In response to mammalian orthoreovirus (MRV) infection, cells initiate a stress response that includes eIF2␣ phosphorylation and protein synthesis inhibition. We have previously shown that early in infection, MRV activation of eIF2␣ phosphorylation results in the formation of cellular stress granules (SGs). In this work, we show that as infection proceeds, MRV disrupts SGs despite sustained levels of phosphorylated eIF2␣ and, further, interferes with the induction of SGs by other stress inducers. MRV interference with SG formation occurs downstream of eIF2␣ phosphorylation, suggesting the virus uncouples the cellular stress signaling machinery from SG formation. We additionally examined mRNA translation in the presence of SGs induced by eIF2␣ phosphorylation-dependent and -independent mechanisms. We found that irrespective of eIF2␣ phosphorylation status, the presence of SGs in cells correlated with inhibition of viral and cellular translation. In contrast, MRV disruption of SGs correlated with the release of viral mRNAs from translational inhibition, even in the presence of phosphorylated eIF2␣. Viral mRNAs were also translated in the presence of phosphorylated eIF2␣ in PKR ؊/؊ cells. These results suggest that MRV escape from host cell translational shutoff correlates with virus-induced SG disruption and occurs in the presence of phosphorylated eIF2␣ in a PKR-independent manner.The nonfusogenic mammalian orthoreoviruses (MRV) are members of a large family of animal and plant viruses (Reoviridae) that includes many members that are of considerable importance in human, animal, and plant disease. Following infection with many viruses from this family, including MRV, the host cell initiates a stress response that culminates in shutoff of protein translation (10,34,43). Viral mRNAs are able to escape this inhibition and continue to be translated in the shutoff environment (40,41,52). In the case of MRV, the innate immune response has been implicated in host translational shutoff via activation of the double-stranded RNA (dsRNA) kinase PKR (12,37,40,45). PKR is activated by binding dsRNA, at which point it homodimerizes and undergoes autophosphorylation (51). Activated PKR phosphorylates serine 51 on the alpha subunit of the cellular translation initiation factor eIF2 (23). In the absence of cellular stress, eIF2␣ binds to GTP and initiator methionyl-tRNA (met-tRNA i ) to form a ternary complex, which subsequently binds to the 40S ribosomal complex to form the 43S preinitiation complex. As translational initiation proceeds, eIF2-bound GTP is hydrolyzed to release initiation factors from the ribosome. The released GDP bound to eIF2 must be exchanged for GTP in a reaction catalyzed by the guanine nucleotide exchange factor, eIF2B. Upon this exchange, eIF2-GTP can again bind mettRNA i to initiate a new round of translation. When phosphorylated, eIF2␣ changes from a substrate to a competitive inhibitor of eIF2B, preventing the exchange of GDP for GTP. This results in global inhibition of protein synthesis (reviewed in r...
At early times in Mammalian Orthoreovirus (MRV) infection, cytoplasmic inclusions termed stress granules (SGs) are formed as a component of the innate immune response, however, at later times they are no longer present despite continued immune signaling. To investigate roles of MRV proteins in SG modulation we examined non-structural protein μNS localization relative to SGs in infected and transfected cells. Using a series of mutant plasmids, we mapped necessary μNS residues for SG localization to amino acids 78 and 79. We examined the capacity of a μNS(78-79) mutant to associate with known viral protein binding partners of μNS and found that it loses association with viral core protein λ2. Finally, we show that while this mutant cannot support de novo viral replication, it is able to rescue replication following siRNA knockdown of μNS. These data suggest that μNS association with SGs, λ2, or both play roles in MRV replication.
Immune privilege helps protect the cornea from damaging inflammation but can also impair pathogen clearance from this mucosal surface. Programmed death-ligand 1 (PD-L1 or B7-H1) contributes to corneal immune privilege by inhibiting the function of a variety of immune cells. We asked whether programmed death-1 (PD-1)/PD-L1 interaction regulates HSV-1 clearance from infected corneas. We show that PD-L1 is constitutively expressed in the corneal epithelium and is upregulated upon HSV-1 corneal infection, with peak expression on CD45 cells NK cells, dendritic cells, neutrophils, and macrophages and CD45 corneal epithelial cells at 4 d postinfection (dpi). As early as 1 dpi, HSV-1-infected corneas of B7-H1 mice as compared with wild-type mice showed increased chemokine expression and this correlated with increased migration of inflammatory cells into the viral lesions and decreased HSV-1 corneal titers. Local PD-L1 blockade caused a similar increase in viral clearance, suggesting a local effect of PD-1/PD-L1 in the cornea. The enhanced HSV-1 clearance at 2 dpi resulting from PD-1/PD-L1 blockade is mediated primarily by a monocyte/macrophage population. Studies in bone marrow chimeras demonstrated enhanced viral clearance when PD-L1 was absent only from nonhematopoietic cells. We conclude that PD-L1 expression on corneal cells negatively impacts the ability of the innate immune system to clear HSV-1 from infected corneas.
Herpes simplex virus 1 (HSV-1) causes a lifelong infection of neurons that innervate barrier sites like the skin and mucosal surfaces like the eye. After primary infection of the cornea, the virus enters latency within the trigeminal ganglion (TG), from which it can reactivate throughout the life of the host. Viral latency is maintained, in part, by virus-specific CD8+ T cells that nonlethally interact with infected neurons. When CD8+ T cell responses are inhibited, HSV-1 can reactivate, and these recurrent reactivation events can lead to blinding scarring of the cornea. In the C57BL/6 mouse, CD8+ T cells specific for the immunodominant epitope from glycoprotein B maintain functionality throughout latency, while CD8+ T cells specific for subdominant epitopes undergo functional impairment that is associated with the expression of the inhibitory checkpoint molecule programmed death 1 (PD-1). Here, we investigate the checkpoint molecule T cell immunoglobulin and mucin domain-containing 3 (Tim-3), which has traditionally been associated with CD8+ T cell exhaustion. Unexpectedly, we found that Tim-3 was preferentially expressed on highly functional ganglionic CD8+ T cells during acute and latent HSV-1 infection. This, paired with data that show that Tim-3 expression on CD8+ T cells in the latently infected TG is influenced by viral gene expression, suggests that Tim-3 is an indicator of recent T cell stimulation, rather than functional compromise, in this model. We conclude that Tim-3 expression is not sufficient to define functional compromise during latency; however, it may be useful in identifying activated cells within the TG during HSV-1 infection. IMPORTANCE Without an effective means of eliminating HSV-1 from latently infected neurons, efforts to control the virus have centered on preventing viral reactivation from latency. Virus-specific CD8+ T cells within the infected TG have been shown to play a crucial role in inhibiting viral reactivation, and with a portion of these cells exhibiting functional impairment, checkpoint molecule immunotherapies have presented a potential solution to enhancing the antiviral response of these cells. In pursuing this potential treatment strategy, we found that Tim-3 (often associated with CD8+ T cell functional exhaustion) is not upregulated on impaired cells but instead is upregulated on highly functional cells that have recently received antigenic stimulation. These findings support a role for Tim-3 as a marker of activation rather than exhaustion in this model, and we provide additional evidence for the hypothesis that there is persistent viral gene expression in the HSV-1 latently infected TG.
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