Department of Protein Engineering
Biomedical Research and Study Centre
University of Latvia
Ratsupites Str., 1 Riga
LV 1067, Latvia
Reconstruction of Hepatitis B virus in vivo
Author:
Anna Zajakina
Supervisor:
Dr.habil.biol. Tatyana Kozlovska, BMC, Riga, Latvia
Opponents:
Prof. Viesturs Baumanis, University of Latvia, Riga, Latvia
Prof. Wolfram H. Gerlich, University of Gissen, Germany
Prof. Kestutis Sasnauskas, Institute of Biotechnology, Vilnius,
Lithuania
Summary of Academic Dissertation
Riga, 2004
Actuality of the work
Despite unceasing efforts of the medical community, hepatitis B (HBV) remains serious type of
viral hepatitis and one of the major problems of global public health. An effective recombinant
vaccine based on yeast-derived HBsAg is available for about 20 years. However, problems of
non-responding individuals and emergence of vaccine escape mutants remain unsolved.
Moreover, attempts at treatment of chronic infections have had only limited success.
Unfortunately, the progress in the development of more effective prophylactic and therapeutic
vaccines as well as the finding of specific antiviral treatment were hampered because of many
aspects in HBV biology have not been investigated in details.
The lack of a convenient cell culture and animal model for efficient production of HBV particles
is a central problem in elucidation of fine mechanisms of virus assembly and secretion. The
structural features of the virion are still unknown. All hepadnaviruses have very narrow host
ranges. Efficient infection by HBV is well documented for only humans and chimpanzees and,
in cell culture, for primary hepatocytes from these hosts. No other cell lines can be infected by
HBV. Therefore, the HBV interaction with cell surface molecules, which mediate virus entry,
virtually, remains unexplored.
In the absence of suitable infection system for HBV, different experimental systems are in use
to study HBV, however, all of them have distinct disadvantages. Many aspects of HBV biology
have been unravelled by studying related hepadnaviruses, such as the duck hepatitis B virus
(DHBV), which is capable of infection of cultured eukaryotic cells (Tuttleman et al., 1986), and
the woodchuck hepatitis virus (WHV), which allows the study in an animal (woodchuck) model
(Tyler et al, 1986). However, many significant differences exist among HBV and animal
hepadnaviruses.
Several HBV expressing cell lines have been established by transfecting viral DNA into liverderived human cell lines and by selecting novel cell lines containing stably integrated HBV
genomes (Weiss et al, 1996;Tsurimotoe*a/., 1987;Sellse*a/., 1987). However, there are some
inherent drawbacks which preclude the use of these cell lines in studying some aspects of HBV
biology. For example, the replication in this case is continuous, therefore, it is not possible to
experimentally control the time or conditions, under which these processes are initiated.
For immunological studies the HBV-transgenic mice proved to be very useful (Guidotti et al,
2002;Gao et al, 2004). However, this approach is very laborious.
The most prospective animal model for HBV studies presently seems to be an Asian tree shrew,
or tupaia, small primate-related animal susceptible for infection with human HBV (Walter et al.,
1996;Yan et al., 1996). However, the main disadvantage of this animal model is the low
efficiency of infection and HBV replication making difficult to prove formation of the complete
progeny virions.
Several approaches have been proposed for delivery of cloned HBV DNA into eukaryotic cells.
However, none of the established methods is ideal. Naked DNA (Will et al, 1985) and
liposomes (Takahashi et al, 1995) reach only a limited number of hepatocytes. More
perspective seems to be the virus mediated transfer of HBV genome proposed in last decade.
Recombinant baculovirus (Delaney & Isom, 1998;Delaney et al., 1999) and replicationdefective adenovirus (Delaney & Isom, 1998;Delaney et al, 1999) models were examined for
this purpose. There are, however, certain disadvantages of baculoviruses and adenoviruses as
vectors for studying HBV. The large genomes of baculoviruses and adenoviruses did not allow
the direct cloning of HBV genomes into these vectors, and production of recombinants is
necessary. Therefore, it is not possible to generate, within a reasonable time, vectors carrying
HBV genomes with defined genetic alterations. Additionally, the production of recombinant
baculoviruses and adenoviruses of suitable titres is time consuming process (5 -10 days),
including particles reamplification, purification, and concentration.
Therefore, the search for new, optimal and easy handled recombinant viruses for cell
transduction with the HBV genome is important, especially since the fine mechanisms of HBV
morphogenesis and release remain unclear. To study these points, and also for purely
biotechnological purposes including generation of new vaccine and gene therapy tools, as well
as for construction and selection of new antiviral drugs, we suggest here the well -known
Semliki Forest Virus vectors as an instrument to initiate HBV gene expression and production
of HBV virion-like particles in cultured eukaryotic cell lines. We tried to adopt this system for
efficient expression of the HBV structural and non-structural genes, in order to start detailed
investigations on the assembly, molecular architecture, and entry into the cells (including
specific attachment to cell receptors) of different forms of HBV virion-like particles.
Aims of this thesis
The focus of this study was to reconstruct the production of HBV particles in mammalian cell
culture using SFV-driven expression of HBV genes. Specifically this study set out to address
the following issues:
1) To adopt SFV expression system for efficient production of all HBV structural proteins
in cell culture.
