12, 18 indicated that good controls are pivotal to draw fundamental conclusions. Moreover, the
experimenter has to be confident that this assay provides the answer on his question.
Figure 6 – Comparison between cell-cell fusion and life MHV-A59 infection with furin inhibition (adopted from De
Haan et al. 200418. A Furin inhibitor dramatically reduces cell-cell fusion activity by MHV S. . Cells were supplied with
expression plasmids for MHV-Spike protein (+S) or control plasmid (mock). Cell-Cell fusion was measured using a
luciferase reporter gene under the T7 promoter that is actively transcribed only after fusion with cells expressing the
T7 polymerase. The addition of furin inhibitor (+inhibitor) completely abolishes cell cell fusion. B Infection of LR-7 cells
with MHV-A59 carrying the luciferase reporter gene was not significantly reduced by the addition of furin inhibitor.
5.2 Retrovirus based pseudotyping compared to life virus
As previously mentioned, the retrovirus based pseudoparticle is often used to study virus entry by
fusion proteins. The pseudotyping assay can be considered to be close to natural virus entry, as it is
based on infectious virus particles which only involve membrane fusion at the cell surface. However,
as a retroviral pseudoparticle is different than the original virus, it is still essential to assess its degree
of representation of genuine virus entry.
19
In a study by Huang et al. (2005) the inhibition of SARS-CoV infectivity by cathepsin L inhibitors is
assessed with both retroviral pseudotyped particles and life SARS-CoV (Figure 7). Infections by the
pseudoviruses and genuine virus were decreased due to cathepsin L inhibition, albeit to different
extend. However, the cells used for the infection assays were different between life SARS-CoV (Vero
118) and MLV-pseudotypes (HEK293T), which might also affect the results19.
Shulla et al. utilized a retrovirus based pseudotyping system to study the effects of transmembrane
protease/serine subfamily member 2 (TMPRSS2) on infectivity of SARS-CoV. TMPRSS2 is a serine
protease which co-localizes with the SARS-CoV receptor (ACE2) on the plasma membrane. It was
concluded from this study that TMPRSS2 enhances infectivity of SARS-CoV, as the infectivity of SARS
S pseudotyped MLV was enhanced. Similar results were seen with genuine SARS CoV, validating the
results of the pseudotyping assay37.
Both studies indicate that retroviral pseudotyping assays are suitable for studying coronavirus entry.
However, as mentioned earlier, Giroglou et al.14 find that truncation of the C terminal tail of the S
protein is necessary for efficient incorporation into retrovirus particles. Therefore, the pseudotyping
does not use the wild-type protein coding sequence. Additionally, the quantity of S protein on
pseudotyped particles may not be equal to that on authentic virus due to less efficient
incorporation, as previously mentioned for HPIV3 in section 422. Thus we can assume that assays
based on retrovirus pseudotyping are merely qualitative instead of quantitatively representing the
real virus infection setting.
Figure 7 – Comparison between life SARS-CoV and S pseudotyped MLV in a cathepsin inhibition assay (adopted from
Huang et al. 2005)19. (Left) Vero 118 cells were infected with SARS-CoV (green) in the presence of Cathepsin B and
Cathepsin L inhibitors. Cathepsin L inhibitor reduces infectivity more than cathepsin B inhibitor. (Right) HEK293T cells were
infected with MLV pseudotyped with SARS-S in the presence increasing concentrations of either cathepsin B, L or both
inhibitor. Infectivity is significantly reduced by cathepsin L inhibitor and to some extent by cathepsin B inhibitor.
20
5.3 VSV∆G compared to life virus
Besides cell-cell fusion assays and retrovirus pseudoparticles, VSV∆G based pseudotyping is also used
to study virus entry mediated by fusion proteins. VSV∆G pseudotypes are real infectious virus, thus
involving entry of virus particles instead of only membrane fusion. Nevertheless, VSV∆G pseudotyped
particles are different than the life virus from which the fusion protein originates. Therefore, it is
reasonable that it does not perfectly represent the entry by the original virus.
The representation of life virus by a VSV∆G is shown in a study by Glende et al. in 200815. Infectivity
of VSV∆G-SARS S and life SARS-CoV are shown to decrease in the presence of methyl-β-cyclodextrin
(mβCD). MβCD captures cholesterol and thereby sequesters it from the plasma membrane.
Infectivity of VSV∆G transcomplemented with VSV-G was not decreased in the presence of MβCD,
indicating the cholesterol dependency to be solely related to the function of the S protein. They
specifically chose for the use of pseudotyping to rule out the possibility that the cholesterol
dependency is mediated through a different SARS protein than S. The inhibitory effect of cholesterol
depletion was comparable between life SARS-CoV and VSV∆G-SARS S, as shown in Figures 3 and 4.
This indicates that the VSV∆G-pseudotyping assay resembles entry of life virus in the context of
cholesterol dependency. Therefore, VSV∆G pseudotyping is a promising tool to study coronavirus
entry mechanism. With 10 mM of mβCD, both VSV∆G-S and SARS-CoV infectivity is reduced to
approximately 50%, indicating that VSV∆G pseudotyping assays can be quantitatively representative
for life virus infection in this study.
