Molecular and functional analyses of a human parvovirus B19 infectious clone demonstrates essential roles for NS1, VP1, and the 11-kilodalton protein in virus replication and infectivity

In an attempt to experimentally define the roles of viral proteins encoded by the B19 genome in the viral life cycle, we utilized the B19 infectious clone constructed in our previous study to create two groups of B19 mutant genomes: (i) null mutants, in which either a translational initiation codon for each of these viral genes was substituted by a translational termination codon or a termination codon was inserted into the open reading frame by a frameshift; and (ii) a deletion mutant, in which half of the hairpin sequence was deleted at both the 5' and the 3' termini. The impact of these mutations on viral infectivity, DNA replication, capsid protein production, and distribution was systematically examined. Null mutants of the NS and VP1 proteins or deletion of the terminal hairpin sequence completely abolished the viral infectivity, whereas blocking expression of the 7.5-kDa protein or the putative protein X had no effect on infectivity in vitro. Blocking expression of the proline-rich 11-kDa protein significantly reduced B19 viral infectivity, and protein studies suggested that the expression of the 11-kDa protein was critical for VP2 capsid production and trafficking in infected cells. These findings suggest a previously unrecognized role for the 11-kDa protein, and together the results enhance our understanding of the key features of the B19 viral genome and proteins.     

Complementation analysis of the flavivirus Kunjin NS3 and NS5 proteins defines the minimal regions essential for formation of a replication complex and shows a requirement of NS3 in cis for virus assembly

We have previously reported successful trans-complementation of defective Kunjin virus genomic RNAs with a range of large lethal deletions in the nonstructural genes NS1, NS3, and NS5 (A. A. Khromykh et al., J. Virol. 74:3253-3263, 2000). In this study we have mapped further the minimal region in the NS5 gene essential for efficient trans-complementation of genome-length RNAs in repBHK cells to the first 316 of the 905 codons. To allow amplification and easy detection of complemented defective RNAs with deletions apparently affecting virus assembly, we have developed a dual replicon complementation system. In this system defective replicon RNAs with a deletion(s) in the nonstructural genes also encoded the puromycin resistance gene (PAC gene) and the reporter gene for beta-galactosidase (beta-Gal). Complementation of these defective replicon RNAs in repBHK cells resulted in expression of PAC and beta-Gal which allowed establishment of cell lines stably producing replicating defective RNAs by selection with puromycin and comparison of replication efficiencies of complemented defective RNAs by beta-Gal assay. Using this system we demonstrated that deletions in the C-terminal 434 codons of NS3 (codons 178 to 611) were complemented for RNA replication, while any deletions in the first 178 codons were not. None of the genome-length RNAs containing deletions in NS3 shown to be complementable for RNA replication produced secreted defective viruses during complementation in repBHK cells. In contrast, structural proteins produced from these complemented defective RNAs were able to package helper replicon RNA. The results define minimal regions in the NS3 and NS5 genes essential for the formation of complementable replication complex and show a requirement of NS3 in cis for virus assembly.     

Bluetongue virus RNA binding protein NS2 is a modulator of viral replication and assembly

Background  

Bluetongue virus (BTV) particles consist of seven structural proteins that are organized into two capsids. In addition, BTV also encodes three non-structural (NS) proteins of which protein 2 (NS2) is the RNA binding protein and is also the major component of virus encoded inclusion bodies (VIBs), which are believed to be virus assembly sites. To investigate the contribution of NS2 in virus replication and assembly we have constructed inducible mammalian cell lines expressing full-length NS2. In addition, truncated NS2 fragments were also generated in an attempt to create dominant negative mutants for NS2 function.     

Hepatitis C virus NS2/3 processing is required for NS3 stability and viral RNA replication

The hepatitis C virus NS2/3 protease is responsible for cleavage of the viral polyprotein between nonstructural proteins NS2 and NS3. We show here that mutation of three highly conserved residues in NS2 (His(952), Glu(972), and Cys(993)) abrogates NS2/3 protease activity and that introduction of any of these mutations into subgenomic NS2-5B replicons results in complete inactivation of NS2/3 processing and RNA replication in both stable and transient replication assays. The effect of uncleaved NS2 on the various activities of NS3 was therefore explored. Unprocessed NS2 had no significant effect on the in vitro ATPase and helicase activities of NS3, whereas immunoprecipitation experiments demonstrated a decreased affinity of NS4A for uncleaved NS2/3 as compared with NS3. This subsequently resulted in reduced kinetics in an in vitro NS3 protease assay with the unprocessed NS2/3 protein. Interestingly, NS3 was still capable of efficient processing of the polyprotein expressed from a subgenomic replicon in Huh-7 cells in the presence of uncleaved NS2. Notably, we show that fusion with NS2 leads to the rapid degradation of NS3, whose activity is essential for RNA replication. Finally, we demonstrate that uncleaved NS2/3 degradation can be prevented by the addition of a proteasome inhibitor. We therefore propose that NS2/3 processing is a critical step in the viral life cycle and is required to permit the accumulation of sufficient NS3 for RNA replication to occur. The regulation of NS2/3 cleavage could constitute a novel mechanism of switching between viral RNA replication and other processes of the hepatitis C virus life cycle.     