2) To verify the correct glycosylation of HBs proteins and its secretion in form of subviral
particles; to show the correct intracellular self-assembly of HBc monomers into 28 nm
core particles.
3) As a first step towards elucidation of the HBV assembly mechanisms, to analyse the
possible HBV like particles secretion in the case of simultaneous synthesis of HBV
structural proteins.
4) To analyse the ability of HBV pgRNA to initiate the synthesis of all HBV structural
proteins, which could provide the formation and secretion of native virions.
Novelty of the work
Until this time SFV has never been applied for expression and studying HBV. However, the
universal nature of SFV expression system (Liljestrom & Garoff, 1991) made it ideally fitting to
accurate elucidation of HBV virion-like particles. The main advantage of the SFV vector system
lies in the high-level transient expression of cloned genes and production of recombinant viruses
with high titres, which infect broad range of eukaryotic cells. Moreover, it is convenient to use:
once the required recombinant plasmids have been constructed, recombinant virus stocks can be
prepared within one day. The expression of recombinant proteins driven by SFV replicons is
also rapid and can be detected already within six hours after infection. Besides, the simple
generation of recombinant plasmids, which does not need any recombination steps, represents
an extra significant advantage of the SFV model. The relatively small size of SFV vector (10
kb) allows its easy handling and propagation, as well as the introduction of mutations and
structural changes into the cloned fragments, which is a very obvious advantage for the
modelling of HBV replication and release, and construction of putative biotechnological tools.
Moreover, the SFV vectors have become attractive for rapid and high-level gene delivery for
gene therapy and RNA vaccine purposes (Ehrengruber, 2002;Lundstrom, 1999;Lundstrom et
al, 2001;Fleeton et al, 2000) that could be applied also for HBV treatment. We are the first
who tried to adopt the SFV system for efficient expression of HBV structural genes and
possible production of different forms of HBV VLPs. All four HBV structural genes, including
core (HBc) and three variants of envelope proteins (LHBs, or large; MHBs, or middle; SHBs, or
small), were cloned into SFV expression vectors and expressed in mammalian cell cultures.
Successful synthesis of HBV proteins was confirmed by immunoprecipitation (IP) of specific
HBV products from cell lysates. Analysis of extracellular fractions revealed successful
secretion of the MHBs and SHBs products in the form of subviral particles. Electron
microscopy showed formation of VLPs similar to the native Dane particles in the case of
simultaneous expression of HBV surface and core genes. Moreover, we found that expression of
HBV RNA pregenome (pgRNA), which naturally serves as a template only for HBc and viral
polymerase (Pol) translation, additionally provides the synthesis of all HBV envelope proteins
in SFV-driven expression. In the medium of pgRNA expressing cells we found 42 nm particles
morphologically indistinguishable from the Dane particles. Therefore, for the first time the high
potential of SFV based expression of HBV structural genes and pgRNA in cell culture was
shown conceptually. The development of such SFV based HBV virion producing system will
help efficient modelling of intimate mechanisms of HBV self-assembly and secretion. Possible
application of SFV-derived HBV virion-like structures as vaccines also is planned to be
elucidated carefully in future.
A B B R E VI AT IO N S
BHK
baby hamster kidney (cell type)
cccDNA
covalently closed circular DNA
DR
direct repeat
EM
electron microscopy
ER
endoplasmic reticulum
HBc
HBV core protein
HBeAg
HBV e antigen
HBs
HBV surface protein
HBsAg
HBV surface antigen, or HBs subviral particles
IP
immunoprecipitation
kb
kilobase
LHBs
HBV large surface protein
MHBs
HBV middle surface protein
MW
molecular weight
NP-40
nonidet P-40
nsP
non-structural protein
ORF
open reading frame
PCR
polymerase chain reaction
pgRNA
HBV RNA pregenome (pregenomic RNA)
Pol
HBV polymerase protein
SDS
sodium dodecyl sulphate
SDS-PAGE
SDS-polyacrylamide gel electrophoresis
SFV
Semliki forest virus
SHBs
HBV small surface protein
VLP
virus-like particle
Short description of the methods
General DNA techniques
DNA work was performed using conventional procedures (Sambrook et al. 1989). Reaction
conditions for PCR, DNA restriction and ligation were those recommended by the enzymes
supplier (Fermentas, Vilnius, Lithuania). E. coli strain DH5a (F-, 80dlacZM15, (lacZYAargF) U169, deoR, recAl, endAl, hsdR17(rk-mk+), phoA, supE44, - thi-1, gyrA96, relAl) was
used for cloning and propagation of plasmids. Detailed description of each plasmid can be
found in the individual publications.
SFV expression system
Here follows a brief description of the SFV expression system utilised in this study.
Electroporation of cells with in vitro transcribed RNA
RNA transcripts were synthesised in vitro from NruI -, or Spel-linearised plasmids using SP6
RNA polymerase. The RNAs were transfected into BHK cells, or other cell lines (HepG2, HuH7, COS-7) by electroporation. The transfection efficiency was close to 100 %.