Figure 8 - Infectivity inhibition of VSV∆G-G and VSV∆G-S (left) and life SARC-CoV (right) by cholesterol depletion
(adopted from Glende et al. 2008)15. (Left) Vero cells, either untreated (black) or treated with mβCD (white and dashed)
were incubated in the presence or absence of cholesterol followed by VSV∆G pseudotype infection. Infection was
measured by counting GFP expressing cells. Infection of VSV∆G-S, but not VSV∆G-G is reduced with increasing
concentrations of mβCD and can be rescued by replenishing cholesterol, indicating that infection mediated by SARS-S is
cholesterol dependent. (Right) Untreated, mβCD-treaded and cholesterol replenished Vero cells were infected by SARSCoV. Infectivity is measured by staining with crystal violet and performing a plaque assay. Infectivity of SARS-CoV is
reduced by treatment with mβCD and can be rescued by adding cholesterol, indicating that SARS-CoV infection is
cholesterol dependent.
21
5.4 Virus-virus fusion compared to life virus
The virus-virus fusion assay is based on the fusion between two viruses. Therefore, the fusion occurs
in the absence of cells, making it possible to easier control the conditions of the experiments.
Simmons et al. reported in 2005 that the endosomal protease cathepsin L, which is believed to
mediate cleavage of SARS Spike, is only active at low pH38. A virus-virus fusion assay was utilized to
study fusion between retrovirus particles with SARS Spike and its receptor ACE2 at various pH. Fusion
mediated by exogenous cathepsin L occurred only if low pH is applied. At neutral pH, no fusion was
detected, indicating that cathepsin L does not mediate fusion by SARS S at high pH38. When
acidification of endosomes is blocked, for example by ammonium chloride, infectivity of SARS-CoV is
reduced.
A similar assay by Simmons et al. in 2011 gave comparable results compared to pseudotyped
lentiviruses when the importance of an amino acid (R667) for trypsin cleavage is assessed39. In both
assays, this amino acid residue was dispensable for trypsin enhanced membrane fusion mediated by
SARS spike protein. It was reported that a virus-virus fusion assay is a cell-free assay39, which rules
out the possibility that cleavage by other cellular proteases interferes with the results.
Unfortunately, the virus-virus fusion assay was not compared with authentic virus to validate the
results. However, it was pointed out earlier that retroviral pseudotypes can be a qualitative tools, it
can be assumed that a virus-virus shares the characteristics. The advantage is the exclusion of
interference by other cellular proteins. The cell-free context of this assay makes it easier to control
the conditions of the experiment, such as pH and composition of the medium in which the viruses
fuse. It also allowed studying the role of cathepsin L without interference by cellular proteases. The
pH levels under which the virus-virus fusion was performed by Simmons et al. in 2005 could harm
cells, thereby interfering with the results of the experiments.
5.5 Other virus entry research systems
There are other systems that can be useful for studying entry of coronaviruses but are less used.
These are VLPs, liposomes and a prospective pseudotyping system based on MHV.
VLPs are believed to resemble natural virus since they are basically a virion without viral genetic
material. The context of the VLP can be made similar to that of life virus, as they can be reconstituted
from the structural proteins of the same virus as the spike protein of choice. Since the genetic
material has no obvious role in the activation of the spike protein, it can be assumed that VLPs might
be a very representative system to study virus entry behaviour. This is in contrast with either VSV∆G
or retrovirus based pseudotyping, because both adopt a fusion protein of a foreign virus into the host
virus particle. VLPs based on coronaviruses have not been shown to be infectious yet, but could be a
good tool to study coronavirus entry. However, obtaining coronavirus VLPs is complicated and MLV
based pseudoparticles seem to work, VLPS are not often used for studying coronavirus entry.
Therefore, it is difficult to find publications of studies in which entry of CoV based VLPs are compared
with life virus. For that reason, we chose to search other viruses from which VLPs are studied for
their capability to fuse with target cells.
Wu et al. studied the interaction of influenza VLPs with cells and reported that the VLPs were able to
fuse with cells45. The VLPs were composed of influenza structural proteins hemagglutinin (HA), matrix
(M1), ion channel (M2) and neuramidase (NA). The entry of the influenza VLPs was shown to be
mediated by HA through binding with sialic acid, which is the same for life influenza virus. The entry
22
of the VLPS was also inhibited by an inhibitor of influenza virus entry45. From these observations we
conclude that influenza VLPs represents infection of authentic influenza virus in many aspects.
VLPs based on the Marburg virus (MARV) were reported by Wenigenrath et al. in 2010. The VLPs
were composed of all seven structural proteins of MARV: nucleocapsid (NP), L, VP35, VP30, VP40,
VP24 and glycoprotein GP. They also contained a small sub-genome encoding luciferase as a reporter
gene. Infections with the VLPs are shown to induce luciferase expression in target cells. They are also
suitable for screening of neutralizing antibodies against MARV44.
Unfortunately, both Wu et al. and Wenigenrath et al. did not compare infectivity the VLPs with life
virus in parallel to show that entry by these VLPs resemble entry of genuine virus. Hence, it is difficult
to discuss the quality of VLPs as models for virus entry.
Liposomes and virosomes with incorporated viral envelope fusion proteins can be used to study
membrane fusion as well. However, as they are reconstituted from a lipid solution and purified viral
proteins, the virosome is a very plain structure which does not resemble a real viral envelope.
