The dynamics and strategy of RNA replication in astroviruses

Abstract

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 reads marks the precise 3' terminus of the (-)sgRNA. We investigate the relative abundance and dynamics of positive and negative RNA species during virus replication and virion packaging, 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/700307v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1d71111org.highwire.dtl.DTLVardef@a1a024org.highwire.dtl.DTLVardef@118d126org.highwire.dtl.DTLVardef@4b3c4e_HPS_FORMAT_FIGEXP M_FIG C_FIG