Flaviviral Replication Complex: Coordination between RNA Synthesis and 5′-RNA Capping

Genome replication in flavivirus requires (−) strand RNA synthesis, (+) strand RNA synthesis, and 5′-RNA capping and methylation. To carry out viral genome replication, flavivirus assembles a replication complex, consisting of both viral and host proteins, on the cytoplasmic side of the endoplasmic reticulum (ER) membrane. Two major components of the replication complex are the viral non-structural (NS) proteins NS3 and NS5. Together they possess all the enzymatic activities required for genome replication, yet how these activities are coordinated during genome replication is not clear. We provide an overview of the flaviviral genome replication process, the membrane-bound replication complex, and recent crystal structures of full-length NS5. We propose a model of how NS3 and NS5 coordinate their activities in the individual steps of (−) RNA synthesis, (+) RNA synthesis, and 5′-RNA capping and methylation.     

Characteristics of Classical Swine Fever Virus Variants Derived from Live Attenuated GPE− Vaccine Seed

The GPE⁻ strain is a live attenuated vaccine for classical swine fever (CSF) developed in Japan. In the context of increasing attention for the differentiating infected from vaccinated animals (DIVA) concept, the achievement of CSF eradication with the GPE⁻ proposes it as a preferable backbone for a recombinant CSF marker vaccine. While its infectious cDNA clone, vGPE⁻, is well characterized, 10 amino acid substitutions were recognized in the genome, compared to the original GPE⁻ vaccine seed. To clarify the GPE⁻ seed availability, this study aimed to generate and characterize a clone possessing the identical amino acid sequence to the GPE⁻ seed. The attempt resulted in the loss of the infectious GPE⁻ seed clone production due to the impaired replication by an amino acid substitution in the viral polymerase NS5B. Accordingly, replication-competent GPE⁻ seed variant clones were produced. Although they were mostly restricted to propagate in the tonsils of pigs, similarly to vGPE⁻, their type I interferon-inducing capacity was significantly lower than that of vGPE⁻. Taken together, vGPE⁻ mainly retains ideal properties for the CSF vaccine, compared with the seed variants, and is probably useful in the development of a CSF marker vaccine.     

Biochemical and structural analysis of the NS5B RNA-dependent RNA polymerase of the hepatitis C virus

Hepatitis C virus (HCV), the major causative agent of chronic and sporadic non-A, non-B hepatitis worldwide, is a distinct member of the Flaviviridae virus family. These viruses have in common a plus-strand RNA genome that is replicated in the cytoplasm of the infected cell via minus-strand RNA intermediates. Owing to the lack of reliable cell culture systems and convenient animal models for HCV, the mechanisms governing RNA replication are not known. As a first step towards the development of appropriate in vitro systems, we expressed the NS5B RNA-dependent RNA polymerase (RdRp) in insect cells, purified the protein to near homogeneity and studied its biochemical properties. It is a primer- and RNA template-dependent RNA polymerase able to copy long heteropolymeric templates without additional viral or cellular cofactors. We determined the optimal reaction parameters, the kinetic constants and the substrate specificity of the enzyme, which turned out to be similar to those described for the 3D polymerase of poliovirus. By analysing a series of nucleosidic and non-nucleosidic compounds for their effect on RdRp activity, we found that ribavirin triphosphates have no inhibitory effect, providing direct experimental proof that the therapeutic effect observed in patients is not related to a direct inhibition of the viral polymerase. Finally, mutation analysis was performed to map the minimal NS5B sequence required for enzymatic activity and to identify the 'classical' polymerase motifs important for template and NTP binding and catalysis.     

Monoclonal antibody recognizing N-terminal epitope of hepatitis C virus nonstructural 5B inhibits viral RNA replication

Summary.  The nonstructural 5B (NS5B) protein of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) with a key role in HCV replication. To characterize the functional roles of NS5B in HCV replication, we produced a panel of 10 monoclonal antibodies (mAbs) directed against NS5B protein from mice immunized with functionally active RdRp. The epitopes of eight mAbs are localized in the middle region (amino acid 240–263) of NS5B protein. On the other hand, the epitopes of two mAbs are mapped to amino acids 67–88 at the N-terminus of NS5B protein. To examine the effects of mAbs on HCV-RNA replication, we performed in vitro RdRp assay using either the 3'-untranslated region (UTR) or the full-length of HCV-RNA as a template in the presence of each mAb. mAbs specific for the middle region of NS5B had no effect on RdRp activity. Surprisingly, mAb recognizing the N-terminal region of NS5B inhibited RdRp activity in a dose-dependent manner. We have confirmed the same result using the other subclass of mAb, whose epitope is also localized to the same N-terminal region of NS5B. These data show that NS5B contains a B-cell epitope located between amino acid residues 67 and 88. Binding of this epitope with an antibody interferes with the enzymatic function of NS5B.     

TNF-Mediated Inhibition of Classical Swine Fever Virus Replication Is IRF1-, NF-κB- and JAK/STAT Signaling-Dependent

The sera from pigs infected with virulent classical swine fever virus (CSFV) contain substantial amounts of tumor necrosis factor (TNF), a prototype proinflammatory cytokine with pleiotropic activities. TNF limits the replication of CSFV in cell culture. In order to investigate the signaling involved in the antiviral activity of TNF, we employed small-molecule inhibitors to interfere specifically with JAK/STAT and NF-κB signaling pathways in near-to-primary endothelial PEDSV.15 cells. In addition, we knocked out selected factors of the interferon (IFN) induction and signaling pathways using CRISPR/Cas9. We found that the anti-CSFV effect of TNF was sensitive to JAK/STAT inhibitors, suggesting that TNF induces IFN signaling. Accordingly, we observed that the antiviral effect of TNF was dependent on intact type I IFN signaling as PEDSV.15 cells with the disrupted type I IFN receptor lost their capacity to limit the replication of CSFV after TNF treatment. Consequently, we examined whether TNF activates the type I IFN induction pathway. With genetically modified PEDSV.15 cells deficient in functional interferon regulatory factor 1 or 3 (IRF1 or IRF3), we observed that the anti-CSFV activity exhibited by TNF was dependent on IRF1, whereas IRF3 was dispensable. This was distinct from the lipopolysaccharide (LPS)-driven antiviral effect that relied on both IRF1 and IRF3. In agreement with the requirement of IRF1 to induce TNF- and LPS-mediated antiviral effects, intact IRF1 was also essential for TNF- and LPS-mediated induction of IFN-β mRNA, while the activation of NF-κB was not dependent on IRF1. Nevertheless, NF-κB activation was essential for the TNF-mediated antiviral effect. Finally, we observed that CSFV failed to counteract the TNF-mediated induction of the IFN-β mRNA in PEDSV.15 cells, suggesting that CSFV does not interfere with IRF1-dependent signaling. In summary, we report that the proinflammatory cytokine TNF limits the replication of CSFV in PEDSV.15 cells by specific induction of an IRF1-dependent antiviral type I IFN response.