Analysis of the function of hPIV2 V protein by using reverse genetics.

1.人副流感病毒2型(HPIV2)V蛋白通过降解STAT蛋白抑制干扰素诱导的先天抗病毒反应。我鉴定了在人类细胞中降解STAT2蛋白所必需的hPIV2 V蛋白残基，它们是C-末端V-唯一结构域的7个半胱氨酸残基、富含色氨酸的基序和AA207个苯丙氨酸，以及P/V公共结构域的AA143个苯丙氨酸。2.为了证实V蛋白对病毒生长的功能，我通过灭活P基因mRNA编辑信号(rPIV2/P-EDIT)使V蛋白完全缺乏表达的重组病毒。即使在不能诱导干扰素的Vero细胞中，rPIV2/P-EDIT的生长速度也非常有限。因此，这些结果表明，V蛋白不仅对STAT蛋白的降解至关重要，而且对促进病毒生长也是必不可少的。人副流感病毒4型(HPIV4)V蛋白是否具有逃避干扰素诱导的抗病毒反应的功能。HPIV4V蛋白具有7个保守的半胱氨酸残基和富含色氨酸的基序，并能与STAT蛋白降解重要的DDB1和Cul4A结合。然而，hPIV4V蛋白没有抑制干扰素信号转导的功能。我证明了唯一不能逃避干扰素诱导的抗病毒反应的副粘病毒是hPIV4.4。我发现hPIV2的V-特异区与NP蛋白的N-末端80个氨基酸结合，对自我结合是关键的。此外，我还鉴定了一种宿主蛋白AIP1/Alix，它参与了几种被包裹的病毒的凋亡和有效萌发，是V蛋白和NP蛋白的相互作用伙伴。我的数据还表明，V和AIP1之间的瞬时结合对病毒的生长很重要。

1.The V protein of human parainfluenza virus type 2 (hPIV2) inhibits interferon (IFN) -induced innate antiviral responses through STAT protein degradation. I identified hPIV2 V protein residues essential for STAT2 protein degradation in human cells, that are conserved seven cysteine residues, tryptophan-rich-motif, and aa 207 phenylalanine in the C-terminal V-unique domain, and aa 143 phenylalanine in the P/V common domain.2.To conform the function for virus growth of the V protein, I made the recombinant virus which completely lacks expression of the V protein by inactivating the P gene mRNA-editing signal (rPIV2/P-edit). The growth rate of rPIV2/P-edit is very limited even in the Vero cells that cannot induce IFN. Thus, these results suggest that the V protein is essential not only for STAT protein degradation but also for promoting virus growth. The rhPIV2s which have the mutation in the V-specific domain also grew lower, but rPIV2 which has the mutation in the P/V common domain grew similar to wt.3.I investigated whether the V protein of human parainfluenza virus type 4 (hPIV4) has the function to evade the IFN-induced antiviral responses. The hPIV4 V protein has the conserved seven cysteine residues and tryptophan-rich-motif, and can bind to DDB1 and Cul4A that are important for STAT protein degradation. However, the hPIV4 V protein has no function to inhibit IFN signaling. I show that the only paramyxovirus that can't evade the IFN-induced antiviral responses to data is hPIV4.4.I identified that the V-specific region of hPIV2 binds to the N-terminal 80 amino acids on the NP protein, and is critical for self-association. Furthermore, I have identified a host protein, AIP1/Alix, involved in apoptosis and efficient budding of several enveloped viruses, as an interacting partner of the V and NP proteins. My data also suggest that the transiently binding between V and AIP1 is important for virus growth.