Masterthesis Infection & Immunity
29-12-2012
The determinants of Respiratory Syncytial Virus tropism
Huib Rabouw
Supervisor:
Xander de Haan
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Index
1. Introduction
2. RSV life cycle
2.1 Attachment and entry
2.2 Transcription and replication
2.3 Assembly and release
2.4 Accessory proteins
3. RSV tropism
4. RSV interactions with host cell receptors
4.1 G protein
4.2 F protein
4.3 Receptor interaction
4.3.1 F protein receptor interaction
4.3.2 G protein receptor interaction
5. Intracellular host cell interactions
5.1 Entry
5.2 Transcription and replication
5.3 Intracellular trafficking
5.4 Assembly and release
6. Environmental factors
7. Anti RSV immunity
8. Concluding remarks
9. References
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1. Introduction
Respiratory syncytial viral (RSV) is the leading cause for childhood respiratory tract infections.
Virtually everyone gets infected with RSV at young age. Although RSV symptoms are often no more
severe than those of a common cold, occasionally patients need hospitalisation, and in rare cases
RSV can even be lethal. Groups at risk for a more severe infection are the very young, the elderly, or
the immunocompromised. RSV infected patients are infectious as long as virus shedding is taking
place. Adults are often contagious for no longer than a week, while young children or
immunocompromised individuals can be contagious for weeks1. RSV preferentially infects the
epithelial cells of the respiratory tract, although occasionally virus is detected in PBMCs. In the
pulmonary tract, it causes damage to the epithelium, and chemotaxis of several immune cells. RSV
has also been tested for involvement in development of allergies. These studies have shown that RSV
plays a role in allergic sensitisation during the first years in life, and has also been associated with
increased risk for development of asthma2,3. In addition, RSV infection can induce other
complications caused by coinfecting bacterial species. Haemophilus influenzae and Streptococcus
pneumoniae have even been shown to interact directly with the RSV G protein and to associate with
G protein expressing epithelial cells4. In conclusion, even while RSV itself only causes serious illness in
a minority of the infected individuals, exposure at young age makes this virus an important factor in
the development of the immune system. Maximum severity of RSV related symptoms is often seen
Fig. 1: Overview of a selection of viruses from the Paramyxoviridae family and their genome
structure. Shown are human respiratory syncytial virus and the other members in the Pneumovirus
family, as well as a few closely related human tropic viruses. (A) phylogeny and (B) genome structure.
(Adapted from Rawling et al.)42
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less than a week after first infection, after which the virus is rapidly suppressed. This time window
indicates that innate immunity probably mediates most of the antiviral response. Also, symptomatic
reinfections with RSV can occur later in life in the absence of apparent significant antigenic changes,
which shows the inadequacy of the adaptive immune response. The virus spreads from one person
to another by direct contact or by inhalation of virus containing aerosols. A typical infection
therefore starts and ends in the pulmonary tract. Further spread throughout the body does not
increase the likelihood of transmission.
RSV is a member of order mononegavirales, family paramyxoviridae, subfamily
pneumovirinae, genus pneumovirus. The other members of genus pneumovirus are the bovine
respiratory syncytial virus (BRSV) and pneumonia virus of mice (PVM). There are no closely related
human tropic viruses in the genus pneumovirus, but other human tropic paramyxoviridae are for
example human metapneumovirus (MPV) and human parainfluenza virus (PIV). Pneumoviruses have
a genome size of about 15kb (15,2kb for hRSV). The RSV genome contains 10 genes, which are
transcribed into 11 different mRNAs, resulting in the synthesis of 11 viral proteins. From 3’ end to 5’
the genome encodes non-structural proteins NS1 and NS2, nucleocapsid protein N, phosphoprotein
P, matrix protein M, short hydrophobic protein SH, attachment protein G, fusion protein F, proteins
M2-1 and M2-2, and the long polymerase protein L. Compared to related human viral species, the
RSV genome is complex, with NS1 and NS2 genes as unique features, and the M2 gene being only
present in a select group of paramyxoviridae (Fig. 1). RSV is an enveloped, non-segmented negative
stranded RNA virus (Fig. 2). The genome is encapsidated by protein N. Other proteins associated with
this complex are P, L, and M2-1. Protein M is attached to the inner leaflet of the viral membrane and
has an important function in the assembly. Proteins SH, G, and F are extending into the extracellular
environment. G and F are believed to mediate the attachment and fusion event respectively of RSV
with the host cell. The function of SH is not well described, but it seems to be an ion transporter5 The
last three proteins, NS1, NS2, and M2-2, play regulatory roles during the infection and are not
incorporated into the virion.
Fig. 2: Structure of the RSV virion. (Adapted from Meyer et al.)111
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The natural target for RSV infection in vivo is the respiratory tract epithelium. In vitro however, a
broad spectrum of cell types have been shown to be susceptible to RSV infection. Also, primary cells
in general seem to be more resistant to RSV than cell lines. Since it is still unknown what factors
determine RSV tropism, there is still no working model that explains why RSV does not spread to
other organs during a natural infection. In this review we will discuss what is known about the factors
that could be important in determining RSV host cell tropism.
2. Viral life cycle
2.1 Attachment and entry
On the outside of the virion, RSV proteins F, G and SH are present. The G protein is believed to be
mainly involved in attachment to cells, whereas the F protein can both assist the attachment as well
as perform the membrane fusion step required for cell entry. Although less efficiently, it was shown
that F alone is sufficient for cell entry6. Entry into host cells is pH independent and is therefore
believed to occur at the plasma membrane. Surprisingly, RSV entry could be partially inhibited by
blocking clathrin dependent endocytosis7. The function of the third membrane protein SH has not yet
been determined, though it is believed to act as an ion transporter. The general consensus is that in
cell culture SH can be completely deleted with no effects on RSV infectivity8, although one study has
shown SH to be required for fusion9.
2.2 Transcription and replication
Upon membrane fusion the capsid is released in the cytoplasm. RSV replication and transcription are
complicated processes that are not fully understood. The viral RNA dependent RNA polymerase
machinery associates with the leader region at the 3’ end of the genome. For transcription, the next
step is for the transcription complex to release from this 3’ end structure and scan for the initiation
site of transcription for the first gene (NS1). For replication, the RNA synthesis starts at the first
nucleotide of the genome. Replication products are immediately encapsidated by viral protein N,
which is thought to be important for protection of the genome from degradation, and for minimising
innate immune responses by limiting detection through Pattern Recognition Receptors (PRR).
Transcription products (mRNAs) are capped and released freely in the cytoplasm. The RNA synthesis
complex consists of proteins N, P, L, and M2-1, of which L has the catalytic domains and has the
polymerase and capping functions10. Phosphoprotein P is a homo tetramer and plays an important
role by being cofactor for viral RNA synthesis and inducing correct formation of complexes of N, P,
and L. Clearance of a promoter and RNA strand elongation are dependent on P11. The function of M21 is not fully understood, but it was shown that replication machinery of RSV strains lacking M2-1
stops RNA synthesis too early which leads to incomplete RNA products. This may however be not the
only function of M2-1, since deletion of M2-1 proteins of related viral species do not seem to
influence mRNA synthesis. Still, in vivo these M2-1 deletions had severe attenuating effects on the
infection12. An important difference between the transcription and replication processes is that
during replication a full-length antigenome is made. The polymerase needs to ignore all the gene
stop sequences at the end of each gene. Several theories exist on how the distinction is made
between transcription and replication13 (reviewed in Cowton). M2-2 has a role as a regulatory
protein in this process. An RSV strain with defective M2-2 showed increased tendency for
transcription rather than replication. Still, M2-2 is not the only factor involved and is not even
required for replication, since even in absence of M2-2 a background level of replication always
occurs. During mRNA synthesis polymerase complexes have a chance to fall off the genome after
each gene end signal. Since some polymerases therefore never reach the end of the genome, 5’
genes are less frequently transcribed than 3’ genes. Transcription and replication takes place in the
cytoplasm at local sites of RNA synthesis. These sites are referred to as inclusion bodies, and consist
of accumulations of ribonucleoprotein (RNP) cores composed of viral RNA and associated proteins14.
Inclusion bodies are often seen close to the host cell membrane and are thought to be a site of
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assembly and budding, from which filamentous viral extensions can be seen projecting into the
extracellular matrix14,15.
