A bacterial artificial chromosome (BAC)-vectored noninfectious replicon of SARS-CoV-2.

Vaccines and antiviral agents are in urgent need to stop the COVID-19 pandemic. To facilitate antiviral screening against SARS-CoV-2 without requirement for high biosafety level facility, we developed a bacterial artificial chromosome (BAC)-vectored replicon of SARS-CoV-2, nCoV-SH01 strain, in which secreted Gaussia luciferase (sGluc) was encoded in viral subgenomic mRNA as a reporter gene. The replicon was devoid of structural genes spike (S), membrane (M), and envelope (E). Upon transfection, the replicon RNA replicated in various cell lines, and was sensitive to interferon alpha (IFN-α), remdesivir, but was resistant to hepatitis C virus inhibitors daclatasvir and sofosbuvir. Replication of the replicon was also sensitive overexpression to zinc-finger antiviral protein (ZAP). We also constructed a four-plasmid in-vitro ligation system that is compatible with the BAC system, which makes it easy to introduce desired mutations into the assembly plasmids for in-vitro ligation. This replicon system would be helpful for performing antiviral screening and dissecting virus-host interactions.

Functional mapping of B-cell linear epitopes of SARS-CoV-2 in COVID-19 convalescent population.

ABSTRACT Pandemic SARS-CoV-2 has caused unprecedented mortalities. Vaccine is in urgent need to stop the pandemic. Despite great progresses on SARS-CoV-2 vaccine development, the efficacy of the vaccines remains to be determined. Deciphering the interactions of the viral epitopes with the elicited neutralizing antibodies in convalescent population inspires the vaccine development. In this study, we devised a peptide array composed of 20-mer overlapped peptides of spike (S), membrane (M) and envelope (E) proteins, and performed a screening with 120 COVID-19 convalescent sera and 24 non-COVID-19 sera. We identified five SARS-CoV-2-specific dominant epitopes that reacted with above 40% COVID-19 convalescent sera. Of note, two peptides non-specifically interacted with most of the non-COVID-19 sera. Neutralization assay indicated that only five sera completely blocked viral infection at the dilution of 1:200. By using a peptide-compete neutralizing assay, we found that three dominant epitopes partially competed the neutralization activity of several convalescent sera, suggesting antibodies elicited by these epitopes played an important role in neutralizing viral infection. The epitopes we identified in this study may serve as vaccine candidates to elicit neutralizing antibodies in most vaccinated people or specific antigens for SARS-CoV-2 diagnosis.

Hepatitis C virus promotes hepatocellular carcinogenesis by targeting TIPE2, a new regulator of DNA damage response.

Infection of hepatitis C virus (HCV) is associated with primary hepatocellular carcinoma (HCC). However, its underlying molecular mechanisms remain enigmatic. Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2), a new negative regulator of immunity, plays significant roles in modulating inflammation and tumorigenesis. We hypothesized that TIPE2 might be involved in the development of HCV-induced HCC. To test this hypothesis, the expression of TIPE2 was determined by Western blot in the tumor and pericarcinomatous tissues collected from ten HCV-positive HCC patients; the interaction between TIPE2 and HCV-encoded non-structural proteins was analyzed by immunoprecipitation and immunofluorescence assays, and tumorigenesis and its mechanisms were studied in cell models and nude mice. Our results demonstrated that the expression of TIPE2 was significantly reduced in HCC tissues compared to that in the paracarcinoma tissues. HCV-encoded non-structural protein NS5A could specifically interact with TIPE2 and induce its degradation. Downregulation of TIPE2 by shRNA in cell lines increased genomic DNA damage and promoted cell colony formation in vitro and tumorigenesis in nude mice. In contrast, overexpression of TIPE2 had an opposite effect. Downregulation of TIPE2 by NS5A is associated with genomic DNA instability and HCV-induced HCC development. Thus, TIPE2 may be a new therapeutic target for the treatment of HCV-associated HCC.

Hepatitis C virus replicative double-stranded RNA is a potent interferon inducer that triggers interferon production through MDA5.