Crystal structure and activity of Kunjin virus NS3 helicase; protease and helicase domain assembly in the full length NS3 protein

Flaviviral NS3 is a multifunctional protein displaying N-terminal protease activity in addition to C-terminal helicase, nucleoside 5'-triphosphatase (NTPase), and 5'-terminal RNA triphosphatase (RTPase) activities. NS3 is held to support the separation of RNA daughter and template strands during viral replication. In addition, NS3 assists the initiation of replication by unwinding the RNA secondary structure in the 3' non-translated region (NTR). We report here the three-dimensional structure (at 3.1 A resolution) of the NS3 helicase domain (residues 186-619; NS3:186-619) from Kunjin virus, an Australian variant of the West Nile virus. As for homologous helicases, NS3:186-619 is composed of three domains, two of which are structurally related and held to host the NTPase and RTPase active sites. The third domain (C-terminal) is involved in RNA binding/recognition. The NS3:186-619 construct occurs as a dimer in solution and in the crystals. We show that NS3:186-619 displays both ATPase and RTPase activities, that it can unwind a double-stranded RNA substrate, being however inactive on a double-stranded DNA substrate. Analysis of different constructs shows that full length NS3 displays increased helicase activity, suggesting that the protease domain plays an assisting role in the RNA unwinding process. The structural interaction between the helicase and protease domain has been assessed using small angle X-ray scattering on full length NS3, disclosing that the protease and helicase domains build a rather elongated molecular assembly differing from that observed in the NS3 protein from hepatitis C virus.     

The acidic domain of hepatitis C virus NS4A contributes to RNA replication and virus particle assembly

Hepatitis C virus NS3-4A is a membrane-bound enzyme complex that exhibits serine protease, RNA helicase, and RNA-stimulated ATPase activities. This enzyme complex is essential for viral genome replication and has been recently implicated in virus particle assembly. To help clarify the role of NS4A in these processes, we conducted alanine scanning mutagenesis on the C-terminal acidic domain of NS4A in the context of a chimeric genotype 2a reporter virus. Of 13 mutants tested, two (Y45A and F48A) had severe defects in replication, while seven (K41A, L44A, D49A, E50A, M51A, E52A, and E53A) efficiently replicated but had severe defects in virus particle assembly. Multiple strategies were used to identify second-site mutations that suppressed these NS4A defects. The replication defect of NS4A F48A was partially suppressed by mutation of NS4B I7F, indicating that a genetic interaction between NS4A and NS4B contributes to RNA replication. Furthermore, the virus assembly defect of NS4A K41A was suppressed by NS3 Q221L, a mutation previously implicated in overcoming other virus assembly defects. We therefore examined the known enzymatic activities of wild-type or mutant forms of NS3-4A but did not detect specific defects in the mutants. Taken together, our data reveal interactions between NS4A and NS4B that control genome replication and between NS3 and NS4A that control virus assembly.     

A genetic interaction between hepatitis C virus NS4B and NS3 is important for RNA replication

Hepatitis C virus (HCV) nonstructural protein 4B (NS4B), a poorly characterized integral membrane protein, is thought to function as a scaffold for replication complex assembly; however, functional interactions with the other HCV nonstructural proteins within this complex have not been defined. We report that a Con1 chimeric subgenomic replicon containing the NS4B gene from the closely related H77 isolate is defective for RNA replication in a transient assay, suggesting that H77 NS4B is unable to productively interact with the Con1 replication machinery. The H77 NS4B sequences that proved detrimental for Con1 RNA replication resided in the predicted N- and C-terminal cytoplasmic domains as well as the central transmembrane region. Selection for Con1 derivatives that could utilize the entire H77 NS4B or hybrid Con1-H77 NS4B proteins yielded mutants containing single amino acid substitutions in NS3 and NS4A. The second-site mutations in NS3 partially restored the replication of Con1 chimeras containing the N-terminal or transmembrane domains of H77 NS4B. In contrast, the deleterious H77-specific sequences in the C terminus of NS4B, which mapped to a cluster of four amino acids, were completely suppressed by second-site substitutions in NS3. Collectively, these results provide the first evidence for a genetic interaction between NS4B and NS3 important for productive HCV RNA replication.     

Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus

Hepatitis C virus (HCV) infection is a global health concern affecting an estimated 3% of the world's population. Recently, cell culture systems have been established, allowing recapitulation of the complete virus life cycle for the first time. Since the HCV proteins p7 and NS2 are not predicted to be major components of the virion, nor are they required for RNA replication, we investigated whether they might have other roles in the viral life cycle. Here we utilize the recently described infectious J6/JFH chimera to establish that the p7 and NS2 proteins are essential for HCV infectivity. Furthermore, unprocessed forms of p7 and NS2 were not required for this activity. Mutation of two conserved basic residues, previously shown to be important for the ion channel activity of p7 in vitro, drastically impaired infectious virus production. The protease domain of NS2 was required for infectivity, whereas its catalytic active site was dispensable. We conclude that p7 and NS2 function at an early stage of virion morphogenesis, prior to the assembly of infectious virus.     

A genetic interaction between the core and NS3 proteins of hepatitis C virus is essential for production of infectious virus

By analogy to other members of the Flaviviridae family, the hepatitis C virus (HCV) core protein is presumed to oligomerize to form the viral nucleocapsid, which encloses the single-stranded RNA genome. Core protein is directed to lipid droplets (LDs) by domain 2 (D2) of the protein, and this process is critical for virus production. Domain 1 (D1) of core is also important for infectious particle morphogenesis, although its precise contribution to this process is poorly understood. In this study, we mutated amino acids 64 to 75 within D1 of core and examined the ability of these mutants to produce infectious virus. We found that residues 64 to 66 are critical for generation of infectious progeny, whereas 67 to 75 were dispensable for this process. Further investigation of the defective 64 to 66 mutant (termed JFH1(T)-64-66) revealed it to be incapable of producing infectious intracellular virions, suggesting a fault during HCV assembly. Furthermore, isopycnic gradient analyses revealed that JFH1(T)-64-66 assembled dense intracellular species of core, presumably representing nucleocapsids. Thus, amino acids 64 to 66 are seemingly not involved in core oligomerization/nucleocapsid assembly. Passaging of JFH1(T)-64-66 led to the emergence of a single compensatory mutation (K1302R) within the helicase domain of NS3 that completely rescued its ability to produce infectious virus. Importantly, the same NS3 mutation abrogated virus production in the context of wild-type core protein. Together, our results suggest that residues 64 to 66 of core D1 form a highly specific interaction with the NS3 helicase that is essential for the generation of infectious HCV particles at a stage downstream of nucleocapsid assembly.     

The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication,but essential for the production of infectious virus

Non-coding regions of viral RNA (vRNA) genomes are critically important in the regulation of gene expression. In particular, pseudoknot (PK) structures, which are present in a wide range of RNA molecules, have a variety of roles. The 5′ untranslated region (5′ UTR) of foot-and-mouth disease virus (FMDV) vRNA is considerably longer than in other viruses from the picornavirus family and consists of a number of distinctive structural motifs that includes multiple (2, 3 or 4 depending on the virus strain) putative PKs linked in tandem. The role(s) of the PKs in the FMDV infection are not fully understood. Here, using bioinformatics, sub-genomic replicons and recombinant viruses we have investigated the structural conservation and importance of the PKs in the FMDV lifecycle. Our results show that despite the conservation of two or more PKs across all FMDVs, a replicon lacking PKs was replication competent, albeit at reduced levels. Furthermore, in competition experiments, GFP FMDV replicons with less than two (0 or 1) PK structures were outcompeted by a mCherry FMDV wt replicon that had 4 PKs, whereas GFP replicons with 2 or 4 PKs were not. This apparent replicative advantage offered by the additional PKs correlates with the maintenance of at least two PKs in the genomes of FMDV field isolates. Despite a replicon lacking any PKs retaining the ability to replicate, viruses completely lacking PK were not viable and at least one PK was essential for recovery of infections virus, suggesting a role for the PKs in virion assembly. Thus, our study points to roles for the PKs in both vRNA replication and virion assembly, thereby improving understanding the molecular biology of FMDV replication and the wider roles of PK in RNA functions.     

Y-box-binding protein 1 interacts with hepatitis C virus NS3/4A and influences the equilibrium between viral RNA replication and infectious particle production

The hepatitis C virus (HCV) NS3/4A protein has several essential roles in the virus life cycle, most probably through dynamic interactions with host factors. To discover cellular cofactors that are co-opted by HCV for its replication, we elucidated the NS3/4A interactome using mass spectrometry and identified Y-box-binding protein 1 (YB-1) as an interacting partner of NS3/4A protein and HCV genomic RNA. Importantly, silencing YB-1 expression decreased viral RNA replication and severely impaired the propagation of the infectious HCV molecular clone JFH-1. Immunofluorescence studies further revealed a drastic HCV-dependent redistribution of YB-1 to the surface of the lipid droplets, an important organelle for HCV assembly. Core and NS3 protein-dependent polyprotein maturation were shown to be required for YB-1 relocalization. Unexpectedly, YB-1 knockdown cells showed the increased production of viral infectious particles while HCV RNA replication was impaired. Our data support that HCV hijacks YB-1-containing ribonucleoparticles and that YB-1-NS3/4A-HCV RNA complexes regulate the equilibrium between HCV RNA replication and viral particle production.     

