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      Lentiviral Vpx Accessory Factor Targets VprBP/DCAF1 Substrate Adaptor for Cullin 4 E3 Ubiquitin Ligase to Enable Macrophage Infection

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          Abstract

          Vpx is a small virion-associated adaptor protein encoded by viruses of the HIV-2/SIVsm lineage of primate lentiviruses that enables these viruses to transduce monocyte-derived cells. This probably reflects the ability of Vpx to overcome an as yet uncharacterized block to an early event in the virus life cycle in these cells, but the underlying mechanism has remained elusive. Using biochemical and proteomic approaches, we have found that Vpx protein of the pathogenic SIVmac 239 strain associates with a ternary protein complex comprising DDB1 and VprBP subunits of Cullin 4–based E3 ubiquitin ligase, and DDA1, which has been implicated in the regulation of E3 catalytic activity, and that Vpx participates in the Cullin 4 E3 complex comprising VprBP. We further demonstrate that the ability of SIVmac as well as HIV-2 Vpx to interact with VprBP and its associated Cullin 4 complex is required for efficient reverse transcription of SIVmac RNA genome in primary macrophages. Strikingly, macrophages in which VprBP levels are depleted by RNA interference resist SIVmac infection. Thus, our observations reveal that Vpx interacts with both catalytic and regulatory components of the ubiquitin proteasome system and demonstrate that these interactions are critical for Vpx ability to enable efficient SIVmac replication in primary macrophages. Furthermore, they identify VprBP/DCAF1 substrate receptor for Cullin 4 E3 ubiquitin ligase and its associated protein complex as immediate downstream effector of Vpx for this function. Together, our findings suggest a model in which Vpx usurps VprBP-associated Cullin 4 ubiquitin ligase to enable efficient reverse transcription and thereby overcome a block to lentivirus replication in monocyte-derived cells, and thus provide novel insights into the underlying molecular mechanism.

          Author Summary

          Monocyte-derived tissue macrophages play crucial roles in infection by primate lentiviruses. Human and simian lentiviruses of the HIV-2 and SIVsm/mac lineages encode a virion-bound virulence factor termed Vpx. Vpx is required to establish infection specifically of monocyte-derived cells, but the underlying molecular mechanism is unclear. In this study we characterize how the replication of SIVmac is blocked in the absence of Vpx and how Vpx overcomes this block. We find that Vpx is required for efficient reverse transcription of the incoming RNA genome, suggesting that Vpx acts early following virion entry into the macrophage, probably on events linked to virion uncoating and/or reverse transcription. We also identified a Vpx-associated ternary protein complex that is the key mediator of Vpx function specifically in macrophages. This complex links Vpx to the cellular machinery that mediates protein ubiquitination and degradation. Together, we describe the immediate downstream effector, the molecular machinery and a tentative mechanism that lentiviral Vpx uses to enable reverse transcription in macrophages. Our findings should lead to the conception of new strategies to control macrophage infection by human and simian lentiviruses.

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          Most cited references 34

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          DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics.

          The components of complex peptide mixtures can be separated by liquid chromatography, fragmented by tandem mass spectrometry, and identified by the SEQUEST algorithm. Inferring a mixture's source proteins requires that the identified peptides be reassociated. This process becomes more challenging as the number of peptides increases. DTASelect, a new software package, assembles SEQUEST identifications and highlights the most significant matches. The accompanying Contrast tool compares DTASelect results from multiple experiments. The two programs improve the speed and precision of proteomic data analysis.
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            A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1.

            Cul4 E3 ubiquitin ligases contain the cullin 4 scaffold and the triple beta propeller Ddb1 adaptor protein, but few substrate receptors have been identified. Here, we identify 18 Ddb1- and Cul4-associated factors (DCAFs), including 14 containing WD40 repeats. DCAFs interact with multiple surfaces on Ddb1, and the interaction of WD40-containing DCAFs with Ddb1 requires a conserved "WDXR" motif. DCAF2/Cdt2, which is related to S. pombe Cdt2, functions in Xenopus egg extracts and human cells to destroy the replication licensing protein Cdt1 in S phase and after DNA damage. Depletion of human Cdt2 causes rereplication and checkpoint activation. In Xenopus, Cdt2 is recruited to replication forks via Cdt1 and PCNA, where Cdt1 ubiquitylation occurs. These studies uncover diverse substrate receptors for Cul4 and identify Cdt2 as a conserved component of the Cul4-Ddb1 E3 that is essential to destroy Cdt1 and ensure proper cell cycle regulation of DNA replication.
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              DCAFs, the missing link of the CUL4-DDB1 ubiquitin ligase.

              The CUL4-DDB1 ubiquitin ligase regulates cell proliferation, survival, DNA repair, and genomic integrity through targeted ubiquitination of key regulators, yet the substrate receptors that dictate the specificity of this ubiquitination machinery have been largely unknown. Recent work identified a family of DDB1 and CUL4-associated factors (DCAFs) as substrate receptors, implicating a broad spectrum of cellular processes regulated by CUL4-DDB1.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                May 2008
                May 2008
                9 May 2008
                : 4
                : 5
                Affiliations
                [1 ]Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America
                [2 ]Stowers Institute for Medical Research, Kansas City, Missouri, United States of America
                [3 ]Skirball Institute of Biomolecular Medicine, New York, New York, United States of America
                University of Geneva, Switzerland
                Author notes

                Conceived and designed the experiments: SS JS. Performed the experiments: SS SKS JS. Analyzed the data: SS SKS LF MW JS. Contributed reagents/materials/analysis tools: SS SKS NM LF MW JS. Wrote the paper: SS JS.

                Article
                07-PLPA-RA-0950R2
                10.1371/journal.ppat.1000059
                2330158
                18464893
                Srivastava et al. 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.
                Page count
                Pages: 12
                Categories
                Research Article
                Biochemistry/Macromolecular Assemblies and Machines
                Cell Biology/Chemical Biology of the Cell
                Chemical Biology/Protein Chemistry and Proteomics
                Infectious Diseases/HIV Infection and AIDS
                Virology/Immunodeficiency Viruses
                Virology/Virulence Factors and Mechanisms

                Infectious disease & Microbiology

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