Therefore, it is doubtful if a virosome can represent the fusion of a virus envelope with a cellular
membrane, since no co-factors are present. At the same time this can be an advantage. If membrane
fusion occurs with only the viral protein, the main component of fusion is found. Reconstituted lipid
vesicles also allow identifying multiple proteins which are together involved in fusion. This can be
performed by reconstituting liposomes with different combinations of proteins and assessing which
set of proteins is able to mediate fusion. Also, the lipid composition of the liposome and the
experimental conditions can be strictly controlled. Hence, it can be useful to study membrane fusion
on protein level alone. Another advantage is that since there is no use of micro-organisms or cells, no
biosafety laboratory would be necessary for studying membrane fusion with this type of assay.
The coronavirus based pseudotyping system has yet to be set up, thus no hard conclusions can be
made about this system at the moment. However, since MHV will be used as a pseudotyped vector,
it would resemble the natural context more than VSV∆G or retroviral pseudoparticles would for
coronavirus entry. That way, the natural infection of a coronavirus can be mimicked more accurately,
giving a more representative assay. But it is still unknown if the fusion protein of interest will
incorporate efficiently enough, as other structural proteins are involved in proper assembly of virus
and specificity of fusion protein incorporation, as observed by Godeke et al.16.
Spike protein of FIPV (feline infectious peritonitis virus) was found to incorporate into MHV. The
produced fMHV grow well on feline cells, indicating that at least some Spike proteins can be
efficiently incorporated into MHV∆S.
Although this system is not operational yet, we think it is very promising for studying coronavirus
entry mechanisms of viruses like SARS-CoV. If it works as proposed, it could also bring a more
quantitative than rather qualitative assay as with the other systems commonly in use for SARS
research.
23
6 Relevance for the research questions and conclusions
All studies start with a question and a hypothesis which is to be proven to come to a conclusion. The
experimental set ups chosen by the experimenter depend on the research questions. Every assay has
advantages and drawbacks, thereby differing in the biological questions which can be approached
with it. Therefore, the experimenter has to carefully decide which systems are suitable to gain
conclusive answers to his questions.
In general, the experimental systems presented in this thesis enable easy studying of multiple
mutants, serotypes and subclasses of viral glycoproteins, because the procedures for mutagenesis on
pseudotyping systems are widely available. For entry research, this is very valuable because the
significance of specific domains and amino acid residues can only be studied by using reverse
genetics. The different genotypes can be studied in the same context, since the pseudotyped particle
remains the same. Pseudotyping also allows taking along fusion proteins with known characteristics,
like HIV gp160 or influenza HA. These reference proteins of known viruses are good benchmarks for
the studied fusion protein and display if the assay conditions are correct, because the experimenter
should be able to reproduce observations from other publications. The same applies to the cell-cell
and virus-virus fusion, since both can be performed with the same backbone material with different
mutant proteins.
As described earlier in this essay, pseudotyping systems are useful for infectivity assays. These
systems seem to represent infectivity of the life virus in various studies. As entry is involved, they
mimic natural virus entry more than other experimental systems. VSV∆G and retrovirus pseudotypes
enter the cell by endocytosis, as most viruses do. However, VSV enters the cell by clathrin-dependent
endocytosis, probably due to its size10, while endocytosis of coronaviruses is believed to be clathrinand caveolae-independent42, indicating that entry routes are still different. The results from VSV∆G
and retrovirus based pseudotyping assays are comparable to genuine virus in various studies.
Therefore, pseudotyping systems are sufficient to study whether a certain mutation or condition
results in a different function of the fusion protein in infectivity, as they seem not to provide
contradictory results about virus entry when compared with life virus in many studies. However,
pseudotyped viruses contain only the fusion protein of the virus that is studied. Therefore, the role of
other viral proteins in fusion and virus entry is excluded in the experiments, so these cannot be
identified by using pseudotyped viruses. Also, as mentioned in chapter 4, pseudotyping assays gave a
more qualitative rather than quantitative outcome, having different kinetics in infection. For
example, the effect of a certain amount of inhibitor or antibodies on infectivity can be different
between life and pseudotyped virus.
As previously mentioned in chapter 4 for HPIV3 retroviral pseudotypes, fusion proteins may
incorporate into pseudoparticles with a different efficiency than into genuine virus22. Thus the
incorporation of fusion proteins into pseudotyped viruses does not resemble natural incorporation
into authentic virus.
Based on these findings, we conclude that pseudotyping is suitable for studying mediation of virus
entry by a single fusion protein, but that it also has its limitations, as described in chapter 4.
Therefore, life virus should always be taken along to prove the observations with the pseudotypes.
Also VLPs can be used as they are believed to resemble life virus entry the most.
24
The cell-cell fusion assay allows easy studying of the basic function of fusion proteins or receptor
specificity. It can thus be used to test if a fusion protein is still functional after introduction of
mutations, or if antibodies against the fusion protein or receptor block membrane fusion. A cell-cell
fusion assay can also be used to investigate whether the fusion protein is expressed at the plasma
membrane. A cell-cell fusion assay does not represent virus fusion behaviour and might even give
contradictory results, because as previously mentioned in section 5.1, the interaction context is
different than viral fusion. Therefore, this assay is not suitable to find clues about a possible
involvement of intracellular or endosomal factors in fusion or activation of the fusion proteins.
