Regulation of cholesterol biosynthesis by human parainfluenza virus type 1

项目总结/摘要 人副流感病毒1型（hPIV 1）是幼儿哮吼的主要原因之一， 对公众健康产生重大影响。人类是唯一已知的hPIV 1感染宿主， 模式显示季节性和明显的两年期感染高峰。目前尚不清楚hPIV 1是如何在 在淡季的人口。到目前为止，关于持续性hPIV 1的研究有限 感染人类呼吸道细胞。在这项研究中，我们调查了病毒的细胞病理学，复制和后代病毒粒子 从感染hPIV 1的人气道细胞释放，以确定hPIV 1是否可以建立持续感染。 我们发现，与其他呼吸道RNA病毒不同，hPIV 1感染不会引起人类细胞病变 呼吸细胞感染的细胞甚至在感染后15天继续产生病毒结构蛋白。然而，在这方面， 感染性病毒的组装和释放随时间减少，在第5天达到100倍减少，并保持在 甚至在15 dpi时也很低，表明hPIV 1容易建立静止感染。有趣的是，大量的 在感染后晚期检测到病毒核衣壳（RNP）聚集体，表明病毒RNP受损 感染性病毒体在质膜上的运输和组装。这种表型与我们 在胆固醇耗尽的细胞中观察到，这特别限制了病毒的组装和释放。引人注目的是，我们发现 hPIV 1感染通过抑制参与甾醇生物合成的基因表达降低胆固醇水平， 3-羟基-3-甲基戊二酰辅酶A还原酶（HMGCR）是一种在大肠杆菌中的限速酶， 胆固醇生物合成这些数据首次提供了hPIV 1调节胆固醇的证据 内稳态，这可能有助于在人呼吸道上皮细胞中建立静止感染。在 本研究计划，我们将阐明hPIV 1调节胆固醇生物合成的机制， 建立静止感染。特别是，我们将分析Insig-1/2蛋白的作用，这是已知的是一个蛋白质。 HMGCR转录调节和泛素化关键调节因子。我们的假设是hPIV 1 通过靶向参与胆固醇生物合成的关键调节剂来操纵细胞胆固醇水平。我们将 鉴定负责hPIV 1诱导的胆固醇合成抑制的细胞和病毒蛋白，并分析 分子间的相互作用我们还将确定它如何促进hPIV 1的静止感染。成功 该提案将提供关于hPIV 1如何控制细胞环境以建立静止 感染

PROJECT SUMMARY/ABSTRACT Human parainfluenza virus type 1 (hPIV1) is one of the major causes of croup among young children and has a significant impact on public health. Humans are the only known host for hPIV1 infection, and the epidemiological patterns show seasonal and distinct biennial peaks of infection. It is not known how hPIV1 is maintained among the human population during the off season. So far, limited studies have been done regarding persistent hPIV1 infection in human airway cells. In this study, we investigated viral cytopathology, replication, and progeny virion release from human airway cells infected with hPIV1 to determine whether hPIV1 can establish persistent infection. We found that, unlike other respiratory RNA viruses, hPIV1 infection did not cause any cytopathic effects in human respiratory cells. Infected cells continue to produce viral structural proteins even at 15 days post infection. However, assembly and release of infectious virus decreased over time reaching 100-fold reduction on day 5 and remained low even at 15 dpi, showing that hPIV1 readily establishes quiescent infection. Interestingly, accumulation of large aggregates of viral nucleocapsid (RNP) was detected at late times post infection, suggesting impaired viral RNP trafficking and assembly of infectious virions at the plasma membrane. This phenotype is similar to what we observed in cholesterol-depleted cells, which specifically limited virus assembly and release. Strikingly, we found that hPIV1 infection reduces cholesterol levels by inhibiting expression of genes involved in sterol biosynthesis and ubiquitination and degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), a rate-limiting enzyme in cholesterol biosynthesis. These data provide evidence for the first time that hPIV1 regulates cholesterol homeostasis, which likely contributes to the established quiescent infection in human respiratory epithelial cells. In this research proposal, we will elucidate the mechanism by which hPIV1 regulates cholesterol biosynthesis to establish a quiescent infection. Especially, we will analyze the role of Insig-1/2 proteins, which are known to be a key regulator for both transcriptional regulation and ubiquitination of HMGCR. Our hypothesis is that hPIV1 manipulates cellular cholesterol levels by targeting key regulators involved in cholesterol biosynthesis. We will identify the cellular and viral proteins responsible for hPIV1-induced inhibition of cholesterol synthesis, and analyze their molecular interactions. We will also determine how it contributes to quiescent infection of hPIV1. Success of this proposal will provide key data regarding how hPIV1 controls the cellular environment to establish quiescent infection.