Pheasant, K. | bioRxiv Science

bioRxiv2022

Dysregulated nuclear export of the late herpes simplex virus 1 transcriptome through the vhs-VP22 axis uncouples virus cytopathic effect and virus production

Herpes simplex virus 1 (HSV1) expresses its genes in a classical cascade culminating in the production of large amounts of structural proteins to facilitate virus assembly. HSV1 lacking the virus protein VP22 ({Delta}22) exhibits late translational shutoff, a phenotype that has been attributed to the unrestrained activity of the virion host shutoff (vhs) protein, a virus-encoded endoribonuclease which induces mRNA degradation during infection. We have previously shown that vhs is also involved in regulating the nuclear-cytoplasmic compartmentalisation of the virus transcriptome, and in the absence of VP22 many virus transcripts are sequestered in the nucleus late in infection. Here we show that despite expressing minimal amounts of structural proteins and failing to plaque on human fibroblasts, the strain 17 {Delta}22 virus replicates and spreads as efficiently as Wt virus, but without causing cytopathic effect (CPE). Nonetheless, CPE-causing virus spontaneously appeared on {Delta}22-infected human fibroblasts, and four viruses isolated in this way had all acquired point mutations in vhs which rescued viral mRNA export and late protein translation. However, unlike a virus deleted for vhs, these viruses still induced the degradation of cellular mRNA, suggesting that vhs mutation in the absence of VP22 is necessary to overcome a disturbance in mRNA export rather than mRNA degradation. The ultimate outcome of secondary mutations in vhs is therefore the rescue of virus-induced CPE caused by late protein synthesis, and while there is a clear selective pressure on HSV1 to mutate vhs for optimal production of late structural proteins, the purpose of this is over and above that of virus production. Author SummaryHSV is a human pathogen that lytically infects cells of the epidermis. Following viral genome replication, structural proteins are produced in abundance to enable the rapid assembly and release of large quantities of infectious progeny. Infected cells also exhibit cytopathic effect (CPE), morphological changes that are exemplified by cell rounding and the breakage of cell-to-cell contacts, facilitating virus dissemination. Here we show that HSV1 with a mutation that results in the nuclear retention of viral mRNA and concomitant shutdown of late protein synthesis, also fails to cause CPE. However, unexpectedly, we found that this virus is still able to release large numbers of infectious virus which can spread between cells without any evidence of cell damage. Nonetheless, despite efficient virus productivity, this virus spontaneously mutates to rescue late protein production and CPE, with mutations mapping to the process of mRNA export. There is therefore a clear selective pressure on HSV1 to optimize the synthesis of late structural proteins, but the purpose of this is over and above that of virus production, a result that has implications for why viruses in general express such large amounts of structural proteins.

microbiology↗

bioRxiv2018

Nuclear-cytoplasmic compartmentalization of the herpes simplex virus 1 infected cell transcriptome is co-ordinated by the viral endoribonuclease vhs and cofactors to facilitate the translation of late proteins

HSV1 encodes an endoribonuclease termed virion host shutoff (vhs) that is produced late in infection and packaged into virions. Paradoxically, vhs is active against not only host but also virus transcripts, and is involved in host shutoff and the temporal expression of the virus transcriptome. Two other virus proteins - VP22 and VP16 - are proposed to regulate vhs to prevent uncontrolled and lethal mRNA degradation but their mechanism of action is unknown. We have performed dual transcriptomic analysis and single-cell mRNA FISH of human fibroblasts, a cell type where in the absence of VP22, HSV1 infection results in extreme translational shutoff. In Wt infection, host mRNAs exhibited a wide range of susceptibility to vhs ranging from resistance to 1000-fold reduction, a variation that was independent of their relative abundance or transcription rate. However, vhs endoribonuclease activity was not found to be overactive against any of the cell transcriptome in {Delta}22-infected cells but rather was delayed, while its activity against the virus transcriptome and in particular late mRNA was minimally enhanced. Intriguingly, immediate-early and early transcripts exhibited vhs-dependent nuclear retention later in Wt infection but late transcripts were cytoplasmic. However, in the absence of VP22, not only early but also late transcripts were retained in the nucleus, a characteristic that extended to cellular transcripts that were not efficiently degraded by vhs. Moreover, the ability of VP22 to bind VP16 enhanced but was not fundamental to the rescue of vhs-induced nuclear retention of late transcripts. Hence, translational shutoff in HSV1 infection is primarily a result of vhs-induced nuclear retention and not degradation of infected cell mRNA. We have therefore revealed a new mechanism whereby vhs and its co-factors including VP22 elicit a temporal and spatial regulation of the infected cell transcriptome, thus co-ordinating efficient late protein production.\n\nAuthor SummaryHerpesviruses are large DNA viruses that replicate in the nucleus and express their genes by exploiting host cell mRNA biogenesis mechanisms including transcription, nuclear export, translation and turnover. As such, these viruses express multiple factors that enable the appropriation of cellular pathways for optimal virus production, and work in concert to shut off host gene expression and to overexpress virus genes in a well-described cascade that occurs in a temporal pattern of immediate-early, early and late proteins. We have analysed global and single cell changes in the host and virus transcriptome to uncover a novel mechanism by which the viral endoribonuclease, termed vhs, turns off early virus gene expression. This is achieved through the vhs-induced nuclear retention of the entire infected cell transcriptome at the onset of late gene expression. To enable the switch from early to late protein production the virus then requires a second factor called VP22 to specifically inhibit the nuclear retention of late transcripts allowing their translation in the cytoplasm. In this way, HSV1 elicits a temporal and spatial regulation of the infected cell transcriptome to co-ordinate efficient late protein production, a process that may be relevant to herpesviruses in general.

microbiology↗