Production of hepatitis B virus nucleocapsidlike core particles in Xenopus oocytes: assembly occurs mainly in the cytoplasm and does not require the nucleus.

The location of hepatitis B virus (HBV) nucleocapsid (core particle) assembly in infected cells remains controversial. Some lines of evidence implicate the nucleus; others favor the cytoplasm. Via injection of a synthetic mRNA encoding the HBV nucleocapsid protein (p21.5), we have expressed both unassembled p21.5 and nucleocapsidlike core particles in Xenopus oocytes. Subcellular fractionation reveals that approximately 91% of the unassembled p21.5 and 95% of the core particles are cytoplasmic, with only 9 and 5%, respectively, in the nucleus. We present evidence showing that unassembled p21.5 equilibrates between nucleus and cytoplasm by passive diffusion and that intact core particles do not enter the nucleus. To examine the role of the nucleus in core particle formation, we expressed p21.5 in surgically anucleate oocytes. We show that anucleate oocytes support efficient core particle formation, indicating that (i) the nucleus is not essential for assembly and (ii) the cytoplasm can assemble most core particles found in oocytes. On the basis of our data, we propose that in oocytes, most core particle assembly (up to 95%) occurs in the cytoplasm, but that at least approximately 5% of the cellular core particles are assembled in the nucleus and remain there. We discuss the implications of these findings for the formation of replication-competent core particles in infected cells.     

The hepatitis B virus (HBV) precore protein inhibits HBV replication in transgenic mice.

In this study, we examined the ability of the hepatitis B virus (HBV) precore, envelope, and X gene products to modulate HBV replication in the livers of transgenic mice that replicate the virus. Hepatic HBV replication was not affected by overexpression of the envelope or X gene products when these animals were crossed with transgenic mice that express the corresponding viral genes in the hepatocyte. Overexpression of the precore protein, however, eliminated nucleocapsid particles from the cytoplasm of the hepatocytes and abolished HBV replication without affecting the hepatic steady-state content of pregenomic HBV RNA. These observations suggest that the precore protein can exert a dominant negative effect on HBV replication, presumably at the level of nucleocapsid particle maturation or stability, suggesting an important role for this enigmatic viral protein in the HBV life cycle.     

Transforming growth factor-β1 suppresses hepatitis B virus replication by the reduction of hepatocyte nuclear factor-4α expression

Several studies have demonstrated that cytokine-mediated noncytopathic suppression of hepatitis B virus (HBV) replication may provide an alternative therapeutic strategy for the treatment of chronic hepatitis B infection. In our previous study, we showed that transforming growth factor-beta1 (TGF-β1) could effectively suppress HBV replication at physiological concentrations. Here, we provide more evidence that TGF-β1 specifically diminishes HBV core promoter activity, which subsequently results in a reduction in the level of viral pregenomic RNA (pgRNA), core protein (HBc), nucleocapsid, and consequently suppresses HBV replication. The hepatocyte nuclear factor 4alpha (HNF-4α) binding element(s) within the HBV core promoter region was characterized to be responsive for the inhibitory effect of TGF-β1 on HBV regulation. Furthermore, we found that TGF-β1 treatment significantly repressed HNF-4α expression at both mRNA and protein levels. We demonstrated that RNAi-mediated depletion of HNF-4α was sufficient to reduce HBc synthesis as TGF-β1 did. Prevention of HNF-4α degradation by treating with proteasome inhibitor MG132 also prevented the inhibitory effect of TGF-β1. Finally, we confirmed that HBV replication could be rescued by ectopic expression of HNF-4α in TGF-β1-treated cells. Our data clarify the mechanism by which TGF-β1 suppresses HBV replication, primarily through modulating the expression of HNF-4α gene.     

Phosphorylation of rubella virus capsid regulates its RNA binding activity and virus replication

Rubella virus is an enveloped positive-strand RNA virus of the family TOGAVIRIDAE: Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs.     

High-level hepatitis B virus replication in transgenic mice.

