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

Human astroviruses (HAstV) are a 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 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 (TEM) revealed that temporal manipulation of ER membrane corresponds with the emergence of ER-contiguous double membrane vesicles (DMV). The expression of transmembrane nonstructural proteins nsp1a/1, nsp1a/2, and nsp1a/1-2 led to the fragmentation of the ER for both HAstV1 and HAstV-VA1. However, only the expression of nsp1a/1-2 established DMV-like 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 demonstrate the functions of nsp1a/1 and nsp1a/2 in the biogenesis of astrovirus-induced ROs, highlighting these proteins as exploitable targets for the design of antivirals restricting astrovirus replication.