Exploration On Mechanisms Of Inhibition Of IFN-β Expression By Porcine Reproductive And Respirtory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is an economically dev-astating viral disease affecting the swine industry worldwide. The etiological agent of PRRS is porcine reproductive and respiratory syndrome virus (PRRSV), a member of Arteriviridae of Nidovirales. The genome of PRRSV is a positive single strand RNA consisting of more than ten ORFs:ORF1a, ORF1b, ORF2a, ORF2b, ORF3-7and ORF5a. ORF1encodes all14known nonstructural proteins (Nsps), including Nspla, Nsp1β, Nsp2-6, Nsp7a, Nsp7β and Nsp8-12. Nsp2is a multi-functional protein com-posed of regions functioning as cysteine proteinase, hypervariable region, and trans-membrane domain. It is involved in regulation of host innate immunity and pathogen-esis. This study was attempted to examine the genetic diversity of PRRSV isolates in Zhejiang, localization of Nsp2in infected cells and the possible pathways of Nsp2in affecting IFN-β expression.PRRSV isolates from Zhejiang and Shanghai regions were used for analysis of the NSP2, GP5and N genes. The results indicate that most strains belong to the North American genotype and were diverging from the earliest Chinese strain CHI a. Se-quence analysis of Nsp2suggests that twenty-two PRRSV strains have30discontin-uous amino acids deletion (38.6%,22/57). Sequence analysis of GP5show that de-coying epitope at position29, glycosylation site at position34, extracellular domain at position58and59had substitutions, indicating high variation of GP5gene during evolution. Prevalent strains in the region were divided into three subgroups (subtype I is European type, subtype II, III are North American type) according to the phyloge-netic tree of N gene. The results show that American type is the domain type in Zhejiang region with extensive genetic variation of Nsp2, GP5and N.Eukaryotic expression of Nsp2in Marc-145or HEK293cells was seen in the cy-toplasm, and Nsp2protein was seen as two individual bands. In PRRSV infected Marc-145cells, Nsp2could be detected at4hours post-infection (hpi) and mainly lo-cated around the nucleus, and then distributed to the whole cytoplasm. Putative Nsp2 protein bands of varying sized were seen at different time points post-infection,120kDa at8hpi,70or50kDa at12hpi, and the amount increased significantly as time extended. These data indicate that Nsp2is produced early in viral replication, but the roles of different subtypes of Nsp2in PRRSV pathogenesis remain to be elucidat-ed.The RNA-associate protein LSm14A has been found as anti-viral factors via IRN-β signaling. Since PRRSV have strategies to escape host innate immunity by an-tagonizing IFN-β expression. To see if porcine LSm14A (pLSm14A) is involved in PRRSV-mediated immune evasion strategy. Western-blot, immunofluorescence and luciferase reporter assay were used to examine the expression and function of pLSm14A. Porcine LSm14A transcript was detected in all examined tissues of Land-race pig by semi-quantitative RT-PCR. Overexpression of pLSm14A in HEK293and Marc-145cells could significantly activate the promoter activity of NF-κB and IFN-β, and the activation of IFN-β promoter is in a dose-dependent manner under the syn-ergistic effect of poly(I:C). The activity of IFN-β promoter induced by LSm14A was significantly inhibited by PRRSV infection. These data suggest that PRRSV could escape the innate immune activity induced by LSm14A.A cell line stabling expressing Nsp2and an eukaryotic plasmid expressing MAVS were constructed. Our results show that MAVS could significantly activate the pro-moter of IFN-β and its transcription. The viral Nsp2inhibited the activity of poly (I:C)-, MAVS-and LSm14A-induced activation of IFN-P promoter. Indirect immuno-fluorescence assay revealed that Nsp2did not inhibit translocation of IRF3or NF-κB into the cell nuclei, although co-localization of Nsp2with MAVS and LSm14A was seen. These findings might suggest that the mechanisms of PRRSV evasion from in-nate immunity could occur in the nuclei.In conclusion, pLSm14A is functional in activating IFN-β expression. Infection of Marc-145cells could inhibit poly(I:C) and pLSm14A-induced IFN-β promoter ac-tivity and expression. Because Nsp2in infected cells distributes throughout the cyto-plasm, colocalize with MAVS or pLSm14A. but does not inhibit translocation of IRF3and NF-κB, we propose that the events that PRRSV interfere with type I IFN expres- sion could be within the nuclei, most probably by binding the antagonizing the activi-ty of IFN-β transcription complex. The study lays a foundation to decipher the mech-anism of how PRRSV avoid host innate immune responses.