1 Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 S. Grand Blvd, Saint Louis, MO 63104, USA
2 Saint Louis University Liver Center, Saint Louis University School of Medicine, 1402 S. Grand Blvd, Saint Louis, MO 63104, USA
Correspondence
John E. Tavis
tavisje{at}slu.edu
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ABSTRACT |
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INTRODUCTION |
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Encapsidation is the process in which the pgRNA and P are incorporated specifically into nascent viral core particles. In this ordered process, P binds to an RNA stemloop () at the 5' end of the pgRNA (Bartenschlager et al., 1990
; Hirsch et al., 1990
; Junker-Niepmann et al., 1990
) and then capsids form, presumably through polymerization of C protein dimers around the P : pgRNA complex. The HBV capsid contains 120 dimers of C in an icosahedral arrangement (Bottcher et al., 1997
; Crowther et al., 1994
), this core particle serves to protect the genome from hazards such as nucleases and to provide an optimal environment for reverse transcription.
The pgRNA is not only an intermediate in DNA replication, but it is also a bicistronic mRNA that encodes C and P. Although the P open reading frame (ORF) is downstream of the C ORF on the pgRNA, it is translated by de novo initiation from its own AUG codon, rather than by frame-shifting such as is employed by retroviruses (Chang et al., 1989; Fouillot & Rossignol, 1996
; Hwang & Su, 1999
; Lin & Lo, 1992
; Ou et al., 1990
; Schlicht et al., 1989
). The unusual bicistronic structure of the pgRNA and difficulties in detecting P by enzymic or physical methods led to the impression that P was inefficiently translated and rapidly encapsidated. This in turn led to the belief that P did not accumulate to detectable levels outside core particles. However, we found recently that the Duck hepatitis B virus (DHBV) P is translated rapidly, is encapsidated inefficiently, accumulates to easily detectable levels in the cytoplasm and is degraded rapidly (Yao et al., 2000
, 2003
; Yao & Tavis, 2003
). Therefore, DHBV P is primarily a short-lived cytoplasmic protein. Coupled with the extremely limited genetic capacity of the hepadnaviruses, this observation led us to speculate that P may have additional roles in virus replication or pathology beyond replication of the viral genome, such as regulating cellular or viral processes (Yao et al., 2000
).
Although DHBV and HBV are both hepadnaviruses with highly similar genetic organizations and replication cycles, the existence of HBV P as a short-lived cytoplasmic protein cannot be assumed because DHBV is an Avihepadnavirus while HBV is an Orthohepadnavirus, and hence there are significant differences between the viruses. DHBV produces two fewer proteins than does HBV (DHBV lacks the middle surface antigen and the X protein), there is very little primary sequence identity between the two viruses and DHBV is non-pathogenic in its natural host whereas HBV is a major human pathogen. Therefore, we investigated whether HBV produces a cytoplasmic, non-encapsidated form of its reverse transcriptase similar to that of DHBV, and if so, how its biochemical characteristics compared with non-encapsidated DHBV P.
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METHODS |
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Cell culture, transfection and isolation of intracellular HBV cores.
Huh7 (human hepatoma) and HepG2 (human hepatoblastoma) cells were maintained in Dulbecco's modified Eagle's medium/F12 with 10 % fetal bovine serum. Transfections employed FuGENE (Roche Diagnostics) according to the manufacturer's instructions. HBV cores were isolated from Huh7 or HepG2 cells transfected with pCMV-HBV-LE by lysis in core particle preparation lysis buffer [CPLB; 10 mM Tris (pH 7·5), 1 mM EDTA, 0·25 % NP40, 50 mM NaCl, 8 % sucrose] followed by sedimentation through a 30 % sucrose cushion as described (Tavis et al., 1998).
Immunoprecipitation.
Transfected Huh7 cells were lysed in 0·75x radioimmunoprecipitation assay buffer [RIPA; 1x RIPA is 20 mM Tris (pH 7·2), 1 % sodium deoxycholate, 1 % Triton X-100, 0·1 % sodium dodecyl sulfate, 150 mM NaCl], CPLB or PEB [phosphate buffered saline (PBS; 137 mM NaCl, 2·7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7·4) plus 0·5 % NP40 and 10 % glycerol] for 10 min on ice. Anti-HBV P antibodies [rabbit polyclonal antibodies against aa 1198 or mouse monoclonal antibodies (zu Putlitz et al., 1999)] or anti-HBV C antibodies (Austral Biologicals) were bound to protein A/G beads (Calbiochem), and the antibody-bead complexes were incubated with cell lysates overnight. The immunocomplexes were washed four times with 1 ml of the appropriate buffer, and P was released by boiling in Laemmli buffer. Following SDS-PAGE, radioactive P was detected and quantified by phosphorimager analysis.
Metabolic labelling and pulsechase determination of half-life of P.
Transfected Huh7 cells were washed twice with Dulbecco's modified Eagle's medium lacking methionine and cysteine (labelling medium) and pulsed with labelling medium supplemented with 120 µCi (4·44 MBq) [35S]methionine/cysteine ml1 (EasyTag Express; PerkinElmer Life Sciences) and 1 % fetal bovine serum. For pulsechase experiments, cells were labelled for 1 h, rapidly washed twice with Dulbecco's modified Eagle's medium/F12, and then fed with Dulbecco's modified Eagle's medium/F12 containing 10 % fetal bovine serum. The transition from the labelling to chase periods was preformed as rapidly as possible (typically under 2 min), and all media were equilibrated to 37 °C and 5 % CO2 before use.
Southern and Western Blots.
Southern blotting was performed as described (Staprans et al., 1991) with internally 32P-labelled monomeric HBV DNA as a probe and detected by phosphorimage analysis. Western blots were performed as described (Yao et al., 2000
) employing commercial anti-HBV C antibodies (Austral Biologicals).