Hepatitis B virus (HBV) transgenic mice whose hepatocytes replicate the virus at levels comparable to that in the infected livers of patients with chronic hepatitis have been produced, without any evidence of cytopathology. High-level viral gene expression was obtained in the liver and kidney tissues in three independent lineages. These animals were produced with a terminally redundant viral DNA construct (HBV 1.3) that starts just upstream of HBV enhancer I, extends completely around the circular viral genome, and ends just downstream of the unique polyadenylation site in HBV. In these animals, the viral mRNA is more abundant in centrilobular hepatocytes than elsewhere in the hepatic lobule. High-level viral DNA replication occurs inside viral nucleocapsid particles that preferentially form in the cytoplasm of these centrilobular hepatocytes, suggesting that an expression threshold must be reached for nucleocapsid assembly and viral replication to occur. Despite the restricted distribution of the viral replication machinery in centrilobular cytoplasmic nucleocapsids, nucleocapsid particles are detectable in the vast majority of hepatocyte nuclei throughout the hepatic lobule. The intranuclear nucleocapsid particles are empty, however, suggesting that viral nucleocapsid particle assembly occurs independently in the nucleus and the cytoplasm of the hepatocyte and implying that cytoplasmic nucleocapsid particles do not transport the viral genome across the nuclear membrane into the nucleus during the viral life cycle. This model creates the opportunity to examine the influence of viral and host factors on HBV pathogenesis and replication and to assess the antiviral potential of pharmacological agents and physiological processes, including the immune response.     

Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A

Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles.     

Characterization of hepatitis B virus capsid particle assembly in Xenopus oocytes.

Little is known about the assembly of the 28-nm nucleocapsid or core particle of hepatitis B virus. Here we show that this assembly process can be reconstituted in Xenopus oocytes injected with a synthetic mRNA encoding the hepatitis B virus capsid protein (p21.5). Injected oocytes produce both a nonparticulate p21.5 species (free p21.5) and capsid particles. We describe rapid and simple methods for fractionating these species on a small scale either with step gradients of 10 to 60% (wt/vol) sucrose or by centrifugation to pellet the particles, and we characterize the oocyte core particles. Free p21.5 exhibits chemical and physical properties distinctly different from those of particles. Free p21.5 is partially cleaved by proteinase K, whereas core particles are almost completely resistant to cleavage. This suggests that the carboxyl-terminal protamine region, the main target for proteases within p21.5, is exposed in free p21.5 but faces the interior of the p21.5 core particle. Finally, pulse-chase experiments demonstrated that free p21.5 can be chased almost quantitatively into core particles, establishing that free p21.5 is fully competent to form particles and represents an assembly intermediate on the pathway for core particle formation. However, core particle assembly appears very dependent on p21.5 concentration and is rapidly compromised if the p21.5 concentration is lowered. The advantages of oocytes for studying assembly are discussed.     

Does a cdc2 Kinase-Like Recognition Motif on the Core Protein of Hepadnaviruses Regulate Assembly and Disintegration of Capsids?

Hepadnaviruses are enveloped viruses, each with a DNA genome packaged in an icosahedral nucleocapsid, which is the site of viral DNA synthesis. In the presence of envelope proteins, DNA-containing nucleocapsids are assembled into virions and secreted, but in the absence of these proteins, nucleocapsids deliver viral DNA into the cell nucleus. Presumably, this step is identical to the delivery of viral DNA during the initiation of an infection. Unfortunately, the mechanisms triggering the disintegration of subviral core particles and delivery of viral DNA into the nucleus are not yet understood. We now report the identification of a sequence motif resembling a serine- or threonine-proline kinase recognition site in the core protein at a location that is required for the assembly of core polypeptides into capsids. Using duck hepatitis B virus, we demonstrated that mutations at this sequence motif can have profound consequences for RNA packaging, DNA replication, and core protein stability. Furthermore, we found a mutant with a conditional phenotype that depended on the cell type used for virus replication. Our results support the hypothesis predicting that this motif plays a role in assembly and disassembly of viral capsids.     

Mapping of functionally important residues of a cysteine-histidine box in the human immunodeficiency virus type 1 nucleocapsid protein.