The cytoplasmic RNA sensors, retinoic acid-inducible gene I and melanoma differentiation-associated gene 5, play crucial roles in innate sensing of hepatitis C virus (HCV). However, the exact identity of the IFN inducer generated during HCV infection is poorly understood. To identify the IFN inducer, we extracted the RNAs from HCV-replicating cells and introduced these into IFN signalling-competent cells to examine IFN production. RNAs isolated from HCV-replicating cells triggered robust IFN-ß and IFN-λ production in Huh7 cells in a viral replication-dependent manner, preferentially through the melanoma differentiation-associated gene 5 but not through the retinoic acid-inducible gene I-mediated pathway. The IFN-inducing capacity of HCV RNA survived after calf intestinal alkaline phosphatase and ssRNA-specific S1 nuclease treatment, but was completely eliminated by dsRNA-specific RNase III digestion, suggesting that viral replicative dsRNA is an IFN inducer. Furthermore, HCV viral RNA extracted from replicating cells was sensitive to 5'-monophosphate-dependent 5'→3' exonuclease (TER) digestion, suggesting that the HCV genome lacks a 5'-triphosphate or -diphosphate. In semi-permeabilized cells, the HCV IFN inducer primarily resided in an enclosed membranous structure that protects the IFN inducer from RNase digestion. Taken together, we identified HCV replicative dsRNA as a viral IFN inducer enclosed within the viral replication factory.

Affinity Purification of the Hepatitis C Virus Replicase Identifies Valosin-Containing Protein, a Member of the ATPases Associated with Diverse Cellular Activities Family, as an Active Virus Replication Modulator.

Like almost all of the positive-strand RNA viruses, hepatitis C virus (HCV) induces host intracellular membrane modification to form the membrane-bound viral replication complex (RC), within which viral replicases amplify the viral RNA genome. Despite accumulated information about how HCV co-opts host factors for viral replication, our knowledge of the molecular mechanisms by which viral proteins hijack host factors for replicase assembly has only begun to emerge. Purification of the viral replicase and identification of the replicase-associated host factors to dissect their roles in RC biogenesis will shed light on the molecular mechanisms of RC assembly. To purify the viral replicase in the context of genuine viral replication, we developed an HCV subgenomic replicon system in which two different affinity tags were simultaneously inserted in frame into HCV NS5A and NS5B. After solubilizing the replicon cells, we purified the viral replicase by two-step affinity purification and identified the associated host factors by mass spectrometry. We identified valosin-containing protein (VCP), a member of the ATPases associated with diverse cellular activities (AAA+ATPase) family, as an active viral replication modulator whose ATPase activity is required for viral replication. A transient replication assay indicated that VCP is involved mainly in viral genome amplification. VCP associated with viral replicase and colocalized with a viral RC marker. Further, in an HCV replicase formation surrogate system, abolishing VCP function resulted in aberrant distribution of HCV NS5A. We propose that HCV may co-opt a host AAA+ATPase for its replicase assembly. IMPORTANCE: Almost all of the positive-strand RNA viruses share a replication strategy in which viral proteins modify host membranes to form the membrane-associated viral replicase. Viruses hijack host factors to facilitate this energy-unfavorable process. Understanding of this fundamental process is hampered by the challenges of purifying the replicase because of the technical difficulties involved. In this study, we developed an HCV subgenomic replicon system in which two different affinity tags were simultaneously inserted in frame into two replicase components. Using this dual-affinity-tagged replicon system, we purified the viral replicase and identified valosin-containing protein (VCP) AAA+ATPase as a pivotal viral replicase-associated host factor that is required for viral genome replication. Abolishing VCP function resulted in aberrant viral protein distribution. We propose that HCV hijacks a host AAA+ATPase for its replicase assembly. Understanding the molecular mechanism of VCP regulates viral replicase assembly may lead to novel antiviral strategies targeting the most conserved viral replication step.

Hepatitis B virus polymerase disrupts K63-linked ubiquitination of STING to block innate cytosolic DNA-sensing pathways.

