Positive-sense single-stranded RNA(+ssRNA)viruses,the most abundant viruses of eukaryotes in nature,require the synthesis of negative-sense RNA(-RNA)using their genomic(positive-sense)RNA(+RNA)as a template for replic...Positive-sense single-stranded RNA(+ssRNA)viruses,the most abundant viruses of eukaryotes in nature,require the synthesis of negative-sense RNA(-RNA)using their genomic(positive-sense)RNA(+RNA)as a template for replication.Based on current evidence,viral proteins are translated via viral+RNAs,whereas-RNA is considered to be a viral replication intermediate without coding capacity.Here,we report that plant and animal+ssRNA viruses contain small open reading frames(ORFs)in their-RNA(reverse ORFs[rORFs]).Using turnip mosaic virus(TuMV)as a model for plant+ssRNA viruses,we demonstrate that small proteins encoded by rORFs display specific subcellularlocalizations,and confirm the presence of rORF2 in infected cells through mass spectrometry analysis.The protein encoded by TuMV rORF2 forms punctuate granules that are localized in the perinuclear region and co-localized with viral replication complexes.The rORF2 protein can directly interact with the viral RNA-dependent RNA polymerase,and mutation of rORF2 completely abolishes virus infection,whereas ectopic expression of rORF2 rescues the mutant virus.Furthermore,we show that several rORFs in the-RNA of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)have the ability to suppress type l interferon production and facilitate the infection of ve-sicular stomatitis virus.In addition,we provide evidence that TuMV might utilize internal ribosome entry sites to translate these small rORFs.Taken together,these findings indicate that the-RNA of+ssRNA vi-ruses can also have the coding capacity and that small proteins encoded therein play critical roles in viral infection,revealing a viral proteome larger than previously thought.展开更多
Positive-sense RNA viruses modify intracellular calcium stores,endoplasmic reticulum and Golgi apparatus(Golgi)to generate membranous replication organelles known as viral factories.Viral factories provide a conducive...Positive-sense RNA viruses modify intracellular calcium stores,endoplasmic reticulum and Golgi apparatus(Golgi)to generate membranous replication organelles known as viral factories.Viral factories provide a conducive and substantial enclave for essential virus replication via concentrating necessary cellular factors and viral proteins in proximity.Here,we identified the vital role of a broadspectrum antiviral,peruvoside in limiting the formation of viral factories.Mechanistically,we revealed the pleiotropic cellular effect of Src and PLC kinase signaling via cyclin-dependent kinase 1 signaling leads to Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1(GBF1)phosphorylation and Golgi vesiculation by peruvoside treatment.The ramification of GBF1 phosphorylation fosters GBF1 deprivation consequentially activating downstream antiviral signaling by dampening viral factories formation.Further investigation showed signaling of ERK1/2 pathway via cyclin-dependent kinase 1 activation leading to GBF1 phosphorylation at Threonine 1337(T1337).We also showed 100%of protection in peruvoside-treated mouse model with a significant reduction in viral titre and without measurable cytotoxicity in serum.These findings highlight the importance of dissecting the broad-spectrum antiviral therapeutics mechanism and pave the way for consideration of peruvoside,host-directed antivirals for positive-sense RNA virus-mediated disease,in the interim where no vaccine is available.展开更多
Hepatitis C virus(HCV)causes chronic infection in over 75%of the HCV-infected individuals,resulting in liver cancer in substantial patients.Since its discovery in 1989,HCV experiences a journey from discovery to cure,...Hepatitis C virus(HCV)causes chronic infection in over 75%of the HCV-infected individuals,resulting in liver cancer in substantial patients.Since its discovery in 1989,HCV experiences a journey from discovery to cure,largely due to the virology studies and success of direct antiviral agent(DAA)development.We reviewed the HCV research journey,from the discovery of this virus to the development of DAAs for cure.Learning the methodology used in HCV studies and the knowledge of developing DAAs against HCV may inspire the studies of other difficult-to-culture viruses,such as hepatitis E virus and norovirus,as well as the development of DAAs for other single-stranded positive-sense RNA viruses,including the pandemic-causing SARS-CoV-2 virus,which shares the common replication strategy of forming a membrane-bound viral replicase.展开更多
基金funded by the National Key Research and Development Program of China(2021YFD1400400)to F.L.the National Natural Science Foundation of China(31930089 and 31972244)to X.Z.and F.L.+2 种基金a startup grant for High-level Talents of Fujian Medical University(XRCZX2019019)the Natural Science Foundation of Fujan Province,China(2020J01604)to Q.S.Work in the R.L.-D.lab is partially funded by the ERC-COG grant GemOmics(101044142)to R.L.-D.
