摘要：The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis.

摘要：Hepatitis B virus (HBV) is a global major health problem, with over one million deaths annually caused by chronic liver damage. Understanding host factors that modulate HBV replication may aid the development of anti-HBV therapies. Our recent genome-wide small interfering RNA screen using recombinant HBV demonstrated that TIP60 inhibited HBV infection. Here, we show that TIP60 complex contributes to anti-HBV defense. The TIP60 complex bound to the HBV promoter and suppressed HBV transcription driven by the precore/core promoter. The silencing of EP400, TRRAP, BAF53a, RUVBL1, and RUVBL2, which form the TIP60 complex, also resulted in increased HBV transcription. These results contribute to our enhanced understanding of the molecular mechanism of HBV transcription associated with the chromatin structure of HBV covalently closed circular DNA (cccDNA). Exploiting these intrinsic cellular defenses might help develop new anti-HBV agents.IMPORTANCE Investigating the molecular mechanism of HBV replication is important to understand the persistent nature of HBV infection and to aid the development of new HBV agents, which are currently limited to HBV polymerase inhibitors. Previously, we developed a new reporter HBV. By screening host factors using this recombinant virus, we identified several gene products that regulate HBV infection, including TIP60. Here, we showed that TIP60, a catalytic subunit of the NuA4 complex, inhibited HBV replication. Depletion of TIP60 increased the level of HBV mRNA. Moreover, TIP60 localized in the HBV cccDNA chromatin complex catalyzed the acetylation of histone H4 to recruit Brd4. These results suggest that TIP60, in concert with other cellular factors, plays an important role in the regulation of the HBV chromatin structure by acting as a critical component of the intrinsic antiviral defense, which sheds new light on the regulation of HBV replication.

摘要：Current models of cell-intrinsic immunity to RNA viruses centre on virus-triggered inducible antiviral responses initiated by RIG-I-like receptors or Toll-like receptors that sense pathogen-associated molecular patterns, and signal downstream through interferon regulatory factors (IRFs), transcription factors that induce synthesis of type I and type III interferons1. RNA viruses have evolved sophisticated strategies to disrupt these signalling pathways and evade elimination by cells, attesting to their importance2. Less attention has been paid to how IRFs maintain basal levels of protection against viruses. Here, we depleted antiviral factors linked to RIG-I-like receptor and Toll-like receptor signalling to map critical host pathways restricting positive-strand RNA virus replication in immortalized hepatocytes and identified an unexpected role for IRF1. We show that constitutively expressed IRF1 acts independently of mitochondrial antiviral signalling (MAVS) protein, IRF3 and signal transducer and activator of transcription 1 (STAT1)-dependent signalling to provide intrinsic antiviral protection in actinomycin D-treated cells. IRF1 localizes to the nucleus, where it maintains the basal transcription of a suite of antiviral genes that protect against multiple pathogenic RNA viruses, including hepatitis A and C viruses, dengue virus and Zika virus. Our findings reveal an unappreciated layer of hepatocyte-intrinsic immunity to these positive-strand RNA viruses and identify previously unrecognized antiviral effector genes.

摘要：Bioluminescence is a powerful tool in the study of viral infection both in vivo and in vitro. Foot-and-mouth disease virus (FMDV) has a small RNA genome with a limited tolerance to foreign RNA entities. There has been no success in making a reporter FMDV expressing a luciferase in infected cell culture supernatants. We report here for the first time a replication-competent FMDV encoding Nanoluciferase, named as Nano-FMDV. Nano-FMDV is genetically stable during serial passages in cells and exhibits growth kinetics and plaque morphology similar to its parental virus. There are applications for the use of Nano-FMDV such as real-time monitoring of FMDV replication in vitro and in vivo.

