Defining the critical functions of the stem-loop II motif in the lifecycle of astrovirus VA1

About This Grant

Project Summary/Abstract Astroviruses are RNA viruses that commonly cause disease in humans, including gastroenteritis and fatal cases of encephalitis. Despite their broad human impact, astroviruses are understudied and many of the critical steps of the viral lifecycle are poorly characterized. There is limited understanding of host-pathogen interactions that facilitate viral replication, including the role of RNA motifs in the viral genome. Our lab was the first to cultivate astrovirus VA1 (VA1), the most common cause of astrovirus encephalitis to date. In a region overlapping with ORF2 and the 3' untranslated region, VA1 is predicted to encode a stem-loop II motif (s2m). Similar motifs with the same secondary structure have been identified in astroviruses and viruses of other viral families, with its importance in the viral lifecycle being virus-dependent. Using SHAPE-MaP on the full-length VA1 genome, we confirmed the formation of the s2m and its secondary structure. Mutagenesis of the s2m in a novel reverse genetics system for VA1 revealed the s2m to be essential. Deletion of the s2m or mutations that disrupt guanine- cytosine base pairs (GC-bp) that are critical for the secondary structure of the s2m result in virus that cannot be propagated. The mutant genomes can be rescued when complementary mutations are introduced into the s2m that restore GC-bp in the secondary structure. Mutagenesis of a position not involved in GC-bp was important but not essential for the function of the s2m. Capsid expression could not be detected from transfected genomes containing s2m mutations. Translation of capsid was also reduced by mutations of the s2m using a reporter system, and we have identified putative proteins involved in translation that may also bind to the s2m. We are now uniquely positioned to study the mechanism of action for the s2m in promoting the VA1 lifecycle using our novel tools that we have developed. Our central hypothesis is that the s2m facilitates viral translation through RNA-protein interactions, mediated by the s2m sequence, structure, and location in the genome. To test this hypothesis, we will take a combination of genetic and biochemical approaches to mechanistically understand why the s2m is essential. In other viral stem-loop structures, the loop region often serves as an important interaction site. We will define the role of the VA1 pentaloop for the function of the s2m by mutagenesis. Next, we will determine whether the function of the s2m is dependent on location in the genome. We will also assess whether the s2m must be encoded on the expressed RNA strand or if it can function independently. Using an RNA-pulldown, we have identified putative proteins that bind to the s2m that also mediate translation. We will confirm s2m-protein interactions and determine the effects of loss of function of these candidate proteins on the viral lifecycle. The findings of this project will provide important insights into the function of the VA1 s2m, address gaps in our knowledge of the VA1 lifecycle, and contribute to our larger understanding in RNA motifs in viral biology. These results will set the foundation for further dissection of the molecular biology of VA1, ultimately accelerating development of antivirals and vaccine-based approaches.