HCV core protein and virus assembly: what we know without structures

Chronic hepatitis C virus (HCV) infection results in a progressive disease that may end in cirrhosis and, eventually, in hepatocellular carcinoma. In the last several years, tremendous progress has been made in understanding the HCV life cycle and in the development of small molecule compounds for the treatment of chronic hepatitis C. Nevertheless, the complete understanding of HCV assembly and particle release as well as the detailed characterization and structure of HCV particles is still missing. One of the most important events in the HCV assembly is the nucleocapsid formation which is driven by the core protein, that can oligomerize upon interaction with viral RNA, and is orchestrated by viral and host proteins. Despite a growing number of new factors involved in HCV assembly process, we do not know the three-dimensional structure of the core protein or its topology in the nucleocapsid. Since the core protein contains a hydrophobic C-terminal domain responsible for the binding to cellular membranes, the assembly pathway of HCV virions might proceed via coassembly at endoplasmic reticulum membranes. Recently, new mechanisms involving viral proteins and host factors in HCV particle formation and egress have been described. The present review aims to summarize the advances in our understanding of HCV assembly with an emphasis on the core protein as a structural component of virus particles that possesses the ability to interact with a variety of cellular components and is potentially an attractive target for the development of a novel class of anti-HCV agents.     

Selective binding of hepatitis C virus core protein to synthetic oligonucleotides corresponding to the 5' untranslated region of the viral genome

Although it is assumed that hepatitis C virus (HCV) core protein binds with viral RNA to form a nucleocapsid, little is known about the resulting molecular interactions. We utilized surface plasmon resonance technology to study the structural basis of the affinity and the preference of the interaction between HCV core protein and oligonucleotides derived from the viral genome. Among the 10 oligonucleotides corresponding to the 5' untranslated region (5'UTR) of the tested HCV genome, the real-time analysis of sensorgrams indicated that the core protein binds most efficiently and stably to the 31-nucleotide-long sequence of the loop IIId domain, whose secondary structure is highly conserved not only among different HCV genotypes but also among pestiviruses. There also could be some interactions of the core protein with the loop I domain and the region of nt 23-41. The kinetic profiles, together with those obtained in experiments using single- and double-stranded polymeric oligonucleotides, suggest a multimerization of the core protein in solution. These newly characterized properties could provide a basis for understanding the pathway of the viral nucleocapsid assembly.     

Specific in vitro association between the hepatitis C viral genome and core protein.

Little is known about the molecular interactions required for hepatitis C virion assembly. The 5' noncoding region (5'NCR) of the RNA genome is highly conserved and has extensive secondary structure. The highly basic core protein is rich in arginine and lysine residues. We postulate that a specific interaction between these structures may be important for virion assembly. Using an RNA gel mobility shift assay, a specific interaction has been demonstrated between the RNA of the 5'NCR and recombinant core protein. Proteins from other regions of the virus do not interact with the viral RNA. The interaction is inhibited competitively by unlabelled sense polarity RNA, but antisense 5'NCR RNA and nonspecific RNAs compete only at much higher concentrations. These data suggest that there is a specific interaction between the 5'NCR of the hepatitis C virus (HCV) genome and HCV core protein. This interaction may be important for the specific encapsidation of the viral genome during HCV replication.     

Core protein-mediated 5'-3' annealing of the West Nile virus genomic RNA in vitro

Genome cyclization through conserved RNA sequences located in the 5' and 3' terminal regions of flavivirus genomic RNA is essential for virus replication. Although the role of various cis-acting RNA elements in panhandle formation is well characterized, almost nothing is known about the potential contribution of protein cofactors to viral RNA cyclization. Proteins with nucleic acid chaperone activities are encoded by many viruses (e.g., retroviruses, coronaviruses) to facilitate RNA structural rearrangements and RNA-RNA interactions during the viral replicative cycle. Since the core protein of flaviviruses is also endowed with potent RNA chaperone activities, we decided to examine the effect of West Nile virus (WNV) core on 5'-3' genomic RNA annealing in vitro. Core protein binding resulted in a dramatic, dose-dependent increase in 5'-3' complex formation. Mutations introduced in either the UAR (upstream AUG region) or CS (conserved sequence) elements of the viral RNA diminished core protein-dependent annealing, while compensatory mutations restored the 5'-3' RNA interaction. The activity responsible for stimulating RNA annealing was mapped to the C-terminal RNA-binding region of WNV core protein. These results indicate that core protein - besides its function in viral particle formation - might be involved in the regulation of flavivirus genomic RNA cyclization, and thus virus replication.     