UNLABELLED: The cellular innate immune system recognizing pathogen infection is essential for host defense against viruses. In parallel, viruses have developed a variety of strategies to evade the innate immunity. The hepatitis B virus (HBV), a DNA virus that causes chronic hepatitis, has been shown to inhibit RNA helicase RIG-I-mediated interferon (IFN) induction. However, it is still unknown whether HBV could affect the host DNA-sensing pathways. Here we report that in transiently HBV-transfected Huh7 cells, the stably HBV-producing cell line HepAD38, and HBV-infected HepaRG cells and primary human hepatocytes, HBV markedly interfered with IFN-ß induction and antiviral immunity mediated by the stimulator of interferon genes (STING), which has been identified as a central factor in foreign DNA recognition and antiviral innate immunity. Screening analysis demonstrated that the viral polymerase (Pol), but not other HBV-encoded proteins, was able to inhibit STING-stimulated interferon regulatory factor 3 (IRF3) activation and IFN-ß induction. Moreover, the reverse transcriptase (RT) and the RNase H (RH) domains of Pol were identified to be responsible for the inhibitory effects. Furthermore, Pol was shown to physically associate with STING and dramatically decrease the K63-linked polyubiquitination of STING via its RT domain without altering the expression level of STING. Taken together, these observations suggest that besides its inherent catalytic function, Pol has a role in suppression of IFN-ß production by direct interaction with STING and subsequent disruption of its K63-linked ubiquitination, providing a new mechanism for HBV to counteract the innate DNA-sensing pathways. IMPORTANCE: Although whether and how HBV infection induces the innate immune responses are still controversial, it has become increasingly clear that HBV has developed strategies to counteract the pattern recognition receptor-mediated signaling pathways. Previous studies have shown that type I IFN induction activated by the host RNA sensors could be inhibited by HBV. However, it remains unknown whether HBV as a DNA virus utilizes evasion mechanisms against foreign DNA-elicited antiviral signaling. In recent years, the cytosolic DNA sensor and key adaptor STING has been demonstrated to be essential in multiple foreign DNA-elicited innate immune signalings. Here, for the first time, we report STING as a new target of HBV to antagonize IFN induction and identify the viral polymerase responsible for the inhibitory effect, thus providing an additional molecular mechanism by which HBV evades the innate immunity; this implies that in addition to its inherent catalytic function, HBV polymerase is a multifunctional immunomodulatory protein.

Tetrazole and triazole as bioisosteres of carboxylic acid: discovery of diketo tetrazoles and diketo triazoles as anti-HCV agents.

A series of diketo tetrazoles and diketo triazoles were designed and synthesized as bioisosteres of α,γ-diketo acid, the active site inhibitor of HCV (Hepatitis C virus) polymerase NS5B. Among the synthesized compounds, 4-(4-fluorobenzyloxy)phenyl diketo triazole (30) exhibited anti-HCV activity with an EC50 value of 3.9 µM and an SI value more than 128. The reduction of viral protein and mRNA levels were also validated, supporting the anti-HCV activity of compound 30. These results provide convincing evidence that the diketo tetrazoles and diketo triazoles can be developed as bioisosteres of α,γ-diketo acid to exhibit potent inhibitory activity against HCV.

Viperin inhibits hepatitis C virus replication by interfering with binding of NS5A to host protein hVAP-33.

Viperin is a type-I and -II interferon-inducible intracytoplasmic protein that mediates antiviral activity against several viruses. A previous study has reported that viperin could limit hepatitis C virus (HCV) replication in vitro. However, the underlying mechanism remains elusive. In the present study, we found that overexpression of viperin could inhibit HCV replication in a dose-dependent manner in both the replicon and HCVcc systems. Furthermore, through co-immunoprecipitation and laser confocal microscopic analysis, viperin was found to interact with the host protein hVAP-33. Mutagenesis analysis demonstrated that the anti-HCV activity of viperin was located to its C terminus, which was required for the interaction with the C-terminal domain of hVAP-33. Competitive co-immunoprecipitation analysis showed that viperin could interact competitively with hVAP-33, and could therefore interfere with its interactions with HCV NS5A. In summary, these findings suggest a novel mechanism by which viperin inhibits HCV replication, possibly through binding to host protein hVAP-33 and interfering with its interaction with NS5A.

Hepatitis C virus co-opts Ras-GTPase-activating protein-binding protein 1 for its genome replication.

We recently reported that Ras-GTPase-activating protein-binding protein 1 (G3BP1) interacts with hepatitis C virus (HCV) nonstructural protein (NS)5B and the 5' end of the HCV minus-strand RNA. In the current study we confirmed these observations using immunoprecipitation and RNA pulldown assays, suggesting that G3BP1 might be an HCV replication complex (RC) component. In replicon cells, transfected G3BP1 interacts with multiple HCV nonstructural proteins. Using immunostaining and confocal microscopy, we demonstrate that G3BP1 is colocalized with HCV RCs in replicon cells. Small interfering RNA (siRNA)-mediated knockdown of G3BP1 moderately reduces established HCV RNA replication in HCV replicon cells and dramatically reduces HCV replication-dependent colony formation and cell-culture-produced HCV (HCVcc) infection. In contrast, knockdown of G3BP2 has no effect on HCVcc infection. Transient replication experiments show that G3BP1 is involved in HCV genome amplification. Thus, G3BP1 is associated with HCV RCs and may be co-opted as a functional RC component for viral replication. These findings may facilitate understanding of the molecular mechanisms of HCV genome replication.