The astrovirus N-terminal nonstructural protein anchors replication complexes to the perinuclear ER membranes

An essential aspect of viral replication is the anchoring of the replication complex (RC) to cellular membranes. Positive-sense RNA viruses employ diverse strategies, including co-translational membrane targeting through signal peptides and co-opting cellular membrane trafficking components. Often, N-terminal nonstructural proteins play a crucial role in linking the RC to membranes, facilitating the early association of the replication machinery. Astroviruses utilize a polyprotein strategy to synthesize nonstructural proteins, relying on subsequent processing to form replication-competent complexes. In this study, we provide evidence for the perinuclear ER membrane association of RCs in five distinct human astrovirus strains. Using tagged recombinant classical human astrovirus 1 and neurotropic MLB2 strains, we establish that the N-terminal domain guides the ER membrane association. Through mutational analysis of the N-terminal domain in replicon and reverse genetics systems, we identified di-arginine motifs responsible for the perinuclear ER retention and formation of functional RCs. Our findings highlight the intricate virus-ER interaction mechanism employed by astroviruses, potentially leading to the development of novel antiviral intervention strategies. ### Competing Interest Statement The authors have declared no competing interest.