Endoplasmic reticulum-anchored nonstructural proteins drive human astrovirus replication organelle formation.

Human astroviruses (HAstV) are major cause of acute, non-bacterial gastroenteritis and have been implicated in severe infections of the nervous system. Despite global prevalence, there are no established treatments for HAstVs due to a lack of understanding of the fundamental biology of infection, including mechanisms of viral replication. Like all positive-stranded RNA (+ssRNA) viruses, infection induces remodeling of host membranes into replication organelles (ROs). However, the intracellular membrane source and viral proteins involved in the coordination of HAstV ROs remain poorly defined. Using immunofluorescence microscopy, we determined that HAstV1 infection drives extensive restructuring of the endoplasmic reticulum (ER) to concentrate RNA replication and virus packaging. Long-term, time-lapse imaging of the ER and time point transmission electron microscopy revealed that temporal manipulation of ER membrane corresponds with the emergence of ER-contiguous double membrane vesicles (DMV). The co-expression of transmembrane nonstructural proteins nsp1a/1 and nsp1a/2 established similar DMV networks in the absence of an active infection. Further, super resolution microscopy revealed the organization of these two viral proteins in RO-like arrangements within the perinuclear region of infected cells. Together, these findings enhance our understanding of HAstV1-induced RO biogenesis, highlighting nsp1a/1 and nsp1a/2 as exploitable targets for the design of antivirals restricting astrovirus replication. Significance: Human astroviruses (HAstV) are understudied, globally prevalent pathogens capable of causing potentially fatal infections of the central nervous system in children, the elderly, and immunocompromised individuals. Despite the capacity to cause devastating disease, there are no established vaccines, antivirals, or therapeutics available combat HAstV infections, in part due to a lack of knowledge for the functions of several viral proteins. This study furthers our understanding of HAstV manipulation of the host cell by identification of the viral proteins responsible for the biogenesis of virus-induced double membrane vesicles, which are a hallmark of replication.