Preparation of recombinant SFV stocks
SFV stocks with packaged recombinant SFV genomes were produced in BHK cells that had
been co-transfected by in vitro transcribed, recombinant RNA and a helper RNA. The
recombinant SFV preparations were harvested after 20 h incubation. The titers of SFV stocks
were determined by infecting BHK cells with serial dilutions of the stocks followed by indirect
immunocytochemistry assay for the expressed HBV protein (Salminen et al 1992). The
infection of cells was carried out with appropriate dilution of virus stocks, which infects 100 %
of cells.
Radio-labelling of synthesised proteins and preparation of cell lysate
[ 35S]-Methionme was used to metabolically label (pulse) synthesised proteins in infected
or
transfected cells for 1-2 h. Chase time 1-3 h was chosen to follow the intracellular transport
(secretion) of the radio-labelled MHBs and SHBs proteins. The chase medium with secreted
particles was analysed directly by SDS-PAGE, or by specific immunoprecipitation with
following analysis in SDS-PAGE.
A Nonidet P-40 (NP-40) containing buffer was used to lyse cells (Helenius and Soderlund
1973). This detergent solubilises lipids and membrane proteins. Cell nuclei were removed by
low-speed centrifugation.
Analysis of cellular RNA
The total cellular RNA isolated from infected cells by TriReagent (Sigma Labochema UAB,
Vilnius, Lithuania) was examined for the presence of HBV specific templates.
Immunoprecipitation (IP) of proteins from cell lysates
For the IP of HBc and preC proteins, rabbit polyclonal anti-HBc Ab were used. The IP of HBs
proteins was performed with goat polyclonal anti-HBs Ab. The attempts for the Pol protein IP
were done by polyclonal rabbit anti-Pol Ab. The HBV proteins were precipitated from cell
lysates prepared in NP-40 containing lysis buffer by incubation with corresponding Ab as
described in corresponding papers and analysed by 12 % S0S-PAGE. Gels were dried and
exposed to autoradiography film overnight or longer.
Immunocytochemical detection of intracellular HBV antigens by mAb
Infected BHK cells were fixed and incubated with corresponding anti-HBV mAb. Then cells
were incubated with anti-mouse IgG conjugated with alkaline phosphatase, the activity of which
was developed as described in corresponding papers. The evaluation was done using a light
microscope.
Concentration of extracellular particles
BHK cells were infected with SFVl/pgRNA virus and incubated for 48 h. After incubation, the
medium containing putative HBV-like particles was collected and the particles were pelleted
through the sucrose cushion by high-speed centrifugation as described in paper n. The pellet
was resuspended and used for EM analysis.
Cryo-lysate preparation and electron microscopy of HBV VLPs, HBc, and HBsAg
Since the NP-40 detergent used in the lysis buffer solubilizes the membrane proteins and
destroy, therefore, HBV envelope, we prepared cryo-lysate to visualize intracellular virion-Hke
and HBsAg particles in EM. BHK cells in 20-24 h post-infection were washed with PBS and
scraped from plates in PBS. Then cell suspentions were subjected to lysis by freezing/thawing
technique (three times), where the liquid nitrogen was used for rapid freezing of cells, the
thawing of cells was done at room temperature.
The aliquots of cryo-lysates, concentrated and untreated cell media, and standard NP-40 lysates
were absorbed on carbon-formvar coated grids, stained with 2% phosphotungstic acid (pH 6,8),
and analysed by EM performed in a JEM 100C electron microscope (JEOL Ltd., Tokyo, Japan)
at 80 kV accelerating voltage.
Overview of the results
Expression of HBV structural genes by SFV replicon. Evaluation of different kinds
of H BV particles formation
We were interested to reconstitute assembly and secretion of HBV-like particles (VLPs) via
effective synthesis of its proteins. Two SFV-derived vectors were used for expression of HBV
structural genes (fig. 1): (1) pSFVl, providing direct expression of HBV gene under the control
of 26S SFV subgenomic promoter; (2) pSFV-C, providing the SFV core-dependent expression
of HBV gene, where the target product was obtained after processing of SFV core-HBV protein
fusion.
Fig. 1. Schematic representation of HBV expressing constructs. The HBV structural genes
including HBc and HBV envelope protein (L-, M-, andSHBs) genes were cloned into pSFV1 (A)
and pSFV-C (B) vectors under control of SFV subgenomic 26S promoter. In pSFV-C vector the
HBV proteins were fused with the original SFV core protein, which cleaves itself after
translation. Only SFV recombinant region is shown. NsP1-4 - SFV non-structural genes
encoding replicases for transcription of subgenomic RNA. SP6 RNA polymerase promoter was
used for in vitro transcription of recombinant RNA with following its transfection into
eukaryotic cells.
The corresponding recombinant SFV/HBV viruses were produced by cotransfectio n of
transcribed in vitro recombinant RNA and a helper RNA, the latter providing the synthesis of
SFV structural proteins. The infection of BHK cells with these recombinant viruses led to the
synthesis of HBV proteins that was confirmed by IP analysis of B HK cell lysates, or by
immunocytochemistry. In contrast to the relatively inefficient synthesis of HBV proteins driven
by the pSFVl vector, especially in the case of SFVl/MHBs, the SFV-C system assured high
level expression of all structural HBV proteins, which is shown on figure 2 using the HBc
protein synthesis in both vectors as an example.
fig. 2. Immunocytochemical detection of HBc protein produced by pSFV1 and pSFV-C
vectors in BHK cells. The cells were infected with appropriate recombinant SFV/HBc virus. At
20 h postinfection, the cells were fixed and processed as described in Materials and Methods.