2.3 Assembly and release
Assembly of new virions takes place at the plasma membrane. Important for this step is RSV protein
M. This protein is associated with the inner surface of the viral membrane. It consists of an Nterminal and a C-terminal group, linked together with a short linker. Large positively charged areas
cover this protein, probably assisting in correct assembly of the capsid to the viral membrane. It also
appears to play a central role in budding16 and blocking RNA synthesis during assembly17. An actin
link has been shown between inclusion bodies and sites of virus budding at the plasma membrane.
This budding is believed to mainly occur at lipid raft microdomains. Budding from the plasma
membrane can result in two morphologically distinct phenotypes: a spherical shape with a diameter
of about 200nm, or a filamentous form with a length that often varies between 1 and 4 μm 18. All RSV
phenotypes (spherical or filamentous, and also electron-dense or empty under the electron
microscope) can be seen within one cell culture. The filamentous particles seem to outnumber
spherical particles18. It was shown that RSV budding happens with at least F, M, N and P proteins16.
RSV does not seem to require the ESCRT machinery for its release. Instead, this release is dependent
on apical recycling endosomal (ARE) pathway. A key player in this process is FIP-2, without which the
virions remain associated to the host cell19.
2.4 Accessory proteins
Having their genes located at the 3’ end of the genome, RSV accessory proteins NS1 and NS2 are
most abundantly synthesised. These proteins are non-structural and are mainly important for
immune evasion. Especially the type I IFN system is being down regulated by these accessory
proteins. NS1 and NS2 inhibit both the production and the signalling of type I IFNs by interfering with
several proteins involved in these pathways20. The last non-structural protein is M2-2. The M2-2 gene
is less frequently transcribed and is thought to cause accumulation of M2-2 protein over time during
an infection. An important role for this protein is mediating the switch from transcription of viral
genes towards replication of the genome21. Once enough viral proteins are created, this focus on
replication is needed for synthesis of viral genetic material for new virions.
3. RSV Tropism
Over the years many different types of cells have been used to culture RSV. Many of these cell lines
have been shown to be permissive to some extend to RSV infection. See table 1 for a complete
overview of used cell lines and their permissiveness to RSV. The search for an RSV non-permissive cell
line proves difficult22. The primary hosts of RSV are humans. Other species that are infectable with
hRSV are for example primates, rats, mice, and hamsters. Infection of these species is significantly
less efficient in comparison to infection in human, and requires exposure to higher titers of virus. In
agreement with this, cell lines with human origin seem to be more susceptible to RSV on average
than other cell lines. Being able to infect so many different cell types in vitro could indicate that RSV
is hardly specific in its cell tropism. Yet, during a normal RSV infection in a human host, the virus is
mainly limited to the ciliated epithelial cells in the respiratory tract. Other cells that can get infection
in vivo are alveolar macrophages and PBMCs. Over the last decades only a handful of studies have
reported systemic spread of RSV to the heart, liver, or cerebrospinal fluid. Somehow, while RSV
occasionally exits the pulmonary tract, the virus hardly spreads at all. In the respiratory tract, RSV has
been shown to specifically infect the ciliated epithelial cells, while basal and intermediate cells
without proper differentiation are no RSV targets ex vivo23.
The tropism of a virus is determined mainly by the interaction of viral proteins with factors
on the host cell membrane. This interaction is the first contact between the virus and the host cell
and is a way for the virus to select cells for infection. In addition, also intracellular factors or
environmental factors may restrict a viral infection to a certain cell type or location in the body.
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Intracellular factors play a role after the initial cell entry has taken place and may cause progeny virus
production to be less efficient or completely absent. An example of a virus for which its tropism is
largely determined by an extracellular factor is the avian influenza virus. In humans, this virus can
only infect the respiratory tract, since an extracellular virion maturation step is mediated by
proteases in the respiratory tract24. Another important factor in cell type tropism is immunity. The
respiratory tract is poorly accessible for many immune cells. Also, in order to avoid excessive immune
activity due to constant exposure to inhaled pathogens, respiratory tract cells have a relatively high
tendency for immune tolerance. The last factor of importance in RSV tropism is the physical barrier
between the respiratory tract and the blood stream. RSV virions are shed from the apical side of
epithelial cells and are therefore likely to encounter no other potential targets than yet another
epithelial cell. Presence of RSV in alveolar macrophages and even PBMCs proves this barrier only
partially blocks RSV spread. Important to note is that RSV theoretically has no benefit from spreading
systemically. The infection starts in the upper respiratory tract and transmission occurs from the
upper respiratory tract. Systemic spread probably does not increase the likelihood of RSV
transmission. For this reason, there will be no positive selection for RSV mutants with expanded cell
type tropisms.
Fig. 3: Factors potentially involved in determining the RSV lung tropism in vivo. (1) A certain environmental factor
specifically present in the lung lumen may influence infectivity of free virions. This factor not necessarily needs to
impact receptor conformation as depicted in the figure, but may also impact for example cell entry efficiency or
stability of the virions. (2) Presence or absence of specific receptors could determine cell type tropism. This
principle is extensively used by viruses to select proper host cells and is likely to also affect RSV tropism. Either a
single receptor, or a combination of receptors may be used. (3) Intracellular factors could influence efficiency of
replication. If crucial host factors are absent or present in concentrations other than the optimal concentration in a
certain cell type, this cell will produce fewer or no infectious progeny virus. (4) The respiratory tract lumen is
relatively badly infiltrated with immune cells. This is partially due to viral factors influencing the chemokine
expression. This may cause the respiratory tract to be a safer place to replicate than other locations and thus limit
the virus to the lungs. (5) RSV is shed from the apical side of epithelial cells. This makes spread through the basal
membrane difficult. Only few RSV particles will therefore ever reach other tissues.
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Taken together, the tropism of a virus can be determined by a range of different factors such
as environmental factors, receptor usage, intracellular factors, immunological factors, or physical
confinement (Fig. 3). For these factors the possible importance in RSV tropism in vivo and in vitro will
be discussed below.
Table 1: Tropism properties of human respiratory syncytial virus
Cell type
Species
Cell line tropism
HEp-256,94,30,19,103,95
Human
HeLa56,27,95
Human
MT-456
Human
293T56,94,30
Human
NCI-H29256,95
Human
A54956,96,103
Human
THP194,104
Human
MRC-530,102,103
Human
BEAS 2B96,106
Human
9HTE96,107
Human
Vero56,30,103
African Green Monkey
CV-130,9,96
African Green Monkey
AGMK-2130,105
African Green Monkey
BSC-1102,103
African Green Monkey
PMK103,20
Rhesus Macaque
P388D98,108
Mouse
NIH/3T356,42,97
Mouse
MHS109
Mouse
BHK-2156,101
Hamster
CHO29,35,103
Hamster
RK-1356,100
Rabbit
MDBK56,30
Cow
BT30
Cow
AK-D56
Cat
FEA102
Cat
MDCK56,6
Dog
E. Derm56
Horse
XLK-WG56
Frog
QT656
Quail
Tb1Lu56
Bat
LLC-PK156
Pig
Mv1Lu56,102
Mink
In vivo species tropism (reviewed in [99])
Primates
Cotton rat
Hamster
Mouse
Guinnea pig
Ferret
Human primary cells
Human
Human
Human
Human
Tissue
RSV Infectivity
Larinx
Cervix
T cell
Kidney
Lung
Lung
Monocyte
Fetal lung
Broncheal epithelium
Tracheal epithelium
Kidney
Kidney
Kidney
Renal epithelium
Kidney
Macrophage
Fibroblast
Macrophage
Kidney
Ovary
Kidney
Kidney
Turbinates
Fetal epithelium
Embryonic fibroblast
Kidney
Skin
Kidney
Fibroblast
Lung
Kidney
Lung
++
++
++
++
+
++
+
n.d.*
n.d.*
n.d.*
+
+
+
n.d.*
n.d.*
+
+
n.d.*
++
n.d.*
-*
-*
n.d.*
n.d.*
+
++
+
++
++
+
+
-*
++
Alveolar Macrophages72,110
Ciliated epithelial23
Mucus cells23
Basal/Intermediate cells23
+
++
---
Indications ++, +, or - are given only if a quantitative measure for the amount of infectivity or replication have
been published, or if the cell line was compared to other cell lines for which a quantitative measure was
published elsewhere.