Reconstituted systems, such as the virus-virus fusion and liposome based assays, have the advantage
of studying membrane fusion without interference by cellular factors. Therefore, the basic function
of a single fusion protein can be studied on a protein biochemical level. Also the conditions such as
pH can be well regulated, since cells influence the acidity of the medium. With a virus-virus fusion
assay, the amount of background signal is greatly reduced, because the virus-virus fusion assay can
be performed in a two-step model. First intervirion fusion is performed and target cells are
subsequently infected with fused virus, increasing the resolution of the assay. However, these assays
do not give information about possible co-factors involved in the fusion process. With a liposome
assay other proteins can be incorporated into the membranes as well, allowing determining whether
multiple viral or cellular proteins are together involved in membrane fusion. However, the context in
which the membranes in these assays fuse is different from that in which a virus envelope interacts
with a cellular membrane. As mentioned before, protein density and lipid composition of
membranes are important for the membrane fusion. Therefore, we conclude that these systems are
not suitable to pursue questions membrane fusion in the context of a viral particle, as with
pseudotyping assays, or cellular aspects as with the cell-cell fusion.
Another important aspect is that most studies on virus entry and fusion protein mechanisms are
performed on cultured cell lines, which are highly susceptible to infection as they expresses large
amount of receptor. Also, most natural infected cells, such as epithelial cells and neurons, are
polarized, restricting virus entry to either apical or basolateral side of the cell, while cultured cells are
most likely not. It is also uncertain whether the composition of cell culture medium affects entry, as
it contains foetal calf serum. Hence, it is questionable if infection in cell cultures resembles entry of
viruses in vivo. In a whole animal, many other factors influence viral infection, such has accessibility
for the virus to susceptible cells, immune responses against the virus and environmental conditions
as pH and temperature. Therefore, conclusions from in vitro studies cannot be translated to virus
entry in an in vivo context, making them less suitable for answering cell biological questions.
Based on these observations, the experimental system has to be chosen carefully since not every
system is suitable for gaining answer to a specific question. Even better, multiple systems will be
used in conjunction with each other to control the observations.
25
7 Possible improvements to pseudotyping systems for virus entry research
Although the previous described systems are well established tools for virus entry research, they are
not perfect and can be improved.
Both VSV∆G and retrovirus bases pseudotyping systems give a good picture of fusion behaviour in
virus entry, as they used the same fusion mechanism as the virus from which the fusion protein was
adopted. For fusion of two membranes, the amounts of fusion proteins on one membrane and
receptor on the other membrane might be important. However, as mentioned before, the amount of
envelope glycoproteins at the surface of pseudoviruses is different than that of the genuine virus and
thus could results from pseudotyping assays only qualitatively compared to authentic virus. As
reported by Giroglou and Fukushi et al., cytoplasmic truncation of SARS-CoV spike protein increased
incorporation into host virus particles MLV and VSV. This shows that manipulations of the fusion
protein of interest can support the effort of pseudotyping. It will be difficult to overcome negative
aspects of this modification, because, the efficiency with which the spike proteins are incorporated
into the host virus particles is related to the structure of the viruses itself and cannot be changed
easily.
As mentioned previously, cell-cell fusion assays in different studies gave contradictory results to real
coronavirus infection. As coronaviruses naturally enter cells by fusion between viral envelope and
endosomal membrane and cell-cell fusion entirely occurs at the cell-surface, the context of the fusion
is different. Possible ways to reduce the discrepancy are simulating intracellular circumstances like
acidity by performing the assay under different pH, exogenously adding endosomal proteases like
cathepsin L while suppressing endogenous proteases. This could make the spike cleavage and fusion
at the cell surface more like during natural infection.
As mentioned before, VLPs are the closest to genuine virus and is thus believed to resemble natural
virus entry. The main difficultie of VLPs is that infection is not amplificated, so detection is difficult.
This might be partially overcome by labelling the VLPs with fluorescent stains like R18 or pyrene-PC,
which visualize the fusion between VLP envelope and plasma membrane.
26
Table 1 - Comparison of different virus entry research systems: Overview of time consumption, advantages, disadvantages
and representation of real virus entry. * The coronavirus based pseudotyping assay has yet to be set up, so given
statements are assumed.
System
Time consumption
Advantages
Disadvantages
Representation of live
virus
VSV∆G
48 hours transfection +
16 hours infection
Real virus entry; protocols are
available;
Use of real virus; different
quantities of glycoprotein in
envelope; cytoplasmic truncation
necessary for efficient
incorporation into pseudoparticles,
background signal due to small
amounts of G protein incorporated
into pseudoparticles
Similar tropism of host
species and cell types;
only quantitative
comparison to genuine
virus
Retrovirus /
lentivirus
72 hours transfection +
48 hours infection
Real virus entry, directly
without background signal
from residual fusion protein,s
commonly used for virus entry
research, so protocols are
available, transduction of
reporter gene into host cell
genome
Different quantities of glycoprotein
in envelope; cytoplasmic truncation
necessary for efficient
incorporation into pseudoparticles
Similar tropism of host
species and cell types;
only qualitative comparison
to genuine virus
Cell-cell
fusion
1 day transfection, 48
hours after cocultivation
measuring fusion
No virus necessary;
commonly used for virus entry
research, so protocols are
available, quite simple assay
Membranes are different; fusion
occurs only at plasma membrane;
Observations differs in
some studies compared to
virus entry; plasma
membrane fusion differs
from virus fusion in
intracellular compartments
Virus-virus
fusion
120 hours to make
retroviruses, 2 hours
inter-virus binding, 40
hours after infection of
target cells luciferase
activity measurement
Two-step assay reduces
“debris” signal
Time consuming; laborious
Different interaction
context; results
comparable to retroviral
pseudotypes
No virus or cell used, so no
BSL/VMT lab necessary,
membrane compositions
strictly controlled
Not used many times, so no real
protocol available
Different amount of
glycoprotein; different
membrane composition
than virus/cell (protein,
lipid, density)
Virosome /
liposome
Virus-like
particle
VLPs can be harvested
after 8h
No real virus;
Difficult to measure “infection”
Might resemble real virus,
depending on expressed
structural proteins
Coronavirus*
Yet under development
More representative for
coronavirus entry, just as easy
as the VSV∆G and retrovirus
systems once it has been set
up
For so far not yet done, so no proof
of concept
Resembles coronavirus
27
Summary
In this thesis, we discussed multiple experimental systems used to study virus entry. We evaluated
the limitations of these systems, what biological questions can be answered by them and also what
conclusions can be drawn from experiments involving these systems. We found that different
experimental set-ups allow studying different aspects of virus fusion proteins.