摘要：Enterovirus 71 (EV71) is one of the causative agents of hand, foot and mouth disease (HFMD) associated with severe neurological disease. EV71's pathogenesis remains poorly understood and the lack of approved antiviral has led to its emergence as a clinically important neurotropic virus. The goals of this study were to: (i) identify novel anti-EV71 compounds that may serve as lead molecules for therapeutics; and (ii) investigate their targets in downstream studies. We screened a 502-compound library of highly purified natural products for anti-EV71 activities in a cell-based immunofluorescence assay that were then confirmed in viral plaque reduction assays. Along with known antivirals, novel inhibitors of EV71 were also identified. We selected camptothecin for downstream studies and found that it is a limited spectrum enterovirus inhibitor that inhibits coxsackievirus A16 but not ECHOvirus 7. Camptothecin, a DNA topoisomerase 1 (TOP1) inhibitor, inhibits both viral RNA replication and translation based on luciferase replicon studies. Depletion of TOP1 using siRNA was then able to rescue EV71 infection from camptothecin inhibition. Interestingly, EV71 viral RNA replication and translation were also in TOP1 depleted cells. We found that nuclear TOP1 was relocalized to cytoplasmic replication vesicles during EV71 infection and localized with viral 3CD using confocal microscopy and proximity-ligation assays. Our findings reveal camptothecin to be a limited spectrum antiviral against enteroviruses that functions in a TOP1-dependent but cytotoxicity-independent manner. TOP1 is in turn needed for maximal EV71 viral RNA replication and viral protein synthesis.

摘要：Alphaviruses are a group of widely distributed human and animal pathogens. It is well established that their replication is sensitive to type I IFN treatment, but the mechanism of IFN inhibitory function remains poorly understood. Using a new experimental system, we demonstrate that in the presence of IFN-β, activation of interferon-stimulated genes (ISGs) does not interfere with either attachment of alphavirus virions to the cells, or their entry and nucleocapsid disassembly. However, it strongly affects translation of the virion-delivered virus-specific RNAs. One of the ISG products, IFIT1 protein, plays a major role in this translation block, although an IFIT1-independent mechanism is also involved. The 5'UTRs of the alphavirus genomes were found to differ significantly in their ability to drive translation in the presence of increased concentration of IFIT1. Prior studies have shown that adaptation of naturally circulating alphaviruses to replication in tissue culture results in accumulation of mutations in the 5'UTR, which increase the efficiency of the promoter located in the 5'end of the genome. Here, we show that these mutations also decrease resistance of viral RNA to IFIT1-induced translation inhibition. In the presence of higher levels of IFIT1, alphaviruses with wt 5'UTRs became potent inducers of type I IFN, suggesting a new mechanism of type I IFN induction. We applied this knowledge of IFIT1 interaction with alphaviruses to develop new attenuated variants of Venezuelan equine encephalitis and chikungunya viruses that are more sensitive to the antiviral effects of IFIT1, and thus could serve as novel vaccine candidates.

摘要：Ebola virus (EBOV) VP40 is a major driving force of nascent virion production and a negative regulator of genome replication/transcription. Here, we showed that the YIGL sequence at the C-terminus of EBOV VP40 is important for virus-like particle (VLP) production and the regulation of genome replication/transcription. Accordingly, a mutation in the YIGL sequence caused defects in VLP production and genome replication/transcription. The residues I293 and L295 in the YIGL sequence were particularly critical for VLP production. Furthermore, an in silico analysis indicated that the amino acids surrounding the YIGL sequence contribute to intramolecular interactions within VP40. Among those surrounding residues, F209 was shown to be critical for VLP production. These results suggested that the VP40 YIGL sequence regulates two different viral replication steps, VLP production and genome replication/transcription, and the nearby residue F209 influences VLP production.

摘要：Duck Tembusu virus (DTMUV) is a novel flavivirus that has caused an outbreak of severe duck egg-drop syndrome since 2010. It has spread rapidly to other avian species, causing enormous economic loss. In the present study, we generated a reporter virus expressing NanoLuc luciferase, which was stable after 10 rounds of continuous propagation without reporter gene deletion. Moreover, we generated two types of replicons driven by the T7 promoter or CMV promoter, both of which worked well in BHK21 cells. Furthermore, we developed the first packaging system for DTMUV by co-transfection into BHK21 cells of a replicon (containing mature C) and a plasmid encoding C16-prM-E, which resulted in the production of single round infectious particles (SRIPs). We also generated a packaging cell line for DTMUV to produce SRIPs. We believe that these multicomponent platform tools are important for DTMUV pathogenesis research and novel vaccine development.

摘要：Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3' untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and β-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies.IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as "vesicle packets" (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools.

摘要：Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV.