Effect Of Respiratory Syncytial Virus Nonstructural Proteins And Persistent Infection On The Suppressor Of Cytokine Signaling1/3(SOCS1/3)

Objective To reconstitute RSV nonstructural protein (NS) expression plasmids (pNS1, pNS2and pNSl/2). To investigate the effect of NSl and NS2on SOCS1and SOCS3expression.Methods Recombinant plasmids for RSV NSl, NS2and co-expressed NSl and NS2(NS1/2) expression plasmids were designed according to the original NS1and NS2open reading frames (ORFs) of the wild type RSV A2strain. The plasmids sequences were identified using sequencing, enzyme digestion and the series concentrations of plasmids were transfected into A549cells to detect target genes expression by using Western blot analysis; we performed qPCR and western blot to analyze SOCS1and SOCS3mRNA levels and the protein expression; Examined the mRNA level of TLR3and RIG-I by using qPCR and to test SOCS protein expression after silencing the target genes of TLR3or RIG-I. The STATs phosphorylation were analyzed by using Western blots; mRNA expression from IFN-a dependent genes2,5-OASl and MxA were measured by qPCR; Western blots were performed to analyze the nuclear accumulation of NF-?B p65; ELISA was performed to measure the concentration of Rantes, Mip-a and IL-6in the supernatants of pNS1, pNS2.Results The expression of NS1-flag, NS2-HA, as well as the co-expression of NSl-flag and NS2-HA, could be detected in the concentration dependent manner. RSV NS1and NS2are both key molecules in the induction of SOCS1expression, while NS1is mainly responsible for SOCS3transcription and proteins expression in the early phase. Both SOCS1and SOCS3were induced during the first hour of NS1expression, while NS2has no effect on SOCS3expression but was able to enhance its level induced by NS1. pNS1and pNS2induced SOCS1and SOCS3mRNA earlier than RIG-I or TLR3mRNA were induced and there was no change of SOCS1, SOCS3proteins expression when RIG-I or TLR3were inhibited. Both NS1and NS2suppressed IFN-a inducible STAT2phosphorylation by8h and completely by24h, compared to NS1, NS2demonstrated a stronger and earlier suppressive effect. Importantly, combined expression proteins NS1and NS2led to an even stronger decreased effect; The expression of either NS1or NS2inhibited MxA and2,5-OAS1mRNA transcription to near background levels. NF-kB p65decreased dramatically in the case of pNS1, pNS2transfection at2h post transfection and NS2had a gradual inhibitory effect on NF-?B p65accumulation.When NS1and NS2were co-expressed, the nuclear transposition of NF-?B p65during the first4h after plasmid transfection was almost eliminated. NS1and NS2obviously decrease the levels of Mip-a, Rantes and IL-624h after transfection.Conclusion Our results indicate that NS1and NS2, which induce the expression of SOCS1and SOCS3, are independent of the endogenous IFN signaling pathway to suppressing innate antiviral response. Objective To establish epithelial cells that were persistently infected with RSV and to investigate the putative role of SOCS in the regulation of the antiviral response and inflammatory status.Methods The surviving HEp-2cells were isolated using clone cylinders for collection after RSV infection, and dilution selection were used for clonal RSV persistence, the RSV N gene was detected through Real-time PCR and the viral antigen was detected using an immunofluorescence assay. To analyze RSV propagation and virulence during persistence, the tenth generation of clones with or without RSV was selected for testing. The supernatants of the cultures of clones with or without RSV persistence were collected for plaque assays at various times. The expression of IFN-? and SOCS1, SOCS3in the persistent infected cells were detected by using ELISA and Western blots respectively. RIG-I or TLR3mRNA expression were detected by using Real-time PCR in the persistently infected cells and siRNAs against RIG-I and TLR3at a final concentration of20nmol/ml were used to analyze SOCS1protein expression. SOCS1was silenced using siRNA at a final concentration of20nmol/ml for24h, and the STAT1/2and pSTAT1/2expression were detected by using western blot.Results The Each generation of clone with RSV-persistent cells showed that half of the cells were RSV antigen-positive and the other half was RSV antigen-negative. The RSV-positive persistent cells grew slowly and formed syncytia. The N gene copies of each clone ranged from2.5x102to3.5x105copies/ml and the viral antigen was detected using an immunofluorescence assay. The viral persistent cells produced a low viral titer ranged from1x103to1.4x104plaque forming units (pfu)/ml, resisted wild-type RSV superinfection, and secreted high levels of IFN-?, Mip-a, IL-8and Rantes(p<0.05). Both innate immune response signals, such as TLR3and RIG-I, and negative regulatory molecules SOCS1were found to be upregulated(p<0.05). The silence of TLR3mRNA decreased the expression of SOCS1and the secretion of cytokines(p<0.05). The silence of SOCS1didn't change the expression of cytokines(p>0.05), but increased the phosphorylation of STAT1and STAT2(p<0.05).Conclusion These results demonstrate that RSV persistent cells are in an inflammatory state and that the upregulation of SOCSl is related to the TLR3induced signaling pathway, modulates the level of STAT1/2phosphorylation, and is associated with viral persistence.