To investigate the role of a single fusion protein in the context of virus particles, pseudotyping
systems are the tools of choice. As discussed in sections 5.2 and 5.3 for retrovirus and VSV∆G based
pseudotypes respectively, pseudotyped viruses resemble entry of authentic virus qualitatively but
entry may be dependent of the fusion protein incorporated in the pseudoparticle. Relevant
conclusion can often be drawn from experiments with pseudotyped virus, but the transfer of
concepts to genuine virus is still required as final prove. Many examples in literature show that
pseudotyping systems are a widely accepted tool and allow comparison of new results to
representative studies. The cell-cell fusion assay is a simple and commonly used assay for studying
functional aspects of fusion proteins. Activation mechanisms, such as receptor binding and
proteolytic cleavage, can be investigated with this assay. It does not represent real virus entry,
because the interaction context is different as mentioned in section 5.1. The same applies to the
virus-virus fusion assay, although the latter can be more strictly controlled due to the cell-free
condition of the assay.
Other systems that can be used, the VLPs, liposomes and a coronavirus based pseudotyping system,
might also be useful to study fusion of coronaviruses with the host cells membrane, but as they are
less often used for studying coronaviruses or even not developed yet, we cannot conclude if they
would be a good addition to systems generally used.
Based on the different publications we studied, we conclude that it depends on the research
questions which system is most suitable for studying fusion during coronavirus entry, since each
system are used to study different aspects of viral fusion proteins.
28
Introduction experiments
For our coronavirus based pseudotyping system a MHV∆S is needed. Therefore, we use the
procedure described by Yount et al.47 to generate icMHV-A59 de novo. Once this is successful,
homologous recombination can be used to delete the Spike gene.
To maintain an infectious MHV∆S stock, a cell line which stably expresses recombinant MHV spike
protein (MHV-Srec) will be created as well. To transcomplement MHV∆S, infectious virus particles are
required. By electroporation of full-length infectious MHV-A59 transcripts into the stable cells,
MHV∆S-Srec will be produced.
Material and Methods
Full-length icMHV-A59 cDNA generation
Seven plasmids containing subsequent fragments A to G (see Table 2) of the MHV-A59 genome are
obtained from the laboratory of Ralph S. Baric (fragment A only) and Mark Denison47. Plasmid A is
transformed into E. Coli PC2495 (fragment A) and plasmids B-G are transformed into SURE
Competent Cells (Stratagene, La Jolla, CA, USA) are transformed with the plasmids and grown on agar
plates supplemented with ampicillin (100 µg/ml) overnight at 30°C. Preparations of the plasmids are
prepared using the ZR Plasmid Miniprep-Classic kit from Zymo Research (Irvine, California, USA.)
according to the manufacturer’s protocol. The bacterial pellets are resuspended in 200 µL P1 buffer
(50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 µg/mL RNase A). 200 µL P2 buffer (200mM NaOH, 1%
SDS) is added to lyse the cells and denature DNA. 400 µL P3 buffer (3.0 M KOAc) is added for
neutralization. The samples are centrifuged to pellet bacterial debris. The supernatants are applied
on Zymo-Spin IIN Columns to bind plasmid DNA. 200 µL of Endo-Wash buffer and 400 µL Plasmid
Wash buffer are subsequently added on the columns to wash the plasmid DNA from proteins bound
to it. DNA is eluted with 30 µL MilliQ water.
Digestion of the plasmids is performed to retrieve the MHV-A59 cDNA fragments. Plasmid containing
fragment A is digested by adding restriction enzymes MluI and BsmBI together with buffer NEB#3
(100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9), followed by incubation
at 37°C for 1 hour and subsequently at 55°C for 1 hour. Plasmids containing B and C are digested with
enzymes BglI and BsmBI with buffer NEB#3 at 37°C and 55°C, 1 hour for both temperatures
respectively. For digestion of plasmid containing fragment D, restriction enzymes BsmBI and AhdI are
added with NEB#4 (50 mM KOAc, 20 mM Tris-OAc, 10 mM MgOAc, 1mM Dithiothreitol, pH 7.9).
Plasmids E and F are digested by adding BsmBI with NEB#3 and incubation at 55°C for 2 hours.
