16
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT), is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex – the reverse transcription complex (RTC) – consisting of viral genomic RNA associated with viral proteins (including RT) and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein – the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF). We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the C-terminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT.

          Related collections

          Most cited references40

          • Record: found
          • Abstract: found
          • Article: not found

          HuR and mRNA stability.

          An important mechanism of posttranscriptional gene regulation in mammalian cells is the rapid degradation of messenger RNAs (mRNAs) signaled by AU-rich elements (AREs) in their 3' untranslated regions. HuR, a ubiquitously expressed member of the Hu family of RNA-binding proteins related to Drosophila ELAV, selectively binds AREs and stabilizes ARE-containing mRNAs when overexpressed in cultured cells. This review discusses mRNA decay as a general form of gene regulation, decay signaled by AREs, and the role of HuR and its Hu-family relatives in antagonizing this mRNA degradation pathway. The influence of newly identified protein ligands to HuR on HuR function in both normal and stressed cells may explain how ARE-mediated mRNA decay is regulated in response to environmental change.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases the in vivo stability of ARE-containing mRNAs.

            The messenger RNAs of many proto-oncogenes, cytokines and lymphokines are targeted for rapid degradation through AU-rich elements (AREs) located in their 3' untranslated regions (UTRs). HuR, a ubiquitously expressed member of the Elav family of RNA binding proteins, exhibits specific affinities for ARE-containing RNA sequences in vitro which correlate with their in vivo decay rates, thereby implicating HuR in the ARE-mediated degradation pathway. We have transiently transfected HuR into mouse L929 cells and observed that overexpression of HuR enhances the stability of beta-globin reporter mRNAs containing either class I or class II AREs. The increase in mRNA stability parallels the level of HuR overexpression, establishing an in vivo role for HuR in mRNA decay. Furthermore, overexpression of HuR deletion mutants lacking RNA recognition motif 3 (RRM 3) does not exert a stabilizing effect, indicating that RRM 3 is important for HuR function. We have also developed polyclonal anti-HuR antibodies. Immunofluorescent staining of HeLa and L929 cells using affinity-purified anti-HuR antibody shows that both endogenous and overexpressed HuR proteins are localized in the nucleus. By forming HeLa-L929 cell heterokaryons, we demonstrate that HuR shuttles between the nucleus and cytoplasm. Thus, HuR may initially bind to ARE-containing mRNAs in the nucleus and provide protection during and after their export to the cytoplasmic compartment.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Identification of a target RNA motif for RNA-binding protein HuR.

              HuR, a protein that binds to specific mRNA subsets, is increasingly recognized as a pivotal posttranscriptional regulator of gene expression. Here, HuR was immunoprecipitated under conditions that preserved HuR-RNA interactions, and HuR-bound target mRNAs were identified by cDNA array hybridization. Analysis of primary sequences and secondary structures shared among HuR targets led to the identification of a 17- to 20-base-long RNA motif rich in uracils. This HuR motif was found in almost all mRNAs previously reported to be HuR targets, was located preferentially within 3' untranslated regions of all unigene transcripts examined, and was conserved in >50% of human and mouse homologous genes. Importantly, the HuR motif allowed the successful prediction and subsequent validation of novel HuR targets from gene databases. This study describes an HuR target RNA motif and presents a general strategy for identifying target motifs for RNA-binding proteins.
                Bookmark

                Author and article information

                Journal
                Retrovirology
                Retrovirology
                BioMed Central
                1742-4690
                2008
                10 June 2008
                : 5
                : 47
                Affiliations
                [1 ]Institut Cochin, Université Paris Descartes, CNRS (UMR8104), Paris, France
                [2 ]Inserm, U567, Paris, France
                [3 ]Architecture et réactivité de l'ARN, UPR 9002 CNRS, 15 rue René Descartes, 67084 Strasbourg, France
                [4 ]Hybrigenics S.A., F-75014 Paris, France
                [5 ]current address : University Children's Hospital, Division of Immunology, Steinwiesstrasse 75, CH-8032, Zürich, Switzerland
                [6 ]current address : CellVir, 4 rue Pierre Fontaine, 9100 Evry, France
                [7 ]Current Address: Institutes of Life and Health Engineering, Jinan University, 601 Huang Pu Avenue West, Guangzhou 510632, China.
                Article
                1742-4690-5-47
                10.1186/1742-4690-5-47
                2441633
                18544151
                f98374c9-c046-4dac-b5cb-5ff4d2956590
                Copyright © 2008 Lemay et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 9 January 2008
                : 10 June 2008
                Categories
                Research

                Microbiology & Virology
                Microbiology & Virology

                Comments

                Comment on this article