Unknown,Transcriptomics,Genomics,Proteomics

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Sequencing of HSV-1 genomic DNA to identify genomic variation resulting from selective pressure imposed by mutation of the PP1 binding site in the pUL21 gene

ABSTRACT: The pUL21 gene is essential for efficient replication and dissemination in herpes simplex virus 1 (HSV-1) but the molecular basis for its function is not well understood. Experimental work revealed that pUL21 is a virus-encoded adaptor of protein phosphatase 1 (PP1). Mutation of the specific motif in pUL21 responsible for PPI recruitment, followed by propagation in cell culture, was performed in order to identify the impact of preventing PP1 recruitment on the viral genome. This allowed identification of compensatory mutations disrupting the viral kinase gene pUS3, which restored viral growth and spread, revealing that pUL21 directly antagonises pUS3. Three mutant strains of HSV-1 were generated, mutating the conserved residues F242 and V243 in the PP1 binding sequence, individually and in combination (pUL21F242E, pUL21V243D, pUL21FV242AA), along with a knockout strain lacking pUL21 (ΔpUL21). These four virus stocks, plus wild-type HSV-1, were propagated in Vero cells for three passages. Genomic DNA was extracted from the P3 viruses and sequenced with Illumina. Reads were trimmed and mapped to an edited version of the HSV-1 genome (based on JQ673480.1) and read depth and variants were quantified.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Human alphaherpesvirus 1

SUBMITTER: Katherine Brown

PROVIDER: E-MTAB-10788 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The overall goal of the study was to use in vivo data combined with functional genomics to define gene expression signatures representative of a spectrum of HSV CNS infections. Innate immune deficiencies result in a spectrum of severe clinical outcomes following infection. In particular, there is a strong association between loss of the signal transducer and activator of transcription (Stat) pathway, breach of the blood-brain barrier (BBB), and virus-induced neuropathology. The gene signatures that characterize resistance, disease, and mortality in the virus-infected nervous system have not been defined. Herpes simplex virus type 1 (HSV-1) is commonly associated with encephalitis in humans, and humans and mice lacking Stat1 display increased susceptibility to HSV central nervous system (CNS) infections. In this study, two HSV-1 strains were used, KOS (wild type [WT]), and Δvhs, an avirulent recombinant lacking the virion host shutoff (vhs) function. In addition, two mouse strains were used: strain 129 (control) and a Stat1-deficient (Stat1(-/-)) strain. Using combinations of these virus and mouse strains, we established a model of infection resulting in three different outcomes: viral clearance without neurological disease (Δvhs infection of control mice), neurological disease followed by viral clearance (Δvhs infection of Stat1(-/-) mice and WT infection of control mice), or neurological disease followed by death (WT infection of Stat1(-/-) mice). Through the use of functional genomics on the infected brain stem and liver, we determined gene signatures that were representative of the three infection outcomes. Gender matched, 6- to 8- week old immunocompetent, control 129S6 and 129S6 Stat1 knockout mice were infected corneally with 2x10^6 PFU of either wild type HSV-1, a vhs-null HSV virus, or mock-infected. Brain stems and liver of individual mice were isolated at days 1, 3, 5 and 7 post-inoculation for microarray analysis. For microarray analysis, samples were collected from n=2 animals (1 male, 1 female) per mouse strain and virus strain for each time point. Equal masses of tissue were pooled from two mock-infected mice per time point and run on microarray.

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Sequencing of HSV-1 genomic DNA to identify genomic variation resulting from selective pressure imposed by mutation of the PP1 binding site in the pUL21 gene

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