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The latency-associated transcript locus of herpes simplex virus 1 is a virulence determinant in human skin.

ABSTRACT: Herpes simplex virus 1 (HSV-1) infects skin and mucosal epithelial cells and then travels along axons to establish latency in the neurones of sensory ganglia. Although viral gene expression is restricted during latency, the latency-associated transcript (LAT) locus encodes many RNAs, including a 2 kb intron known as the hallmark of HSV-1 latency. Here, we studied HSV-1 infection and the role of the LAT locus in human skin xenografts in vivo and in cultured explants. We sequenced the genomes of our stock of HSV-1 strain 17syn+ and seven derived viruses and found nonsynonymous mutations in many viral proteins that had no impact on skin infection. In contrast, deletions in the LAT locus severely impaired HSV-1 replication and lesion formation in skin. However, skin replication was not affected by impaired intron splicing. Moreover, although the LAT locus has been implicated in regulating gene expression in neurones, we observed only small changes in transcript levels that were unrelated to the growth defect in skin, suggesting that its functions in skin may be different from those in neurones. Thus, although the LAT locus was previously thought to be dispensable for lytic infection, we show that it is a determinant of HSV-1 virulence during lytic infection of human skin.

SUBMITTER: Vanni EAH

PROVIDER: S-EPMC7794027 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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The latency-associated transcript locus of herpes simplex virus 1 is a virulence determinant in human skin.

Vanni Emilia A H EAH Foley Joseph W JW Davison Andrew J AJ Sommer Marvin M Liu Dongmei D Sung Phillip P Moffat Jennifer J Zerboni Leigh L Arvin Ann M AM

PLoS pathogens 20201228 12

Herpes simplex virus 1 (HSV-1) infects skin and mucosal epithelial cells and then travels along axons to establish latency in the neurones of sensory ganglia. Although viral gene expression is restricted during latency, the latency-associated transcript (LAT) locus encodes many RNAs, including a 2 kb intron known as the hallmark of HSV-1 latency. Here, we studied HSV-1 infection and the role of the LAT locus in human skin xenografts in vivo and in cultured explants. We sequenced the genomes of o ...[more]

PMID: 33370402

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Project description:During herpes simplex virus (HSV) latency, most viral genes are silenced, with the exception of one region of the genome encoding the latency-associated transcript (LAT). This long noncoding RNA was originally described as having a role in enhancing HSV-1 reactivation. However, subsequent evidence showing that the LAT blocked apoptosis and promoted efficient establishment of latency suggested that its effects on reactivation were secondary to establishment. Here, we utilized an adeno-associated virus (AAV) vector to deliver a LAT-targeting hammerhead ribozyme to HSV-1-infected neurons of rabbits after the establishment of HSV-1 latency. The rabbits were then induced to reactivate latent HSV-1. Using this model, we show that decreasing LAT levels in neurons following the establishment of latency reduced the ability of the virus to reactivate. This demonstrates that the HSV-1 LAT RNA has a role in reactivation that is independent of its function in establishment of latency. In addition, these results suggest the potential of AAV vectors expressing LAT-targeting ribozymes as a potential therapy for recurrent HSV disease such as herpes stromal keratitis, a leading cause of infectious blindness.IMPORTANCE Herpes simplex virus (HSV) establishes a lifelong infection and remains dormant (latent) in our nerve cells. Occasionally HSV reactivates to cause disease, with HSV-1 typically causing cold sores whereas HSV-2 is the most common cause of genital herpes. The details of how HSV reactivates are largely unknown. Most of HSV's genes are silent during latency, with the exception of RNAs made from the latency-associated transcript (LAT) region. While viruses that make less LAT do not reactivate efficiently, these viruses also do not establish latency as efficiently. Here we deliver a ribozyme that can degrade the LAT to the nerve cells of latently infected rabbits using a gene therapy vector. We show that this treatment blocks reactivation in the majority of the rabbits. This work shows that the LAT RNA is important for reactivation and suggests the potential of this treatment as a therapy for treating HSV infections.

Project description:The HSV-1 latency-associated transcript (LAT) locus contains two small noncoding RNA (sncRNA) sequences (sncRNA1 and sncRNA2) that are not microRNAs (miRNAs). We recently reported that sncRNA1 is more important for in vitro activation of the herpesvirus entry mediator than sncRNA2, but its in vivo function is not known. To determine the role, if any, of sncRNA1 during herpes simplex virus 1 (HSV-1) infection in vivo, we deleted the 62-bp sncRNA1 sequence in HSV-1 strain McKrae using dLAT2903 (LAT-minus) virus, creating ΔsncRNA1 recombinant virus. Deletion of the sncRNA1 in ΔsncRNA1 virus was confirmed by complete sequencing of ΔsncRNA1 virus and its parental virus (i.e., McKrae). Replication of ΔsncRNA1 virus in tissue culture or in the eyes of infected mice was similar to that of HSV-1 strain McKrae and dLAT2903 viruses. However, the absence of sncRNA1 significantly reduced the levels of ICP0, ICP4, and gB but not LAT transcripts in infected rabbit skin cells in vitro. In contrast, the absence of sncRNA1 did reduce LAT expression in trigeminal ganglia (TG), but not in corneas, by day 5 postinfection (p.i.) in infected mice. Levels of eye disease in mice infected with ΔsncRNA1 or McKrae virus were similar, and despite reduced LAT levels in TG during acute ΔsncRNA1 infection, McKrae and ΔsncRNA1 viruses did not affect latency or reactivation on day 28 p.i. However, mice infected with ΔsncRNA1 virus were more susceptible to ocular infection than their wild-type (WT) counterparts. Expression of host immune response genes in corneas and TG of infected mice during primary infection showed reduced expression of beta interferon (IFNβ) and IFNγ and altered activation of key innate immune pathways, such as the JAK-STAT pathway in ΔsncRNA1 virus compared with parental WT virus. Our results reveal novel functions for sncRNA1 in upregulating the host immune response and suggest that sncRNA1 has a protective role during primary ocular HSV-1 infection. IMPORTANCE HSV-1 latency-associated transcript (LAT) plays a major role in establishing latency and reactivation; however, the mechanism by which LAT controls these processes is largely unknown. In this study, we sought to establish the role of the small noncoding RNA1 (sncRNA1) encoded within LAT during HSV-1 ocular infection. Our results suggest that sncRNA1 has a protective role during acute ocular infection by modulating the innate immune response to infection.

Project description:Herpes simplex virus type 1 (HSV-1) establishes latency in human sensory ganglia, during which time the viral genome is transcriptionally silent with the exception of the latency-associated transcripts (LATs). The most abundant LAT is a 2-kb RNA whose biosynthesis is poorly characterized. The 2-kb LAT may be a primary transcript, or its synthesis may involve splicing and/or other forms of processing. Two potential RNA polymerase II promoters (LAP1 and LAP2) upstream of the 2-kb LAT 5' end have been identified. To investigate the role played by LAP1 and LAP2 in the synthesis of the 2-kb LAT under lytic and latent conditions, we analyzed HSV-1 mutants which contain deletions of one or both of these promoters. During lytic infection in cell culture, the cis elements critical for the normal accumulation of the 2-kb LAT were mapped to LAP2, while LAP1 sequences were largely dispensable. The 5' ends of the major 2-kb LATs produced by the wild-type and LAP deletion viruses were examined by primer extension analysis and were all found to be identical (+/- 2 bp). The accumulation of the 2-kb LAT during latent infections of murine trigeminal ganglia was examined by Northern (RNA) blot and by reverse transcription-PCR. In contrast to the results found in lytic infections, the critical cis elements needed for 2-kb LAT accumulation during latency were mapped to LAP1. Deletion of LAP1 resulted in a 500-fold reduction in 2-kb LAT accumulation, whereas deletion of LAP2 resulted in only a 2- to 3-fold reduction. Deletion of both LAP1 and LAP2 resulted in undetectable levels of the 2-kb LAT. Our results indicate that both LAP1 and LAP2 are critical for 2-kb LAT expression but under different conditions. LAP1 is essential for LAT expression during latency, while LAP2 is primarily responsible for LAT expression in lytic infections in cell culture. LAP1 and LAP2 may prove to be functionally independent promoter elements that control 2-kb LAT expression during different stages of HSV-1 infections.

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The latency-associated transcript locus of herpes simplex virus 1 is a virulence determinant in human skin.

Publications

The latency-associated transcript locus of herpes simplex virus 1 is a virulence determinant in human skin.

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