++ infection or replication happens at a good efficiency
+ infection or replication occur to some extend
- infection or replication may occur at very low levels or be absent
*Parameter was observed but not quantified. N.d.* means that RSV infection or replication occurs but no
quantification or comparison with other cell lines was determined; -* means RSV infection/replication was
shown to be very low or absent, but was observed to some extend in at least one study. Since host cells with
indications ++ or + by definition have been shown to support infection, they are not given an additional *
indication.
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4. RSV interactions with host cell receptors
4.1 G protein
The G protein is expressed as a type II transmembrane protein that is highly variable between RSV
strains. It consists of a short cytoplasmic tail, a transmembrane region, and two extracellular highly
variable and highly glycosylated domains flanking a short central conserved domain25. G exists as a
membrane bound (mG) protein and as a soluble protein lacking the transmembrane region (sG) (Fig.
4). Cell entry is mediated by proteins G and F. G is considered the RSV attachment protein26,27, while
F protein has the fusogenic properties28. The G protein has been shown to interact with
glucosaminoglycans (GAGs) on the surface of target cells. For efficient attachment to cells, GAGs
need to be N-sulfated and at least have a length of ten saccharide groups29. This selective use of
GAGs demonstrates that it is not simply electrostatic interactions that take place. Some degree of
host cell selection may occur as a result of these interactions. Noteworthy however, is that depletion
of these GAGs, or blocking G proteins with soluble GAG never completely inhibits cell entry29.
Moreover, RSV can successfully infect cell lines only through the F protein30 even in the complete
absence of G protein attachment.
Fig. 4: protein structure of the G protein. Indicated are the hypervariable domains, the central
conserved domain, the heparin binding domain (HBD) and the CX3C motif on membrane bound G (mG)
and soluble G (sG).
Apart from its attachment function, G protein also has substantial immune modulatory
functions. It can significantly alter chemokine expression in the lungs31 and therefore immune cell
infiltration, inhibit the type I IFN system, cause Th2 skewing, and induce potent but ineffective
adaptive immune responses. Soluble G protein is thought to play a dual role both by being a
smokescreen for G specific antibodies and by influencing immune cells by direct interaction. The
exact roles of G in vivo has proven difficult to determine, since many different truncations on G
hardly seem to affect the fitness of the virus (deletion of CX3C domain and heparin binding domain25,
C terminal truncations32), while complete absence of G severely attenuates RSV in vivo. Also, in
mouse studies it was shown that presence of soluble G was sufficient to restore a significant amount
of virus infectivity in comparison to ∆G RSV25. Also in vitro the roles of G are uncertain. As mentioned
above, ∆G RSV is able to infect cell lines and blocking G protein interactions of WT RSV does not
completely inhibit infectivity. This means that, while G plays a role, it is not absolutely required. In
one cell line (vero) it was even shown that WT and ∆G RSV are equally infectious and a cold-passaged
RSV strain isolated from these vero cells completely lost a functional G protein33. Vero cells are
known for their inability to produce type I IFN, which implies that the function of G protein is (in
vitro) immune suppression rather than attachment.
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The importance of GAGs in the attachment of RSV is a point of discussion. Many studies have
given proof of the involvement of GAGs in attachment, although no study has ever shown a 100%
dependency on GAGs in any cell type. Blocking GAGs by GAG binding proteins or antibodies, blocking
GAG interactions by addition of soluble GAGs, or proteolytic cleavage of GAGs have never been able
to completely inhibit RSV infection. A very crucial point about which studies disagree is whether the
GAG-like structures are present on the virion or on host cells. The G protein contains heparin-like
structures. A study in 1998 showed that antibodies to heparin-like structures or heparinase
proteolytic cleavage on RSV virions could inhibit infection in HEp-2 cells34. Interestingly,
preincubation of HEp-2 cells with heparinase did not reduce RSV infectivity in the study, indicating
that only the RSV virion associated heparin-like structures play a role in attachment. Completely the
opposite was shown by another study in which heparinase activity on HEp-2 cells prior to incubation
with RSV significantly inhibited infection35. The importance of cell-associated GAGs was also
demonstrated by the reduced infectivity in CHO cells deficient in GAG synthesis35. Also F has been
shown to interact with cell associated GAGs36. Still, a G protein deficient RSV strain was less
dependent, but not completely independent, on GAG interactions37. This G deficient RSV strain had
an overall reduced infectivity in HEp-2 cells when compared to WT RSV virus. In vero cells though,
RSV strains with or without G protein grew to equally high titers25. It was even shown that infection
by G deficient RSV could not be inhibited by addition of soluble heparin, while infection by WT RSV
could be blocked by soluble heparin25. This implies that for RSV attachment to vero cells neither G
protein, nor cell associated heparin play a role. F protein uses a heparin independent attachment
mechanism, and can even be hindered by attachments made by G.
Apart from G protein attachment to GAGs, G has also been shown to interact with L-selectin
like proteins. These L-selectins bind sugar structures like heparins. As mentioned above, the G
protein is highly glycosylated and contains heparin-like structures, which may be the explanation for
this. L-selectin is a surface bound protein that is present on immune cells in the respiratory tract, but
not on epithelial cells38. Other L-selectin like molecules that have been identified that are present on
the epithelium are annexin II and nucleolin. Blocking these receptors with fucoidan (polysaccharide
consisting mainly of fucose sulphate) potently inhibited RSV entry38. Later, nucleolin was identified as
an important F interaction candidate. Note that, while nucleolin was not specifically analysed further
for interaction with G in this study, these data might mean that F and G use the same receptors or
interact together with a single receptor.
Another candidate receptor for G attachment to immune cells is the CX3C chemokine
receptor (CX3CR1). This receptor normally binds fractalkine, the only known member of the CX3C
chemokine family, to which the G protein has remarkable similarities. This interaction could be
important as an attachment39 but is probably mostly involved in immune modulation40. The CX3CR1
is involved in chemotaxis of leukocytes, and it is not clear whether it is present on the apical surface
of respiratory tract epithelium. One of the similarities with fractalkine is a GAG binding domain.
Assuming that immune evasion is a critical role for G, even the often described GAG binding feature
may be no more than a side effect of its CX3C mimicry. G modulates chemokine expression31 en can
disrupt normal chemotaxis of immune cells. G was also shown to be important in causing a Th2
orientation of the immune system31, which is relatively ineffective against RSV and can cause
increased symptom severity. In vivo the role of this interaction is unclear. Virus production in CX3CR1
knockout C57BL/6 mice was shown to be equal to WT40, or significantly lower than WT (Peeples,
unpublished data). Such contradictions, together with the apparent lack of effect when the CX3C
motif is deleted32, make the exact role of the CX3C domain hard to predict.
A study in 2011 in which whole genomes or RSV strains were sequenced41 showed
glycoprotein G to be the most variable gene in the RSV genome. Two different factors can contribute
to variability in a gene: active environmental pressure e.g. by the immune system, or passive
mutations that are not eliminated because of a lack of selection. The first factor is thought to be very
important for G variability given its location on the outside of the virion and the immunological
response to G protein epitopes. The latter factor may however also be of importance. In contrast to
many other paramyxoviridae, RSV does not require its attachment protein for the attachment
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function. This lack of a strict function for G may have given it more freedom to evolve. Sequencing of
G genes isolated from RSV patients has shown that, in addition to minor sequence variations, some
RSV strains had severe truncations in the G protein41. This indicates that also in vivo large parts of the
G protein are not crucial for sustained infection. On the other hand, the results from this study gave
indications that a fraction of the virus obtained from these patients did have functional G. This could
mean that presence of any G protein is sufficient during RSV infection, and that these G proteins do
not necessarily need to be attached to the RSV virion.
Although the RSV G protein named the RSV attachment protein, it seems to have multiple
important functions, of which even the importance of the attachment function is uncertain. There
have been indications that G protein interacts with GAGs (chondroitin B, heparan sulfate, heparin), Lselectin like molecules38 (L-selectin, annexin II, nucleolin), CX3CR139, L-SIGN42, and DC-SIGN42. For
none of these interactions it has been undisputedly determined whether it has a function in
attachment and/or immune modulation.
