The polypyrimidine tract-binding protein (PTB) is required for efficient replication of hepatitis C virus (HCV) RNA

Our earlier work found that the polypyrimidine tract-binding protein (PTB) specifically interacts with the poly(U/C) tract of the hepatitis C virus (HCV) 3' untranslated region (3'UTR) [Luo, G., 1999. Cellular proteins bind to the poly(U) tract of the 3' untranslated region of hepatitis C virus RNA genome. Virology 256, 105-118]. We report here that the phosphorylated form of PTB is associated with the membrane-bound HCV replication complex. To determine whether the PTB is required for HCV RNA replication, synthetic small interfering RNAs (siRNAs) were used to specifically knockdown PTB expression in the HCV replicon-harboring Huh7 cells. The level of PTB expression was efficiently reduced by PTB-specific siRNAs. Consequently, the levels of HCV proteins as well as HCV RNA were proportionally decreased by increasing concentrations of PTB siRNA. However, the translation mediated by the encephamyocarditis virus internal ribosome entry site was unaffected, suggesting that the inhibition of HCV RNA replication by PTB siRNA was not due to its effect on the EMCV IRES-mediated translation. Collectively, these findings demonstrate that PTB is required for efficient HCV RNA replication in the cell.     

Replication of the genome RNAs of defective interfering particles of vesicular stomatitis and Sendai viruses using heterologous viral proteins

We have tested the ability of heterologous viral proteins to support the in vivo and in vitro replication of the RNA of defective interfering (DI) particles of two serotypes of VSV and of Sendai virus. In all the combinations of heterologous coinfections in vivo, DI particle replication was observed only in the coinfection with the VSV-Indiana DI particle and wild-type VSV-New Jersey. By quantitating RNA synthesis in reconstitution experiments we showed that with DI nucleocapsids isolated from infected cells, however, the soluble protein fraction from heterologous wild-type virus-infected cells could substitute in vitro to varying degrees for the homologous proteins in the elongation reaction of RNA replication and encapsidation. In these cases successful replication was confirmed by demonstrating the specific association of the heterologous N protein with the product nucleocapsid RNA. The initiation step, that is, the initial binding of the nucleocapsid protein to the leader RNA, in contrast, requires the homologous protein, since heterologous viral proteins could not support RNA replication and encapsidation from purified DI particles.     

Primary structure and translation of a defective interfering RNA of murine coronavirus

An intracellular defective-interfering (DI) RNA, DIssE, of mouse hepatitis virus (MHV) obtained after serial high multiplicity passage of the virus was cloned and sequenced. DIssE RNA is composed of three noncontiguous genomic regions, representing the first 864 nucleotides of the 5' end, an internal 748 nucleotides of the polymerase gene, and 601 nucleotides from the 3' end of the parental MHV genome. The DIssE sequence contains one large continuous open reading frame. Two protein products from this open reading frame were identified both by in vitro translation and in DI-infected cells. Sequence comparison of DIssE and the corresponding parts of the parental virus genome revealed that DIssE had three base substitutions within the leader sequence and also a deletion of nine nucleotides located at the junction of the leader and the remaining genomic sequence. The 5' end of DIssE RNA was heterogeneous with respect to the number of UCUAA repeats within the leader sequence. The parental MHV genomic RNA appears to have extensive and stable secondary structures at the regions where DI RNA rearrangements occurred. These data suggest that MHV DI RNA may have been generated as a result of the discontinuous and nonprocessive manner of MHV RNA synthesis.     

Mutagenesis of the murine hepatitis virus nsp1-coding region identifies residues important for protein processing, viral RNA synthesis, and viral replication

Despite ongoing research investigating mechanisms of coronavirus replication, functions of many viral nonstructural proteins (nsps) remain unknown. In the current study, a reverse genetic approach was used to define the role of the 28-kDa amino-terminal product (nsp1) of the gene 1 polyprotein during replication of the coronavirus murine hepatitis virus (MHV) in cell culture. To determine whether nsp1 is required for MHV replication and to identify residues critical for protein function, mutant viruses that contained deletions or point mutations within the nsp1-coding region were generated and assayed for defects in viral replication, viral protein expression, protein localization, and RNA synthesis. The results demonstrated that the carboxy-terminal half of nsp1 (residues K(124) through L(241)) was dispensable for virus replication in culture but was required for efficient proteolytic cleavage of nsp1 from the gene 1 polyprotein and for optimal viral replication. Furthermore, whereas deletion of nsp1 residues amino-terminal to K(124) failed to produce infectious virus, point mutagenesis of the nsp1 amino-terminus allowed recovery of several mutants with altered replication and RNA synthesis. This study identifies nsp1 residues important for protein processing, viral RNA synthesis, and viral replication.     

