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      D471G Mutation in LCMV-NP Affects its Ability to Self-associate and Results in a Dominant Negative Effect in Viral RNA Synthesis


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          Arenaviruses merit significant interest because several family members are etiological agents of severe hemorrhagic fevers, representing a major burden to public health. Currently, there are no FDA-licensed vaccines against arenaviruses and the only available antiviral therapy is limited to the use of ribavirin that is partially effective. Arenavirus nucleoprotein (NP) is found associated with the genomic RNA forming the viral ribonucleoproteins (vRNPs) that together with the polymerase (L) direct viral replication and transcription. Virion formation requires the recruitment of vRNPs into budding sites, a process in which the arenavirus matrix-like protein (Z) plays a major role. Therefore, proper NP-NP and NP-Z interactions are required for the generation of infectious progeny. In this work we demonstrate the role of the amino acid residue D471 in the self-association of lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP). Amino acid substitutions at this position abrogate NP oligomerization, affecting its ability to mediate replication and transcription of a minigenome reporter plasmid. However, its ability to interact with the Z protein, counteract the cellular interferon response and bind to dsRNA analogs was retained. Additionally, we also document the dominant negative effect of D471G mutation on viral infection, suggesting that NP self-association is an excellent target for the development of new antivirals against arenaviruses.

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          Transmission of lymphocytic choriomeningitis virus by organ transplantation.

          In December 2003 and April 2005, signs and symptoms suggestive of infection developed in two groups of recipients of solid-organ transplants. Each cluster was investigated because diagnostic evaluations were unrevealing, and in each a common donor was recognized. We examined clinical specimens from the two donors and eight recipients, using viral culture, electron microscopy, serologic testing, molecular analysis, and histopathological examination with immunohistochemical staining to identify a cause. Epidemiologic investigations, including interviews, environmental assessments, and medical-record reviews, were performed to characterize clinical courses and to determine the cause of the illnesses. Laboratory testing revealed lymphocytic choriomeningitis virus (LCMV) in all the recipients, with a single, unique strain of LCMV identified in each cluster. In both investigations, LCMV could not be detected in the organ donor. In the 2005 cluster, the donor had had contact in her home with a pet hamster infected with an LCMV strain identical to that detected in the organ recipients; no source of LCMV infection was found in the 2003 cluster. The transplant recipients had abdominal pain, altered mental status, thrombocytopenia, elevated aminotransferase levels, coagulopathy, graft dysfunction, and either fever or leukocytosis within three weeks after transplantation. Diarrhea, peri-incisional rash, renal failure, and seizures were variably present. Seven of the eight recipients died, 9 to 76 days after transplantation. One recipient, who received ribavirin and reduced levels of immunosuppressive therapy, survived. We document two clusters of LCMV infection transmitted through organ transplantation. Copyright 2006 Massachusetts Medical Society.
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            Quantification of lymphocytic choriomeningitis virus with an immunological focus assay in 24- or 96-well plates.

            Titers of lymphocytic choriomeningitis virus (LCMV) were determined on adherent fibroblast cell lines in 24- or 96-well plates. After absorption of virus by cells and 48 h incubation under a methylcellulose overlay, cell monolayers were fixed with 4% formaldehyde in phosphate-buffered saline, permeabilized by incubation in 0.5% Triton X-100 in balanced salt solution and then stained with a monoclonal rat anti-LCMV and a peroxidase-labeled second stage antibody. The sensitivity of the assay is within a factor of 2-4 of conventional plaquing methods. The method also detects poorly or non-plaquing LCMV isolates, and therefore drastically reduces the need for titration of LCMV in mice. The method is quicker (2-3 days), as compared to conventional methods (4-6 days) and less expensive in terms of work and materials.
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              The Lassa virus glycoprotein precursor GP-C is proteolytically processed by subtilase SKI-1/S1P.

              The surface glycoprotein of the Lassa virus, a member of the arenaviridae family, is synthesized as a 76-kDa precursor (GP-C) that is posttranslationally cleaved into an N-terminal 44-kDa subunit and a C-terminal membrane-anchored 36-kDa subunit. Cleavage occurs at the C-terminal end of the unusual recognition motif R-R-L-L. We show here that GP-C is cleaved in the endoplasmic reticulum by the cellular subtilase SKI-1/S1P, an enzyme that has so far been observed to be involved in cholesterol metabolism. Furthermore, we present evidence that only cleaved glycoprotein is incorporated into virions and that this is necessary for the formation of infectious virus. To our knowledge, there have been no previous reports of this type of viral glycoprotein processing, one that may be an interesting target for antiviral therapy.

                Author and article information

                16 October 2012
                October 2012
                : 4
                : 10
                : 2137-2161
                [1 ]Department of Microbiology and Immunology, University of Rochester, 601 Elmwood Avenue, Rochester, New York 14642; Email: eortizriano@ 123456urmc.rochester.edu (E.O-R); benson_cheng@ 123456urmc.rochester.edu (B.Y.H.C); luis_martinez@ 123456urmc.rochester.edu (L. M-S)
                [2 ]Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037; Email: juanct@ 123456scripps.edu (J.C. dlT)
                Author notes
                [* ] To whom correspondence should be addressed; Email: luis_martinez@ 123456urmc.rochester.edu (L.M-S), Tel.: +1-585-276-4733; juanct@ 123456scripps.edu (J.C. dlT), Tel.: +1-858-784-9462.
                © 2012 by the authors; licensee MDPI, Basel, Switzerland.

                This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/).

                : 09 August 2012
                : 21 September 2012
                : 26 September 2012

                Microbiology & Virology
                nucleoprotein,lymphocytic choriomeningitis virus,self-association,type i interferon,minigenome,dominant negative,z matrix protein,viral-like particles,double-stranded rna


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