Purification of E. coli-expressed HIS-tagged hepatitis B core antigen by Ni2+ -chelate affinity chromatography

Hepatitis B virus is a major cause of human liver disease. In the case of chronic infection the virus can lead to liver cancer and cirrhosis. The virion consists of an outer envelope containing lipids of the endoplasmic reticulum and virally-encoded surface proteins. This lipoprotein shell encloses the nucleocapsid or core antigen (HBcAg), which contains the viral genome. The capsid consists of dimers of a 183-residue protein, which can be divided into an assembly (residues 1-149) and a protamin-like domain (residues 150-183), responsible for polymerization into particles and RNA packaging, respectively. Upon expression of the core gene in bacteria the products are assembled into capsids resembling those of wild type particles. A purification protocol was developed for unpolymerised (dimeric) and polymerized HBcAg by fusion of six histidine residues to a C-terminal deletion mutant of the core protein allowing the isolation of the respective antigens after denaturing Ni2+-chelate affinity chromatography and renaturing dialysis. The possible incorporation of E. coli proteins during the assembly process and the inclusion of nucleic acids can be avoided. The method might be an attractive alternative to common purification protocols of hybrid virus-like particles (VLPs) for vaccine use.     

Kinetics and functional studies on interaction between the replicase proteins of Tomato Bushy Stunt Virus: requirement of p33:p92 interaction for replicase assembly

The assembly of the functional replicase complex via protein:protein and RNA:protein interactions among the viral-coded proteins, host factors and the viral RNA on cellular membranes is a key step in the replication process of plus-stranded RNA viruses. In this work, we have characterized essential interactions between p33:p33 and p33:p92 replication proteins of Tomato bushy stunt virus (TBSV), a tombusvirus with a non-segmented, plus-stranded RNA genome. Surface plasmon resonance (SPR) measurements with purified recombinant p33 and p92 demonstrate that p33 interacts with p92 in vitro and that the interaction requires the S1 subdomain, whereas the S2 subdomain plays lesser function. Kinetic SPR analyses showed that binding of S1 subdomain to the C-terminal half of p33 takes place with moderate binding affinity in the nanomolar range whereas S2 subdomain binds to p33 with micromolar affinity. Using mutated p33 and p92 proteins, we identified critical amino acid residues within the p33:p92 interaction domain that play essential role in replication and the assembly of the tombusviral replicase. In addition, we show that interaction takes place between replication proteins of TBSV and the closely related Cucumber necrosis virus but not between TBSV and the more distantly related Turnip crinkle virus, suggesting that selective protein interactions might prevent the assembly of chimeric replicases carrying replication proteins from different viruses during mixed infections.     

The small delta antigen of hepatitis delta virus is an acetylated protein and acetylation of lysine 72 may influence its cellular localization and viral RNA synthesis

Hepatitis delta virus (HDV) is a single-stranded RNA virus that encodes two viral nucleocapsid proteins named small and large form hepatitis delta antigen (S-HDAg and L-HDAg). The S-HDAg is essential for viral RNA replication while the L-HDAg is required for viral assembly. In this study, we demonstrated that HDAg are acetylated proteins. Metabolic labeling with [(3)H]acetate revealed that both forms of HDAg could be acetylated in vivo. The histone acetyltransferase (HAT) domain of cellular acetyltransferase p300 could acetylate the full-length and the N-terminal 88 amino acids of S-HDAg in vitro. By mass spectrometric analysis of the modified protein, Lys-72 of S-HDAg was identified as one of the acetylation sites. Substitution of Lys-72 to Arg caused the mutant S-HDAg to redistribute from the nucleus to the cytoplasm. The mutant reduced viral RNA accumulation and resulted in the earlier appearance of L-HDAg. These results demonstrated that HDAg is an acetylated protein and mutation of HDAg at Lys-72 modulates HDAg subcellular localization and may participate in viral RNA nucleocytoplasmic shuttling and replication.     

Transcription from the gene encoding the herpesvirus entry receptor nectin-1 (HveC) in nervous tissue of adult mouse

Hepatitis C virus core protein, in addition to being a component of the viral capsid, has a number of regulatory functions. Here we showed two bodies of evidence indicating that a fraction of the core protein species is a substrate of the ubiquitin (Ub)-proteasome pathway of targeted proteolysis. First, the core protein processing the C-terminal hydrophobic region is metabolically unstable, and incubation with a proteasome inhibitor led to a significant accumulation of the protein. Second, an in vivo ubiquitylation assay indicates conjugation of multi-Ub chain to the unstable core protein. In contrast, a stable form of core protein, p21, is also able to be ubiquitylated, but it links to a single or only a few Ub moiety. Therefore, processing event(s) at the C-terminal hydrophobic domain of HCV core protein may affect the ubiquitylation pathway, particularly the efficiency of the multi-Ub chain assembly, resulting in stable, matured core proteins.     

The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.     

