Herpes Simplex Virus 2 Virion Host Shutoff Endoribonuclease Activity Is Required To Disrupt Stress Granule Formation

Abstract

We previously established that cells infected with herpes simplex virus 2 (HSV-2) are disrupted in their ability to form stress granules (SGs) in response to oxidative stress and that this disruption is mediated by virion host shutoff protein (vhs), a virionassociated endoribonuclease. Here, we test the requirement for vhs endoribonuclease activity in disruption of SG formation. We analyzed the ability of HSV-2 vhs carrying the point mutation D215N, which ablates its endoribonuclease activity, to disrupt SG formation in both transfected and infected cells. We present evidence that ablation of vhs endoribonuclease activity results in defects in vhs-mediated disruption of SG formation. Furthermore, we demonstrate that preformed SGs can be disassembled by HSV-2 infection in a manner that requires vhs endoribonuclease activity and that, befitting this ability to promote SG disassembly, vhs is able to localize to SGs. Together these data indicate that endoribonuclease activity must be maintained in order for vhs to disrupt SG formation. We propose a model whereby vhs-mediated destruction of SG mRNA promotes SG disassembly and may also prevent SG assembly. IMPORTANCEStress granules (SGs) are transient cytoplasmic structures that form when a cell is exposed to stress. SGs are emerging as potential barriers to viral infection, necessitating a more thorough understanding of their basic biology. We identified virion host shutoff protein (vhs) as a herpes simplex virus 2 (HSV-2) protein capable of disrupting SG formation. As mRNA is a central component of SGs and the best-characterized activity of vhs is as an endoribonuclease specific for mRNA in vivo, we investigated the requirement for vhs endoribonuclease activity in disruption of SG formation. Our studies demonstrate that endoribonuclease activity is required for vhs to disrupt SG formation and, more specifically, that SG disassembly can be driven by vhs endoribonuclease activity. Notably, during the course of these studies we discovered that there is an ordered departure of SG components during their disassembly and, furthermore, that vhs itself has the capacity to localize to SGs. Stress granules (SGs) are dynamic, non-membrane-bound cytoplasmic compartments that contain translationally silent mRNAs that remain associated with a cadre of translation initiation proteins, poly(A) binding protein (PABP), and the small ribosomal subunit in the form of messenger ribonucleoprotein complexes (mRNPs) (1). SGs assemble rapidly in response to a variety of stressful conditions and likewise can disassemble rapidly when stress is alleviated. Natural stresses that eukaryotic cells encounter include starvation, elevated temperature (heat shock), oxidation, and virus infection, all of which can be sensed by four distinct eukaryotic initiation factor 2 (eIF2) kinases. Following the sensing of stress, these kinases become activated and phosphorylate the alpha subunit of eIF2 (eIF2␣), leading to a failure to initiate new rounds of translation (2). The ensuing stoppage in prote...

KSHV inhibits stress granule formation by viral ORF57 blocking PKR activation

“…In the course of drafting our manuscript for publication, Finnen and colleagues reported that viral vhs protein encoded by UL41 in herpes simplex virus type 2 (HSV-2) suppresses SG formation [ 78 ]. Although α-herpesvirus vhs and γ-herpesvirus vSOX are not homologs, both are RNA endonucleases and exert their host shutoff function by digesting host mRNAs [ 79 – 82 ] which are fundamental for SG formation [ 8 ].…”

“…Although α-herpesvirus vhs and γ-herpesvirus vSOX are not homologs, both are RNA endonucleases and exert their host shutoff function by digesting host mRNAs [ 79 – 82 ] which are fundamental for SG formation [ 8 ]. In assumption of KSHV vSOX encoded by viral ORF37 in blocking SG formation via a mechanism similar to HSV-2 vhs [ 78 ], we transfected both HEK293 and HeLa cells with a Flag-tagged KSHV vSOX expression vector for 24 h and stained the cells for TIA-1-specific SG formation in the presence or absence of vSOX after induction by arsenite for 30 min. We confirmed vSOX expression in HEK293 cells by anti-Flag antibody staining of HEK293 cells ( Fig 9A ) and by anti-Flag antibody Western blot ( Fig 9B ), but had difficulty to express vSOX in HeLa cells.…”

NUDT21 links mitochondrial IPS-1 to RLR-containing stress granules and activates host antiviral defense

“…Inhibition of SG formation by depletion of G3BP1 indeed suppressed type I IFN expression in response to infection with influenza A virus or Newcastle disease virus (NDV) (25, 26). The fact that many viral factors interfere with SG formation (27–31) also supports an antiviral function of SGs.…”

The Endonucleolytic RNA Cleavage Function of nsp1 of Middle East Respiratory Syndrome Coronavirus Promotes the Production of Infectious Virus Particles in Specific Human Cell Lines

“…Viral proteins that inhibit host gene expression, including nsp1 of CoV (49)(50)(51), are often major virulence factors (70)(71)(72)(73)(74)(75)(76)(77)(78)(79). Accordingly, it is likely that MERS-CoV nsp1 also plays a critical role in MERS-CoV pathogenesis.…”

“…Herpes simplex virus 1 and 2 virion host shutoff protein, Kaposi's sarcomaassociated herpesvirus SOX, and influenza A virus PA-X are virus-encoded RNases that induce endonucleolytic cleavage of host mRNAs, leading to host mRNA degradation (84,85). These viral endonucleases contribute to evasion of host antiviral responses, including IFN response and stress granule formation, and contribute to viral pathogenesis (70)(71)(72)(73)(74)(75)(76)(77)(78)(79), while roles of these virus-encoded endonucleases in production of virus particle have not been explored. To our knowledge, MERS-CoV nsp1 represents the first viral protein whose RNA cleavage-inducing function promotes virus assembly/budding.…”