Integrative functional genomics decodes herpes simplex virus 1

Funder: Alexander von Humboldt-Stiftung (Alexander von Humboldt Foundation); doi: https://doi.org/10.13039/100005156Abstract: The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution

Targeting RNA–Protein Interactions within the Human Immunodeficiency Virus Type 1 Lifecycle

RNA–protein interactions are vital throughout the HIV-1 life cycle for the successful production of infectious virus particles. One such essential RNA–protein interaction occurs between the full-length genomic viral RNA and the major structural protein of the virus. The initial interaction is between the Gag polyprotein and the viral RNA packaging signal (psi or Ψ), a highly conserved RNA structural element within the 5′-UTR of the HIV-1 genome, which has gained attention as a potential therapeutic target. Here, we report the application of a target-based assay to identify small molecules, which modulate the interaction between Gag and Ψ. We then demonstrate that one such molecule exhibits potent inhibitory activity in a viral replication assay. The mode of binding of the lead molecules to the RNA target was characterized by ¹H NMR spectroscopy

The complex of MCMV proteins and MHC class I evades NK cell control and drives the evolution of virus-specific activating Ly49 receptors

CMVs efficiently target MHC I molecules to avoid recognition by cytotoxic T cells. However, the lack of MHC I on the cell surface renders the infected cell susceptible to NK cell killing upon missing self recognition. To counter this, mouse CMV (MCMV) rescues some MHC I molecules to engage inhibitory Ly49 receptors. Here we identify a new viral protein, MATp1, that is essential for MHC I surface rescue. Rescued altered-self MHC I molecules show increased affinity to inhibitory Ly49 receptors, resulting in inhibition of NK cells despite substantially reduced MHC I surface levels. This enables the virus to evade recognition by licensed NK cells. During evolution, this novel viral immune evasion mechanism could have prompted the development of activating NK cell receptors that are specific for MATp1-modified altered-self MHC I molecules. Our study solves a long-standing conundrum of how MCMV avoids recognition by NK cells, unravels a fundamental new viral immune evasion mechanism, and demonstrates how this forced the evolution of virus-specific activating MHC I–restricted Ly49 receptors

Subcellular localization of read-through transcripts in HSV-1 infection.

<p>(a) Boxplots indicating the distribution of the percentage of transcription downstream of genes identified before infection in total, cytoplasmic, nucleoplasmic and chromatin-associated RNA. Numbers in boxes indicate median values. (b) Boxplots indicating the distribution of read-through at 8h p.i. HSV-1 infection in all RNA fractions. Numbers in boxes indicate median values. (c) Boxplots indicating the distribution of log2 ratios of nucleoplasm enrichment (= gene FPKM in nucleoplasmic RNA/ gene FPKM in cytoplasmic RNA) at 8h p.i. compared to uninfected cells. Ratios are shown separately for groups of genes with different amounts of read-through in 7-8h p.i. 4sU-RNA. The value for IRF1 is highlighted in red. (d) Heatmap of read counts (sum of 2 replicates) in total, cytoplasmic, nucleoplasmic and chromatin-associated RNA for the intergenic splicing events shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006954#ppat.1006954.g003" target="_blank">Fig 3C</a>. Results for the intergenic splicing junction connecting SRSF2 and JMJD6 are highlighted by a blue box. (e) Spearman correlation between read-through (calculated from 4sU-seq data) in all conditions and the percentage of transcription downstream of genes identified in chromatin-associated RNA of uninfected/untreated cells. Correlation to mock read-through values is shown below. Mock read-through values were calculated as described in methods and correlations were averaged for each condition. (f) Mapped sequencing reads (negative strand) for total (light/dark pink), cytoplasmic (light/dark green), nucleoplasmic (light/dark red) and chromatin-associated RNA (light/dark cyan) in uninfected cells (light colors) and at 8h p.i. (dark colors) for the IRF1 gene. Read coverage ranges and RefSeq gene annotation are indicated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006954#ppat.1006954.g001" target="_blank">Fig 1C</a>. (g) Scatterplot of read-through at 2-3h p.i. against the percentage of transcription downstream of genes identified in chromatin-associated RNA of uninfected/untreated cells. Colors indicate density of points (red = highest density, blue = lowest density).</p

Experimental set-up and read distribution downstream of genes.

<p>(a-b) Experimental set-up of our original 4sU-seq time-course for HSV-1 infection [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006954#ppat.1006954.ref010" target="_blank">10</a>] (a) and for the analysis of DoG transcription in salt and heat stress (b). 4sU-tagging was performed in 1h intervals before infection and stress as well as for the first 8h of HSV-1 infection and for the first 2h of salt and heat stress. Two biological 4sU-RNA replicates of each condition were subjected to Illumina sequencing (4sU-seq). (c-d) 4sU-seq read coverage (= number of mapped sequencing reads, sum of 2 replicates) for the genes SRSF3 (c) and GAPDH (d) in uninfected/untreated samples (gray), during HSV-1 infection (cyan) and in salt (yellow) and heat (red) stress. Read coverage ranges are indicated in square brackets on the y-axis. Only reads mapping to the corresponding strand are shown. RefSeq gene annotation is indicated below (blue). Boxes indicate coding regions and untranslated regions (UTRs; narrow boxes) and lines intronic regions. The transcribed strand is indicated by the direction of the arrowheads. (e-f) Distribution of reads mapping in sense direction downstream of annotated gene 3’ ends in HSV-1 infection (e) and salt and heat stress (f) (shown separately for the two replicates: solid lines = replicate 1, dashed lines = replicate 2). Only gene 3’ ends with no gene on either strand within the 100kb downstream region were considered. Read counts in sense direction to the gene were determined in 2kb windows downstream of gene 3’ ends and divided by window length and the total number of mapped reads. Reads counts mapping to the antisense strand are shown in Fig B in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006954#ppat.1006954.s003" target="_blank">S3 File</a>.</p

Extensive increase of downstream open chromatin during HSV-1 infection.