The H/ACA RNP assembly factor SHQ1 functions as an RNA mimic

SHQ1 is an essential assembly factor for H/ACA ribonucleoproteins (RNPs) required for ribosome biogenesis, pre-mRNA splicing, and telomere maintenance. SHQ1 binds dyskerin/NAP57, the catalytic subunit of human H/ACA RNPs, and this interaction is modulated by mutations causing X-linked dyskeratosis congenita. We report the crystal structure of the C-terminal domain of yeast SHQ1, Shq1p, and its complex with yeast dyskerin/NAP57, Cbf5p, lacking its catalytic domain. The C-terminal domain of Shq1p interacts with the RNA-binding domain of Cbf5p and, through structural mimicry, uses the RNA-protein-binding sites to achieve a specific protein-protein interface. We propose that Shq1p operates as a Cbf5p chaperone during RNP assembly by acting as an RNA placeholder, thereby preventing Cbf5p from nonspecific RNA binding before association with an H/ACA RNA and the other core RNP proteins.     

A 5' RNA element promotes dengue virus RNA synthesis on a circular genome

The mechanisms of RNA replication of plus-strand RNA viruses are still unclear. Here, we identified the first promoter element for RNA synthesis described in a flavivirus. Using dengue virus as a model, we found that the viral RdRp discriminates the viral RNA by specific recognition of a 5' element named SLA. We demonstrated that RNA-RNA interactions between 5' and 3' end sequences of the viral genome enhance dengue virus RNA synthesis only in the presence of an intact SLA. We propose a novel mechanism for minus-strand RNA synthesis in which the viral polymerase binds SLA at the 5' end of the genome and reaches the site of initiation at the 3' end via long-range RNA-RNA interactions. These findings provide an explanation for the strict requirement of dengue virus genome cyclization during viral replication.     

Analysis of the retrovirus capsid interdomain linker region

In structural studies, the retrovirus capsid interdomain linker region has been shown as a flexible connector between the CA N-terminal domain and its C-terminal domain. To analyze the function of the linker region, we have examined the effects of three Moloney murine leukemia virus (M-MuLV) capsid linker mutations/variations in vivo, in the context of the full-length M-MuLV structural precursor protein (PrGag). Two mutations, A1SP and A5SP, respectively, inserted three and seven additional codons within the linker region to test the effects of increased linker lengths. The third variant, HIV/Mo, represented a chimeric HIV-1/M-MuLV PrGag protein, fused at the linker region. When expressed in cells, the three variants reduced the efficiency of virus particle assembly, with PrGag proteins and particles accumulating at the cellular plasma membranes. Although PrGag recognition of viral RNA was not impaired, the capsid linker variant particles were abnormal, with decreased stabilities, anomalous densities, and aberrant multiple lobed and tubular morphologies. Additionally, rather than crosslinking as PrGag dimers, particle-associated A1SP, A5SP, and HIV/Mo proteins showed an increased propensity to crosslink as trimers. Our results suggest that a wild-type retrovirus capsid linker region is required for the proper alignment of capsid protein domains.     

The polar alkaline disassembly of papaya mosaic virus

The disassembly of papaya mosaic virus (PMV) at alkaline pH (pH 10) was monitored by following the decrease of turbidity at 320 nm and by electron microscopy. Most of the virion particle population undergoes a sequential loss of protein subunits starting exclusively from the 3'OH end of the RNA, but a small fraction resists degradation. The progressive decrease in size distribution suggests a sequential process and the presence of "brush-like" structures at one end of the virus particle favors a polar model for disassembly. Furthermore, after treatment with nucleases to digest the exposed RNA tails, the 5' cap structure (m7GpppGp) was found to be conserved in the remaining nucleoprotein intermediates. This indicates that the disassembly of PMV occurs from the 3' terminus to the 5' end, a polarity opposite to that of viral assembly. These RNA fragments (containing the 5' end) retain their ability to reassemble with the viral protein. This result is consistent with earlier evidence that the initiation site for virus assembly is located at the 5' end of PMV-RNA. Electron microscope examination suggests the presence of meta-stable size classes during the disassembly process. This observation might indicate a variability of the RNA-protein interactions along the RNA molecules.     

Hepatitis C virus core protein interacts with a human DEAD box protein DDX3.

