Regulation of cholesterol biosynthesis by human parainfluenza virus type 1

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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.