The uninfected BHK cells were used as a negative control. The red staining shows the
distribution of HBc protein detected with monoclonal anti-HBc antibody. The figure
demonstrates the high production efficiency of HBc in pSFV-C vector, whereas the pSFV1
vector-driven expression revealed the low level of recombinant protein synthesis.
Products of LHBs, MHBs, and SHBs genes appeared also in glycosylated forms in the same
way as during HBV infection in human (Stibbe & Gerlich, 1983). SHBs products existed as
non-glycosylated p24 and mono-glycosylated gp27 molecules. MHBs product showed three
bands: non-glycosylated p31 (not detected during viral infection), mono- and double
glycosylated forms gp33, and gp36. LHBs product appeared in forms of non-glycosylated p39
and mono-glycosylated gp42. Surprisingly, the expression of pSFV1/LHBs construct provided
the synthesis of all three variants of HBs proteins. The possible internal translation of the HBs is
described below (fig. 8 A, page 19). Analysis by IP and EM of extracellular fractions revealed
the successful secretion of MHBs protein in form of subviral particles (fig. 3 C). The EM
analysis of cryolysate of BHK cells producing SHBs showed the presence of 22-nm SHBs
subviral particles (fig. 3 A), which were found also in extracellular fractions (not shown).
Interestingly, we observed more efficient SHBs secretion of ayw subtype of HBV, whereas the
SHBs of adw subtype demonstrated rather low secretion ability. As expected, LHBs products
were not secretable. By EM we found also the 28 nm HBc particles in the lysate of HBc producing cells (fig. 3 B) and intracellular rod-like particles in LHBs producing cells (not
shown).
Fig. 3. Electron micrographs of HBV particles produced in BHK cells upon
SFV driven expression, (A) HBsAg-22-nm-like particles in cryo-lysate of
pSFV1/SHBs transfected BHK cells. (B) HBV core particles (HBc) in the NP-40
containing lysate of BHK cells transfected with pSFVl/HBc. (C) Secreted HBsAg22-nm particles in the medium of pSFV-C/MHBs transfected BHK cells. (D) HBV
VLPs in medium of BHK cells co-transfected with SFV-C/HBc and SFV-C/MHBs.
(E) The native HBV virion. Bars, 20 nm.
The BHK cells, which were used in these experiments, are not natural host cells for HBV.
However, they appeared optimal for infection with and production of recombinant SFV
particles, allowing the highest yields of recombinant proteins synthesis. Beside the BHK cells,
we established similar expression patterns of the HBV proteins for all studied constructs in
others cell lines (HuH-7, HepG2, COS-7), only the level of production was lower. The results of
the expression of HBV structural genes are summarised in Table I.
Therefore, we have demonstrated the expression of all HBV structural genes by SFV replicon.
According to the idea of HBV particles reconstruction composed from different HBV structural
proteins, we tried to co-express them with different combinations using more effective SFV-C
vector (Table 2.). Co-transfection of BHK cells with different combinations of RNAs led to
efficient equimolar production of HBV structural proteins, even if all four genes were expressed
simultaneously. Since the LHBs product was considered to be an inhibiting secretion agent, we
limited its production level by a two-fold decrease of its RNA for cell transfection comparing
with other RNAs, which were used equimolary. The electron microscopy analysis of the
medium of cells co-transfected with MHBs and HBc showed the presence of not only 22-nm
HBsAg particles, but also HBV virion-like particles similar to the native Dane particles from the
human blood (fig. 3 D, E). We observed also that the co-expression of MHBs and HBc led to
the increase of NP-40 soluble
Table 1. The HBV structural genes expressed by SFV repticon. (a) Variants of the constructs
used in this study, (b) Expression level of the HBV proteins found in BHK and HuH-7 (human
hepatoma) cell lines. Cells were infected with recombinant SFV preparation of appropriate
constructs, pulse-labeled with 35S-methioninefor 2 hours, and cell lysates were used for specific
HBV protein IP, which were analysed in SDS-PAGE. (c) Intracellular and extracellular particle
formation. Expression of the HBc gene led to formation of 28 nm HBc particles; SHBs and
MHBs - 18-22 nm HBsAg particles; LHBs - rod-like particles of 22 nm in diameter the same
as described by Xu (Xu et al, 1997). The corresponding particles were found by EM of infected
cell lysates, cell cryolysates, or in the media of infected cells. Perceptible level of protein
production indicated with +; high level with ++; very high level with +++; ND = not
determined; +/- = traces; - =not found Apart of the HBc protein was insoluble in NP-40
lysate.
Table 2. Co-expression ofHBV structural proteins produced by SFV-Cvector, (a) BHKcells
were transfected with RNAs of corresponding constructs using different combinations, (b) The
synthesis of each protein in the appropriate combination was proved by JP analysis of
metabolically labelled cell lysates in SDS-PAGE. Formation of VLPs was estimated by EM of
the medium of transfected cells.
fraction of the HBc protein, which usually was partially insoluble, when produced in pSFV-C
vector. This additionally points out the interaction of MHBs and HBc proteins inside the cell.
