Comparative Transcriptome Analyses Of PRRSV-infected And IFN-stimulated Porcine Alveolar Macrophages

Porcine reproductive and respiratory syndrome virus?PRRSV?is one of the most crucial viral infectious pathogens that severely harm the pig industry all around the world.It mainly infects porcine alveolar macrophage?PAMs?,which belong to monocyte/macrophage lineage,leading to aberrant biological function of PAMs.Here the transcriptome landscapes of PAMs in response to PRRSV were performed as to provide scientific basis for further investigating the effect of PRRSV on PAMs.In this study high-throughput sequencing technology was used to reveal the dynamic transcriptional profiling of PAMs in response to PRRSV and interferon?IFN?,and the differences between PAMs in response to PRRSV and IFN were compared at the transcriptional level.Meanwhile,the regulation mechanism of nsp1?nucleus accumulation was investigated as to reveal its effect on biological function of nsp1?and PRRSV replication,providing scientific evidences for aberrant immunological function of PAMs during PRRSV infection and new clue for illustrating immune evasion strategy of PRRSV.The transcriptome analyses of PAMs in response to PRRSV in vitro at different time points were performed using high throughput sequencing.Within sus scrofa reference genome,38222 annotated mRNAs,12987 annotated long noncoding RNAs?lncRNAs?and 17624 predicted novel IncRNAs were identified in PRRSV-infected PAMs.In comparion with mock-infected groups,the numbers of differentially expressed mRNAs,annotated IncRNAs and novel lncRNAs were 1220,241 and 439 in PAMs after 6 h post-infection respectively,the numbers of differentially expressed mRNAs,annotated lncRNAs and novel lncRNAs were 3903,974 and 2608 in PAMs after 9 h post-infection respectively,the numbers of differentially expressed mRNAs,annotated lncRNAs and novel IncRNAs were 10330,3534 and 7562 in PAMs after 12 h post-infection respectively.And differentially expressed mRNAs,annotated lncRNAs and novel lncRNAs with the same change tendency in PRRSV-infected PAMs were 868,132 and 241,respectively.The statistical significance of mRNAs?GBP1,HPDL,PRCP and CD163?,annotated IncRNAs?XR₃01539.1 and XR₂97549.1?and novel IncRNAs?TCONS₀0048171 and TCONS 00154605?were confirm using quantitative real-time quantitative PCR?qRT-PCR?.Using bioinformatics technology,GO term annotation and KEGG pathway enrichment analyses were performed focusing on these differentially expressed protein-coding genes in each PRRSV-infected group.The results showed that many genes involved in proinflammatory cytokines,chemokines and signaling pathways related to IFN,phagocytosis and antigen processing and presentation were significantly down-regulated,indicating aberrant immune function of PAMs PRRSV infection.Moreover,among the differentially and highly expressed annotated lncRNAs,IncRNA XR₂97549.1 was predicted to both cis-and trans-regulate its neighboring gene PTGS2,which is related to inflammatory response.Parallelly,the transcriptome analyses of IFN-stimulated PAMs at different time points were also performed using high throughput sequencing.In comparion with mock-infected groups,the numbers of differentially expressed mRNAs,annotated lncRNAs and novel lncRNAs were 3951,423 and 1318 in PAMs after 6 h post-stimulation respectively,and the numbers of differentially expressed mRNAs,annotated lncRNAs and novel lncRNAs were 9305,902 and 1975 in PAMs after 9 h post-stimulation respectively.And differentially expressed genes?mRNAs,annotated lncRNAs and novel lncRNAs?with the same change tendency in IFN-stimulated PAMs were 1801,311 and 693,respectively.The statistical significance of mRNAs?HPDL,DPYSL2,PRCP and NAAA?,annotated lncRNAs?XR₃01539.1 and XR 297549.1?and novel lncRNAs?TCONS₀0048171 and TCONS₀0154605?were confirm using quantitative real-time quantitative PCR?qRT-PCR?.Using bioinformatics technology,GO term annotation and KEGG pathway enrichment analyses were performed focusing on these differentially expressed protein-coding genes in each IFN-stimulatd group.The results showed that the over-represented biological process terms include immune system process and cell cycle.And these differentially expressed protein-coding genes were enriched in cytokine-cytokine receptor interaction in environmental information processing,influenza A in human diseases and chemokine signaling pathway in organismal systems.Through functional prediction of IFN-stimulated annotated lncRNAs,it showed that lncRNA XR₃07435.1 and XR₃04488.1 may cis-regulate the expression of OAS1 and PML genes respectively,both of which encode antiviral proteins;lncRNA₃08080.1 may cis-regulate the expression of IFIH1,which encodes an essential pattern recognition receptor;and lncRNA₂97412.1 may trans-regulate the expression of ISG15,which is also essential for antiviral response.Based on the transcriptome landscapes of PAMs in response to PRRSV and IFN,the differentiallyexpressed mRNAs and lncRNAs were compared between PRRSV-infected and IFN-stimulated groups.The results showed that 80 mRNAs,16 annotated lncRNAs and 34 novel lncRNAs were up-regulated in both groups;169 mRNAs,17 annotated IncRNAs and 42 novel lncRNAs were down-regulated in both groups;1 mRNA and no lncRNAs were up-regulated in PRRSV-infected groups but down-regulated in IFN-stimulated groups;10 mRNA,1 novel lncRNA and no annotated lncRNAs were down-regulated in PRRSV-infected groups but up-regulated in IFN-stimulated groups.Among them SIGLEC-1 gene,which encodes PRRSV receptor CD169;VCAM1,which encodes cell surface protein