Plasmid G is digested with SfiI and BsmBI with NEB#4, incubated at 50°C and 55°C for 1 hour at both
temperatures. BSA (100 µg/mL) is added to all reactions. The enzymes and buffers are ordered from
New England Biolabs® Inc. (Ipswich, UK). After incubation, the digestion products are separated by
agarose gel electrophoresis (Figure 9).The bands with the proper size of the required fragments were
recovered from the gel.
29
Table 2- Plasmids used for full-length cDNA clone of icMHV-A59
Fragment
A
B
C
D
Plasmid
pTopo
pSMART
pSMART
pSMART
Genes
Nsp1,nsp2, 5’-nsp3
3’-nsp3, 5’-nsp4
3’-nsp4, nsp5, 5’-nsp6
3’-nsp6, nsp7, nsp8, nsp9
Expected size on gel (bp)
5000
4676
1952
1451
Restriction enzymes
MluI + BsmBI
BglI + BsmBI
BglI + BsmBI
BsmBI + AhdI
Buffer
NEB#3
NEB#3
NEB#3
NEB#4
E
F
G
pSMART
pSMART
pMH54
Nsp10, nsp11, 5’-nsp12
3’-nsp12, nsp13-16
2a, HE, 4a, 4b, 5a, E, M, N
2793
6985
8711
BsmBI
BsmBI
SfiI + BsmBI
NEB#3
NEB#3
NEB#4
To extract the DNA from the gel pieces, the QIAquick Gel Extraction Microcentrifuge and Vacuum kit
from QIAGEN, Venlo, the Netherlands, is used according to the supplied protocol. In short, the gel
slices are solved in Buffer QG by incubation at 50°C and vortexing. The solubilised DNA is applied to
the QIAquick columns and centrifuged. The bound DNA is washed by adding Buffer PE to the
QIAquick columns and centrifuging at 13.000 rpm for 1 minute. The columns are then transferred to
a 1.5 mL microcentrifuge tube. 30 µl MilliQ water is applied on the columns to elute the DNA.
To assemble a full-length cDNA clone of MHV-A59, the single fragments are ligated to each other.
DNA concentrations are determined by NanoDrop 3300 (second column Table 3). In order to equalize
the quantities of each fragment, the size ratio’s to fragment B is calculated (third column Table 2).
Taken 500 ng for fragment B, the required ng of the other fragments are determined (fourth column
Table 2). Fragments were added in equimolar amounts to the ligation reaction (most right column
Table 2). The ligation was performed overnight at 4°C By adding 3.4 µL T4 ligase and 6.7 µL T4 ligase
buffer (both from New England Biolabs® Inc., Ipswich, UK),. The ligation product was run on a 0.35%
agarose gel to check assembly.
Infectious RNA transcripts are produced from the full-length MHV-A59 cDNA. Therefore, 4.0 µL GTP,
15 µL Cap, 3.0 µL buffer, 5.0 µL template (full-length cDNA) and 2.0 µL T7 polymerase are mixed
together. To support infection, a cDNA coding for the nucleocapsid (N) protein (pCK70) was
transcribed. The samples were placed in a PCR machine for 4 hours at 37°C. Agarose gel
electrophoresis is performed with aliquots of the transcripts to check RNA production.
To produce virus from the RNA transcripts, the transcription products are introduced into BHK-21
cells by electroporation. Approximately 1*10^7 BHK-21 cells were detached from cell culture vessel
and washed extensively in PBS (without Ca/Mg). Cells were finally resuspended in 1 ml PBS (without
Ca/Mg). 15 µL of pCK-70 transcription sample were mixed with 30 µL MHV-A59 transcription mix.
This mix was added to 800 µL cell suspension. Electroporation occurred in 4 mm electroporation
cuvette by pulsing two times at 0.3 kV/ 975 μF. The electroporated cells were immediately transferred
to 10 mL warm (37°C) cell culture medium.
The total amount of electroporated cell suspension is transferred to flasks with LR7 cells. The flasks
were placed in an incubator at 37°C.
Recombinant MHV-Spike protein expressing cells
We use the MLV retrovirus system to generate stable cell lines. The pQCXIX (Clonetech) vector set
allows the incorporation of the gene of interest into the host cell genome by retroviral expression
vectors. Different selection markers such as puromycin resistance gene (pQCXIP) and hygromycin
30
resistance gene (pQCXIH) are available. We introduced the MHV-A59-Srec fusion protein into the
expression cassette of the vectors, transduced cells and started the selection procedure.
Plasmids pQCXIH and pQCXIP are digested to obtain the target vector by mixing 28 µL MilliQ water,
5.0 µL NEB#1, 5 µL BSA, 1 µL PacI and 1 µL AgeI with 10 µL MilliQ water containing 3 µg of the
respective plasmid. Plasmid pCAGGS-MHV-Srec was digested to obtain the donor insert by mixing 27
µL MilliQ water, 5.0 µL NEB#4, 5.0 µL BSA, 1.0 µL XmaI, 1.0 µL PacI and 1.0 µL AhdI with 10 µL MilliQ
water in which 3 µg DNA is suspended. All digestions are performed at 37°C for 2 hours.
From the Srec insert, the total volume was loaded on an agarose gel, from the target vector plasmids
(pQCXIH and pQCXIP) only a small volume is loaded to check for proper digestion.
After separation by agarose gel electrophoresis, the 4022 bp band of pCAGGS is excised from the gel.
