Non-structural protein (nsp) 1 of PRRS virus is a viral antagonist for type I interferons (IFNs), and in cells expressing nsp1, CREB-binding protein (CBP) is degraded. nsp1 is auto-processed into nsp1α and nsp1β subunits and in the present study we show that the nsp1α subunit was responsible for CBP degradation. The nsp1α subunit contains three distinct functional motifs; a papain-like cysteine protease α (PCPα) motif, an N-terminal zinc finger motif (ZF1), and a newly reported C-terminal zinc finger motif (ZF2). To study the structure function of nsp1α and its IFN antagonism, these motifs were individually mutated and the mutants were examined for their IFN suppression ability. The mutations that destroyed the PCPα activities (C76S, H146Y, and C76S/H146Y) did not affect the IFN suppressive activity of nsp1α, indicating that the cysteine protease activity did not participate in IFN suppression. The mutations of C70S, C76S, H146Y, and/or M180I which coordinated the ZF2 motif also did not alter IFN suppression. However, the mutations of C8S, C10S, C25S, and/or C28S for the ZF1 motif impaired the IFN antagonism of nsp1α, demonstrating that ZF1 was the essential element of nsp1α for IFN suppression. Wild-type nsp1α localized in the both nucleus and cytoplasm, but the ZF1 mutants that lost the IFN suppressive activity did not localize in the nucleus and remained in the cytoplasm. Consistent with their cytoplasmic distribution, CBP was not degraded by these mutants. Our results indicate that the ZF1 motif of nsp1α plays an important role for IFN regulation and further demonstrate that the CBP degradation is likely the key mechanism for IFN suppression mediated by the nsp1α subunit protein of PRRS virus.

译文

PRRS病毒的非结构蛋白 (nsp) 1是I型干扰素 (ifn) 的病毒拮抗剂,在表达nsp1的细胞中,CREB结合蛋白 (CBP) 被降解。nsp1被自动加工成nsp1α 和nsp1β 亚基,在本研究中,我们表明nsp1α 亚基是CBP降解的原因。nsp1α 亚基包含三个不同的功能基序; 木瓜蛋白酶样半胱氨酸蛋白酶 α (pcp α) 基序,N端锌指基序 (ZF1) 和新报道的C端锌指基序 (ZF2)。为了研究nsp1α 的结构功能及其IFN拮抗作用,对这些基序进行了单独突变,并检查了突变体的IFN抑制能力。破坏pcp α 活性的突变 (C76S,H146Y和C76S/H146Y) 不影响nsp1α 的IFN抑制活性,表明半胱氨酸蛋白酶活性不参与IFN抑制。协调ZF2基序的C70S,C76S,H146Y和/或M180I的突变也不会改变IFN抑制。然而,ZF1基序的C8S,C10S,C25S和/或C28S突变损害了nsp1α 的IFN拮抗作用,表明ZF1是nsp1α 抑制IFN的基本要素。野生型nsp1α 位于细胞核和细胞质中,但是失去IFN抑制活性的ZF1突变体未定位在细胞核中,而保留在细胞质中。与它们的细胞质分布一致,这些突变体不会降解CBP。我们的结果表明,nsp1α 的ZF1基序在IFN调节中起着重要作用,并进一步证明CBP降解可能是PRRS病毒nsp1α 亚基蛋白介导的IFN抑制的关键机制。

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