<p>(a) 4sU-seq read coverage for SRSF3 (strand-specific, grey = uninfected/unstressed, cyan = selected time-points of HSV-1 infection, yellow = 2h salt stress, red = 2h heat stress) as well as ATAC-seq data (no strand specificity, green = HSV-1 infection, brown = salt and heat stress) and identified open chromatin regions (OCRs, black lines). The OCRs shown here were derived from replicate 1. Read coverage ranges and RefSeq gene annotation are indicated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006954#ppat.1006954.g001" target="_blank">Fig 1C</a>. (b) Numbers of identified OCRs (y-axis) with a certain minimum length (x-axis) for all ATAC-seq samples. Results for replicates are shown separately (solid lines = replicate 1, dashed lines = replicate 2). (c) Empirical cumulative distribution functions indicating the fraction of genes (y-axis) with at most a certain dOCR length (average between two replicates) at 6h p.i. (x-axis). Genes were grouped according to read-through in 7-8h p.i. as described in methods. (d) 4sU-seq and ATAC-seq read coverage and identified OCRs in HSV-1 infection for the FBN2 and SLC12A2 genes. 4sU-seq reads are shown separately for positive [+] and negative [–] strand. ATAC-seq reads are not strand-specific. Read coverage ranges and RefSeq gene annotations are indicated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006954#ppat.1006954.g001" target="_blank">Fig 1C</a>. (e) Boxplots indicating the distribution of gene expression (FPKM) values for genes with >80% read-through at 7-8h p.i. and dOCR length of either ≥5kb (white) or <5kb (gray.) (f) Empirical cumulative distribution functions indicating the fraction of genes (y-axis) with at most a certain downstream dOCR length at 6h p.i. (x-axis) for highly-expressed genes (FPKM at 7-8h p.i. ≥2). Genes were grouped according to read-through in 7-8h p.i. as described in methods.</p

Global characteristics of DoTT/DoG transcription in salt and heat stress and HSV-1 infection.

<p>(a) Boxplots showing the distribution of read-through in salt (orange) and heat stress (red) and HSV-1 infection (cyan). Results for individual replicates are shown in Fig D in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006954#ppat.1006954.s003" target="_blank">S3 File</a>. (b) Median read-through values for each condition and time-point are plotted against the standard deviation in gene expression (= gene FPKM) fold-changes (log2). The gray curve indicates the result of a locally weighted polynomial regression (LOWESS) on all HSV-1 infection time-points. Spearman correlation (Cor) between median read-through and standard deviation in log2 expression fold-changes across all samples is also indicated. (c) 6-mers whose frequency in the 100nt up- or downstream of gene 3’ ends is significantly correlated to read-through in at least one sample (FDR adjusted p<0.0001). FDR adjusted p-values for all samples are color-coded (red for negative correlations, blue for positive correlations). (d) Boxplots showing the distribution of read-through in 2h salt and heat stress and 4-5h and 7-8h p.i. for genes without (w/o) or with (w/) at least one occurrence of the AAUAAA motif in the 100nt upstream of gene 3’ends. P-values of Wilcoxon rank sum tests comparing read-through in each sample between the two groups are indicated above the x-axis.</p

Comparison of DoTT/DoG transcription and association with aberrant splicing.

<p>(a) Spearman correlation for read-through values between all samples. (b) Heatmap of read-though for all 3,682 analyzed genes in salt and heat stress and HSV-1 infection (excluding the first two time-points with very low levels of read-through). Colors indicate read-though >5%. Hierarchical clustering was performed using average linkage clustering based on Euclidean distances. (c) Heatmap of read counts (sum of 2 replicates) for intergenic splicing events connecting exons of neighboring genes on the same strand. Junctions are annotated with the upstream and downstream gene symbol. Results for the intergenic splicing junction connecting SRSF2 and JMJD6 are highlighted by a blue box. Only junctions are shown with >2 reads covering at least 5bp of both exons in either 2h salt stress, 2h heat stress or 7-8h p.i. HSV-1 infection. Hierarchical clustering was performed as for (b). (d) Percentage of splicing junctions that are part of protein-coding transcripts, novel (using either 2 or 1 known exon boundary), nonsense-mediated-decay (NMD)-associated or only observed in a processed transcript. Results are shown separately for non-regulated, up-regulated and down-regulated junctions (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006954#sec012" target="_blank">methods</a> for definition) for 2h salt and heat stress and 4–5hs and 7-8h p.i. HSV-1 infection.</p