Mechanisms of astrovirus infection

Astroviruses are small, non-enveloped, positive-sense single-stranded RNA viruses (+ssRNA) that are prevalent in bird and animal populations. Human astrovirus (HAstV) infection has historically been known as a leading cause of non-bacterial gastroenteritis. However, in recent years, divergent HAstVs have been found in cases of encephalitis. Despite their prevalence, there is limited information regarding the mechanisms of virus infection. The virus is known to produce two nonstructural polyproteins required for infection, which are cleaved into functional subunits by host signal peptidase and the virus-encoded serine protease (Pro). However, the specific sites of Pro cleavage in the polyprotein have not been discovered. Thus, we do not know the specific sequences of viral proteins, which has made it difficult to assign functions to these proteins. Similarly, lack of information and tools has made studying virus-host interactions challenging. Thus, few host proteins that regulate HAstV infection have been identified. Furthermore, like all +ssRNA viruses, HAstV infection leads to dramatic remodeling of intracellular membranes to form replication organelles (ROs), which concentrate viral host factors required for infection. However, the source of the membranes for these critical structures is not well understood. Overall, there are major gaps in our knowledge of all aspects of HAstV biology and there is a need for tools that will allow us to build a foundation to expand our research on fundamental mechanisms that regulate HAstV infection. The goal of this proposal is to utilize tools we have developed for +ssRNA viruses and HAstV to understand the mechanisms of (1) viral nonstructural polyprotein processing and Pro activity, (2) host protein regulation of virus infection, and (3) viral protein manipulation of host organelles. We have made significant progress to be well-suited to accomplish these proposed projects. We have developed a library of polyprotein expression constructs and a cDNA infectious clone to investigate Pro-dependent cleavage. Additionally, we have successfully developed a versatile viral protease activity reporter that has been shown to work for enteroviruses and flaviviruses, which we will adapt for HAstV to study intracellular Pro activity. We have generated tools to study the microenvironment of the viral nonstructural proteins in living cells and to perform a CRISPR screen for pro- and anti-viral proteins. Lastly, we have innovative tools and strategies to visualize the manipulation of host organelles upon viral protein expression or infection using long-term, time-lapse imaging of living cells. Overall, the proposed projects are designed to significantly advance the field of cell biology of HAstV infection through investigation of the molecular virology and virus-host interactions.