| Porcine reproductive and respiratory syndrome virus(PRRSV)is an important pathogen impacting the global swine industry.Nonstructural protein 9(nsp9)of PRRSV specifies the function of viral RNA-dependent RNA polymerase(RdRp),involving in the formation of viral replication and transcription complex(RTC).The nsp9 of PRRSV plays important roles in viral replication regulation and pathogenicity.To date,the biological functions of PRRSV nsp9 have not been elucidated fully.The present study focuses on the identification of the nsp9 constitution,the intracellular localization of nsp9 and its interaction with intracellular membranes,and the dissection of potential replication-nonessential sites for PRRSV in nsp9,in an attempt to provide valuable evidence for better understanding the biological functions of nsp9.Whether PRRSV nsp9 contains nsp8 in virus-infected cells is not determined yet.In order to confirm nsp8 serves the N-terminus of PRRSV nsp9,in this study,fused GST-nsp8 protein was expressed and purified,and used to prepare specific rabbit anti-nsp8 polyclonal antibody(pAb).Meanwhile,specific antibodies to nsp7,nsp9 and nsp10 were used to perform immunoprecipitation(IP)assay.The results show that PRRSV nsp9 is identified as a major product with a size of between 72-95 kDa,which exhibits the similar mobility on the gel to the in vitro expressed nsp8-9ORF1b,but not nsp9ORF1b.The antibody to nsp8,but not to nsp7 or nsp10,can detect and immunoprecipitate a major product with the similar mobility to the 72-95 kDa form of nsp9.It was further proved to contain nsp9ORF1b by mass spectrometry.Moreover,neither PLP2 nor nsp4 is able to cleave nsp8-9ORF1b in vitro.Together,our findings suggest that nsp8 is an N-terminal extension of nsp9.The interaction between nsp9 and intracellular membranes was then explored.The secondary structure of PRRSV nsp9 was predicted by Globplot,and then confocal microscopy revealed that the nsp9 of PRRSV shares three regions exhibiting feature of membrane-association structure,namely aal-139,aa237-333 and aa494-685,and the former two can partially associate with cellular mitochondria.Consistently,fractionation analysis indicate that most of the isolated crude nsp9 locate in crude mitochondrial fraction.What is more,neither high salt nor high pH solution exaction lead to the extraction of nsp9 from the mitochondria of PRRSV-infected cells,suggesting that the nsp9 of PRRSV can be integrated into the mitochondrial membrane.Furthermore,nsp2 and nsp10 are also shown to be mainly present in crude mitochondrial fraction.The crude mitochondrial fraction of PRRSV-infected MARC-145 cells was fractionated by 30%Percoll medium and then subjected to Western blot.The results show that nsp9,nsp2 and nsp10 are co-fractionated with mitochondrial proteins.However,nsp9 cannot co-localize with cellular mitochondria in PRRSV-infected MARC-145 cells by confocal microscopy.Finally,siRNA was used to demonstrate that the knockdown of the expression of MFN2,a mitochondrial outer membrane protein,can significantly inhibit PRRSV replication.Collectively,our results suggest that the PRRSV RTC with nsp9 as its core component is tightly associated with mitochondrial membrane.The potential nonessential sites in nsp9 for the replication of PRRSV were analyzed in this study.Sixteen representative strains of PRRSV 2(type 2)were chosen to analyze the amino acid similarity of nsp9.The results show that the nsp9 proteins of the PRRSV strains exhibit 94.9%～99.7%homology and only the nsp9 of PRRSV HB-1/3.9 has a deletion of G109 compared with other strains.In order to analyze the effect of G109 of nsp9 on the viral replication,RvHBn9+109G(insertion)and RvJXn9△G109(deletion)were constructed and rescued in MARC-145 cells using full-length cDNA clones pWSK-HB-1/3.9 and pWSK-JXwn respectively.The growth kinetics of the two cloned viruses were examined and compared.The results show that RvHBn9+109G exhibits similar replication efficiency to RvHB-1/3.9 in MARC-145 cells.However,RvJXn9△G109 shows higher titers in MARC-145 cells than RvJXwn,with significant differences at 12 hpi(36.67-fold,p<0.001),24 hpi(17.02-fold,p<0.01)and 36 hpi(8.38-fold,p<0.5),respectively.Meanwhile,RvJXn9△G109 displays higher titers in pulmonary alveolar macrophages(PAMs)than RvJXwn,with significant differences at 12 hpi(17.52-fold,p<0.001)and 24 hpi(14.68-fold,p<0.001),respectively.Furthermore,pWSK-JXwn was used to construct a serial of full-length clones with corresponding amino acid sites deletion at the N-terminal/C-terminal of nsp9 G109,linker of N-terminal and C-terminal domains of nsp9,or C-terminal or N-terminal of nsp9,but no virus was rescued.Our data reveal that the G109 of nsp9 is a nonessential site for PRRSV replication,and its deletion has no impact on the replication of PRRSV.In summary,our findings indicate that:(1)the nsp8 of PRRSV serves as the N-terminal portion of nsp9;(2)the nsp9 of PRRSV associates with mitochondrial membrane in the infected cells;(3)the G190 of nsp9 is a replication-nonessential site for PRRSV.Our present study provide evidence for understanding biological functions of PRRSV nsp9,and a basis for further exploring the molecular mechanism in relation to the regulation of PRRSV’s replication and pathogenicity. |
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