Although visualisation of the HBV VLPs by EM was convincing, their real number was low.
According to earlier studies, the HBV particles secretion involves genomic DNA synthesis
(Gerelsaikhan et al, 1996). Therefore, the VLP formation in absence of DNA replication could
be inefficient. Probably, this reason was decisive in detection of VLPs in media of cells cotransfected by structural protein genes in different combinations, since only MHBs and HBc cosynthesis demonstrated the small amount of VLP secretion. Nevertheless, this result in contrast
to other attempts (Shiosaki et al., 1991;Takehara et al., 1988) shows the principal possibility of
HBV assembly in condition of high level expression of structural genes, but in absence of DNA
synthesis, and provides new views on the HBV assembly process. Moreover, the further
adaptation of SFV-driven production of HBV VLPs could promote the development of
improved vaccine on the basis of such particles.
SFV-driven expression of HBV pgRNA
There is evidence that HBV pgRNA plays a key role in HBV biology. Once the pgRNA is
synthesised, it serves as a template for the translation of viral HBc and Pol proteins (Ganem &
Varmus, 1987). On the other hand, the pgRNA is a target for encapsidation into the HBV
nucleocapsids together with the Pol for further reverse transcription (Pollack & Ganem, 1993).
The newly synthesised partially double-stranded HBV DNA may be transported by mature
nucleocapsids back to the cell nucleus (Kann et al, 1999;Rabe et al, 2003) and enrich the pool
of intranuclear transcribed HBV DNA for the synthesis of all the viral mRNAs. We were
interested in determining if the SFV system could be used to reconstitute assembly and secretion
of HBV VLPs via effective cytoplasmic synthesis of the HBV pgRNA. Moreover, the coexpression of pgRNA with HBV structural genes, theoretically, may promote the VLP
formation by the mimic of the native assembly process. Our idea is schematically represented on
Figure 4.
Fig. 4 The principl of HBV virion productio by infection of the cell with recombinant SFV
containing HBVpgRNA. After cell infection with recombinant SFV1/pgRNA virus, the SFV replicases
provide the replication ofRNA and synthesis ofpgRNAfrom SFV subgenomic RNA promoter. pgRNA
serves as a template for HBV core and polymerase translation (Pol, TP - terminal protein - indicates the
Pol domain covalently linking to the (-)DNA strand) with following encapsidation of pgRNA and Pol
into core particles. These nucleocapsids transport the newly synthesised HBV DNA, which appeared via
reverse transcription (RT) of pgRNA, into the nucleus, where the cccDNA is formed. The specific HBV
transcripts transcribed from HBV cccDNA provide the synthesis of envelope proteins that form the
envelope of secreted HBV particles.
A DNA copy of the full-length HBV pgRNA was cloned into the pSFV1 expression vector
under the control of the subgenomic 26S SFV promoter. The resulting plasmid pSFVl/pgRNA
was used for production of corresponding recombinant SFVl/pgRNA virus. This virus, used for
further infection of the BHK cells, induced effective cytoplasmic synthesis of the pgRNA. The
IP analysis of cell lysate showed the presence of HBV structural proteins as soluble products in
the NP-40 lysis buffer. The HBc protein was detected as a clear 21 kDa band. As expected, we
found the synthesis of all three envelope proteins (LHBs, MHBs, and SHBs). The SHBs
products existed as non-glycosylated p24 and mono-glycosylated gp27 molecules. The MHBs
products showed three bands: non-glycosylated p31, mono- and double glycosylated forms gp33
and gp36, correspondingly. The LHBs products occurred as non-glycosylated p39 and monoglycosylated gp42 forms. However, the level of HBs production was low as compared with
direct expression of HBV structural genes in the same vector. This was confirmed by
immunocytochemical analysis of infected BHK cells (fig. 5).
Fig, 5. Immunocytochemical detection of HBs proteins in BHK cells. Cells were
infected with appropriate recombinant virus (SFV1/pgRNA and SFV1/LHBs). At 20 h
postinfection, the cells were fixed and processed as described in Materials and Methods.
Proteins were detected with monoclonal anti-preS1, anti-preS2 or anti-HBs antibodies
(indicated in white rectangles). Uninfected BHK cells incubated with the same
antibodies were used as a negative control (not shown). The figure shows the high
production level of HBs provided by the SFV1/LHBs construct, comparing with the
same ensured by the SFV1/pgRNA construct.
EM was used to analyze the production of extracellular HBV particles. The BHK cells were
infected with SFVl/pgRNA virus, incubated for 48 h, and putative HBV virion-like particles
from the cell culture medium were pelleted through the sucrose cushion. We observed in these
patterns the presence of 42-nm particles morphologically similar to the native HBV virions.
Moreover, the cryolysates of SFVl/pgRNA infected cells contained such Dane-like particles as
well, which probably were retained inside the cell (fig. 6).