Immunofluorescence.
Huh7 cells were grown on glass coverslips and transfected with pCMV-HPol, pCMV-HPol, pCMV-HBV-LE, pCMV-HBV-LEC, pCMV-HBV-LEe, pCMV-HBV-LECe or pBS. Two days post-transfection, cells were fixed with 3·7 % formalin in PBS for 10 min at room temperature followed by ice-cold methanol for 7 min and were blocked by incubation with PBS containing 5 % goat serum at 37 °C for 50 min. Primary and secondary antibodies were diluted in PBS/5 % goat serum, and incubated for 2 h or 50 min, respectively. Coverslips were washed three times in PBS. Standard immunofluorescence images were captured digitally at 600x with a SPOT camera attached to an Olympus fluorescence microscope. Confocal microscopy was performed at a magnification of 600x on a Bio-Rad MRC 1024 confocal system attached to a Nikon Optiphot microscope.
Digital images.
Digital images were processed with Adobe Photoshop and assembled in Adobe Illustrator.
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RESULTS |
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Half-life of non-encapsidated HBV P
The half-life of P was determined by transfecting Huh7 cells with expression vectors for P and performing pulsechase experiments 3 days after transfection. Transfected cells were metabolically labelled with [35S]methionine/cysteine for 1 h, washed with non-radioactive medium, supplied with non-radioactive medium and incubated for various times. At each time point, cells were lysed, P was immunoprecipitated and radioactivity in P was quantified by phosphorimage analysis. The half-life of HBV P expressed from pCMV-HPol was 87 +/ 8 min (Fig. 4), and the half-life was unchanged when P was expressed from the pgRNA in the presence or absence of C (i.e. from pCMV-HBV-LE or pCMV-LEC; data not shown). Therefore, non-encapsidated HBV P is considerably more stable than the DHBV P, whose half-life varies from 1525 min depending on the time post-transfection (Yao & Tavis, 2003
).
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DISCUSSION |
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The biochemical characteristics of non-encapsidated P from DHBV and HBV are similar but not identical (Table 1). Similarities include a granular distribution in the cytoplasm without obvious co-localization with the endoplasmic reticulum or Golgi apparatus. Differences include an apparently lower accumulation level and a longer half-life for HBV P. Furthermore, non-encapsidated HBV P migrates at its predicted mass, without the slower mobility isoforms found for DHBV P on denaturing SDS-PAGE (Yao et al., 2000
, 2003
). The slower mobility isoforms presumably result from post-translational modification, and hence we have no evidence for extensive post-translational modification of HBV P. An additional difference is that co-expression of HBV C with P in Huh7 cells greatly reduces the sensitivity of detection of P by immunofluorescence, despite equivalent expression of P as determined by immunoprecipitation (Fig. 6
). In contrast, no effect of DHBV C on P can be detected beyond encapsidation itself.
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Our data indicate that non-encapsidated P accumulates in the cytoplasm, but do not preclude possible interactions between P and the exterior of capsids. However, if P is associated with the exterior of capsids, our data indicate that the binding is too weak to survive immunoprecipitation (Fig. 2). Lott et al., (2000)
have reported extensive interactions between HBV P and C when the proteins are expressed in insect cells from baculovirus vectors, or in Huh7 cells when the proteins are expressed from vaccinia virus vectors. We have not detected these complexes, even when using identical lysis conditions. However, our experiments employed P and C expressed from the pgRNA and used anti-HBV P or C antibodies instead of epitope-tagged P and anti-FLAG antibodies, and so the differing results could be attributable to the alternate techniques employed.
HBV P has been previously detected in the nucleus of diseased livers by immunofluorescence (McGarvey et al., 1996). We were unable to detect unambiguously HBV P by immunofluorescence in 26 paraffin-embedded HBV-infected liver tissues (data not shown) either with our polyclonal antibodies or with the anti-HBV P monoclonal antibody 2C8 (zu Putlitz et al., 1999
). This may be due to either low levels of P in the tissues and/or masking of P by C as we have observed in Huh7 cells (Fig. 6
).
The detection of non-encapsidated HBV P in cultured cells transfected with a pgRNA expression vector indicates that accumulation of P in the cytoplasm is not limited to the avian hepadnaviruses, but is also found in the mammalian hepadnaviruses. This implies that any function(s) non-encapsidated P may have in virus replication or pathogenesis could be conserved between the hepadnaviral genera. HBV establishes a persistent infection lasting decades, and the mechanism(s) employed by the virus to usurp the cell and to evade the immune system are not fully understood. We have speculated that P might be a dual-function protein that replicates the viral genome when encapsidated and regulates cellular or viral processes when free in the cytoplasm (Yao et al., 2000). Other HBV genes are known to have dual roles: the C ORF encodes the C protein and the e-antigen, which is proposed to be a neonatal tolerogen (Milich et al., 1990
), and the surface glycoproteins encoded by the S ORF serve both as elements of the viral envelope and as decoy antigens when released on subviral particles. Plausible (but unproven) roles for the nonencapsidated P include regulating responsiveness to interferon, regulating cellular gene expression, or regulating a viral function such as gene expression or virion assembly. The results presented here support the hypothesis of a dual role for P by demonstrating that HBV P also exists in the cytoplasm, where it has multiple opportunities to interact with cellular regulatory pathways. Therefore, further analysis of non-encapsidated P could increase our understanding of outstanding issues in hepadnavirology such as the mechanisms of immune evasion or carcinogenesis.
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ACKNOWLEDGEMENTS |
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Received 20 May 2004;
accepted 21 July 2004.
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