Biogenesis and function of astrovirus replication organelles

About This Grant

PROJECT SUMMARY/ABSTRACT Human astroviruses (HAstV) are positive sense, single-stranded RNA (+ssRNA) viruses and an understudied, globally prevalent cause of non-bacterial gastroenteritis. The recent isolation of novel HAstVs from children with severe or fatal infections of the nervous system revealed close phylogenetic clustering with animal-restricted viruses, highlighting a potential for zoonosis and an urgent, unmet need for effective treatments. However, gaps in knowledge for the basic biology of astrovirus replication and pathogenesis complicate the design of targeted antiviral interventions. An essential step for all productive +ssRNA virus infections is the manipulation of intracellular membranes to construct replication organelles (RO), or specialized compartments that concentrate factors for viral replication. Using immunofluorescence (IF) and transmission electron microscopy (TEM), we identified that the activities of both endoplasmic reticulum (ER)-anchored HAstV nonstructural proteins, nsp1a/1 and nsp1a/2, facilitate the remodeling of ER membrane into networks of double membrane vesicles (DMV) that isolate replicating RNA from the cytoplasm. However, the mechanisms that direct RO formation and the process by which replicated RNA reaches the cytoplasmic compartment for virion packaging remain elusive. Thus, this proposal seeks to understand the structural and molecular determinants of astrovirus RO formation and function by characterizing the independent and mutual interactions of nsp1a/1 and nsp1a/2. Aim 1 will test the hypothesis that dimerization enabled by a well-conserved leucine zipper motif drives nsp1a/1-mediated ER remodeling. This will be addressed using microscopy, biochemical assays, and reverse genetics approaches to probe the influence of dimerization on nsp1a/1-mediated ER remodeling and viral fitness. In Aim 2, we will test the hypothesis that nsp1a/2 performs a similar role to the coronavirus nsp4, forming an oligomer capable of inducing membrane curvature and assembling a DMV pore complex involved in RNA transport. Using cryo-FIB-SEM and electron tomography, we will elucidate the structure of nsp1a/2 in situ to reveal residues essential for oligomerization and interactions with viral RNA. The importance of these residues in nsp1a/2-mediated ER remodeling and viral fitness will be validated by site-directed mutagenesis, reverse genetics, and microscopy. In Aim 3, we will test the hypothesis that nsp1a/1 and nsp1a/2 interact to coordinate RO biogenesis. The domains of interaction will be mapped by co-immunoprecipitation and truncation of each viral protein in a co-expression system, and the influence of this interaction on productive DMV biogenesis will be assessed by IF and TEM. The indispensable role of ROs in the replication and assembly of +ssRNA viral progeny establishes the interactions of nsp1a/1 and nsp1a/2 as attractive, potentially druggable targets for effective therapeutics. Thus, understanding the specific domains and residues required for nsp1a/1- and nsp1a/2-mediated RO biogenesis will aid in the rational design of antivirals targeting this essential step of astrovirus infection.