Autophagy plays an important role in the interaction between viruses and host cells.SARS-CoV-2 infection can disrupt the autophagy process in target cells.However,the precise molecular mechanism is still unknown.In th...Autophagy plays an important role in the interaction between viruses and host cells.SARS-CoV-2 infection can disrupt the autophagy process in target cells.However,the precise molecular mechanism is still unknown.In this study,we discovered that the Nsp8 of SARS-CoV-2 could cause an increasing accumulation of autophagosomes by preventing the fusion of autophagosomes and lysosomes.From further investigation,we found that Nsp8 was present on mitochondria and can damage mitochondria to initiate mitophagy.The results of experiments with immunofluorescence revealed that Nsp8 induced incomplete mitophagy.Moreover,both domains of Nsp8 orchestrated their function during Nsp8-induced mitophagy,in which the N-terminal domain colocalized with mitochondria and the C-terminal domain induced auto/mitophagy.This novel finding expands our understanding of the function of Nsp8 in promoting mitochondrial damage and inducing incomplete mitophagy,which helps us to understand the etiology of COVID-19 as well as open up new pathways for creating SARS-CoV-2 treatment methods.展开更多
During severe acute respiratory syndrome coronavirus(SARS-CoV)infection,the activity of the replication/transcription complexes(RTC)quickly peaks at 6 hours post infection(h.p.i)and then diminishes significantly in th...During severe acute respiratory syndrome coronavirus(SARS-CoV)infection,the activity of the replication/transcription complexes(RTC)quickly peaks at 6 hours post infection(h.p.i)and then diminishes significantly in the late post-infection stages.This“down-up-down”regulation of RNA synthesis distinguishes different viral stages:primary translation,genome replication,and finally viron assembly.Regarding the nsp8 as the primase in RNA synthesis,we confirmed that the proteolysis product of the primase(nsp8)contains the globular domain(nsp8C),and indentified the resectioning site that is notably conserved in all the three groups of coronavirus.We subsequently crystallized the complex of SARS-CoV nsp8C and nsp7,and the 3-D structure of this domain revealed its capability to interfuse into the hexadecamer super-complex.This specific proteolysis may indicate one possible mechanism by which coronaviruses to switch from viral infection to genome replication and viral assembly stages.展开更多
Porcine reproductive and respiratory syndrome virus (PRRSV) is a member within the family Arteriviridae of the order Nidovirales. Replication of this positive-stranded RNA virus within the host cell involves expressio...Porcine reproductive and respiratory syndrome virus (PRRSV) is a member within the family Arteriviridae of the order Nidovirales. Replication of this positive-stranded RNA virus within the host cell involves expression of viral replicase proteins encoded by two ORFs, namely ORF1 a and ORF1 b. In particular, translation of ORF1 b depends on a-1-ribosomal frameshift strategy. Thus, nonstructural protein 9 (nsp9), the first protein within ORF1 b that specifies the function of the viral RNA-dependent RNA polymerase, is expressed as the C-terminal extension of nsp8, a small nsp that is encoded by ORF1 a. However, it has remained unclear whether the mature form of nsp9 in virus-infected cells still retains nsp8,addressing which is clearly critical to understand the biological function of nsp9. By taking advantage of specific antibodies to both nsp8 and nsp9, we report the following findings. (1) In infected cells, PRRSV nsp9 was identified as a major product with a size between 72 and 95 k Da (72–95 KDa form), which exhibited the similar mobility on the gel to the in vitro expressed nsp8–9 ORF1 b, but not the ORF1 b-coded portion (nsp9 ORF1 b). (2) The antibodies to nsp8, but not to nsp7 or nsp10, could detect a major product that had the similar mobility to the 72–95 KDa form of nsp9. Moreover, nsp9 could be co-immunoprecipitated by antibodies to nsp8, and vice versa. (3) Neither nsp4 nor nsp2 PLP2 was able to cleave nsp8–nsp9 in vitro. Together, our studies provide experimental evidence to suggest that nsp8 is an N-terminal extension of nsp9.Our findings here paves way for further charactering the biological function of PRRSV nsp9.展开更多
基金supported by the National Natural Science Foundation of China (grant numbers 32100131 and 31670716)Wuhan Science and Technology Bureau[grant numbers 2020020601012318]+1 种基金Hubei Key Laboratory of Wudang Local Chinese Medicine Research (Hubei University of Medicine) (grant numbers WDCM2022008)Jianghan University (grant numbers 08190006,06210035,2021yb138 and 2019037).
