Cell Entry-Independent Role for the Reovirus μ1 Protein in Regulating Necroptosis and the Accumulation of Viral Gene Products

Abstract

The reovirus outer capsid protein 1 regulates cell death in infected cells. To distinguish between the roles of incoming, capsid-associated, and newly synthesized 1, we used small interfering RNA (siRNA)-mediated knockdown. Loss of newly synthesized 1 protein does not affect apoptotic cell death in HeLa cells but enhances necroptosis in L929 cells. Knockdown of 1 also affects aspects of viral replication. We found that, while 1 knockdown results in diminished release of infectious viral progeny from infected cells, viral minus-strand RNA, plus-strand RNA, and proteins that are not targeted by the 1 siRNA accumulate to a greater extent than in control siRNA-treated cells. Furthermore, we observed a decrease in sensitivity of these viral products to inhibition by guanidine hydrochloride (GuHCl) (which targets minus-strand synthesis to produce double-stranded RNA) when 1 is knocked down. Following 1 knockdown, cell death is also less sensitive to treatment with GuHCl. Our studies suggest that the absence of 1 allows enhanced transcriptional activity of newly synthesized cores and the consequent accumulation of viral gene products. We speculate that enhanced accumulation and detection of these gene products due to 1 knockdown potentiates receptor-interacting protein 3 (RIP3)-dependent cell death.Citation Roebke KE, Danthi P. 2019. Cell entryindependent role for the reovirus μ1 protein in regulating necroptosis and the accumulation of viral gene products. J Virol 93:e00199-19. RESULTSNewly synthesized 1 does not affect reovirus-induced apoptosis. The reovirus outer capsid protein 1 regulates apoptotic cell death following infection (9)(10)(11)(12)(13)20). However, whether this is a function of incoming capsid-associated 1 or newly synthesized 1 protein has not been evaluated directly. To determine whether newly synthesized 1 is responsible for this described role in apoptosis, we knocked down the levels of the T3D strain 1 protein in reovirus-infected HeLa cells using siRNA. Because Downloaded from 100%. (B) Cell death was quantified 24 h following infection by AOEB staining. Cell death for each independent infection and means are shown. Error bars indicate SDs. P values were determined by Student's t test. *, P Ͻ 0.05; **, P Ͻ 0.005.

Reovirus σ3 Protein Limits Interferon Expression and Cell Death Induction

“…The levels of outer capsid protein µ1 modulate cell death (10). Our work indicates that newly synthesized µ1 negatively regulates the induction of necroptosis in L929 cells by preventing excessive accumulation of viral gene products.…”

“…These data suggest that newly synthesized σ3 negatively regulates cell death in L929 cells following reovirus infection. L929 cells infected with reovirus undergo RIP3-dependent necroptosis (9,10). It is possible, however, that σ3 knockdown changes the nature of cell death in these cells.…”

“…We recently identified a role for the reovirus µ1 protein in regulating viral replication and cell death (10). Knockdown of this essential outer capsid protein with small interfering RNA (siRNA) results in enhanced viral gene expression, protein production, and minus strand RNA synthesis to generate new genome.…”

Reovirus σ3 protein limits interferon expression and cell death induction

“…replication and cell death demonstrated that this outer capsid protein limits the cell death response by negatively regulating viral replication events (10). We observed that knockdown of newly synthesized µ1 results in increased production of secondary transcripts and dsRNA genome.…”

Isolation and characterization of mammalian orthoreoviruses using a cell line resistant to sapelovirus infection

“…Many cell lines, including L929, Hela, LLC‐MK2 and MDCK cells, are susceptible to MRV infection and used for virus isolation (Desrosiers & Lengyel, 1979; Ooms, Kobayashi, Dermody, & Chappell, 2010; Roebke & Danthi, 2019; Rozee & Easterbrook, 1970). However, the general ubiquity of infectious microbes in swine faecal specimens has made the virus isolation difficult.…”