4.2 F protein
The F protein mediates fusion of the virions membrane with a target cell, and is also responsible for
formation of syncytia by infected cells. Like fusion proteins of other members of the paramyxoviridae
family, F proteins form homotrimeric complexes. It is a class I fusion protein that consists of a
hydrophobic fusion peptide, two heptad repeat regions (HRA and HRB), a transmembrane domain
and a C terminal intracellular domain. It is initially translated as an F0 precursor protein, which has
two closely located cleavage sites. A cleavage step by a furin like protease is required for maturation
of the protein. A small residue (p27) between the two cleavage sites is released, and the two
remaining parts of F (F1 and F2) are linked by two disulfide bonds. F1-F2 forms the active fusion
protein (Fig. 5).
Fig. 5: Protein structure of the F protein. Indicated are subunits F1 and F2, the two heptad repeat
domains (HRA & HRB), and the fusion peptide (FP) at the end of HRA.
In the paramyxoviridae family the double cleavage is a unique feature of the RSV F protein.
The importance of it was demonstrated by showing that introduction of this double cleavage site in a
sendai virus strain significantly increased infectivity. This virus was now able to induce membrane
fusion in absence of its attachment protein (HN)43. This is in agreement with the finding that RSV can
be infectious even without its attachment protein G30. This means that RSV F protein has an
attachment function as well as fusion function. During fusion, the HRA domains attack the host cell
membrane with their N-terminal fusion peptide, after which they fold back over the HRB regions in
an antiparallel manner to form a six helix bundle. This brings viral and host cell membranes in close
proximity, which allows the exchange of lipids followed by fusion43. The direct triggers initiating this
fusion event are not known. There have been indications that F interacts with several different
cellular factors, including GAGs36 (heparan sulfate), RhoA GTPase44, ICAM-145, nucleolin22, Surfactant
protein A46, Lactoferrin46, and MD-247. The F proteins C terminal intracellular tail has been shown to
be required for proper assembly of infectious progeny virus48. The fusion capacity of F however,
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seems not to be hindered by truncations of the intracellular tail, which could be seen by normal or
even increased syncytia formation in vitro48. The authors propose that either lack of the intracellular
domain causes the F protein to become less stable and mediate premature fusion events before
virion formation, or the F protein loses its proper localisation to lipid rafts for virion assembly and
instead is spread over the cellular surface resulting in increased cell-cell fusion. Compared to G, the
functions of F are clear and well defined. Without F there is no budding of new virions and no fusion
at the plasma membrane. There have been no indications of immune regulation properties other
than antigenic variation or antigenic shielding by glycosylations in F itself, which are normal features
for any viral surface exposed proteins.
4.3 Receptor interaction
Key to predicting the cell tropism of RSV entry is understanding the process of cell entry from the
first interaction of virion with host cell, to the initiation of fusion. Although it is believed that at least
one, or possibly even a combination of specific cellular RSV receptors are required for cell entry, RSV
virions were shown to fuse even with artificial membranes that contain no more than egg PC and
cholesterol49. This leads to the question how RSV avoids uncontrolled fusion with random
membranes, including those of cells that cannot support replication or even of neighbouring RSV
virions.
As fusion and attachment proteins respectively, F and G are the determinants of the
efficiency of host cell entry. These two proteins are therefore very important factors in the RSV
tropism by host cell selection. The search for the host cell surface protein that acts as RSV receptor
has taken much effort over the last decades, and has resulted in the identification of several
candidates. Recently, nucleolin has been shown to be a functional RSV receptor. This was the first
protein for which so extensively its effect in RSV infection was tested. RSV infectivity was reduced
after inoculating cells with nucleolin specific antibodies, by binding competition with soluble
nucleolin, and by RNAi knockdown of cellular nucleolin. Also, RSV non-permissive cells could be
infected after transfection with nucleolin and in vivo knockdown of nucleolin seriously inhibited RSV
infection in mice22. Unfortunately, nucleolin as RSV receptor does not explain the complex RSV
tropism. Its surface expression is not limited to the ciliated airway epithelium, and is widely present
in lung tissue in mice and therefore possibly also in human50. Also, it is expressed on many other cell
types elsewhere in the body51-53. As the name suggests, it is mainly associated with the cell nucleus. A
few years ago, it was established that nucleolin is also present in the cytoplasm and at the plasma
membrane, thus allowing it to function as an RSV receptor54. Furthermore, on human alveolar
epithelial cells nucleolin was found to colocalise with RSV in a low temperature binding assay on
primary human alveolar epithelial cells22. Nucleolin expression was shown to be increased in asthma
or cystic fibrosis patients55. Theoretically it is possible that nucleolin mediated increased
susceptibility to RSV at young ages made these individuals more prone to develop such conditions2,3.
Nucleolin is well established to be excessively expressed in cancer cells and therefore also
immortalised cell lines. This may be an important determinant causing RSV to successfully infect so
many different cell lines in vitro. It seems that RSV is very capable in infecting cell lines, but that
completing the full RSV life cycle requires more specific host factors56. Replication is seen mainly in
cell lines derived from human, or from semipermissive hosts such as primates or hamsters. Since
entry and completing the complete replication cycle do not correlate that well, nucleolin (or any
other RSV receptor) cannot be the only factor contributing to the cell type tropism of RSV.
Many host cell membrane bound molecules have been shown to interact with one of the
RSV membrane proteins. These molecules range from aspecific factors such as cholesterol rich
domains57 or sulfated saccharide groups29, to more specific protein factors like nucleolin22,38, annexin
II38, ICAM-145, and TLR4 coreceptors58. Question remains, which ones of these interactions are
important for cell entry and thus for host cell tropism.
13
4.3.1 F protein receptor interaction
It was shown that species specificity of the RSV virus is mediated by the F2 fragment of the F
protein. A human RSV strain with its F2 fragment replaced with bovine F2 was unable to infect
human primary cells and had its tropism shifted towards bovine cells. The opposite happened to
bovine RSV strains with human F2 fragments59. Although species specificity is not comparable to cell
type specificity, this principle indicates that whatever combination of interactions is important in
determining cell type tropism of the RSV, the interaction of the F2 fragment with a specific host cell
factor is always necessary. Glycosylations in the F protein have been shown to be important for its
function. Incomplete glycosylations as obtained by specifically inhibiting glycosylation enzymes in the
golgi resulted in less infective virus60. Three N-linked glycosylation sites are present on the mature F
protein (with its p27 fragment cleaved out): At N27 and N70 in the F2 subunit, and at N500 in the F1
subunit. Some studies have been looking at the function of F protein single residues by site-directed
mutagenesis. Focus of these studies were the conserved cysteine residues (required for
intramolecular disulfide bonds) and the N-linked glycosylation sites (possibly required for fusion
activity). The F protein contains 14 cysteine residues (C37, C69, C212, C313, C322, C333, C343, C358,
C367, C393, C416, C422, and C439) in the extracellular domain of which C37-C382 and C69-C212
form disulfide bonds between F1 and F2. The other disulfide bonds are within the F1 subunit. Figure
6A shows the F protein with coloured indications at every cysteine residue and asparagine residue
indicating the severity of the attenuation upon single amino acid mutation leading to loss of either a
disulphide bond or an N-linked glycosylation (green = mutation of residue has no effect, or very little
effect on RSV infectivity, yellow = mutation of residue has intermediate effect of RSV infectivity, red =
severe (close to 100%) attenuation upon mutation). Specific mutations in many of the cysteine
residues resulted in severe reductions in expression of mature F. Interesting are the cysteine residues
at 69 and 212, and the asparagine residue at 70. If removed, these residues affect, but not
completely inhibit fusion capacity. The cys212 is located at the base of the heptad repeat A (HRA)
region, which is extended towards the host cell membrane to allow insertion of the fusion peptide in
the host cell membrane during the first step of the fusion event. This means that residues cys212cys69-asp70 would be a perfect place to attach to F2 and transfer a conformational change to the F1
HRA domain. Also, in the 3D conformation these three residues are located on the outside of the
protein which allows interactions with other proteins (Fig 6B-D). Unfortunately, only the post fusion
3D crystal structure has been determined for the RSV F protein, so the pre fusion orientation of these
residues cannot be determined. For human parainfluenza 5 (PIV-5) however, the F protein pre fusion
crystal structure is known (Fig. 6E). A comparable F1-F2 disulfide bond (cys64-cys185) can be found in
this protein, also with a potential N-linked glycosylation site directly adjacent to this disulfide bond
on the F2 fragment (asn65). The fact that this region is conserved is by itself already an indication of
its importance for the function of the protein. In this pre fusion 3D structure these cys64-asn65cys185 residues can be found well exposed and directed away from the virion pointing towards the
host cell.