It was interesting to know, whether these VLPs contain the DNA genome, which could appear
by reverse transcription of pgRNA by the Pol protein. The pilot experiments including the PCR
analysis of intracellular and extracellular fractions of BHK cells infected with SFVl/pgRNA
showed the presence of HBV DNA genome in the lysate and in the medium (not shown). The
fractions were treated with DNase in order to extract only the nucleic acids protected by
particles. The protected nucleic acids were extracted by phenol/chloroform. Our PCR results
show the production of active Pol protein from the pgRNA and synthesis of at least (-)-DNA
strand of the HBV genome. Moreover, there have been shown already that Sindbis virus-based
expression of the pgRNA of Duck hepatitis B virus (DHBV) provides the DNA genome
synthesis (Huang & Summers, 1991). This finding indirectly indicates the competence of
Alfavirus-based cytoplasmic expression of the pgRNA for initiation of HBV replication.
Fig. 6. Electron microscopy of HBV 42 nm virion-Hke particles in the cryolysate and
concentrated cell culture medium of the BHK cells infected with the SFV1/pgRNA virus. The
28 nm core particles were also found in concentrated medium of infected cells (s een in the
medium). Native HBVvirion from infected human blood was used as a control
Since the mature nucleocapsids should be transported to the nucleus, we applied the
immunocytochemical technique to examine its possible nuclear localization. As expected, we
found the nuclear anti-HBc staining of BHK cells expressing pgRNA (not shown). Contrary to
this, the direct HBc protein expression from pSFVl/HBc construct, where no DNA synthesis is
possible, did not display any nuclear localization of HBc protein. However, we found the
nuclear localization the HBc deletion variants produced by SFV replicon (Bruvere R. et al,
2004). The reason of this phenomenon is not clear, and is the subject for further investigations.
We plan to use the HBc deletion variants for detailed studies of morphogenesis and release of
HBV virions under condition of co-expression with pgRNA.
The development of animal model (mouse, tupaia et al.) for advanced HBV studies also can be
considered in the context of this study. Our preliminary results show that intravenous injection
of recombinant SFV virus containing an EGFP (Enhanced Green Fluorescent Protein) gene as a
marker can target the virus to the liver of infected mice. Therefore, we plan to use the mouse
model for the one-step infection with the SFV/pgRNA virus, in order to initiate production of
the HBV-like virions in vivo. However, since we have shown here that the non-human, HBVnon-specific, cell line (BHK) can support production of the HBV-like particles, the SFV model
can be used without obligatory liver targeting.
Internal translation of HBs proteins from HBV pgRNA-like templates in SFV-based
expression
As was described above, the pgRNA initiates the synthesis of all three variants of HBs proteins.
We supposed that this is the result of specific transcription of the HBs mRNAs in the nucleus,
where the mature DNA genome could be transported by the nucleocapsid. However, the HBV
sequence specific Northern blot analysis of total RNA isolated from cells infected with the
SFVl/pgRNA virus did not revealed the presence of specific HBs transcripts (2-4 and 2-1 kb).
Only the recombinant genomic and subgenomic RNAs were detected. This result prompted us
to suppose the internal translation initiation of HBs proteins. To verify this idea, we expressed
in SFV1 expression vector the other HBV templates, such as pcRNA, pgRNA3' and Pol gene,
internally containing the HBs sequences (fig. 7). These mRNAs can not provide the HBV DNA
replication.
Fig. 7. Schematic diagram of recombinant SFV-driven constructs for analysis of internal
translation of HBs proteins (L-, M-, SHBs). (A) HBs ORF and its products. The box
representing ORF is divided into three domains (preSl, preS2 and S) by located in frame AUG
codons (vertical bars) for the translation of the LHBs (L), MHBs (M), and SHBs (S) proteins
(depicted above as horizontal bars), respectively. (B) Constructs used in this study. Only the
SFV recombinant region of each construct is shown. The nsPl-4 genes encode the SFV
replication complex. pSFV1/LHBs, pSFV1/pgRNA, and pSFV1/Pol contain the HBV
sequences of LHBs gene, pgRNA (with direct repeat elements DR1 and DR2), and Pol gene,
respectively. pSFV1/pcRNA has preC region on the 5' end of pgRNA. pSFV1/pgRNA3' 
represents the pgRNA with deleted 3' DR1. The vertical interrupted lines restrict the
approximate position of HBs genes in all constructs. The recombinant RNAs for transfection
were transcribed in vitro by SP6 RNA polymerase following linearisation of the DNA construct
with Nrul (not shown). Note that coding regions indicated are not to scale.
Surprisingly, the expression of all these templates showed the synthesis of three variants of HBs
proteins (fig. 8 D, E, F) in the same proportion as was described for pgRNA (fig. 8 C).
Moreover, we revealed the extreme correlation between the length of the 5' end of mRNA and
the level of the HBs protein production. The translation of them decreases from the highest level
for the pSFV1/L construct to the pSFV1/Pol and finally to the pSFV1/pgRNA,
pSFV1/pgRNA3', pSFV1/pcRNA constructs. The latter three constructs supported similar low
synthesis of the HBs proteins, all having long 5' end preceding the start codon of the L gene.
