mi R-29a promotes hepatitis B virus replication and expression by targeting SMARCE1 in hepatoma carcinoma

AIM To investigate the functional role and underlying molecular mechanism of mi R-29 a in hepatitis B virus(HBV) expression and replication.METHODS The levels of mi R-29 a and SMARCE1 in HBV-infected Hep G2.2.15 cells were measured by quantitative real-time PCR and western blot analysis. HBV DNA replication was measured by quantitative PCR and Southern blot analysis. The relative levels of hepatitis B surface antigen and hepatitis B e antigen were detected by enzyme-linked immunosorbent assay. The Cell Counting Kit-8(CCK-8) was used to detect the viability of Hep G2.2.15 cells. The relationship between mi R-29 a and SMARCE1 were identified by target prediction and luciferase reporter analysis.RESULTS mi R-29 a promoted HBV replication and expression, w h i le S MA R C E 1 r e p r e s s e d H B V r e p lic a t io n a n d expression. Cell viability detection indicated that mi R-29 a transfection had no adverse effect on the host cells. Moreover, SMARCE1 was identified and validated to be a functional target of mi R-29 a. Furthermore, restored expression of SMARCE1 could relieve the increased HBV replication and expression caused by mi R-29 a overexpression.CONCLUSION mi R-29 a promotes HBV replication and expression through regulating SMARCE1. As a potential regulator of HBV replication and expression, mi R-29 a could be a promising therapeutic target for patients with HBV infection.

HIF-1αpromotes virus replication and cytokine storm in H1N1 virus-induced severe pneumonia through cellular metabolic reprogramming

The mortality of patients with severe pneumonia caused by H1N1 infection is closely related to viral replication and cytokine storm.However,the specific mechanisms triggering virus replication and cytokine storm are still not fully elucidated.Here,we identified hypoxia inducible factor-1α(HIF-1α)as one of the major host molecules that facilitates H1N1 virus replication followed by cytokine storm in alveolar epithelial cells.Specifically,HIF-1αprotein expression is upregulated after H1N1 infection.Deficiency of HIF-1αattenuates pulmonary injury,viral replication and cytokine storm in vivo.In addition,viral replication and cytokine storm were inhibited after HIF-1αknockdown in vitro.Mechanistically,the invasion of H1N1 virus into alveolar epithelial cells leads to a shift in glucose metabolism to glycolysis,with rapid production of ATP and lactate.Inhibition of glycolysis significantly suppresses viral replication and inflammatory responses.Further analysis revealed that H1N1-induced HIF-1αcan promote the expression of hexokinase 2(HK2),the key enzyme of glycolysis,and then not only provide energy for the rapid replication of H1N1 virus but also produce lactate,which reduces the accumulation of the MAVS/RIG-I complex and inhibits IFN-α/βproduction.In conclusion,this study demonstrated that the upregulation of HIF-1αby H1N1 infection augments viral replication and cytokine storm by cellular metabolic reprogramming toward glycolysis mainly through upregulation of HK2,providing a theoretical basis for finding potential targets for the treatment of severe pneumonia caused by H1N1 infection.

ISG20 inhibits bluetongue virus replication

ISG20 is an interferon-inducible exonuclease that inhibits virus replication.Although ISG20 is thought to degrade viral RNA,the antiviral mechanism and specificity of ISG20 remain unclear.In this study,the antiviral role of ovine ISG20(o ISG20)in bluetongue virus(BTV)infection was investigated.It was found that BTV infection upregulated the transcription of ovine ISG20(o ISG20)in a time-and BTV multiplicity of infection(MOI)-dependent manner.Overexpression of o ISG20 suppressed the production of BTV genome,proteins,and virus titer,whereas the knockdown of o ISG20 increased viral replication.o ISG20 was found to co-localize with BTV proteins VP4,VP5,VP6,and NS2,but only directly interacted with VP4.Exonuclease defective o ISG20 significantly decreased the inhibitory effect on BTV replication.In addition,the interaction of mutant o ISG20 and VP4 was weakened,suggesting that binding to VP4 was associated with the inhibition of BTV replication.The present data characterized the anti-BTV effect of o ISG20,and provides a novel clue for further exploring the inhibition mechanism of double-stranded RNA virus by ISG20.

Nucleolin interacts with the rabbit hemorrhagic disease virus replicase RdRp, nonstructural proteins p16 and p23, playing a role in virus replication

Rabbit hemorrhagic disease virus(RHDV)is a member of the Caliciviridae family and cannot be propagated in vitro,which has impeded the progress of investigating its replication mechanism.Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells.Here,aided by this replicon system and using two-step affinity purification,we purified the RHDV replicase and identified its associated host factors.We identified rabbit nucleolin(NCL)as a physical link,which mediating the interaction between other RNA-dependent RNA polymerase(Rd Rp)-related host proteins and the viral replicase Rd Rp.We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 cells,respectively.NCL was identified to directly interact with RHDV Rd Rp,p16,and p23.Furthermore,NCL knockdown severely impaired the binding of Rd Rp to Rd Rp-related host factors.Collectively,these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins.