Several studies have implicated hepatitis C virus (HCV) core in influencing the expression of host genes. To identify cellular factors with a possible role in HCV replication and pathogenesis, we looked for cellular proteins that interact with the viral core protein. A human liver cDNA library was screened in a yeast two-hybrid assay to identify cellular proteins that bind to core. Several positive clones were isolated, one of which encoded the C-terminal 253 amino acids of a putative RNA helicase, a DEAD box protein designated DDX3. Bacterially expressed glutathione-S-transferase-DDX3 fusion protein specifically pulled down in vitro translated and radiolabeled HCV core, confirming a direct interaction. Immunofluorescent staining of HeLa cells with a polyclonal antiserum showed that DDX3 is located predominantly in nuclear speckles and at low levels throughout the cytoplasm. In cells infected with a recombinant vaccinia virus expressing HCV structural proteins (core, E1, and E2), DDX3 and core colocalized in distinct spots in the perinuclear region of the cytoplasm. The regions of the proteins involved in binding were found by deletion analysis to be the N-terminal 59 amino acid residues of core and a C-terminal RS-like domain of DDX3. The human DDX3 is a putative RNA helicase and a member of a highly conserved DEAD box subclass that includes murine PL10, Xenopus An3, and yeast Ded1 proteins. Their role in RNA metabolism or gene expression is unknown. The significance of core-helicase interaction in HCV replication and pathogenesis is discussed.     

Assembly of HIV GAG-B-Galactosidase Fusion Proteins into Virus Particles

We have studied the assembly of human immunodeficiency virus (HIV-1) Gag-B-galactosidase (Gag-B-gal; GBG) fusion proteins into HIV particles in the presence of HIV Gag proteins. Release of fusion proteins from cells was measured by assay of media versus cellular B-gat activities and was dependent on co-expression of unfused Gag proteins. Gag-B-gal incorporation into virus particles was demonstrated by detergent treatment and density gradient fractionation studies and was dependent on protein-protein interactions requiring the C-terminal two-thirds of the HIV CA domain. The central MA domain appeared unimportant for fusion protein incorporation; a nonmyristylated GBG protein was incorporated but at a relatively reduced level, while the NC and p6 domains slightly affected the assembly of fusion proteins into particles. Subcellular fractionation studies showed that all fusion proteins including the nonmyristylated one were enriched in the cytoplasmic pellet fraction. However, assembly into particles did not correlate with subcellular fractionstions patterns. Similarly, virion incorporation levels of Gag-B-gel proteins did not correlate with their immunofluorescence localization patterns. However, we observed that while most fusion proteins displayed a perinuclear ring with heterogeneous staining throughout cells, short fusion proteins appeared enriched on the intracellular membranes, and fusion proteins with intact MA but deleted NC domains showed an enhanced surface staining without a clear perinuclear ring. Altogether, our data suggest that the CA domain is the primary determinant for assembly of HIV fusion proteins into virus particles.     

Defective rotavirus particle assembly in lovastatin-treated MA104 cells

Rotavirus is a non-enveloped virus that depends on cellular lipids for cell entry and associates with lipid rafts during assembly. However, the effects of cellular lipids on rotavirus assembly are still not fully understood. The present study analyzes the effects of lovastatin, an inhibitor of cholesterol biosynthesis, during rotavirus infection in MA104 cells with regard to viral growth and particle assembly. Following viral infection, a 2-log relative reduction of viral titers was observed in drug-treated cells, while viral mRNA levels in infected cells remained unaltered in both groups. Furthermore, the levels of some viral proteins in drug-treated cells were elevated. The observed discordance between the viral RNA and protein levels and the decrease in infectivity titers of viral progeny in the drug-treated cells suggested that the drug affects viral assembly, the viral proteins not being properly incorporated into virions. Transmission electron microscopic (TEM) analysis revealed that in drug-treated cells there was an increase in “empty-looking” rotavirus particles devoid of an electron-dense core as compared to the normal, electron-dense particles seen in untreated infected cells. The present study thus provides visual evidence of defective rotavirus particle assembly as a result of cholesterol depletion. The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination policy.     

Nucleotide sequence and coding capacity of the large (L) genomic RNA segment of Seoul 80-39 virus, a member of the hantavirus genus.

The nucleotide sequence of the large (L) genomic RNA segment of Seoul 80-39 virus was determined from overlapping cDNA clones. The virion L RNA segment is 6530 nucleotides long. The 3' and 5' terminal sequences are inversely complementary for 15 bases. The viral complementary-sense RNA contains a single open reading frame from an AUG codon at nucleotide position 37-39 to a UAA stop codon at nucleotide position 6490-6492. This ORF could encode a polypeptide of 2151 amino acids (246,662 kDa) which likely corresponds to the L protein detected in purified viral particles (Elliott et al., 1984) and is assumed to be an RNA-dependent RNA polymerase molecule (Schmaljohn and Dalrymple, 1983). Comparison of the L protein of the Seoul 80-39 virus with the polymerase proteins encoded by other negative-stranded RNA viruses revealed 44% similarity only with the part of the Bunyamwera virus L protein (Elliott, 1989) and a very weak homology with the PB1 protein of influenza virus.