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      Membrane Binding and Subcellular Localization of Retroviral Gag Proteins Are Differentially Regulated by MA Interactions with Phosphatidylinositol-(4,5)-Bisphosphate and RNA

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          ABSTRACT

          The matrix (MA) domain of HIV-1 mediates proper Gag localization and membrane binding via interaction with a plasma-membrane (PM)-specific acidic phospholipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2]. HIV-1 MA also interacts with RNA, which prevents Gag from binding to membranes containing phosphatidylserine, a prevalent cellular acidic phospholipid. These results suggest that the MA-bound RNA promotes PM-specific localization of HIV-1 Gag by blocking nonspecific interactions with cellular membranes that do not contain PI(4,5)P 2. To examine whether PI(4,5)P 2 dependence and RNA-mediated inhibition collectively determine MA phenotypes across a broad range of retroviruses and elucidate the significance of their interrelationships, we compared a panel of Gag-leucine zipper constructs (GagLZ) containing MA of different retroviruses. We found that in vitro membrane binding of GagLZ via HIV-1 MA and Rous sarcoma virus (RSV) MA is both PI(4,5)P 2 dependent and susceptible to RNA-mediated inhibition. The PM-specific localization and virus-like particle (VLP) release of these GagLZ proteins are severely impaired by overexpression of a PI(4,5)P 2-depleting enzyme, polyphosphoinositide 5-phosphatase IV (5ptaseIV). In contrast, membrane binding of GagLZ constructs that contain human T-lymphotropic virus type 1 (HTLV-1) MA, murine leukemia virus (MLV) MA, and human endogenous retrovirus K (HERV-K) MA is PI(4,5)P 2 independent and not blocked by RNA. The PM localization and VLP release of these GagLZ chimeras were much less sensitive to 5ptaseIV expression. Notably, single amino acid substitutions that confer a large basic patch rendered HTLV-1 MA susceptible to the RNA-mediated block, suggesting that RNA readily blocks MA containing a large basic patch, such as HIV-1 and RSV MA. Further analyses of these MA mutants suggest a possibility that HIV-1 and RSV MA acquired PI(4,5)P 2 dependence to alleviate the membrane binding block imposed by RNA.

          IMPORTANCE

          MA basic residues in the HIV-1 structural protein Gag interact with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2] and RNA. RNA inhibits HIV-1 MA binding to non-PI(4,5)P 2 acidic lipids. This inhibition may promote PM specificity of Gag membrane binding, an early essential step in virus assembly. However, whether and how relationships between these interactions have developed among retroviruses are poorly understood. In this study, by comparing diverse retroviral MA domains, we elucidated a strong correlation among PI(4,5)P 2 dependence, susceptibility to RNA-mediated inhibition, and cellular behaviors of Gag. Mutagenesis analyses suggest that a large basic patch on MA is sufficient to confer susceptibility to RNA-mediated inhibition but not for PI(4,5)P 2-dependent membrane binding. Our findings highlight RNA’s role as a general blocker of large basic patches and suggest a possibility that some retroviruses, including HIV-1, have evolved to bind PI(4,5)P 2, while others have adopted smaller basic patches on their MA domains, to overcome the RNA-mediated restriction of membrane binding.

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          Most cited references78

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          Protein structure homology modeling using SWISS-MODEL workspace.

          Homology modeling aims to build three-dimensional protein structure models using experimentally determined structures of related family members as templates. SWISS-MODEL workspace is an integrated Web-based modeling expert system. For a given target protein, a library of experimental protein structures is searched to identify suitable templates. On the basis of a sequence alignment between the target protein and the template structure, a three-dimensional model for the target protein is generated. Model quality assessment tools are used to estimate the reliability of the resulting models. Homology modeling is currently the most accurate computational method to generate reliable structural models and is routinely used in many biological applications. Typically, the computational effort for a modeling project is less than 2 h. However, this does not include the time required for visualization and interpretation of the model, which may vary depending on personal experience working with protein structures.
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            The distribution and function of phosphatidylserine in cellular membranes.

            Phosphatidylserine (PS) is the most abundant negatively charged phospholipid in eukaryotic membranes. PS directs the binding of proteins that bear C2 or gamma-carboxyglutamic domains and contributes to the electrostatic association of polycationic ligands with cellular membranes. Rather than being evenly distributed, PS is found preferentially in the inner leaflet of the plasma membrane and in endocytic membranes. The loss of PS asymmetry is an early indicator of apoptosis and serves as a signal to initiate blood clotting. This review discusses the determinants and functional implications of the subcellular distribution and membrane topology of PS.
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              Membrane phosphatidylserine regulates surface charge and protein localization.

              Electrostatic interactions with negatively charged membranes contribute to the subcellular targeting of proteins with polybasic clusters or cationic domains. Although the anionic phospholipid phosphatidylserine is comparatively abundant, its contribution to the surface charge of individual cellular membranes is unknown, partly because of the lack of reagents to analyze its distribution in intact cells. We developed a biosensor to study the subcellular distribution of phosphatidylserine and found that it binds the cytosolic leaflets of the plasma membrane, as well as endosomes and lysosomes. The negative charge associated with the presence of phosphatidylserine directed proteins with moderately positive charge to the endocytic pathway. More strongly cationic proteins, normally associated with the plasma membrane, relocalized to endocytic compartments when the plasma membrane surface charge decreased on calcium influx.
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                Author and article information

                Journal
                mBio
                MBio
                mbio
                mbio
                mBio
                mBio
                American Society of Microbiology (1752 N St., N.W., Washington, DC )
                2150-7511
                9 December 2014
                Nov-Dec 2014
                : 5
                : 6
                : e02202-14
                Affiliations
                [1]Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA
                Author notes
                Address correspondence to Akira Ono, akiraono@ 123456umich.edu .

                Editor Vinayaka R. Prasad, Albert Einstein College of Medicine

                Article
                mBio02202-14
                10.1128/mBio.02202-14
                4324246
                25491356
                9506ee0d-cbb4-4966-ac80-ceaa70cfa25e
                Copyright © 2014 Inlora 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
                : 27 October 2014
                : 7 November 2014
                Page count
                Pages: 13
                Categories
                Research Article
                Custom metadata
                November/December 2014

                Life sciences
                Life sciences

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