In several different virus species (Mumps virus, SV5, NDV, senday, nipah, and hendra) the F2
region surrounding the disulfide bond with F1 showed remarkable conservation and site-directed
mutagenesis on some residues in this area affected the fusion capacity of these viruses61. In only one
of these abovementioned six viruses the F protein an asparagine residue was located next to the
disulfide bond however. Also, as indicated by the yellow colour in Fig. 6A-C, removal of the
asparagine at position 70 does not completely block RSV fusion. These data indicate that N70 on its
own is not the host cell binding site but might be part of a bigger region around the F2-F1 disulfide
bond (cys69-cys212 for RSV) that is important for initiation of the fusion step. The exact amino acid
sequence in this area may be determining which host cell receptors are required for this interaction,
and it would be interesting to see whether tropism characteristics of one virus can be mimicked in
another viral species by exchanging these F2 regions. Alternatively, the glycosylation could also be
involved in shielding the surrounding interaction region from recognition by the immune system, and
not be part of the interaction itself. Given the 3D structure of the pre fusion F protein of PIV-5 it
could be hypothesised that attachment to the F2 cysteine or asparagine residues (Fig. 6D and 6E;
14
Fig. 6: 2D and 3D positioning of important cys and asn residues in the F protein. (A) 2D structure of
RSV F protein, with indications at potential N-linked glycosylation sites and disulfide bond forming cys
residues representing the severity of attenuation upon loss of the residue. Green: no effect. Yellow:
partial effect, no full inhibition of infection. Red: close to 100% inhibition. (B) 3D structure of
monomeric F protein in which the same residues are highlighted in the same colour coding as in A. (C)
3D structure of RSV F protein trimer (post fusion state). The same residues are highlighted in the same
colour coding as in A. (D) Cys69-cys212 disulfide bridge, and the N70 glycosylation site are shown in
spacefill lay-out. The adjacent F2 region is shown in green, the adjacent F1 region is shown in blue.
The Asn70 residue is shown red. (E) Pre fusion conformation of PIV-5 fusion protein. The same F1-F2
disulfide bond (cys64-cys185 for PIV-5 F), as well as the surrounding regions on F1 and F2, including
the asn65 residue are shown in the lame lay-out as in D.
highlighted by spacefill layout) or the surrounding F2 region (shown in green), followed by
mechanical force pulling this region away from the protein core, would pull free the F1 region (shown
in blue) at the base of the HRA domain through the disulfide bond. Probably, since these F2 and F1
regions seem to be positioned parallel to each other over a long stretch of amino acids (Fig. 6E), the
disulfide bond is only part of the F1-F2 interaction. This would explain why mutations in the cys69
15
and cys212 residues, which cause loss of the F1-F2 disulfide bond, do not fully inhibit membrane
fusion60. Instead, removal of the disulfide bond gives this F1 domain a chance to break interaction
with F2 rather than with the F protein core, thus giving F proteins a chance not to activate upon
receptor binding. A schematic overview of this process is given in figure 7. Preincubation of RSV
virions with surfactant protein A (SP-A) has been shown to increase the infectivity of these virions in
a dose dependent manner46,62. This phenomenon is not caused by SP-A crosslinking RSV to host cells,
since host cell incubation with SP-A, followed by exposure to RSV did not result in increased
infectivity46. The SP-A molecule interacts with the F2 subunit of the F protein. Furthermore, this
interaction was found to be N-linked glycosylation dependent46. This effect of SP-A may be a result of
interaction with the N70 glycosylation of F2, possibly by initiating the fusion event before a host cell
membrane is even present. Interaction of SP-A with F2 peptide might result in intermediate fusion
complexes with released HRA domains (Fig. 7 stage 2). Interestingly, others have shown an inhibitory
effect of SP-A binding to F protein63.
Above, a mechanical force is described as a possible cause for pulling the HRA domain from
the protein core, but it could also be that conformational changes in the F2 following receptor
interaction cause release of HRA.
Fig. 7: Possible model for initiation of fusion by RSV F protein. (1) The Asn70 residue with its
surrounding amino acids are responsible for attaching to the cell surface. (2) This region confers a
conformational change or a physical pulling force to the F1 HRA domain through the cys69-cys212
disulfide bond, pulling the HRA with the fusion peptide free from the protein core. (3) the fusion peptide
is now free to insert in the host cell membrane. (4) HRA is now folding back in an antiparallel manner
over the HRB domain to form a six-helix bundle, (5) which brings host cell membrane and viral
membrane together for fusion. The upper panel shows only the involved F1 and F2 strands as well as the
cys69-cys212 disulfide bond (-s-s-) and the asn70 glycosylation (•). The lower panel shows a simplified
structure of the F protein during these steps.
16
The surface expression of potential F receptor nucleolin depends on N-linked
glycosylations64. These N-linked glycosylations are also required for optimal self-interaction of
nucleolin molecules64. Clustering of nucleolin molecules could be important if RSV requires
interaction with more than one receptor molecule at a time for cell entry. The mechanisms by which
N-glycolysations influence self-affinity are not known. It is possible, that these glycosylations not only
increase self-affinity, but also affinity for other N-glycolysated proteins such as RSV F protein.
In summary, the above mentioned data give indications that F2 interaction with the host cell
is required for fusion59 and that this interaction is N-linked glycosylation dependent60. Two N-linked
glycosylation sites are present in F2, of which mutations in the N70 residue influence fusion activity60.
This N70 residue is present at the outer surface of the molecule (Fig. 6B-D) and is probably
positioned at a perfect position for host cell interaction in the pre-fusion conformation (Fig. 6E). This
N70 residue is in direct contact with the F1 HRA region which attacks the host cell membrane during
the initial phase of the fusion event through a cys69-cys212 disulfide bond. Furthermore, the
adjacent F2 region (but not necessarily the asn residue) is conserved in related viruses and is
important in F function61.
4.3.2 G protein receptor interaction
At least part of the F protein content of a virion is in complex with G65. For both these
receptors several potential receptors have been found and the importance of each of these
interactions in vivo and in vitro for cell entry is not well defined. The major problem is that the
mechanism by which fusion is triggered is unknown. Since RSV is thought to enter cells through
fusion at the plasma membrane, it is highly likely that interaction with a specific cell surface bound
receptor triggers fusion. This direct trigger for fusion is probably an interaction mediated by the F
protein, since ∆G RSV strains can still enter host cells. On the other hand, the presence of F-G
complexes on RSV virions means G might play a more active role in the fusion step other than just
initial attachment. If G has such a role, the interaction with F could be either inhibitory or stimulating.
Host cell entry by viral proteins G and F could be happening through several different mechanisms
(Fig. 8).
1 – The G protein has no other function than attachment. Binding of G to a cellular receptor
increases the time spent in close proximity to the host cell membrane and thereby gives F
time to find its receptor. The direct trigger for fusion is interaction with F.
2 – The G protein is in complex with F. In addition to its attachment function, interaction with
G can also assist F fusion by bringing F in a (more) active state. The direct trigger for fusion is
interaction with F.
3 – The G protein is initially interacting with F. Upon binding with a cellular receptor, G
releases F, thus allowing F to initiate fusion. The direct trigger for fusion is interaction with G.
4 – The G protein is initially interacting with F. Upon binding with a cellular receptor, G
releases F, thus allowing F to find its specific receptor. The direct trigger for fusion is
interaction with F.
5 – The G protein has little or no function in attachment. Irrespective of what G binds, F
needs to find its receptor and mediates both attachment and fusion.
For each of these options goes that F and G interactions with the host cell might be specific, requiring
a single specific receptor, or be nonspecific, requiring interaction with a protein with ‘sufficient’
affinity. In addition, rather than one entry mechanisms, RSV might also use a combination of two or
more of these mechanisms, perhaps depending on the type of cell infected.
17
Fig. 8: Possible models of interaction between G and F leading to fusion initiation. (1) G is
attachment protein, interaction of F with its receptor leads to fusion. (2) G is attachment protein
and helps F find the proper receptor to initiate fusion. (3) G is attachment protein and upon binding
releases F to allow fusion. (4) G is attachment protein and upon binding releases F to allow scanning
for the F binding receptor. This secondary interaction initiates fusion. (5) G does not function as an
attachment protein. Independent of G, F both attaches and initiates fusion.