The DNA is extracted with the QIAquick Gel Extraction kit as previously mentioned. The pQCXIH and
pQCXIP DNA is purified with the QIAquick PCR purification kit according to the supplied protocol. In
short, 5 volumes of Buffer PB were added to 1 volume of the digestion samples. The samples are
applied on a QIAquick column and centrifuged. The bound DNA was washed by adding PE buffer to
the column and subsequent centrifuging. The DNA was eluded by adding Buffer EB (10mM Tris-Cl, pH
8.5).
To ligate the MHV-Srec insert into the pQCXIH or pQCXIP target vector, different reaction mixes are
made, each with a different ratio between QCXIH/P and insert MHV-Srec (1:0, 1:2, and 1:5). The
ligations are performed with a volume of 10 µL: 1 µL T4 ligase, 1 µL 10X T4 ligase buffer, 1 µL pQCXIH
or pQCXIP, and either 0, 2.0 or 5.0 µL MHV-Srec. These samples are filled out to a volume of 10 µL
with MilliQ water. The samples were mixed and spun down before incubation at room temperature
for 1 hour.
50 µL of E. coli pc2495 is added to ligation products. For transformation, the samples were incubated
15 minutes on ice, followed by 2 minutes at 42°C and 2 minutes on ice. 1 mL LB medium was added
to each sample and all samples were incubated at 37°C while shaking for 30 minutes. The bacteria
were put on agar plates supplemented with ampicillin (100 µg/mL). The plates were wrapped in
aluminium foil and incubated at room temperature until colonies were apparent.
From the plates with E. coli pc2495 transformed with ligation mixes pQCXIH or pQCXIP and pCAGGSMHV-Srec in a ratio of 1:2, two colonies are picked to inoculate 10 mL ampicillin-containing (100
µg/mL) medium. Incubation is performed at 37°C overnight while shaking. The plasmids are miniprepped by using the same procedure as previously described for icMHV-A59 and test digestions are
performed to check the plasmids. For each sample 0.5 µL SpeI, 1.5 µL NEB#4 (bot New England
Biolabs), 1.5 µL BSA (10x) and 6.5 µL MilliQ water are mixed with 500 ng plasmid DNA. The digestion
mixes are incubated for 2 hours at 37°C. After agarose gel electrophoresis, DNA fragments were
checked with a UV transilluminator.
A second transformation and midi prepping is performed to obtain sufficient pQCXIH- and pQCXIPMHV-Srec, as the mini prep gave a low yield. A test digestion is performed with 500 ng plasmid, 1 µL
SpeI, 5 µL NEB#4 and 5µL BSA 10x. Incubation is performed overnight. The digestion is run on a gel to
check the plasmids.
For MLVpp production, 70% confluent HEK293T cells in 10 cm dishes are transfected with 10 µL of
either pQCXIH-MHV-Srec or pQCXIP-MHV-Srec packaging construct, 10 µL MLV-GagPol, 1.0 µL VSV-G,
769 µL DMEM-/- and 210 µL PEI (polyethyleneimine). This is performed in duplicate. As a control for
31
transduction, a transfection was performed with 10.64 µL pQeGFP packaging construct, 10 µL MLVgag-pol, 1.0 µL VSV-G, 768.4 µL DMEM-/- and 210 µL PEI. The producer cells are incubated for 3 days
at 37°C.
The supernatants from the 10 cm dishes with producer cells are filtered on a 0.45 µm filter to
remove debris and cells. The target cells were transduced by adding 5 mL of the supernatants with
either H-Srec, P-Srec or GFP containing MLVpp onto the cells. The target cells are HEK293T passage
58 and HEK293T-CCM1 cells, which stably expressing murine CCM1a from a retroviral expressing
vector pQCXIN grown under 2,5 mg/mL G418 (Geneticin), passage 96/16.
After 4 hours, the mediums of H-Srec and P-Srec and GFP transduced cells were replaced with the
remaining amount of supernatant.
Selection of transfected cells is performed with hygromycin (pQCXIH) and puromycin (pQCXIP).
Puromycin is obtained from a 5 mg/mL stock, G418 from a 250 mg/mL stock and hygromycin B from
a 50 mg/mL stock (Roche, expired in 2006). HEK-pQCXIH-Srec cells are selected under hygromycin
concentrations of 75, 200 and 750 µg/mL. HEK-pQCXIP-Srec cells are selected under puromycin
concentrations of 5.0, 10 and 15 µg/mL. HEK-Ceacam1a-pQCXIH-Srec cells are selected under
hygromycin concentrations of 75, 200 and 750 µg/mL and 2 mg/mL G418. HEK-Ceacam1a-pQCXIPSrec cells are selected under puromycin concentrations of 5.0, 10 and 15 µg/mL and 2 mg/mL G418.
The medium was replaced with fresh DMEM+/+ supplemented with antibiotics after two days.
Results
MHV-A59 generation
Agarose gel electrophoresis is performed to separate the digestion products (Figure 9). The bands
corresponding to the size of the icMHV-A59 cDNA fragments were visible and are excised.
Figure 9 – Agarose gel of digestion of icMHV-A59 fragments. The samples of the digestion reactions were separated by
agarose gel electrophoresis. The bands corresponding to the size of each fragment (A: 5000, B: 4676, C: 1952, D: 1451, E:
2793, F: 6985 and G: 8711 bp) were excised from the gel. The yellow box at fragment D indicates the proper band which
was cut out afterwards.