Replication of Tomato bushy stunt virus RNA in a plant in vitro system

An ideal system to investigate individual determinants of the replication process of (+)-strand RNA viruses is a cell-free extract that supports viral protein and RNA synthesis in a synchronized manner. Here, we applied a translation/replication system based on cytoplasmic extracts of Nicotiana tabacum cells to Tomato bushy stunt virus (TBSV) RNA. In vitro translated TBSV proteins p33 and p92 form viral replicase, which, in the same reaction, accomplishes the entire replication cycle on exogenous TBSV DI or full-length RNA. Tests of mutant TBSV RNAs confirmed the template specificity of the in vitro replication reaction. Complementation experiments ascertained the significance of an earlier identified TBSV host factor. Interestingly, formation of the viral replicase occurs also in the absence of concurrent protein synthesis demonstrating that translation and RNA replication are not functionally linked in this system. Our studies with cell-free extracts of a plant host thus confirmed earlier findings and enabled novel insights into the TBSV RNA replication process.     

Role of La autoantigen and polypyrimidine tract-binding protein in HCV replication

To determine if the cellular factors La autoantigen (La) and polypyrimidine tract-binding protein (PTB) are required for hepatitis C virus (HCV) replication, we used siRNAs to silence these factors and then monitored their effect on HCV replication using quantitative RT-PCR. In addition, we determined the influence of PTB on the activity of the 3' noncoding region (NCR) of HCV and investigated its interaction with the components of the HCV replicase complex. We found that La is essential for efficient HCV replication while PTB appears to partially repress replication. PTB does, however, block the binding of HCV RNA-dependent RNA polymerase (RdRp, NS5B) to the 3'NCR. Indirect immunofluorescence microscopy showed co-localization of cytoplasmic PTB with the HCV RdRp in hepatoma cells (Huh-7) expressing HCV proteins, while in vitro translation of viral proteins from the HCV replicon revealed the interaction of PTB isoforms with NS5B polymerase and NS3.     

Translation elongation factor-1alpha,La, and PTB interact with the 3' untranslated region of dengue 4 virus RNA

The 384-nt long 3' untranslated region (3'UTR) of dengue 4 virus (DEN4) is not polyadenylated, but contains the adjacent thermodynamically stable conserved short and long stem-loop structures (L-SL) and the conserved sequences CS1 and CS2. The latter are duplicated (CS2A and CS2B) in DEN4. Dengue virus replication, like that of other RNA viruses, might involve the cis-elements located within the 3'UTR and the trans-acting factors that could interact with the viral replicase to function as a replicase complex. The identification and characterization of viral and cellular proteins involved in the interaction with the 3'UTR of dengue virus will help us to understand the cellular requirements for viral replication. To determine these requirements, mobility shift and cross-linking assays were performed with uninfected and DEN4-infected C6/36 cell extracts as well as the different segments of the 3'UTR. Our results revealed that RNA-protein complexes were formed with the RNAs which involved the domains CS2A, CS2B, CS1, and L-SL. The minimum RNA sequence that was able to form specific and stable complexes with cellular proteins was the CS1-L-SL region. Using UV-induced cross-linking we identified eight proteins with molecular weights of 34, 39, 51, 52, 56, 62, 72, and 84 kDa that bound to the complete 3'UTR. The translation elongation factor-1alpha (EF-1alpha) bound to the complete 3'UTR and to the CS1-L-SL region. In addition, the recombinant GST-human La autoantigen bound to the 3'UTR and to the CS1-L-SL region as demonstrated by mobility shift and cross-linking assays. Although different antibodies against PTB were unable to react with any of the cellular proteins from C6/36, the recombinant His-PTB protein did bind to the complete 3'UTR and to the CS1-L-SL region. The specific binding of La and PTB to the sequences considered essential for viral RNA replication may suggest that these proteins could function as RNA chaperones to maintain RNA structure in a conformation that favors viral replication, while EF-1alpha may function as an RNA helicase.     