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein contains two copies of a sequence motif, the cysteine-histidine box, that is conserved among retroviruses. To identify the functionally relevant positions of a cysteine-histidine box, each amino acid in the proximal copy of the motif was individually substituted by site-directed mutagenesis. Mutations at 5 of 14 positions abolished virus replication and reduced the viral RNA content of mutant particles to between 10 and 20% of parental levels. Mutations at other positions had either no or only a minor effect on virus replication and virion RNA content. In vitro binding of RNA to bacterially expressed mutant Pr55gag polyprotein correlated well with the effects of the mutations on particle-associated viral RNA levels. The two different copies of the motif in the HIV-1 nucleocapsid protein are not functionally equivalent, since the conversion of the proximal motif to an exact copy of the distal motif results in a defect in virus replication and a reduction in the viral RNA content of mutant particles. The simultaneous substitution of functionally relevant positions in both motifs led to a significant decline in gag protein export, indicating that the nucleocapsid domain of the gag precursor is also required for efficient assembly or release of the virion.     

Phenotypic mixing between different hepadnavirus nucleocapsid proteins reveals C protein dimerization to be cis preferential.

Hepadnaviruses encode a single core (C) protein which assembles into a nucleocapsid containing the polymerase (P) protein and pregenomic RNA during viral replication in hepatocytes. We examined the ability of heterologous hepadnavirus C proteins to cross-oligomerize. Using a two-hybrid assay in HepG2 cells, we observed cross-oligomerization among the core proteins from hepatitis B virus (HBV), woodchuck hepatitis virus, and ground squirrel hepatitis virus. When expressed in Xenopus oocytes, in which hepadnavirus C proteins form capsids, the C polypeptides from woodchuck hepatitis virus and ground squirrel hepatitis virus, but not duck hepatitis B virus, can efficiently coassemble with an epitope-tagged HBV core polypeptide to form mixed capsids. However, when two different core mRNAs are coexpressed in oocytes the core monomers show a strong preference for forming homodimers rather than heterodimers. This holds true even for coexpression of two HBV C proteins differing only by an epitope tag, suggesting that core monomers are not free to diffuse and associate with other monomers. Thus, mixed capsids result from aggregation of different species of homodimers.     

A splice hepadnavirus RNA that is essential for virus replication.

According to the current model of hepadnavirus gene expression, the viral envelope proteins are produced from unspliced subgenomic RNAs, in contrast to the retroviral mechanism, where the subgenomic env RNA is generated by RNA splicing. We now describe and characterize a novel duck hepatitis B virus RNA species which is derived from the RNA pregenome by loss of a 1.15 kb intron. This RNA (termed spliced L RNA) codes for the large surface protein (L protein), as does the previously described unspliced mRNA (the preS RNA); however, it differs in 5' leader sequence and promoter control. Mutational analysis indicates that the spliced L RNA is functionally important for virus replication in infected hepatocytes and ducks, but not for virus formation from transfected DNA genomes. This suggests that the newly discovered second pathway for L protein synthesis plays a distinct role in an early step in the viral life cycle.     

Regulation of hepatitis C virus replication by the core protein through its interaction with viral RNA polymerase

The hepatitis C virus (HCV) core protein is a structural component of the nucleocapsid and has been shown to modulate cellular signaling pathways by interaction with various cellular proteins. In the present study, we investigated the role of HCV core protein in viral RNA replication. Immunoprecipitation experiments demonstrated that the core protein binds to the amino-terminal region of RNA-dependent RNA polymerase (RdRp), which encompasses the finger and palm domains. Direct interaction between HCV RdRp and core protein led to inhibition of RdRp RNA synthesis activity of in vitro. Furthermore, over-expression of core protein, but not its derivatives lacking the RdRp-interacting domain, suppressed HCV replication in a hepatoma cell line harboring an HCV subgenomic replicon RNA. Collectively, our results suggest that the core protein, through binding to RdRp and inhibiting its RNA synthesis activity, is a viral regulator of HCV RNA replication.