The most well-defined property of G is its interaction with GAGs. GAGs are long, unbranched
saccharide chains that consist of repeats of saccharide dimers of hexuronic acid (D-glucuronate
(GlcA) or L-iduronate (IdoA)) and hexosamine (N-acetylglucosamine (GlcNAc) or Nacetylgalactosamine (GalNAc)). The different combinations of these saccharide groups results in the
variation of GAGs. This variation is even greater when taking into account that each saccharide
molecule has the potential to be modified in several different ways. N-sulfation is done by enzymes
with the combined properties to remove acetyl groups from nitrogen atoms in the GAGs, and then
replace them with sulphate groups. These so-called N-deacetylase/N-sulfotransferases influence the
amount of sulfation on GAGs and may thereby influence RSV attachment66. Five types of GAGs are
expressed on the surface of a broad spectrum of cells: Heparan Sulfate (HS), Hyaluronic Acid (HA),
and Chondroitin Sulfate A, B, and C (CS-A, CS-B, CS-C)29. Of these five, CS-B and heparan sulfate were
shown to influence RSV infectivity in HEp-2 cells. This was done by pretreating these cells with GAG
specific proteases and comparing infectivity with that of untreated cells29. Also, exposure of RSV to
soluble forms of these GAGs could partially neutralise the virions. Soluble heparin, which is
structurally very similar to heparan sulfate, also had a significant neutralising effect. Heparin is not
expressed on the surface of respiratory epithelial cells though, and is therefore not considered an
important factor for attachment in a natural infection, although it could assist in attachment to some
cell types and in vitro cell cultures. Of the above mentioned GAGs, those that have N-linked
sulfations (HS, heparin) influence RSV infectivity. Of those that have no N-linked sulfations (CS-A, CSB, CS-C, HA) only addition of soluble CS-B was shown to slightly affect RSV infectivity. This indicates
that N-linked sulfations on host cell are indeed important in RSV entry. Still, other interactions are
taking place as well. Even in cell types with severe GAG synthesis deficiency no full inhibition of
infection can be established29. Furthermore, also F was shown to be (partially dependent on GAGs for
attachment37. It seems that RSV has several different potential interactions that lead to proper
attachment which includes GAG interaction by G or by F. GAG independent attachment also takes
place and is probably also mediated by a combination of G and F interactions. In table 2 an overview
is given of some host cell proteins and their viral interaction partners. Undoubtedly, as more studies
are done on this topic, more potential RSV receptors will be found. Although there is no clear
evidence explaining the mechanism behind the fusion step itself, it is good to keep in mind that also
attachment and fusion may be inducible by multiple independent pathways depending on the
surface expression level of both viral and host factors.
18
Table 2: Potential cellular RSV receptors and their viral binding protein
GAGs
ICAM-1
Nucleolin
Lactoferrin
SP-A
RhoA
TLR4
MD-2
Annexin II
L-selectin
DC-SIGN / L-SIGN
CX3CR1
G protein
Hallak et al.
Malhotra et al.
F protein
Karger et al.
Behera et al.
Tayyari et al.
Sano et al.
Sano et al.
Pastey et al.
Lizundia et al.
Rhallabandi et al.
Malhotra et al.
Malhotra et al.
Johnson et al.
Tripp et al.
5. Intracellular interactions with host cells
Intracellularly, interaction with host proteins is required for every step in the viral life cycle. For
successful infection, viruses use a broad spectrum of host proteins. This includes many factors as
diverse as cellular membrane bound proteins for recognition of proper cell targets, cytoskeleton
proteins for transport, and ribosomes for translation of the viral genome. Tropism of viruses for
certain cell types is not only based on their cell entry mechanisms. Cells that do not have the proper
intracellular machinery to produce infective new virions may on small scale get infected, but do not
spread the virus. For RSV several host proteins and protein systems have been described to be
necessary. Understanding interactions between host and virus proteins may give additional
information on the reasons for RSV cell tropism.
5.1 Entry
Entry in the host cell has been shown to occur at the plasma membrane in a pH independent
manner67. Still, blocking the endocytotic clathrin pathway impairs RSV cell entry. Because of this
apparent contradiction it is still not proven beyond doubt that RSV really enters cells only at the
plasma membrane. It has been proposed that RSV cell entry does not occur instantaneously after
attachment. Studies with viral membrane bound fluorophores have shown that membrane fusion
happens earlier than cargo release in the cytoplasm57. Fusion may then happen at the plasma
membrane, yet cargo release could be done from early endosomes. Many viral species replicate their
RNA genomes membrane bound or even membrane encapsulated to shield it from detection by TLRs
or degradation by RNAses. Remaining partially fused to an early endosome for prolongued periods of
time may have similar functions. Upon release of the cargo from the virion into the cytoplasm the
core gets transported to inclusion bodies. Little is known about entry and the following trafficking.
Some host proteins have been shown to be involved in this process somehow. HSP90 depletion leads
to increased association of N with actin14. This may indicate that RSV genomes and associated
proteins travel towards inclusion bodies via actin, and that HSP90 depletion can lead to less efficient
homing to inclusion bodies. Therefore more RSV material remains associated with actin. HSP90 was
also shown to be associated with viral inclusion bodies14.
5.2 Transcription & Replication
The concentration of required intracellular proteins can be of influence on the efficiency of RSV
infection in a specific cell type. If critical host proteins are absent in certain cells, it could even make
these cell completely unable to support RSV infection. It was hypothesised that infection of cells
other than respiratory epithelial cell leads to unproductive infection. Even in this situation infection
of local antigen presenting cells (APC) is not necessarily a waste of virions. Such infections would give
the virus the opportunity to modulate immune responses while avoiding immune activation. Type I
IFN system antagonists could then be transcribed and translated, while absence of replication
intermediates causes the infection to go unnoticed. Type I IFNs are a crucially important factor as a
link between APCs and adaptive immunity68 and blocking this system for APCs from the respiratory
19
tract may explain why immunity to RSV is inefficient and leads to recurring infections even in the
absence of significant antigenic drift. Still, even if this theory is true, it only explains the reasons for
RSV to infect epithelial cells and some APCs, it does not explain how. For VSV, another member in the
order of the mononegavirales (family Rhabdoviridae), two different types of viral RNA synthesis
complexes have been found, of which one is the transcriptase complex (L, P, and host proteins HSP60
and EF-1α) and the other is the replicase complex (L, P, N)69. This does not mean that the same sort
of complexes can be found for RSV, but theoretically this provides a mechanism by which some host
cells may support the full replication cycle, while in some host cells only transcription can occur.
Occasionally, infection of alveolar macrophages or PBMCs has been reported. Infection of RSV in
mice has been shown to result in detectable plasma viral load in addition to virus in the pulmonary
tract70. Interestingly, in contrast to the respiratory tract, no replication was detected in blood, which
is in support for the theory that infections outside the respiratory tract do not result in infective
virion production. The authors pinpointed the limited sensitivity of the assay as a possible
explanation for not finding replication in PBMCs. Still, this shows that replication in the blood is
either absent, or at least low70. A handful of reports have shown infections of organs other than the
lungs71 (reviewed in Eisenhut), such as the liver, the heart, and the cerebrospinal fluid. Apparently,
RSV can infect more types of tissue, which is in agreement with RSV tropism in vitro. The transition
from the lungs to other organs is very rare and could be a matter of chance, possibly linked to
genetic variations of the virus or a consequence of inadequate antiviral immunity outside the
respiratory tract environment.
Alveolar macrophages have been shown to be able to support replication and production of
new virions over prolonged periods of time72. This indicates that neither the correct receptors are
absent, nor some specific intracellular property of epithelial cells is lacking in macrophages. If indeed
macrophages support effective replication, it is remarkable that the virus does not spread
throughout the body. A gradual decline in the production of virions over a period of 10 days in these
macrophages was reported though, which may be a result of suboptimal conditions in
macrophages72. For the in vivo lung tropism this means that virus progeny from macrophages (and
possibly also of other cells that are not in the respiratory tract epithelium) may be less infectious,
which could cause only infection in the lung to persist. Unfortunately, no intracellular cell type
specific factor has been found that could explain the RSV tropism in vivo and in vitro. Some cellular
factors that have been shown to be of importance during replication intracellularly are actin
associated proteins such as cofilin-1 and filamin-1, and others such as HSP70, HSP90, and HSC7014
although the mechanisms involved remain to be elucidated.
