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Relationships between MA-RNA Binding in Cells and Suppression of HIV-1 Gag Mislocalization to Intracellular Membranes.

ABSTRACT: The HIV-1 Gag matrix (MA) domain mediates the localization of Gag to the plasma membrane (PM), the site for infectious virion assembly. The MA highly basic region (MA-HBR) interacts with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], a PM-specific acidic lipid. The MA-HBR also binds RNAs. To test whether acidic lipids alone determine PM-specific localization of Gag or whether MA-RNA binding also plays a role, we compared a panel of MA-HBR mutants that contain two types of substitutions at MA residues 25 and 26 or residues 29 and 31: Lys?Arg (KR) (25/26KR and 29/31KR) and Lys?Thr (KT) (25/26KT and 29/31KT). Consistent with the importance of the HBR charge in RNA binding, both KT mutants failed to bind RNA via MA efficiently, unlike the corresponding KR mutants. Both 25/26KT Gag-yellow fluorescent protein (YFP) and 29/31KT Gag-YFP bound nonspecifically to the PM and intracellular membranes, presumably via the myristoyl moiety and remaining MA basic residues. In contrast, 25/26KR Gag-YFP bound specifically to the PM, suggesting a role for the total positive charge and/or MA-bound RNA in navigating Gag to the PM. Unlike 29/31KT Gag-YFP, 29/31KR Gag-YFP was predominantly cytosolic and showed little intracellular membrane binding despite having a higher HBR charge. Therefore, it is likely that MA-RNA binding blocks promiscuous Gag membrane binding in cells. Notably, the introduction of a heterologous multimerization domain restored PI(4,5)P2-dependent PM-specific localization for 29/31KR Gag-YFP, suggesting that the blocking of PM binding is more readily reversed than that of intracellular membrane binding. Altogether, these cell-based data support a model in which MA-RNA binding ensures PM-specific localization of Gag via suppression of nonspecific membrane binding.IMPORTANCE The PM-specific localization of HIV-1 Gag is a crucial early step in infectious progeny production. The interaction between the MA highly basic region (MA-HBR) of Gag and the PM-specific lipid PI(4,5)P2 is critical for Gag localization to the PM. Additionally, in vitro evidence has indicated that MA-RNA binding prevents nonspecific binding of Gag to non-PI(4,5)P2-containing membranes. However, cell-based evidence supporting a role for HIV-1 MA-RNA binding in PM-specific subcellular localization has been scarce; thus, it remained possible that in cells, just the high basic charge or the PI(4,5)P2 binding ability is sufficient for MA to direct Gag specifically to the PM. The present study reveals for the first time an excellent correlation between RNA binding of the MA-HBR and inhibition of promiscuous Gag localization, both within the cells, and thereby provides cell-based evidence supporting a mechanism in which HIV-1 MA binding to RNA ensures the specific localization of Gag to the PM.

SUBMITTER: Thornhill D

PROVIDER: S-EPMC6854485 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Relationships between MA-RNA Binding in Cells and Suppression of HIV-1 Gag Mislocalization to Intracellular Membranes.

Thornhill Dishari D Olety Balaji B Ono Akira A

Journal of virology 20191113 23

The HIV-1 Gag matrix (MA) domain mediates the localization of Gag to the plasma membrane (PM), the site for infectious virion assembly. The MA highly basic region (MA-HBR) interacts with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], a PM-specific acidic lipid. The MA-HBR also binds RNAs. To test whether acidic lipids alone determine PM-specific localization of Gag or whether MA-RNA binding also plays a role, we compared a panel of MA-HBR mutants that contain two types of substitutions at ...[more]

PMID: 31511376

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Project description:UNLABELLED: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)P2]. 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)P2. To examine whether PI(4,5)P2 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)P2 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)P2-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)P2 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)P2 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)P2] and RNA. RNA inhibits HIV-1 MA binding to non-PI(4,5)P2 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)P2 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)P2-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)P2, while others have adopted smaller basic patches on their MA domains, to overcome the RNA-mediated restriction of membrane binding.

Project description:BackgroundHuman immunodeficiency virus type 1 (HIV-1) uses cellular proteins and machinery to ensure transmission to uninfected cells. Although the host proteins involved in the transport of viral components toward the plasma membrane have been investigated, the dynamics of this process remain incompletely described. Previously we showed that the double-stranded (ds)RNA-binding protein, Staufen1 is found in the HIV-1 ribonucleoprotein (RNP) that contains the HIV-1 genomic RNA (vRNA), Gag and other host RNA-binding proteins in HIV-1-producing cells. Staufen1 interacts with the nucleocapsid domain (NC) domain of Gag and regulates Gag multimerization on membranes thereby modulating HIV-1 assembly. The formation of the HIV-1 RNP is dynamic and likely central to the fate of the vRNA during the late phase of the HIV-1 replication cycle.ResultsDetailed molecular imaging of both the intracellular trafficking of virus components and of virus-host protein complexes is critical to enhance our understanding of factors that contribute to HIV-1 pathogenesis. In this work, we visualized the interactions between Gag and host proteins using bimolecular and trimolecular fluorescence complementation (BiFC and TriFC) analyses. These methods allow for the direct visualization of the localization of protein-protein and protein-protein-RNA interactions in live cells. We identified where the virus-host interactions between Gag and Staufen1 and Gag and IMP1 (also known as VICKZ1, IGF2BP1 and ZBP1) occur in cells. These virus-host interactions were not only detected in the cytoplasm, but were also found at cholesterol-enriched GM1-containing lipid raft plasma membrane domains. Importantly, Gag specifically recruited Staufen1 to the detergent insoluble membranes supporting a key function for this host factor during virus assembly. Notably, the TriFC experiments showed that Gag and Staufen1 actively recruited protein partners when tethered to mRNA.ConclusionsThe present work characterizes the interaction sites of key components of the HIV-1 RNP (Gag, Staufen1 and IMP1), thereby bringing to light where HIV-1 recruits and co-opts RNA-binding proteins during virus assembly.

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Relationships between MA-RNA Binding in Cells and Suppression of HIV-1 Gag Mislocalization to Intracellular Membranes.

Publications

Relationships between MA-RNA Binding in Cells and Suppression of HIV-1 Gag Mislocalization to Intracellular Membranes.

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