Persistent infection with mouse hepatitis virus 3 in mouse lymphoid cell lines

The sensitivity of mice to mouse hepatitis virus 3 (MHV3) varies according to strain, age, and immune status of the animals. In semisusceptible strains, mice surviving the acute phase of infection develop a chronic disease characterized by the occurrence of paralysis, virus persistence, and immunodeficiency. Persistent MHV3 infections established in vitro in YAC and RDM -4 mouse lymphoid cell lines were characterized by virus production, presence of cytoplasmic viral antigens, and cell lysis. The occurrence of cell "crisis" in YAC cells was manifested by a sharp increase in cell lysis and in the number of fluorescent cells and, concomitantly, by a marked decrease in virus titers. A relationship was observed among the percentage of fluorescent cells, cell lysis, and virus yield and was modulated by renewal of culture media, change in temperature, or inhibition of cellular RNA synthesis. Cell cloning and antibody treatment experiments indicated that viral transmission was performed by viral infection of newly permissive cells produced by the division of uninfected cells in the culture and not by transmission of viral information by infected dividing cells. The biological and biochemical properties of MHV3 variants derived from persistently infected YAC lymphoid cells were characterized. Thermosensitivity and thermolability of cloned viruses originating from persistently infected YAC cells, as well as parent virus suspensions, were studied. A similar heterogeneity was observed when YAC-derived cloned substrains (YAC-MHV3) were compared with parent-derived cloned viruses, indicating that no selection of temperature-sensitive mutants was induced in persistently infected YAC cells. However, the capacity of MHV3 to induce a lethal acute disease when injected into susceptible mice was lost very rapidly. The absence of pathogenicity was related to the induction of a subclinical infection which elicited defense mechanisms. These data suggest, therefore, that MHV3 replication in lymphoid cell lines leads to induction or selection of variants which maintain pathogenicity in vitro but display reduced pathogenic effects in vivo.     

SYNCRIP (synaptotagmin-binding, cytoplasmic RNA-interacting protein) is a host factor involved in hepatitis C virus RNA replication

Hepatitis C virus (HCV) RNA replication requires viral nonstructural proteins as well as cellular factors. Recently, a cellular protein, synaptotagmin-binding, cytoplasmic RNA-interacting protein (SYNCRIP), also known as NSAP1, was found to bind HCV RNA and enhance HCV IRES-dependent translation. We investigate whether this protein is also involved in the HCV RNA replication. We found that SYNCRIP was associated with detergent-resistant membrane fractions and colocalized with newly-synthesized HCV RNA. Knock-down of SYNCRIP by siRNA significantly decreased the amount of HCV RNA in the cells containing a subgenomic replicon or a full-length viral RNA. Lastly, an in vitro replication assay after immunodepletion of SYNCRIP showed that SYNCRIP was directly involved in HCV RNA replication. These findings indicate that SYNCRIP has dual functions, participating in both RNA replication and translation in HCV life cycle.     

Viral genome synthesis in BHK 21 cells persistently infected with Sendai virus

We have carried out daily analysis of the viral RNA species replicated by nucleocapsids present in the cytoplasm of two different BHK 21 cell lines persistently infected with Sendai virus. At each day of analysis (both before and after crises of cytopathology, and during long periods of stable growth) DI particle size RNA was the major species of viral RNA replicated within these nucleocapsids. The molar ratio of DI RNA to standard virus (50 S) RNA species within intracellular nucleocapsids was always extremely high (from 10/1 to 340/1). Thus, there is no evidence for cyclic variations in relative predominance of standard virus and DI nucleocapsids in populations of these persistently infected cells, although “cycling” could occur at the individual cell level at infrequent intervals. Furthermore, challenge of persistently infected cultures with standard Sendai virus or incubation at 33° also failed to significantly decrease the ratio of DI nucleocapsids to standard virus nucleocapsids. It was also found that multiple DI RNA species are present (most are not resolvable by sucrose gradient analysis), and that these evolve with time—some arising and increasing with time, while others are outcompeted. No selective advantage for DI RNA of any particular size was observed. Finally, by measuring the total amount of nucleocapsids present in these persistently infected cells (as estimated by level of NP protein in purified nucleocapsids), and the degree of synthesis of viral nucleocapsid (as determined by [³H]uridine incorporation in the presence of actinomycin D), we found that the rate of viral genome replication was 30- to 40-fold lower in the persistently infected cells than it was in St acutely infected cells. These findings may indicate that the accumulated nucleocapsids in persistently infected cells are built up over a period of many weeks of slow synthesis, although turnover times for NP protein and RNA of nucleocapsids have not yet been determined in persistently infected cells.