Reovirus Low-Density Particles Package Cellular RNA

Abstract

Packaging of segmented, double-stranded RNA viral genomes requires coordination of viral proteins and RNA segments. For mammalian orthoreovirus (reovirus), evidence suggests either all ten or zero viral RNA segments are simultaneously packaged in a highly coordinated process hypothesized to exclude host RNA. Accordingly, reovirus generates genome-containing virions and “genomeless” top component particles. Whether reovirus virions or top component particles package host RNA is unknown. To gain insight into reovirus packaging potential and mechanisms, we employed next-generation RNA-sequencing to define the RNA content of enriched reovirus particles. Reovirus virions exclusively packaged viral double-stranded RNA. In contrast, reovirus top component particles contained similar proportions but reduced amounts of viral double-stranded RNA and were selectively enriched for numerous host RNA species, especially short, non-polyadenylated transcripts. Host RNA selection was not dependent on RNA abundance in the cell, and specifically enriched host RNAs varied for two reovirus strains and were not selected solely by the viral RNA polymerase. Collectively, these findings indicate that genome packaging into reovirus virions is exquisitely selective, while incorporation of host RNAs into top component particles is differentially selective and may contribute to or result from inefficient viral RNA packaging.

Mammalian orthoreovirus can exit cells in extracellular vesicles

“…Then, they were freeze-thawed twice at -80°C and room temperature. Virus titers in the resulting lysates were determined by FFA [ 101 ] and calculated based on numbers of infected cells quantified in four countable fields of view per well in duplicate wells, with countable fields containing ~ 50–500 reovirus-positive cells.…”

Mammalian orthoreovirus can exit cells in extracellular vesicles

“…Then, they were freeze-thawed twice at -80°C and room temperature. Virus titers in the resulting lysates were determined by FFA (93) and calculated based on numbers of infected cells quantified in four countable fields of view per well in duplicate wells, with countable fields containing ~ 50-500 reovirus-positive cells.…”