Beside the HBs translation, we revealed the HBc and pre-core (preC) protein synthesis provided
by pc- and pgRNAs (fig. 8 C, D, E). Unfortunately, we could not detect the Pol protein
translation neither in the case of pSFV1/pgRNA expression nor in the direct pSFV1/Pol
expression. Our polyclonal anti-Pol antibodies demonstrated strong non-specificity in
immunoprecipitation and immunoblot experiments. Moreover, the very low level of production
of this protein caused by (/) translation via the ribosome leaky scanning model (Lin & Lo,
1992;Fouillot et al, 1993), and {if) unfavourable sequence context around the Pol start codon
(Kozak, 1987) usually established additional difficulties for the detection of the HBV Pol
protein.
The described HBs synthesis represents a new example of internal translation initiation for three
proteins in the same ORF, the AUG start codons of which are located more than 1000 nt
downstream from the 5' end of the template. We do not know, whether this additional synthesis
of HBs proteins from the pgRNA could play a definite role in HBV biology. Transcription of
specific mRNAs in the nucleus is the initial regulation stage of HBV gene expression. This
process depends on the activation of promoter/enhancer elements, which are sensitive to the
presence of hepatocytespecific factors (Antonucci & Rutter, 1989;Kosovsky et al, 1996;Tang &
McLachlan, 2001). In the case when L and S gene promoters are silent, due to accidental
mutations or deficiency of the cellular factors, the pgRNA may appear as a unique source of HBs
protein translation. Moreover, the capability of HBV to infect cells of other organs, such as
kidney, pancreas (Dejeane* a/., 1984), or some blood cells (Blum & Vyas, 1983;Lobbianief a/.,
1990), where the expression is hampered by the absence of hepatocytespecific transcription
factors, may be provided by additional mechanisms for the HBs protein synthesis including such
from pgRNA. Therefore, the pgRNA alone may be able to initiate viral production by ensuring
synthesis of Pol and all HBV structural proteins.
Nevertheless, the finding of internal translation of HBs proteins from the pgRNA does not
refute the idea of retrograde transport of mature nucleocapsids into the nucleus, where the
specific HBs transcripts have to be synthesized, presented on figure 4 (page 14). This process
of cccDNA formation in hepatoma cell cultures usually takes more then 5 days (Liu et al.,
2004). Since the SFV experimental model extends the strong cytopathic effect to the
transfected/infected cells, there is impossible to incubate cell monolayers longer than three days
and to provide the possible cccDNA formation. Recently, non-cytopathic genomes of SFV and
Sindbis virus (SIN), both representatives of Alphaviruses, were isolated and adopted to foreign
gene expression (Perri et al, 2000). To examine whether the pgRNA may initiate and support
all steps of HBV replication, these non-cytopathic vectors will be applied for pgRNA expression
in future.
Fig. 8. Analysis of internal translation of HBs proteins produced in BHK cells infected with
appropriate recombinant SFV virus. Infected cells were pulse-labeled with 35S-methionine and cell
lysates were used for specific HBV protein IP with goat anti~HBs antibodies (aHBs), or with rabbit antiHBc antibodies (aHBc). The IP was analysed by 12 % SDS-PAGE. (A) pSFV1/LHBs. The positions of
various forms of the S, M, andL proteins are given on the left (lane 1). The band indicated as p31? could
represent a non-glycosylated form of the M protein, although its molecular mass on the gel is smaller
(about 29kD) than reported for the non-glycosylated M protein, (B) Negative controls. The IP of
uninfected BHK cells incubated with rabbit anti-HBc antibodies (lane 1) and goat anti-HBs antibodies
(lane 3). The lysate of uninfected BHK cells-lys (lane 2). (C) pSFV1/pgRNA. HBs proteins marked with
dots/stars here and below (on D, E) consistently from bottom to the top: S (p24, gp27), M (p31, gp33,
gp36), L (p39, gp42) (lane 2). A possible band corresponding to a non-glycosylated form of the M
protein (p31) is very weak on this gel (marked with a star). HBc protein - Cp21 (lane 3). The origin of
an additional band of about 26 kDa (p26 ?) in anti-HBc IP is unclear. The figure is continued on the
next page.
Fig. 8 (continuation). Analysis of internal translation of HBs proteins produced in BHK cells infected
with appropriate recombinant SFV virus. (D) pSFV1/pcRNA. HBs proteins - lane 2. A possible band
corresponding to the non-glycosylated M protein is marked with a star. preC protein (preC p25), its
processed forms preCp22 and HBe pi 7, as well an unknown protein of about 26 kDa (p26 ?), are shown
with arrows (lane 3). (E) pSFV1/pgRNA3'k. HBs proteins - lane I. The p31 and gp36 forms of the M
protein were not found. However their expected positions are indicated with stars. HBc protein (C p21)
and unknown HBc specific protein (p26?) are marked with arrows (lane 3). (F) pSFV1/Pol (lane 2). The
anti-HBs immunoprecipitate ofSFV1/pgRNA infected cells was analysed in lane 3, allowing us compare
HBs production level with the pSFV1/Pol construct. The positions of various forms of the S, M, and L
proteins are given on the right. Double the number of cells were used for the lysis and IP of HBs
proteins produced by pgRNA, pcRNA and pgRNA3'A than in the case of Pol or direct LHBs expression.
Overexposure of the film was necessary to visualise the very low level of translation of the HBs proteins
expressed by pgRNA, pcRNA andpgRNA3'A templates. MW- rainbow 14C-methylated protein marker
(Amersham). The positions of protein size markers (in kDa) are indicated on the sides of the gels.
