Increased eIF2α Phosphorylation Attenuates Replication of Herpes Simplex Virus 2 vhs Mutants in Mouse Embryonic Fibroblasts and Correlates with Reduced Accumulation of the PKR Antagonist ICP34.5

In a previous study, it was observed that cells infected with herpes simplex virus 2 (HSV-2) failed to accumulate stress granules (SGs) in response to oxidative stress induced by arsenite treatment. As a follow-up to this observation, we demonstrate here that disruption of arsenite-induced SG formation by HSV-2 is mediated by a virion component. Through studies on SG formation in cells infected with HSV-2 strains carrying defective forms of UL41, the gene that encodes vhs, we identify vhs as a virion component required for this disruption. Cells infected with HSV-2 strains producing defective forms of vhs form SGs spontaneously late in infection. In addition to core SG components, these spontaneous SGs contain the viral immediate early protein ICP27 as well as the viral serine/threonine kinase Us3. As part of these studies, we reexamined the frameshift mutation known to reside within the UL41 gene of HSV-2 strain HG52. We demonstrate that this mutation is unstable and can rapidly revert to restore wild-type UL41 following low-multiplicity passaging. Identification of the involvement of virion-associated vhs in the disruption of SG formation will enable mechanistic studies on how HSV-2 is able to counteract antiviral stress responses early in infection. In addition, the ability of Us3 to localize to stress granules may indicate novel roles for this viral kinase in the regulation of translation. IMPORTANCEEukaryotic cells respond to stress by rapidly shutting down protein synthesis and storing mRNAs in cytoplasmic stress granules (SGs). Stoppages in protein synthesis are problematic for all viruses as they rely on host cell machinery to synthesize viral proteins. Thus, many viruses target SGs for disruption or modification. Infection by herpes simplex virus 2 (HSV-2) was previously observed to disrupt SG formation induced by oxidative stress. In this follow-up study, we identify virion host shutoff protein (vhs) as a viral protein involved in this disruption. The identification of a specific viral protein involved in disrupting SG formation is a key step toward understanding how HSV-2 interacts with these antiviral structures. Additionally, this understanding may provide insights into the biology of SGs that may find application in studies on human motor neuron degenerative diseases, like amyotrophic lateral sclerosis (ALS), which may arise as a result of dysregulation of SG formation.

Section: Discussionmentioning

confidence: 99%

The Herpes Simplex Virus 2 Virion-Associated Ribonuclease vhs Interferes with Stress Granule Formation

Finnen

Hay

Dauber

et al. 2014

“…In so doing, it helps determine viral mRNA levels and facilitates the sequential expression of different classes of viral mRNAs (38). Significantly, during animal infections, Vhs impedes the establishment of an interferon-induced antiviral state (61)(62)(63)(64)(65)(66)(67). It blocks the activation of dendritic cells (68)(69)(70) and inhibits other components of the innate and adaptive immune responses (41,(71)(72)(73)(74)(75).…”

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confidence: 99%

“…It blocks the activation of dendritic cells (68-70) and inhibits other components of the innate and adaptive immune responses (41,(71)(72)(73)(74)(75). As such, it is an important determinant of HSV virulence (61)(62)(63)(64)(65)(66)(67)(76)(77)(78)(79)(80)(81)(82)(83)(84)(85).Although Vhs degrades many cellular and viral transcripts, not all mRNAs are equally sensitive. For unknown reasons, a number of host mRNAs appear to be refractory, or at least less sensitive to Vhs degradation, and actually increase in abundance following infection (43,(86)(87)(88)(89)(90)(91)(92)(93).…”

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confidence: 99%

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mRNA Decay during Herpes Simplex Virus (HSV) Infections: Mutations That Affect Translation of an mRNA Influence the Sites at Which It Is Cleaved by the HSV Virion Host Shutoff (Vhs) Protein