A cis-acting replication element in the sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C virus RNA replication

RNA structures play key roles in the replication of RNA viruses. Sequence alignment software, thermodynamic RNA folding programs, and classical comparative phylogenetic analysis were used to build models of six RNA elements in the coding region of the hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B. The importance of five of these elements was evaluated by site-directed mutagenesis of a subgenomic HCV replicon. Mutations disrupting one of the predicted stem-loop structures, designated 5BSL3.2, blocked RNA replication, implicating it as an essential cis-acting replication element (CRE). 5BSL3.2 is about 50 bases in length and is part of a larger predicted cruciform structure (5BSL3). As confirmed by RNA structure probing, 5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop, and an 8-base internal loop. Mutational analysis and structure probing were used to explore the importance of these features. Primary sequences in the loops were shown to be important for HCV RNA replication, and the upper helix appears to serve as an essential scaffold that helps maintain the overall RNA structure. Unlike certain picornavirus CREs, whose function is position independent, 5BSL3.2 function appears to be context dependent. Understanding the role of 5BSL3.2 and determining how this new CRE functions in the context of previously identified elements at the 5' and 3' ends of the RNA genome should provide new insights into HCV RNA replication.     

Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures.

The subcellular location of the nonstructural proteins NS1, NS2B, and NS3 in Vero cells infected with the flavivirus Kunjin was investigated using indirect immunofluorescence and cryoimmunoelectron microscopy with monospecific antibodies. Comparisons were also made by dual immunolabelling using antibodies to double-stranded RNA (dsRNA), the putative template in the flavivirus replication complex. At 8 h postinfection, the immunofluorescent patterns showed NS1, NS2B, NS3, and dsRNA located in a perinuclear rim with extensions into the peripheral cytoplasm. By 16 h, at the end of the latent period, all patterns had changed to some discrete perinuclear foci associated with a thick cytoplasmic reticulum. By 24 h, this localization in perinuclear foci was more apparent and some foci were dual labelled with antibodies to dsRNA. In immuno-gold-labelled cryosections of infected cells at 24 h, all antibodies were associated with clusters of induced membrane structures in the perinuclear region. Two important and novel observations were made. First, one set of induced membranes comprised vesicle packets of smooth membranes dual labelled with anti-dsRNA and anti-NS1 or anti-NS3 antibodies. Second, adjacent masses of paracrystalline arrays or of convoluted smooth membranes, which appeared to be structurally related, were strongly labelled only with anti-NS2B and anti-NS3 antibodies. Paired membranes similar in appearance to the rough endoplasmic reticulum were also labelled, but less strongly, with antibodies to the three nonstructural proteins. Other paired membranes adjacent to the structures discussed above enclosed accumulated virus particles but were not labelled with any of the four antibodies. The collection of induced membranes may represent virus factories in which translation, RNA synthesis, and virus assembly occur.     

Coupled translation and replication of poliovirus RNA in vitro: synthesis of functional 3D polymerase and infectious virus.

Poliovirus RNA polymerase and infectious virus particles were synthesized by translation of virion RNA in vitro in HeLa S10 extracts. The in vitro translation reactions were optimized for the synthesis of the viral proteins found in infected cells and in particular the synthesis of the viral polymerase 3Dpol. There was a linear increase in the amount of labeled protein synthesized during the first 6 h of the reaction. The appearance of 3Dpol in the translation products was delayed because of the additional time required for the proteolytic processing of precursor proteins. 3Dpol was first observed at 1 h in polyacrylamide gels, with significant amounts being detected at 6 h and later. Initial attempts to assay for polymerase activity directly in the translation reaction were not successful. Polymerase activity, however, was easily detected by adding a small amount (3 microliters) of translation products to a standard polymerase assay containing poliovirion RNA. Full-length minus-strand RNA was synthesized in the presence of an oligo(U) primer. In the absence of oligo(U), product RNA about twice the size of virion RNA was synthesized in these reactions. RNA stability studies and plaque assays indicated that a significant fraction of the input virion RNA in the translation reactions was very stable and remained intact for 20 h or more. Plaque assays indicated that infectious virus was synthesized in the in vitro translation reactions. Under optimal conditions, the titer of infectious virus produced in the in vitro translation reactions was greater than 100,000 PFU/ml. Virus was first detected at 6 h and increased to maximum levels by 12 h. Overall, the kinetics of poliovirus replication (protein synthesis, polymerase activity, and virus production) observed in the HeLa S10-initiation factor in vitro translation reactions were similar to those observed in infected cells.