Ebola virus(EBOV)is an enveloped negative-sense RNA virus and a member of the filovirus family.Nucleoprotein(NP)expression alone leads to the formation of inclusion bodies(IBs),which are critical for viral RNA synthes...Ebola virus(EBOV)is an enveloped negative-sense RNA virus and a member of the filovirus family.Nucleoprotein(NP)expression alone leads to the formation of inclusion bodies(IBs),which are critical for viral RNA synthesis.The matrix protein,VP40,not only plays a critical role in virus assembly/budding,but also can regulate transcription and replication of the viral genome.However,the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown.Here,we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release.Furthermore,we find four point mutations(L692A,P697A,P698A and W699A)within the C-terminal hydrophobic core of NP result in a stronger VP40–NP interaction within IBs,sequestering VP40 within IBs,reducing VP40–VLP egress,abolishing the incorporation of NC-like structures into VP40–VLP,and inhibiting viral RNA synthesis,suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP.Consequently,the C-terminal hydrophobic core of NP is exposed and binds VP40,thereby inhibiting RNA synthesis and initiating virion assembly/budding.展开更多
Inclusion bodies(IBs)of respiratory syncytial virus(RSV)are formed by liquid-liquid phase separation(LLPS)and contain internal structures termed“IB-associated granules”(IBAGs),where anti-termination factor M2-1 and ...Inclusion bodies(IBs)of respiratory syncytial virus(RSV)are formed by liquid-liquid phase separation(LLPS)and contain internal structures termed“IB-associated granules”(IBAGs),where anti-termination factor M2-1 and viral mRNAs are concentrated.However,the mechanism of IBAG formation and the physiological function of IBAGs are unclear.Here,we found that the internal structures of RSV IBs are actual M2-1-free viral messenger ribonucleoprotein(mRNP)condensates formed by secondary LLPS.Mechanistically,the RSV nucleoprotein(N)and M2-1 interact with and recruit PABP to IBs,promoting PABP to bind viral mRNAs transcribed in IBs by RNArecognition motif and drive secondary phase separation.Furthermore,PABP-eIF4G1 interaction regulates viral mRNP condensate composition,thereby recruiting specific translation initiation factors(eIF4G1,eIF4E,eIF4A,eIF4B and eIF4H)into the secondary condensed phase to activate viral mRNAs for ribosomal recruitment.Our study proposes a novel LLPS-regulated translation mechanism during viral infection and a novel antiviral strategy via targeting on secondary condensed phase.展开更多
基金We acknowledge Dr.Bo Zhang(Wuhan Institute of Virology)and Dr.Heinz Feldmann(National Institutes of Health,Hamilton,Montana,USA)for providing the Ebola cDNA and minigenome assay system,respectivelyThis research is supported by grants from the National Natural Science Foundation of China(81825015)+4 种基金National Key R&D Program of China(2017YFA0505801)National Science and Technology Major Project(2018ZX10101004)National Natural Science Foundation of China(81871650 and 31630086)The Natural Science Foundation of Hubei Province Innovation Group(2017CFA022)Advanced Customer Cultivation Project of Wuhan National Biosafety Laboratory(2019ACCP-MS06).
文摘Ebola virus(EBOV)is an enveloped negative-sense RNA virus and a member of the filovirus family.Nucleoprotein(NP)expression alone leads to the formation of inclusion bodies(IBs),which are critical for viral RNA synthesis.The matrix protein,VP40,not only plays a critical role in virus assembly/budding,but also can regulate transcription and replication of the viral genome.However,the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown.Here,we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release.Furthermore,we find four point mutations(L692A,P697A,P698A and W699A)within the C-terminal hydrophobic core of NP result in a stronger VP40–NP interaction within IBs,sequestering VP40 within IBs,reducing VP40–VLP egress,abolishing the incorporation of NC-like structures into VP40–VLP,and inhibiting viral RNA synthesis,suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP.Consequently,the C-terminal hydrophobic core of NP is exposed and binds VP40,thereby inhibiting RNA synthesis and initiating virion assembly/budding.
基金supported by the grants from National Key R&D Program of China(2021YFC2300702 and 2021YFC2300200)the Hubei Provincial Natural Science Foundation of China(2021CFB364)+1 种基金the National Natural Science Foundation of China(82130064,81825015,U22A20337 and 32000119)the Key Biosafety Science and Technology Program of Hubei Jiangxia Laboratory(JXBS001).
文摘Inclusion bodies(IBs)of respiratory syncytial virus(RSV)are formed by liquid-liquid phase separation(LLPS)and contain internal structures termed“IB-associated granules”(IBAGs),where anti-termination factor M2-1 and viral mRNAs are concentrated.However,the mechanism of IBAG formation and the physiological function of IBAGs are unclear.Here,we found that the internal structures of RSV IBs are actual M2-1-free viral messenger ribonucleoprotein(mRNP)condensates formed by secondary LLPS.Mechanistically,the RSV nucleoprotein(N)and M2-1 interact with and recruit PABP to IBs,promoting PABP to bind viral mRNAs transcribed in IBs by RNArecognition motif and drive secondary phase separation.Furthermore,PABP-eIF4G1 interaction regulates viral mRNP condensate composition,thereby recruiting specific translation initiation factors(eIF4G1,eIF4E,eIF4A,eIF4B and eIF4H)into the secondary condensed phase to activate viral mRNAs for ribosomal recruitment.Our study proposes a novel LLPS-regulated translation mechanism during viral infection and a novel antiviral strategy via targeting on secondary condensed phase.