5.3 Intracellular trafficking
The cytoskeleton, which consists mainly of actin and tubulin, is involved in all sorts of cellular
transport pathways. RSV, as well as any other virus, hijacks parts of this transport system to get to
the right place in a target host cell. It has been shown that RSV does not properly bud from cells with
defects in the apical recycling endosomal pathay (ARE)73. Actin, which is an important factor in this,
was also found in RSV virions, indicating its involvement during the final budding step. A study done
with inhibitors of the actin and microtubule systems also showed actin to be involved mainly in
budding from the plasma membrane. Inhibition of microtubules on the other hand, also affected cell
associated viral titers and are therefore probably influences assembly or trafficking steps early in
infection74. Actin may also play a role even before the RSV infection occurs. It was shown that
potential RSV receptor nucleolin colocalises with, and depends on actin for its deposition at the cell
surface75.
RSV M protein is located on the inner surface of the virion, and does not stick out into the
extracellular matrix and is not considered part of the core (N, P, L, M2-1, and RNA). M proteins
intracellular localisation is also different from that of other viral proteins. Whereas soluble proteins
get transcribed in the cytoplasm and membrane bound proteins follow the ER-golgi secretion route,
M can be detected in the cell nucleus early in infection. Its import is mediated by the β1 nuclear
import receptor76. The probable reason why M is being translocated to the nucleus is host shutoff. M
20
has been shown to be able to inhibit viral replication during the final stages of infection when it has
been exported from the nucleus in sufficient amounts. Also, transcription in RSV infected cells is
lower than in uninfected cells. Temporarily importing M into the host cell nucleus and thus not
exposing the cytoplasmic viral RNA to M also gives the virus time to transcribe sufficient amounts of
viral proteins. The mechanisms by which for M is imported into the nucleus during early stages of
infection, while later in infection the export is dominant, are not yet understood. For export from the
nucleus, M interacts with the Crm1 mediated nuclear export mechanism76. This step is crucial, since
M is necessary for efficient virus assembly in the cytoplasm and inhibition of more viral transcription
when packaging should be priority. Differences in trafficking efficiency of viral products between cell
types could influence the amount of infectious progeny produced by these cells.
5.4 Assembly and release
Assembly and release occur from inclusion bodies close to the cellular apical membrane. Airway
epithelial cells, the primary target cells of RSV infection, need specific trafficking pathways to
maintain the cells polarisation and the epithelial integrity. RSV is released only from the apical
membrane and is therefore likely to be hijacking such a trafficking pathway. In 2008 it was shown
that RSV requires the apical recycling endosomal (ARE) pathway for the final steps in the life cycle73.
The recycling endosome functions as a recycling compartment as well as a destination for proteins
that get transported towards the apical membrane. Blocking the ARE pathway with a dominant
negative form of the ARE associated protein FIP2 severely impairs RSV release. Given the function in
maintaining a cells polarity, the ARE pathway may be less active or absent in non-epithelial cells, thus
causing RSV virion release to be less efficient. Still, replication in non-polarised cells such as
macrophages can be found72,110.
6. Environmental Factors
Apart from receptor interactions and intracellular interactions, RSV might also be influenced by
extracellular factors. In the case of influenza it is known that proteases in the respiratory tract
mediate the activating fusion protein (HA) cleavage24. For some influenza strains, an important part
of the respiratory tract tropism is determined by the inability to cleave HA molecules outside the
lung, where the proper protease is absent. The RSV F protein is translated as an inactive F0 precursor
protein which undergoes activating cleavage by a furin like protease. The RSV F protein cleavage
happens at two distinct locations on the F0 precursor, between amino acid positions 109/110 and
136/137. The fact that two cleavage sites are present might indicate that a couple of specific
proteases are required for F activation. Cleavage of the two sites is necessary for virus infectivity77.
Cleavage of the two sites probably is mediated by two different proteases, since the two cleavage
site sequences are slightly different77. The respiratory tract may contain unique protease
environment. RSV virions may therefore have adapted specifically to this environment and are only
optimally infectious if they have been exposed to this lung specific mix of proteases. The high
specificity for respiratory tract epithelial cells, the occasional infection of respiratory tract APCs, and
the lack of further systemic spread can thereby be explained. This still leaves the matter of the
extensive RSV tropism in vitro unexplained. It seems highly unlikely that so many cell lines mimic
such an environment. Therefore, if the protease composition plays a role in RSV tropism it is a minor
role, influencing only partially the efficiency of infection. It could be that the cleavage event is a
matter of chance, determined by the concentrations of proteases present, or that lack of cleavage
reduces but not abolishes RSV infectivity. The respiratory tract would then be a location of relatively
high local transmission efficiency. Suboptimal protease concentrations could lower infectivity, but
not completely block it. In vitro cell cultures are packed with RSV permissive cells and have no antiRSV immunity. This may compensate for lower RSV infectivity caused by a lack of proteases.
Even though extracellular proteases are known to influence tropism of influenza, this whole
scenario is not very likely to affect RSV. In contrast to influenza HN cleavage, the maturating cleavage
step of RSV F0 takes place intracellularly in the golgi or trans-golgi network. Though not impossible, it
21
is unlikely that extracellular proteases like those in the respiratory tract lumen have a big effect on
the overall infectivity of virions. F0 can be expressed on the cell surface though78, which means that
extracellular proteases might have some effect. Also, the respiratory tract protease mix is maintained
somehow, and perhaps respiratory tract epithelial cells contain and secrete these proteases in high
quantities. This could explain why RSV preferentially infects this cell type for replication in vivo.
However unlikely, environmental factors may play a (small) role in RSV tropism and one has to keep
them in mind when trying to find the determinants for this tropism. So far there have been no
studies that give any evidence to support the existence of such factors. Other potentially influential
environmental factors are for example ion concentrations, since it was shown that RSV fusion is
dependent on Ca2+ 79.
7. Anti RSV immune response
Immune responses to RSV are very complex. No complete immunity to RSV is acquired during
an infection. This gives rise to recurring infections later in life, even in the absence of antigenic
changes80. Also, at a few days post infection, when the severity of the infection is often at its
maximum, the adaptive immune system is not yet active. It seems that innate immunity plays the
most important role. Innate anti RSV immune responses consist of many facets. Infected airway
epithelium cells detect RSV by pattern recognition receptors (PRRs) like TLRs. This results in
establishment of an antiviral environment through the production of cytokines, chemokines,
surfactant proteins and other antiviral compounds. RSV causes upregulation of numerous proinflammatory cytokines such as IL-6, IL-8, TNF-alpha, IFN-alpha, IFN-beta, IL-1beta, and IL-11 shortly
after infection. A variety of chemokines induces attraction and trans epithelial migration of many
different types of immune cells. Important chemokines are members of the CC chemokine family
(RANTES, MCP-1, MIP-1α, MIP-1β) and members of the CXC chemokine family (IP-10, IL-8)31.
Together these chemokines attract neutrophils, leukocytes and monoytes. Antiviral compounds such
as surfactant proteins can bind viral epitopes and assist in activating innate and adaptive immunity81.
Their importance is illustrated by the finding that RSV clearance does not occur in surfactant protein
A (SP-A) knockout mice. This is in apparent contradiction to the fact that SP-A can increase infectivity
of RSV in HEp-2 cells46.
Although both F and G influence chemokine expression levels in the respiratory tract, G
protein was shown to skew the immune response to Th2 orientation. The G protein also has
structural similarities to CX3C chemokine fractalkine and binds to its receptor (CX3CR1). The
fractalkine molecule mediates leukocyte chemotaxis82 and is mainly expressed on Th1 cells83. G
protein competition for the CX3CR1 may therefore hamper infiltration of the lungs with Th1 cells.On
the other hand, Th2 chemokine TARC is upregulated in mice under the influence of G protein84.
Subsequent degranulation of infiltrating cells that respond to G alterations in the lung leads to
enhanced Th2 cytokine release (IL-4, IL-5, IL-10, IL-13), and may be the basis for the Th2 skewing
during as RSV infection. The Th2 inducing function of G may be mediated either by the soluble or
membrane associated G. Importantly, while priming with G induces a Th2 biased response, F protein
induces Th1 immunity84. Th2 responses to RSV are a risk factor for developing more clinical disease,
while Th1 is associated with rapid clearance85. These data indicate that G is not only involved in
immune evasion, but also in eliciting the wrong type of immune response. This is probably the reason
why F alone is enough for RSV infection is many cell cultures, but G is required in vivo to compensate
for F induced Th1 responses.
