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      KSHV 2.0: A Comprehensive Annotation of the Kaposi's Sarcoma-Associated Herpesvirus Genome Using Next-Generation Sequencing Reveals Novel Genomic and Functional Features

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          Abstract

          Productive herpesvirus infection requires a profound, time-controlled remodeling of the viral transcriptome and proteome. To gain insights into the genomic architecture and gene expression control in Kaposi's sarcoma-associated herpesvirus (KSHV), we performed a systematic genome-wide survey of viral transcriptional and translational activity throughout the lytic cycle. Using mRNA-sequencing and ribosome profiling, we found that transcripts encoding lytic genes are promptly bound by ribosomes upon lytic reactivation, suggesting their regulation is mainly transcriptional. Our approach also uncovered new genomic features such as ribosome occupancy of viral non-coding RNAs, numerous upstream and small open reading frames (ORFs), and unusual strategies to expand the virus coding repertoire that include alternative splicing, dynamic viral mRNA editing, and the use of alternative translation initiation codons. Furthermore, we provide a refined and expanded annotation of transcription start sites, polyadenylation sites, splice junctions, and initiation/termination codons of known and new viral features in the KSHV genomic space which we have termed KSHV 2.0. Our results represent a comprehensive genome-scale image of gene regulation during lytic KSHV infection that substantially expands our understanding of the genomic architecture and coding capacity of the virus.

          Author Summary

          Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing agent in immunocompromised patients that establishes long-lasting infections in its hosts. Initially described in 1994 and extensively studied ever since, KSHV molecular biology is understood in broad outline, but many detailed questions are still to be resolved. After almost two decades, specific aspects pertaining to the organization of the KSHV genome as well as the fate of the viral transcripts during the productive stages of infection remain unexplored. Here we use a systematic genome-wide approach to investigate changes in gene and protein expression during the productive stage of infection known as the lytic cycle. We found that the viral genome has a large coding capacity, capable of generating at least 45% more products than initially anticipated by bioinformatic analyses alone, and that it uses multiple strategies to expand its coding capacity well beyond what is determined solely by the DNA sequence of its genome. We also provide an expanded and highly detailed annotation of known and new genomic features in KSHV. We have termed this new architectural and functional annotation KSHV 2.0. Our results indicate that viral genomes are more complex than anticipated, and that they are subject to tight mechanisms of regulation to ensure correct gene expression.

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          Most cited references103

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          Genome-wide analysis of mammalian promoter architecture and evolution.

          Mammalian promoters can be separated into two classes, conserved TATA box-enriched promoters, which initiate at a well-defined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3' UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them.
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            Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans.

            Upstream ORFs (uORFs) are mRNA elements defined by a start codon in the 5' UTR that is out-of-frame with the main coding sequence. Although uORFs are present in approximately half of human and mouse transcripts, no study has investigated their global impact on protein expression. Here, we report that uORFs correlate with significantly reduced protein expression of the downstream ORF, based on analysis of 11,649 matched mRNA and protein measurements from 4 published mammalian studies. Using reporter constructs to test 25 selected uORFs, we estimate that uORFs typically reduce protein expression by 30-80%, with a modest impact on mRNA levels. We additionally identify polymorphisms that alter uORF presence in 509 human genes. Finally, we report that 5 uORF-altering mutations, detected within genes previously linked to human diseases, dramatically silence expression of the downstream protein. Together, our results suggest that uORFs influence the protein expression of thousands of mammalian genes and that variation in these elements can influence human phenotype and disease.
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              Upstream open reading frames as regulators of mRNA translation.

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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                January 2014
                January 2014
                16 January 2014
                : 10
                : 1
                : e1003847
                Affiliations
                [1 ]Novartis Institute for Biomedical Research, Department of Infectious Diseases, Emeryville, California, United States of America
                [2 ]Novartis Vaccines and Diagnostics, Bioinformatics, Emeryville, California, United States of America
                [3 ]Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California, United States of America
                University of North Carolina at Chapel Hill, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: CA JSW DG. Performed the experiments: CA NSG ASM MH PB. Analyzed the data: CA BW AM. Contributed reagents/materials/analysis tools: AM ASM NSG. Wrote the paper: CA DG.

                Article
                PPATHOGENS-D-13-01657
                10.1371/journal.ppat.1003847
                3894221
                24453964
                a4ddd3bf-3712-4ab7-8a81-8d130b290a97
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 21 June 2013
                : 20 October 2013
                Page count
                Pages: 23
                Funding
                CA, ASM and AM were supported by a Novartis Presidential Postdoctoral fellowship. NSG was supported by a human frontier science program postdoctoral fellowship. JSW and DG were supported by the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Computational Biology
                Genomics
                Functional Genomics
                Genome Expression Analysis
                Genome Sequencing
                Sequence Analysis
                Microbiology
                Virology
                Viral Classification
                DNA viruses
                Molecular Cell Biology
                Gene Expression
                DNA transcription
                Protein Translation
                RNA processing

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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