Diverse Regulations of Viral and Host Genes in Tomato Germplasms Responding to Tomato Yellow Leaf Curl Virus Inoculation

Tomato yellow leaf curl virus(TYLCV)is the dominating pathogen of tomato yellow leaf curl disease that caused severe loss to tomato production in China.In this study,we found that a TYLCV-resistant tomato line drastically reduced the accumulation of viral complementary-sense strand mRNAs but just moderately inhibited that of viral DNA and virion-sense strand mRNAs.However,two other resistant lines did not have such virus inhibition pattern.Analysis of differential expressed genes showed that the potential host defense-relevant processes varied in different resistant tomatoes,as compared to the susceptible line,suggesting a diversity of tomato TYLCV-resistance mechanisms.

Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication

Human adenovirus B7(HAdV-B7)causes severe acute lower respiratory tract infections in children.However,neither the child-specific antivirals or vaccines are available,nor the pathogenesis is clear.Autophagy,as part of innate immunity,plays an important role in resistance to viral infection by degrading the virus and promoting the development of innate and adaptive immunity.This study provided evidence that HAdV-B7 infection induced complete autophagic flux,and the pharmacological induction of autophagy decreased HAdV-B7 replication.In this process,the host protein Bcl2-associated athanogene 3(BAG3)mediated autophagy to inhibit the replication of HAdV-B7 by binding to the PPSY structural domain of viral protein pVI through its WW structural domain.These findings further our understanding of the host immune response during viral infection and will help to develop broad anti-HAdV therapies.

Novel and potent inhibitors targeting DHODH are broad-spectrum antivirals against RNA viruses including newly-emerged coronavirus SARS-CoV-2

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide,such as the on-going outbreak of the novel coronavirus SARS-CoV-2.Herein,we identified two potent inhibitors of human DHODH,S312 and S416,with favorable drug-likeness and pharmacokinetic profiles,which all showed broad-spectrum antiviral effects against various RNA viruses,including influenza A virus,Zika virus,Ebola virus,and particularly against SARS-CoV-2.Notably,S416 is reported to be the most potent inhibitor so far with an EC5o of 17 nmol/L and an SI value of 10,505.88 in infec-ted cells.Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells.This work demonstrates that both S312/S416 and old drugs(Leflunomide/Teriflunomide)with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide,no matter such viruses are mutated or not.

Recovery of a Far-Eastern Strain of Tick-Borne Encephalitis Virus with a Full-Length Infectious cDNA Clone

Tick-borne encephalitis virus(TBEV) is a pathogenic virus known to cause central nervous system(CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the endemic countries are increasing. However, there is no effective antiviral against the disease. This underscores the urgent need for tools to study the emergence and pathogenesis of TBEV and to accelerate the development of vaccines and antivirals. In this study, we reported an infectious c DNA clone of TBEV that was isolated in China(the WH2012 strain). A beta-globin intron was inserted in the coding region of nonstructural protein 1(NS1) gene to improve the stability of viral genome in bacteria. In mammalian cells, the inserted intron was excised and spliced precisely, which did not lead to the generation of inserted mutants. High titers of infectious progeny viruses were generated after the transfection of the infectious clone. The cDNA-derived TBEV replicated efficiently, and caused typical cytopathic effect(CPE) and plaques in BHK-21 cells. In addition, the CPE and growth curve of cDNA-derived virus were similar to that of its parental isolate in cells. Together, we have constructed the first infectious TBEV cDNA clone in China, and the clone can be used to investigate the genetic determinants of TBEV virulence and disease pathogenesis, and to develop countermeasures against the virus.

Targeting m6A modification inhibits herpes virus 1 infection

The latent infection by herpes virus type 1(HSV-1)may be lifelong in trigeminal ganglia and a suspected cause of Alzheimer's Disease(AD)and Amyotrophic lateral sclerosis(ALS).Whether and how N6-methyladenosine(m6A)modification of viral RNAs affects virus infection are poorly understood.Here,we report that HSV-1 infection enhanced the expression of m6A writers(METTL3,METTL14)and readers(YTHDF1/2/3)at the early infection stage and decreased their expression later on,while suppressed the erasers'(FTO,ALBKH5)expression immediately upon infection to facilitate viral replication.Inhibiting m6A modification by 3-deazaadenosine(DAA)significantly decreased viral replication and reduced viral reproduction over 1000 folds.More interestingly,depleting the writers and readers by siRNAs inhibited virus replication and reproduction;whereas depleting the erasers promoted viral replication and reproduction.Silencing YTHDF3 strikingly decreased viral replication by up to 90%,leading to reduction of up to 10-fold viral replication and over 100-fold virus reproduction,respectively.Depletion of m6A initiator METTL3(by 60%–70%)by siRNA correlatedly decreased viral replication 60%–70%,and reduced virus yield over 30-fold.Consistently,ectopic expression of METTL3 largely increased virus yield.METTL3 knockdown suppressed the HSV-1 intermediate early and early genes(ICP0,ICP8 and UL23)and late genes(VP16,UL44,UL49 and ICP47);while ectopic expression of METTL3 upregulated these gene expression.Results from our study shed the lights on the importance for m6A modification to initiate HSV-1 early replication.The components of m6A modification machinery,particularly m6A initiator METTL3 and reader YTHDF3,would be potential important targets for combating HSV-1 infections.