Intracellularly in infected cells, virus products are detected by PRRs. This leads to innate
immune responses like production of type I IFNs. The most important type I IFNs are a variety of IFNα subtypes and IFN-β. All type I IFNs signal through the same receptor (IFNAR) and mediate
transcription of a broad spectrum of antiviral interferon stimulated genes (ISGs). Inhibiting type I IFNs
is vitally important during a virus infection. The main function of RSV accessory proteins NS1 and NS2
has been shown to be inhibition of synthesis and signalling of type I IFNs, and also RSV G inhibits the
type I IFN system86. Deletions in RSV proteins NS1 and NS2 proteins result in attenuated growth in
22
vivo and in vitro, showing that these IFNs play an important role even in cell culture87. Interestingly,
deletions in NS1 and/or NS2 hardly affected the growth characteristics of RSV in vero cells. Vero cells
do have the type I IFN receptor, but are known to be unable to produce type I IFNs themselves.
Although the type I IFNs are associated with innate immunity, by conducting a bridging function
between innate and adaptive immunity they also affect the efficiency of adaptive immune
responses68. It has been shown by microarray analysis, that in comparison with influenza, RSV had
very low induction of genes involved in the type I IFN system. This suggests again that the inhibition
of type I IFNs is a specific and effective function of RSV88.
It was shown that cytotoxic T lymphocytes (CTL) in the lung are less responsive to RSV than
other CTLs89. This phenomenon was independent of route of infection or even on the virus species.
Therefore it is likely that it is not caused by RSV, but rather an environmental factor specific to the
lungs. This tendency to tolerance might be required to avoid overreacting to the vast amount of
pathogens or allergens that invade the respiratory tract with every breath. By simply remaining
located in the respiratory tract, RSV exploits this reduced immune responsiveness. The mechanisms
contributing to the reduction in CTL activity are not understood, but may be linked to a specific
cytokine profile in the lung89. As many factors in the immune system are always linked together, CTL
tolerance probably reflects a more general reduced immune activity. The importance of this for the
respiratory tract tropism of RSV infection is unclear. Even in immune compromised individuals RSV
does not often spread systemically. Systemic spread of RSV is extremely rare, but still it was shown
both in mice and human that being immune compromised probably increases the risk of spread.
These data indicate that while the immune system is not the only tropism determinant, it does play a
role.
8. Concluding remarks
The tropism characteristics of RSV are very complex, with on the one hand a strict cell type specificity
in vivo and on the other hand an extensive list of infectable cells in vitro. The simplest approach to
control which cells are infected is by interacting with specific cellular receptors. In the search for the
determinant of RSV tropism many researchers have tried to find this receptor. After many years of
research several potential receptors have been found, but none of them could be pinpointed as the
sole explanation for RSV infection. Ciliated epithelial cells in the respiratory tract, which are the
natural targets in vivo for RSV, either do not express potential receptors or are not uniquely
expressed only in these cells. Since so many different receptors have been found over the years, a
few of which have been shown to be at least partially involved in RSV cell entry, RSV probably uses
multiple receptors for entry. Either one receptor out of a set of possible entry mediators, or a specific
combination of receptors are needed. Also the exact roles of the viral glycoproteins in this is still
unclear. RSV strains without G protein have been shown to be able to infect cell lines and in one cell
type even equally well as WT RSV. In vivo the G protein seems to be absolutely required, although
some studies have shown that several truncations, or deletion of membrane bound G hardly affected
the fitness of the virus. It is possible that the most important function of the G protein is its inhibition
of the type I IFN system. This would explain why soluble G is enough make a virus infectious. Also in
cell culture this is possibly the case, since the only cell line in which G protein is completely
redundant (vero cells) is known for its inability to produce type I IFNs even without the influence of
G. Also, no study yet has been able to completely inhibit RSV infectivity by blocking G protein
interactions. Antibodies to G do seem to affect the level of cell entry, but this effect is unrelated to
where the antibodies bind, and is maximal when using a combinations of antibodies to different
epitopes on G. This gives the impression that entry is not inhibited by blocking specific receptor
interaction with G. Instead, simply covering the virion with a layer of G specific antibodies may also
make it difficult for F to bind specific receptors. This is also supported by the finding that ∆G RSV
infects vero cells better than WT RSV when soluble heparin is added. In this situation no G protein
results in higher infectivity than blocked G protein. On the other hand, some studies have proven
23
that inhibiting G protein interactions without targeting G itself (proteolytic cleavage of G receptors,
knockdown of receptors, etc.) inhibits RSV infectivity. This means that either F is partially dependent
on the same receptors, or G contributes to the cell entry. Given all the ostensibly contradicting data
on RSV entry, it is highly likely that RSV receptors differ depending on the cell type and possibly even
on the RSV strain. For that reason, it is recommendable that further characterisation of RSV
receptors should be performed on primary human epithelial cells since they are the best
representation of the in vivo situation available. Receptors found in other cell types may give
important information about the virus, but should at least be confirmed in a more suitable model
before any definite conclusions can be made about the natural in vivo situation. Notably, even
primary epithelial cells can differ from the in vivo situation90, which makes this type of research very
challenging.
A study in 2005 about the tropism of RSV in several different cell lines has shown that entry
and replication are not necessarily comparable56. Some cell types can be infected by RSV
(determined by F protein mediated cell-cell fusion), while replication is absent. This proves that
presence or absence of replication is not only determined by cell surface receptors. Intracellular
factors are bound to play a role. Unfortunately, like for cell surface receptors, no obvious candidates
that might determine RSV tropism have been found. Intracellular factors may be proteins with which
RSV requires interaction, but might also be the tendency of a certain cell to enter an ‘antiviral’ state.
For example a difference in type I IFN production as a consequence of RSV infection may have a huge
effect on the amount of progeny virus produced. This principle is best illustrated by the equal
replication of ∆G RSV or WT RSV in vero cells which are defective in type I IFN production. In other
cell types ∆G RSV is less infectious, possibly as a result of replication inhibition by type I IFNs. Cell
lines in general often have less innate immune activity when compared to primary cells91. This could
be a reason for lesser permissiveness of these primary cells to RSV when compared to cell lines.
Table 3: Short summary of the determinants of RSV tropism in vivo
Tropism determinant
Receptor usage
Intracellular factors
Immune system
Physical confinement
Contributing factor?
Very likely
Very likely
Very likely
Very likely
Environmental factor
Unlikely
Blocking several receptors partially inhibits infection
Even while entry is possible, some cell types do not support infection
Systemic spread almost only occurs in immune compromised individuals
The vast majority of the RSV virions never encounter cells other than
epithelial cells
Although not impossible, no such factor has ever been found
A factor that is likely to be involved in respiratory tract tropism in vivo is the immune system.
It was shown in mice that RSV infection leads to systemic spread in some immune compromised
animals, and never in immune competent mice92. Also in the human host this seems to be the case93,
although systemic spread has been reported also for patients with a normal immune system in very
rare cases71. The luminal secretion of RSV virions from infected epithelial cells keeps the vast majority
of the RSV confined in the respiratory tract. Probably the respiratory tract tropism of RSV is the result
of a combination of many of the factors described above. Presence of surface receptors and
intracellular factors determine the efficiency of replication in certain tissues, and the immune system
determines the rate with which the virus gets cleared. Most likely, RSV adapted to all these factors is
such a way that the respiratory tract is the optimal tissue to infect. The advantage of this, is that RSV
is present in an organ from which transmission to a new host is easy. The apical secretion of virions,
and the specificity for epithelial cells is in that case a way to maximise the virus titer in the lumen (to
increase chance of transmission) while limiting the amount of infected cells (to reduce immune
activation and thereby clearance). It is possible that RSV infection is limited to the respiratory tract as
a result of multiple factors. That way, no single tropism determinant is likely to ever be found (Table
3). The only tropism determinant described in this review for which no evidence at all has been
found in the case of RSV are extracellular environmental factor. It is therefore highly doubtful, but
not impossible, that such factor exists.
24
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