Shiflett

Read

2013

During lytic infections, the herpes simplex virus (HSV) virion host shutoff (Vhs) endoribonuclease degrades many host and viral mRNAs. Within infected cells it cuts mRNAs at preferred sites, including some in regions of translation initiation. Vhs binds the translation initiation factors eIF4H, eIF4AI, and eIF4AII, suggesting that its mRNA degradative function is somehow linked to translation. To explore how Vhs is targeted to preferred sites, we examined the in vitro degradation of a target mRNA in rabbit reticulocyte lysates containing in vitro-translated Vhs. Vhs caused rapid degradation of mRNAs beginning with cleavages at sites in the first 250 nucleotides, including a number near the start codon and in the 5= untranslated region. Ligation of the ends to form a circular mRNA inhibited Vhs cleavage at the same sites at which it cuts capped linear molecules. This was not due to an inability to cut any circular RNA, since Vhs cuts circular mRNAs containing an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) at the same sites as linear molecules with the IRES. Cutting linear mRNAs at preferred sites was augmented by the presence of a 5= cap. Moreover, mutations that altered the 5= proximal AUG abolished Vhs cleavage at nearby sites, while mutations that changed sequences surrounding the AUG to improve their match to the Kozak consensus sequence enhanced Vhs cutting near the start codon. The results indicate that mutations in an mRNA that affect its translation affect the sites at which it is cut by Vhs and suggest that Vhs is directed to its preferred cut sites during translation initiation. During lytic herpes simplex virus (HSV) infections, viral and cellular gene expressions are regulated at multiple steps of mRNA metabolism (1, 2), including transcription (2-8), splicing (2, 9-12), transport from the nucleus to the cytoplasm (2, 13-20), translation (21-32), and stabilization of mRNAs in the cytoplasm (33-43). Among the posttranscriptional controls are the regulation of mRNA half-lives (36-42) and translation (23,29) by the HSV virion host shutoff (Vhs) protein (UL41). Vhs is an endoribonuclease (33,(44)(45)(46)(47) that is a minor component of virions (48)(49)(50). At early times, copies of Vhs from infecting virions accelerate the degradation of many housekeeping and constitutively expressed cellular mRNAs (40,42,48,(51)(52)(53)(54)(55), with a concomitant downturn in the levels of those mRNAs and in the synthesis of the proteins that they encode (52,56,57). Following the onset of viral transcription, Vhs ensures the rapid turnover of most, if not all, viral mRNAs (36, 37, 57-60). In so doing, it helps determine viral mRNA levels and facilitates the sequential expression of different classes of viral mRNAs (38). Significantly, during animal infections, Vhs impedes the establishment of an interferon-induced antiviral state (61-67). It blocks the activation of dendritic cells (68-70) and inhibits other components of the innate and adaptive immune responses (41,(71)(72)(73)(74)(75). As su...

“…␥34.5 is transcribed as a leaky late (␥1) gene (9). It encodes infected cell protein 34.5 (ICP34.5) (10), whose expression is detected as early as 2 to 3 h postinfection (11)(12)(13)(14). Truncation or stop codon insertion mutants of HSV-1 and HSV-2 ␥34.5 retain the capacity to replicate efficiently in many actively dividing cell types (14)(15)(16) and in footpad tissue of mice (17).…”

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confidence: 99%

Up to Four Distinct Polypeptides Are Produced from the γ34.5 Open Reading Frame of Herpes Simplex Virus 2

Korom

Davis

Morrison

2014

Self Cite

The herpes simplex virus 1 (HSV-1) ICP34.5 protein strongly influences neurovirulence and regulates several cellular antiviral responses. Despite the clinical importance of HSV-2, relatively little is known about its ICP34.5 ortholog. We found that HSV-2 produces up to four distinct forms of ICP34.5 in infected cells: a full-length protein, one shorter form sharing the N terminus, and two shorter forms sharing the C terminus. These forms appeared with similar kinetics and accumulated in cells over much of the replication cycle. We confirmed that the N-terminal form is translated from the primary unspliced transcript to a stop codon within the intron unique to HSV-2 ␥34.5. We found that the N-terminal form was produced in a variety of cell types and by 9 of 10 clinical isolates. ICP27 influenced but was not required for expression of the N-terminal form. Western blotting and reverse transcription-PCR indicated the C-terminal forms did not contain the N terminus and were not products of alternative splicing or internal transcript initiation. Expression plasmids encoding methionine at amino acids 56 and 70 generated products that comigrated in SDS-PAGE with the C1 and C2 forms, respectively, and mutation of these sites abolished C1 and C2. Using a recombinant HSV-2 encoding hemagglutinin (HA)-tagged ICP34.5, we demonstrated that the C-terminal forms were also produced during infection of many human and mouse cell types but were not detectable in mouse primary neurons. The protein diversity generated from the HSV-2 ␥34.5 open reading frame implies additional layers of cellular regulation through potential independent activities associated with the various forms of ICP34.5. IMPORTANCEThe herpes simplex virus 1 (HSV-1) protein ICP34.5, encoded by the ␥34.5 gene, interferes with several host defense mechanisms by binding cellular proteins that would otherwise stimulate the cell's autophagic, translational-arrest, and type I interferon responses to virus infection. ICP34.5 also plays a crucial role in determining the severity of nervous system infections with HSV-1 and HSV-2. The HSV-2 ␥34.5 gene contains an intron not present in HSV-1 ␥34.5. A shorter N-terminal form of HSV-2 ICP34.5 can be translated from the unspliced ␥34.5 mRNA. Here, we show that two additional forms consisting of the C-terminal portion of ICP34.5 are generated in infected cells. Production of these N-and C-terminal forms is highly conserved among HSV-2 strains, including many clinical isolates, and they are broadly expressed in several cell types, but not mouse primary neurons. Multiple ICP34.5 polypeptides add additional complexity to potential functional interactions influencing HSV-2 neurovirulence.