mRNA capping enzymes are masked in reovirus progeny subviral particles

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.

Section: Discussionmentioning

confidence: 99%

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

Roebke

Danthi

2019

“…Nascent viral cores function as secondary transcriptase particles that continue to synthesize positivesense RNAs (7). The guanylyltransferase and methyltransferase activities in secondary cores are inhibited, and viral mRNAs synthesized from these particles are uncapped (20). Late in infection, uncapped reovirus mRNAs are translated with much greater efficiency than capped viral mRNAs (20)(21)(22).…”

Section: Discussionmentioning

confidence: 99%

“…The guanylyltransferase and methyltransferase activities in secondary cores are inhibited, and viral mRNAs synthesized from these particles are uncapped (20). Late in infection, uncapped reovirus mRNAs are translated with much greater efficiency than capped viral mRNAs (20)(21)(22). As replication proceeds, VFs enlarge as additional viral RNAs and proteins are produced and incorporated into the structure.…”

Section: Discussionmentioning

confidence: 99%

“…New cores serve as secondary transcriptase particles that amplify viral transcription, and thereby reovirus protein production, in the late stages of replication. In many cells, late transcripts produced by secondary transcriptase particles lack 5=-cap structures and are also efficiently translated (20)(21)(22). As replication proceeds, viral transcription, translation, genome replication, and virion assembly all continue within the VF (7).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Nonstructural Protein σ1s Is Required for Optimal Reovirus Protein Expression

Phillips

Stuart

Simon

2018

Reovirus non-structural protein σ1s is required for the establishment of viremia and hematogenous viral dissemination. However, the function of the σ1s protein during the reovirus replication cycle is not known. In this study, we found that σ1s was required for efficient reovirus replication in SV40-immortalized endothelial cells (SVECs), mouse embryonic fibroblasts, human umbilical vein endothelial cells (HUVECs), and T84 human colonic epithelial cells. In each of these cell lines, wild type reovirus produced substantially higher viral titers than a σ1s-deficient mutant. The σ1s protein was not required for early events in the reovirus infection, as no difference in infectivity between the wild type and σ1s-null viruses was observed. However, wild type virus produced markedly higher viral protein levels than the σ1s-deficient strain. The disparity in viral replication did not result from differences in viral transcription or protein stability. We further found that the σ1s protein was dispensable for cell killing and induction of type-1 interferon responses. In the absence of σ1s, viral factory (VF) maturation was impaired, but sufficient to support low levels of reovirus replication. Together, our results indicate that σ1s is not absolutely essential for viral protein production, but rather potentiates reovirus protein expression to facilitate reovirus replication. Our findings suggest that σ1s enables hematogenous reovirus dissemination by promoting efficient viral protein synthesis, and thereby reovirus replication, in cells that are required for reovirus spread to the blood.Hematogenous dissemination is critical a step in the pathogenesis of many viruses. For reovirus, nonstructural protein σ1s is required for viral spread via the blood. However, the mechanism by σ1s promotes reovirus dissemination is unknown. Here, we identified σ1s as a viral mediator of reovirus protein expression. We found several cultured cell lines in which σ1s is required for efficient reovirus replication. In these cells, wild type virus produced substantially higher levels of viral protein than a σ1s-deficient mutant. The σ1s protein was not required for viral mRNA transcription or viral protein stability. Owing to reduced levels of viral protein synthesized in the absence of σ1s, maturation of viral factories was impaired and significantly fewer viral progeny were produced. Taken together, our findings indicate that σ1s is required for optimal reovirus protein production, and thereby viral replication, in cells required hematogenous reovirus dissemination.

“…Millward and co-workers have reported (34,35,40) that transcripts synthesized late in the infection by human reovirus progeny virions lack a 5'-terminal cap, while transcripts synthesized by infecting parental virions have a cap. Furthermore, these investigators reported that as the infection proceeds, mRNA translation shifts from a capdependent to cap-independent mode (33).…”

Section: 500 P9mentioning

confidence: 99%

Regulation of expression of the wound tumor virus genome in persistently infected vector cells is related to change in translational activity of viral transcripts

Peterson¹,

Nuss²

1986

The interaction between a plant virus and its insect vector was studied at the molecular level by examining wound tumor virus (WTV) gene expression in cultured cells derived from its leafhopper vector. Infection of vector cells by WTV is noncytopathic and results in an acute phase (through day 5), followed by persistence beginning with the first cell passage. Viral-specific polypeptide synthesis and viral genome RNA accumulation increased to a maximum level during the first 5 days following inoculation and then decreased as infected cells were passaged (to 5 to 20% of the level observed during the acute phase by passages 10 to 15). In contrast, viral-specific mRNAs were present at approximately the same level in the acute phase and in the early stage (passage 10) of the persistent phase of infection. Although viral transcripts isolated at different times after inoculation exhibited identical electrophoretic migration patterns, they had different functional activities in cell-free translation systems. Transcripts isolated from persistently infected cells were inefficiently translated in vitro, reflecting the situation in infected cells. These results indicate that the decline in the level of viral polypeptide synthesis associated with the persistent phase of WTV infection is related to a change in the translational activity of viral transcripts.