文摘Positive-sense single-stranded RNA(+ssRNA)viruses,the most abundant viruses of eukaryotes in nature,require the synthesis of negative-sense RNA(-RNA)using their genomic(positive-sense)RNA(+RNA)as a template for replication.Based on current evidence,viral proteins are translated via viral+RNAs,whereas-RNA is considered to be a viral replication intermediate without coding capacity.Here,we report that plant and animal+ssRNA viruses contain small open reading frames(ORFs)in their-RNA(reverse ORFs[rORFs]).Using turnip mosaic virus(TuMV)as a model for plant+ssRNA viruses,we demonstrate that small proteins encoded by rORFs display specific subcellularlocalizations,and confirm the presence of rORF2 in infected cells through mass spectrometry analysis.The protein encoded by TuMV rORF2 forms punctuate granules that are localized in the perinuclear region and co-localized with viral replication complexes.The rORF2 protein can directly interact with the viral RNA-dependent RNA polymerase,and mutation of rORF2 completely abolishes virus infection,whereas ectopic expression of rORF2 rescues the mutant virus.Furthermore,we show that several rORFs in the-RNA of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)have the ability to suppress type l interferon production and facilitate the infection of ve-sicular stomatitis virus.In addition,we provide evidence that TuMV might utilize internal ribosome entry sites to translate these small rORFs.Taken together,these findings indicate that the-RNA of+ssRNA vi-ruses can also have the coding capacity and that small proteins encoded therein play critical roles in viral infection,revealing a viral proteome larger than previously thought.
基金Ministry of Education Tier 2 grant(MOE2017-T2-1-078 and MOE-2017-T2-2-014,Singapore)National Research Foundation Competitive Research Programme(NRF-CRP21-2018-0004,Singapore)。
文摘Positive-sense RNA viruses modify intracellular calcium stores,endoplasmic reticulum and Golgi apparatus(Golgi)to generate membranous replication organelles known as viral factories.Viral factories provide a conducive and substantial enclave for essential virus replication via concentrating necessary cellular factors and viral proteins in proximity.Here,we identified the vital role of a broadspectrum antiviral,peruvoside in limiting the formation of viral factories.Mechanistically,we revealed the pleiotropic cellular effect of Src and PLC kinase signaling via cyclin-dependent kinase 1 signaling leads to Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1(GBF1)phosphorylation and Golgi vesiculation by peruvoside treatment.The ramification of GBF1 phosphorylation fosters GBF1 deprivation consequentially activating downstream antiviral signaling by dampening viral factories formation.Further investigation showed signaling of ERK1/2 pathway via cyclin-dependent kinase 1 activation leading to GBF1 phosphorylation at Threonine 1337(T1337).We also showed 100%of protection in peruvoside-treated mouse model with a significant reduction in viral titre and without measurable cytotoxicity in serum.These findings highlight the importance of dissecting the broad-spectrum antiviral therapeutics mechanism and pave the way for consideration of peruvoside,host-directed antivirals for positive-sense RNA virus-mediated disease,in the interim where no vaccine is available.
基金This study is supported in part by the National Natural Science Foundation of China(No.81772181,No.81971926).
文摘Hepatitis C virus(HCV)causes chronic infection in over 75%of the HCV-infected individuals,resulting in liver cancer in substantial patients.Since its discovery in 1989,HCV experiences a journey from discovery to cure,largely due to the virology studies and success of direct antiviral agent(DAA)development.We reviewed the HCV research journey,from the discovery of this virus to the development of DAAs for cure.Learning the methodology used in HCV studies and the knowledge of developing DAAs against HCV may inspire the studies of other difficult-to-culture viruses,such as hepatitis E virus and norovirus,as well as the development of DAAs for other single-stranded positive-sense RNA viruses,including the pandemic-causing SARS-CoV-2 virus,which shares the common replication strategy of forming a membrane-bound viral replicase.
