The dynamics and strategy of RNA replication in astroviruses

Astroviruses are positive-sense single-stranded RNA viruses that cause significant disease across avian and mammalian hosts, yet their replication mechanisms remain poorly understood. The replication of astrovirus RNA occurs via a double-stranded RNA intermediate that is used as a template for the synthesis of new positive-sense RNA, which is covalently linked to the virus-encoded protein VPg. These viruses also produce a capsid-encoding subgenomic (sg) RNA that is 3′-coterminal with the genomic RNA. The mechanisms by which the astrovirus sgRNA is produced and regulated during infection have not yet been characterized. Using high-throughput sequencing of RNA from cells infected with each of five different astrovirus strains, we demonstrate that the presence of a (-)sgRNA is a conserved feature of infection, supporting a premature termination model of subgenomic RNA production. A pronounced pile-up in the mapping positions of the 3′ ends of negative-sense RNA fragments marks the precise 3′ terminus of the (-)sgRNA. We investigate the relative abundance and replication dynamics of positive and negative RNA species during the virus life cycle, and perform a mutational analysis of conserved residues in the genomic and subgenomic 5′ termini. Together, this work elucidates the dynamics of genomic and subgenomic RNA synthesis during astrovirus infection.