CONCLUDING REMARKS
1. We have expressed all structural genes of human hepatitis B virus (HBV) in Semliki
forest virus (SFV) expression vectors pSFV1 and pSFV-C allowing direct and SFV coredependent expression of HBV genes, respectively. Three variants of HBV surface genes,
large (LHBs), middle (MHBs) and small (SHBs), as well as core (HBc) gene have been
amplified by PCR technique as independent units and as fusions with SFV core protein
gene, cloned in both SFV vectors and expressed in BHK cell culture as single proteins or
in different combinations. Contrary to relatively inefficient synthesis of HBV proteins in
pSFV1 system, pSFV-C system assured high level expression of all structural HBV
proteins where target products were obtained after processing of SFV core-HBV protein
fusions. All fused SFVC-HBV proteins were split correctly. Products of SHBs, MHBs,
and LHBs genes appeared also in glycosylated forms in the same way as during HBV
infection in human. IP analysis of expressed products demonstrated their immunological
specificity.
2. Analysis of extracellular fractions revealed the successful secretion of subviral HBs
particles form BHK cells expressing MHBs, or SHBs genes. As expected, LHBs as well
as HBc products were not secretable, however, were able to form intracellular LHBs rodlike and core particles, correspondingly. Moreover, we found virion-like particles (VLPs)
in the medium of BHK cells producing HBc and MHBs proteins, which were
morphologically similar to the native virions from the human blood. Therefore, the SFVdriven expression of HBV genes could represent a new model for HBV self-assembly
and secretion of different kinds of HBV subviral and VLPs in cell culture.
3. The complete HBV RNA pregenome (pgRNA) was inserted into the pSFV1 vector and
expressed in BHK cell line. As a result of pgRNA expression, beside the HBc protein
synthesis, the production of all three forms of HBs proteins was detected. The analysis of
concentrated medium of pgRNA expressing BHK cells revealed the secretion of VLPs
similar to the native virions. These observations allow us offer a new approach for
production of HBV in cell culture.
4. The expression of pgRNA-like templates including precore RNA (pcRNA), 3' deleted
pgRNA (pgRNA3'A) and frill length Pol gene in pSFV1 expression vector showed the
internal translation of three forms of HBs proteins. Maximal production of the HBs was
provided by the Pol mRNA, while the pcRNA, the pgRNA3 'A, and the native pgRNA
showed relatively low internal translation of the HBs. These data allow the proposal of a
ribosome leaky scanning model of internal translation initiation for HBs proteins in SFVdriven expression. The putative functional role of such exceptional synthesis of HBs
proteins from pgRNA and pcRNA templates in the natural HBV infection process needs
further evaluation.
5. Reckoning up the results described here, we suppose that SFV-based HBV gene
expression could provide an appearance of new insights on many aspects of HBV
biology. There are a broad spectrum of applications that could be developed on the basis
of the proposed model, including advanced studies of HBV replication, self-assembly,
budding and entry into new cells, and high-resolution structural analysis of different
HBV forms. From the practical point of view, the proposed SFV-driven model opens
new roads for the real generation of HBV-based gene therapy and protein/RNA vaccine
tools, as well as for simple and non-expensive models for the design and screening of
antiviral drugs.
This thesis is based on the following publications:
> Kozlovska T., Zajakina A., Ose V., Bruvere R-, Aleksejeva J-, Pumpens P,, and
Garoff H. 2004. Synthesis of all Hepatitis B structural proteins in Semliki Forest Virus
expression system. Ada Universitatis Latviensis, Biol, 676: 39-51.
> Zajakina A., Ose V., Garoff H., and Kozlovska T. 2004. New experimental approach for
production of HBV virion-like particles in eukaryotic cell lines by SFV-replicon-driven
expression. Proc.Latv.Acad.Sci., 58: 49-54.
> Zajakina A ., Kozlovska T., Bruv ere R., Alekse jeva J., Pumpens P., and Garoff I I .
2004. Translation of the Hepatitis B virus (HBV) surface proteins from the HBV
pregenome and precore RNAs in Semliki Forest virus driven expression. J.Gen. Virol., 85:
3343-3351.
The results were presented on the following conferences:
Zajakina A., Garoff H., and Kozlovska T. Hepatitis B virus assembly in SFV expression
system. "International congress of medical students and young physicians", May 20-22, 2001,
Poznan, Poland.
Zajakina A., Kozlovska T., Aleksejeva E., Ose V., H. Garoff. Synthesis of the Hepatitis B
proteins in Semliki Forest Virus expression system. International Conference 'The cell biology
of virus infection", September 22-26, 2001, EMBL, Heidelberg, Germany.
Kozlovska T., Zajakina A., Aleksejeva J., Bruvere R., Pumpens P. Reconstruction of Hepatitis
B particles in Semliki Forest Virus expression system. "Baltic States Congress on Hepatology",
October 3-5,2002, Riga, Latvia.
Bruvere R., Zajakina A., Garoff H., Kozlovska T. Intracellular localization of hepatitis B virus
core and surface proteins encoded by recombinant Semliki forest virus replicons. 13-th Annual
Conference of the German Society of Cytometry, October 16-18, 2003, Heidelberg, Germany.