For the ligation reaction an equimolar amount of each plasmid is added to the ligation mix (Table 3)
32
Table 3 -Preparations of icMHV-A59 fragments for ligation
Fragment
A
B
C
D
E
F
G
DNA concentration
(ng/µL) (a)
79,1
66,7
47,7
82,9
63,0
111,8
37,5
Ratio length fragment with B (bp B/bp
fragment) (b)
0,94
2,34
3,22
1,67
0,67
0,54
Ng DNA for ligation (c)
(500/b)
532
500
214
155
299
746
925
µL added for ligation
(d) (c/a)
6,7
7,5
4,5
1,9
4,7
6,7
24,7
There was no cytopathic effect visible on the producer cells, indicating that no virus is produced. The
experiment was performed again with electroporation into LR7 cells instead of BHK-21. Additionally,
we repeated the experiment with electroporation at 25 Farad and 850 Volts. No infection was
apparent after 4 days, while medium was changed after 48 hours.
Recombinant MHV-Spike protein expressing cells
The gel of the digestion products of pQCXIH, pQCXIP and pCAGGS-MHV-Srec showed all the expected
bands (7800 bp for pQCXIH, 7200 bp for pQCXIP and 4022, 2655 and 2066bp for pCAGGS-MHVSrec_1), although the intensity of the band of pQCXIP was lower than that of pQCXIH while they
should be the same (see Figure 10). Incompletely digested DNA is visible above the excised bands of
S-rec.
Figure 10 - Agarose gel of digested pQCXIH (H),
pQCXIP (P) and pCAGGS-MHV-Srec. H gave a
distinct band at 7800 bp and P at 7200. Three
bands at 4022, 2655 and 2066 were visible for
pCAGGS-MHV-Srec, with the 4022 band excised
for further use.
The control plates (E. coli pc2495 transformed with ligation products of pQCXIH or pQCXIP and
pCAGGS-MHV-Srec in a ratio of 1:0) showed 7 and 9 colonies for pQCXIH and pQCXIP respectively.
The plates with transformed pc2495 with ligation mix with PQCXIH/pQCXIP and pCAGGS-MHV-Srec in
a 1:2 ratio had 87 and 120 colonies respectively, suggesting a proper ligation and transformation.
The pQeGFP transduced cells shown GFP expression with fluorescence microscope, indicating a
successful transduction (Figure 11). Approximately 10% of the cells expressed GFP.
33
Figure 11 – Transduction control with pQeGFP retroviral vector expressing GFP. HEK293T cells (left) and HEK-Ceacam1a
cells (right) expressing GFP (green) after transduction with pQeGFP retroviral vector. As the transduction conditions are
comparable to pQCXIH- and pQCXIP-Srec, we assume that transduction for these vectors was successful as well.
All HEK-pQCXIH-Srec cells died at hygromycin concentrations of 200 and 750 µg/mL. A single colony
from the dish at hygromycin concentration of 75 µg/mL was visible, but is not picked. All HEKpQCXIP-Srec cells died during the selection process at puromycin concentrations of 15 and 10 µg/mL
but six colonies could be picked at 5 µg/mL puromycin. The whole dish of HEK-CEACAM1-pQCXIHSrec with 75 µg/mL hygromycin was overgrown. 200 polyclonal colonies of HEK-Ceacam1a-pQCXIH
were seen at a hygromycin concentration of 200 µg/mL. 20 colonies were visible in the dish at 750
µg/mL hygromycin and six colonies are picked. Polyclonal cells are taken from the dish with 75 µg/mL
hygromycin. One HEK-CEACAM1-pQCXIP-Srec colony could be picked at 5 µg/mL puromycin, but all
cells died at puromycin concentrations of 10 and 15 µg/mL puromycin.
Discussion experiments
MHV-A59 generation
Unfortunately, no icMHV-A59 is produced from the full-length cDNA clone. It is difficult to determine
why it failed, everything till the transcription seem to work properly. It is uncertain that full-length
infectious transcript is produced, although a defined band was visible at high molecular weight. We
did not use RNA denaturing gels for the transcription products, thus we cannot estimate the size of
this transcript.
As described in the literature part of this thesis in section 1.7, there are many useful applications for
full-length cDNA clones of virus. Full-length cDNA clones allow easier application of reverse genetics
without the need for negative selection, and as they are usually cloned into plasmid vectors, they can
be obtained in high numbers by growing them on bacteria.
34
Recombinant MHV-Spike protein expressing cells
As the pQCXIH-MHV-Srec and pQCXIP-MHV-Srec transduced cells were treated under the same
conditions as pQeGFP, we assume that the transductions of MHV-Srec succeeded. Immunostaining
will be used to be sure that the cells were expressing MHV-Srec.
Creating a cell line stably expressing MHV-Srec makes it possible to use the same cell line for many
different experiments without having to transfect cells for each assay separately, thereby circumvent
possible issues with different levels of transfection efficiency. The stable cell line can also be used for
pseudotyping MLV, by transfecting them with two plasmids: one containing a packaging construct
and the second expressing MLV Gag-Pol poly-protein.
Acknowledgement
I would like to thank Mr. Oliver Wicht, MSc (PhD student, Department of Infectious disease and
Immunology, division Virology, Faculty of Veterinary Medicine, Utrecht University) for bringing me on
this interesting topic and supervising me at writing this thesis and doing my practical work.
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