The Role of Cellular Chaperones in RNA Virus Infection

 描述(申请人提供)：一个有功能的蛋白质动态平衡网络，包括细胞伴侣和泛素-蛋白酶体系统，对细胞生存至关重要。 和人类健康。越来越多的与蛋白质折叠功能障碍有关的疾病就证明了这一点。病毒感染也给细胞蛋白的动态平衡机制带来了很大的负担，因为在感染过程中有大量的结构复杂的蛋白质。我们以前已经证明，90 kDa热休克蛋白Hsp90的化学抑制剂通过抑制其衣壳前体蛋白P1的加工，有效地阻止许多肠道病毒物种的感染。值得注意的是，在所有测试的病毒中，即使在长期传代后也没有出现耐药病毒变异，这表明导致独立于Hsp90的P1折叠的突变降低了病毒的适应性。观察到，选择对选择后持续存在的病毒种群的组成有重大影响，这支持了这一点。这项建议的目标是(I)利用新的深度测序技术和群体遗传学来量化Hsp90抑制剂对病毒群体结构的影响，以及(Ii)从生物物理学的角度描述在所选群体中观察到的伴侣独立性的机制。我们的工作假设是，Hsp90功能的抑制将改变病毒种群结构和病毒基因组序列，以反映病毒蛋白折叠的改变限制。

 DESCRIPTION (provided by applicant): A functioning protein homeostasis network, which includes cellular chaperones and the ubiquitin-proteasome system, is critical to cellular viability and human health. This is evidenced by an ever-growing number of diseases associated with protein folding dysfunction. Viral infection also places a significant burden on the cellular proten homeostasis machinery, due to the large amount of a few structurally complex proteins during infection. We have shown previously that chemical inhibitors of the 90kDa heat-shock protein, Hsp90, potently block infection by numerous enteroviral species by inhibiting the processing of their capsid precursor protein, P1. Notably, in all of the viruses tested, resistant viral variantsdid not emerge, even after long-term passage suggesting that mutations that lead to folding of P1 independent of Hsp90 reduce fitness of the virus. This is supported by the observation that selection has significant effects on the composition of the viral population that persists after selection. The goal of this proposal is (i) to leverage new deep-sequencing technologies and population genetics to quantify the effect of Hsp90 inhibitors on viral population structure, and (ii) to biophysically characterize the mechanism of chaperone independence observed in the selected population. Our working hypothesis is that the inhibition of Hsp90 function will change the viral population structure and viral genome sequence to reflect the altered constraints on viral protein folding.