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      Characterization of a Novel Hepadnavirus in the White Sucker (Catostomus commersonii) from the Great Lakes Region of the United States

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          ABSTRACT

          The white sucker Catostomus commersonii is a freshwater teleost often utilized as a resident sentinel. Here, we sequenced the full genome of a hepatitis B-like virus that infects white suckers from the Great Lakes Region of the United States. Dideoxy sequencing confirmed that the white sucker hepatitis B virus (WSHBV) has a circular genome (3,542 bp) with the prototypical codon organization of hepadnaviruses. Electron microscopy demonstrated that complete virions of approximately 40 nm were present in the plasma of infected fish. Compared to avi- and orthohepadnaviruses, sequence conservation of the core, polymerase, and surface proteins was low and ranged from 16 to 27% at the amino acid level. An X protein homologue common to the orthohepadnaviruses was not present. The WSHBV genome included an atypical, presumptively noncoding region absent in previously described hepadnaviruses. Phylogenetic analyses confirmed WSHBV as distinct from previously documented hepadnaviruses. The level of divergence in protein sequences between WSHBV and other hepadnaviruses and the identification of an HBV-like sequence in an African cichlid provide evidence that a novel genus of the family Hepadnaviridae may need to be established that includes these hepatitis B-like viruses in fishes. Viral transcription was observed in 9.5% (16 of 169) of white suckers evaluated. The prevalence of hepatic tumors in these fish was 4.9%, and only 2.4% of fish were positive for both virus and hepatic tumors. These results are not sufficient to draw inferences regarding the association of WSHBV and carcinogenesis in white sucker.

          IMPORTANCE We report the first full-length genome of a hepadnavirus from fishes. Phylogenetic analysis of this genome indicates divergence from genomes of previously described hepadnaviruses from mammalian and avian hosts and supports the creation of a novel genus. The discovery of this novel virus may better our understanding of the evolutionary history of hepatitis B-like viruses of other hosts. In fishes, knowledge of this virus may provide insight regarding possible risk factors associated with hepatic neoplasia in the white sucker. This may also offer another model system for mechanistic research.

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          Most cited references38

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          ProtTest: selection of best-fit models of protein evolution.

          Using an appropriate model of amino acid replacement is very important for the study of protein evolution and phylogenetic inference. We have built a tool for the selection of the best-fit model of evolution, among a set of candidate models, for a given protein sequence alignment. ProtTest is available under the GNU license from http://darwin.uvigo.es
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            Hepatitis B virus infection initiates with a large surface protein-dependent binding to heparan sulfate proteoglycans.

            Contrary to many other viruses, the initial steps of the hepatitis B virus (HBV) infection, including attachment to hepatocytes, specific receptor interactions, and membrane fusion, are unsolved. Using HepaRG cells as an in vitro cell culture system, we here report that HBV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cell-surface-associated heparan sulfate proteoglycans. Binding to GAGs requires the integrity of the pre-S domain as a part of the large (L-) viral envelope protein. HBV infection was abrogated by incubation of virions with heparin, but not the structurally related GAGs chondroitin sulfate A, B, and C. Infection was also abolished by suramin, a known inhibitor of duck hepatitis B virus infection or highly sulfated dextran sulfate. Polycationic substances such as poly-L-lysine, polybrene, and protamine also prevented infection, however, by addressing cellular components. Enzymatic removal of defined acidic carbohydrate structures from the cell surface using heparinase I/III or the obstruction of GAG synthesis by sodium chlorate inhibited HBV infection of HepaRG cells and, moreover, led to a reduction of HBV cell surface binding sites. The biochemical analysis showed selective binding of L-protein-enriched viral particles (virions or filaments) to heparin. GAG-dependent binding of HBV was improved by polyethylene glycol, a substance that specifically enhances HBV infection. HBV infection requires the initial attachment to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors. This interaction initializes the multistep entry process of HBV and cannot be bypassed by alternative routes.
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              Hepatitis B virus replication.

              Hepadnaviruses, including human hepatitis B virus (HBV), replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription: it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA, epsilon, as template, and depends on cellular chaperones; moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids. This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV (DHBV), now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cell-free systems. At this time, they can, unfortunately, not be complemented by three-dimensional structural information on the involved components. However, at least for the epsilon RNA element such information is emerging, raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal, will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                J Virol
                J. Virol
                jvi
                jvi
                JVI
                Journal of Virology
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                0022-538X
                1098-5514
                16 September 2015
                1 December 2015
                16 September 2015
                : 89
                : 23
                : 11801-11811
                Affiliations
                [a ]West Virginia University, School of Natural Resources, Morgantown, West Virginia, USA
                [b ]U.S. Geological Survey, Leetown Science Center, Kearneysville, West Virginia, USA
                [c ]U.S. Geological Survey, Western Fisheries Research Center, Seattle, Washington, USA
                Author notes
                Address correspondence to Luke R. Iwanowicz, liwanowicz@ 123456usgs.gov .

                Citation Hahn CM, Iwanowicz LR, Cornman RS, Conway CM, Winton JR, Blazer VS. 2015. Characterization of a novel hepadnavirus in the white sucker ( Catostomus commersonii) from the Great Lakes region of the United States. J Virol 89:11801–11811. doi: 10.1128/JVI.01278-15.

                Article
                01278-15
                10.1128/JVI.01278-15
                4645335
                26378165
                f427ba1b-7ac2-48c2-873d-3e9526927d65
                Copyright © 2015 Hahn et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 15 May 2015
                : 31 August 2015
                Page count
                Figures: 8, Tables: 3, Equations: 0, References: 49, Pages: 11, Words: 7996
                Categories
                Genetic Diversity and Evolution
                Spotlight

                Microbiology & Virology
                Microbiology & Virology

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