CD106;and SERPING1,which encodes C1 esterase inhibitor,were significantly up-regulated in IFN-stimulated groups but down-regulated in PRRSV-infected groups.Furthermore,as to analyze the different antiviral status of PAMs in response to PRRSV and IFN,the transcriptome landscapes were compared between PAMs after 9 h post-infection and after 6 h post-stimulation.The differentially expressed mRNAs,annotated IncRNAs and novel IncRNAs were 5549,626 and 1503,respectively,indicating the difference of antiviral status of PAMs at the transcriptional level.The significantly over-represented terms were characterized using GO term analyses,including molecular function terms:immune system process,regulation of immune system process and defense response;cellular component terms:cytoplasm,mitochondrion and organelle membrane;and biological process terms:small conjugating protein ligase binding,ubiquitin protein ligase binding and electron carrier activity.Using KEGG pathway enrichment analyses,it showed that the differentially expressed genes were enriched in herpes simplex infection,influenza A infection,tuberculosis,lysosome,phagosome,antigen processing and presentation and osteoclast differentiation.Among the top 40 protein-coding genes which are highly expressed and significantly upregulated in IFN-stimulated group,multiple genes were known to encode well-characterized antiviral proteins,including OASL,ISG15,GBP1,SAMHD1,IFITM1,IFITM3,IFIT1,IFIT3 and BST2,indicating the transcriptional differences of antiviral genes were an essential marker between normal and aberrant antiviral status of PAMs.Nsp1?is an essential protein in PRRSV replication and suppression of host innate immune response.So far the molecular mechanism of Nsp1?nuclear accumulation during PRRSV infection is still unknown.In this study,lysine?K?residues in PRRSV JXwn06 Nsp1? was mutated to arginine?R?respectively,and these mutants were further characterized using confocal microscopy and immunoprecipitation assay.Cleavage protein bands were detected in K124R mutant,and K200R mutation led to two phenotypes when compared with wild type,including enhanced post-translational modification bands and spotty distribution in both nucleus and cytoplasm.To futher exploring the molecular mechanism underlying K124R induced protein cleavge,site directed mutagenesis was performed on 123-GKYLQRRLQ-131 motif.With Western Blotting it was clear that only R124 led to Nsp1?cleavage.Interestingly,when K124 was mutated into tryptophan?W?or aspartic acid?D?,it showed enhanced posttranslational modification bands similar to those of K200Rmutant.However,only K200R mutant dispersed in both nucleus and cytoplasm and distributed in the cytoplasm.Moreover,K200R mutation significantly blocked the effect of nsp1? to inhibit the transcriptional activity of IFN-?.In order to illustrate the molecular mechanism underlying K200R induced change in nsp1? cellular distribution,K200 was mutated into different types of amino acids using site directed mutagenesis assay.With both Western Blotting and confocal microscopy,it is clear that lysine residue at the 200th is essential for maintaining normal phenotype of Nsp1?.Furthermore,using K200 as an entry point,scanning mutagenesis of the C terminal extension?CTE?domain of Nsp1?was performed and confocal microscopy and luciferase reporter assay were used to characterize the key amino acids involved in Nsp1? cellular distribution and antagonizing transcriptional activity of IFN-?.It was shown that F194,F196,K200,W201,Y202 and G203 affect Nsp1? cellular distribution to varying degrees,and each mutant significantly blocked the ability of Nsp1? to inhibit the transcriptional activity of IFN-?.Using sequencing alignment,a FxFxxxKWYG motif embeded in the end of CTE domain of PRRSV was identified.Confocal microscopy and luciferase reporter assay were used to investigate the effect of FxFxxxKWYG motif to Nsp1?cellular distribution and inhibit IFN-? transcriptional activity.It was shown that Nsp1? with FxFxxxKWYG motif deletion almost dispersed in the cytoplasm,and the ability of Nsp1? to inhibit IFN-? transcriptional activity was significantly suppressed.Infectious clone and TCID50 were used to explore the effect of key amino acids on PRRSV infection,the results showed that while the virus titer of RvJXNsp1?K124R was similar to that of RvJXwn at different time points in MARC-145 cells,the virus titer of RvJXNs1?K200R was significantly lower than that of RvJXwn at 24 and 36 h post-infection.Of note,the virus titer of RvJXNsp1?F194A was significantly lower than that of RvJXwn at each time point,indicating the importance of FxFxxxKWYG motif in Nsp1?nuclear accumulation and virus multiplication.Moreover,using confocal microscopy the dynamic distribution of Nsp1?of RvJXNsp1?K200R and RvJXNsp1?F194A in MARC-145 cells were characterized,the results showed that while both K200R and F194A mutants could translocate into the nuclear,the nuclear translocation process was significantly blocked in F194A mutant,leading to cytoplasmic distribution of Nsp1?F194A..In summary,differentially expressed genes in PAMs in response to either PRRSV or IFN were characterized through comparative transcriptional analyses,revealing the potential immune invasion mechanism of PRRSV at the transcriptional level and the molecular mechanism of Nsp1? nuclear accumulation.These findings help to provide scientific evidences for further investigation of immunological function of PAMs and immune evasion mechanism of PRRSV,also provide an insight in novel vaccine design and control of PRRS disease.