文摘Autophagy plays an important role in the interaction between viruses and host cells.SARS-CoV-2 infection can disrupt the autophagy process in target cells.However,the precise molecular mechanism is still unknown.In this study,we discovered that the Nsp8 of SARS-CoV-2 could cause an increasing accumulation of autophagosomes by preventing the fusion of autophagosomes and lysosomes.From further investigation,we found that Nsp8 was present on mitochondria and can damage mitochondria to initiate mitophagy.The results of experiments with immunofluorescence revealed that Nsp8 induced incomplete mitophagy.Moreover,both domains of Nsp8 orchestrated their function during Nsp8-induced mitophagy,in which the N-terminal domain colocalized with mitochondria and the C-terminal domain induced auto/mitophagy.This novel finding expands our understanding of the function of Nsp8 in promoting mitochondrial damage and inducing incomplete mitophagy,which helps us to understand the etiology of COVID-19 as well as open up new pathways for creating SARS-CoV-2 treatment methods.
基金This work was supported by Project 973 of the Ministry of Science and Technology of China(Nos.2006CB806503,2007CB914301)the National Natural Science Foundation of China(Grant Nos.30221003,30730022).
文摘During severe acute respiratory syndrome coronavirus(SARS-CoV)infection,the activity of the replication/transcription complexes(RTC)quickly peaks at 6 hours post infection(h.p.i)and then diminishes significantly in the late post-infection stages.This“down-up-down”regulation of RNA synthesis distinguishes different viral stages:primary translation,genome replication,and finally viron assembly.Regarding the nsp8 as the primase in RNA synthesis,we confirmed that the proteolysis product of the primase(nsp8)contains the globular domain(nsp8C),and indentified the resectioning site that is notably conserved in all the three groups of coronavirus.We subsequently crystallized the complex of SARS-CoV nsp8C and nsp7,and the 3-D structure of this domain revealed its capability to interfuse into the hexadecamer super-complex.This specific proteolysis may indicate one possible mechanism by which coronaviruses to switch from viral infection to genome replication and viral assembly stages.
基金supported by the National Key Basic Research Plan Grant from the Chinese Ministry of Science and Technology(2014CB542700)the China National Thousand Youth Talents program(1051-21986001)the earmarked fund for China Agriculture Research System(CARS-35)from the Chinese Ministry of Agriculture
文摘Porcine reproductive and respiratory syndrome virus (PRRSV) is a member within the family Arteriviridae of the order Nidovirales. Replication of this positive-stranded RNA virus within the host cell involves expression of viral replicase proteins encoded by two ORFs, namely ORF1 a and ORF1 b. In particular, translation of ORF1 b depends on a-1-ribosomal frameshift strategy. Thus, nonstructural protein 9 (nsp9), the first protein within ORF1 b that specifies the function of the viral RNA-dependent RNA polymerase, is expressed as the C-terminal extension of nsp8, a small nsp that is encoded by ORF1 a. However, it has remained unclear whether the mature form of nsp9 in virus-infected cells still retains nsp8,addressing which is clearly critical to understand the biological function of nsp9. By taking advantage of specific antibodies to both nsp8 and nsp9, we report the following findings. (1) In infected cells, PRRSV nsp9 was identified as a major product with a size between 72 and 95 k Da (72–95 KDa form), which exhibited the similar mobility on the gel to the in vitro expressed nsp8–9 ORF1 b, but not the ORF1 b-coded portion (nsp9 ORF1 b). (2) The antibodies to nsp8, but not to nsp7 or nsp10, could detect a major product that had the similar mobility to the 72–95 KDa form of nsp9. Moreover, nsp9 could be co-immunoprecipitated by antibodies to nsp8, and vice versa. (3) Neither nsp4 nor nsp2 PLP2 was able to cleave nsp8–nsp9 in vitro. Together, our studies provide experimental evidence to suggest that nsp8 is an N-terminal extension of nsp9.Our findings here paves way for further charactering the biological function of PRRSV nsp9.
