Study of uncoating program of nuclear replicating virus

病毒进入宿主细胞后，衣壳的解体和随后的病毒基因组的释放是SV40感染的必要步骤，尽管其机制尚不清楚。当病毒颗粒缺乏次要的衣壳蛋白Vp2和Vp3(统称为Vp2/3)时，通常由细胞提示诱导的病毒分解没有得到适当的调控：缺乏Vp2/3的颗粒在进入细胞时过早地释放病毒DNA(Nakanishi等人)。J·维罗尔。2007)。为了研究Vp1和Vp2/3相互作用在病毒分解调控中的作用，在Vp1和Vp2/3结合的残基上引入突变，并分析它们对病毒感染周期的影响。这些突变的目的是(1)改变VP1与Vp2/3、VP1 K116R与VP3 Q174D之间的离子相互作用界面，以及(2)在VP1与Vp2/3、VP1 V230C-VP3 E159C和VP1 E257C-VP3 Q174C之间形成二硫键。另外(3)结合三个不同温度…突变体VP3P103S-M110I-Q113K等更多的性征敏感突变(TDS)也被包括在分析中。突变的病毒DNA在转染后，衣壳蛋白表达和病毒DNA复制正常进行，所有突变株形成的颗粒组成与野生型病毒粒子相似。然而，除VP1K116R外，突变体在37℃或限制温度(VP3 P103S-M110I-Q113K)下形成斑块的能力严重缺陷。突变体颗粒明显能够进入细胞，与Importinα/β异二聚体相互作用，但在启动病毒早期基因表达方面存在缺陷或延迟，表明突变颗粒的缺陷存在于与病毒颗粒的核输入机制相互作用之后。这些结果表明，病毒衣壳的主要构象变化包括Vp1-Vp2/3相互作用的移动，发生在细胞进入过程的后期，包括核后进入事件。较少

Upon viral entry into the host cell disassembly of the capsid and subsequent release of the viral genome is the essential steps for SV40 infection, though mechanism underlies of which is poorly defined. The viral disassembly, normally induced by cellular cue, was not properly regulated when the viral particles lack the minor capsid proteins, Vp2 and Vp3 (collectively Vp2/3) : the Vp2/3-less particles prematurely released the viral DNA upon cell entry (Nakanishi et al. J.Virol. 2007). In order to examine the contribution of Vp1 and Vp2/3 interaction on the regulation of viral disassembly, mutations were introduced to the residues involving Vp1 and Vp2/3 binding, and analyzed their impact on viral infectious cycle. The mutations are designed to (1) alter the ionic interaction interface between Vp1 and Vp2/3, Vp1 K116R and Vp3 Q174D, and to (2) form disulfide bond between Vp1 and Vp2/3, Vp1 V230C-Vp3 E159C and Vp1 E257C-Vp3 Q174C. In addition (3) a mutant combined with three distinct temp … More erature sensitive mutations (tsDs), each reported to exhibit 'uncoating defect', Vp3 P103S-M110I-Q113K, was also included in the analysis. Upon transfection of the mutant viral DNA, capsid protein expression and viral DNA replication takes place normally, and all the mutants form particles with similar composition with that of wild-type virion. However, the mutants, except Vp1 K116R, were severely defective in their ability to form plaques at 37C or at the restrictive temperature (Vp3 P103S-M110I-Q113K). The mutant particles are apparently be able to enter the cells, interact with importin alpha/beta heterodimer, though either defective or delayed in initiating viral early gene expression, indicating that the defect of the mutants lies after interaction with nuclear import machinery of the viral particles. The results imply that major conformational change of viral capsid involving shift of Vp1-Vp2/3 interaction occurs at the later phase of cell entry processes, including the post-nuclear entry events, during the infection. Less