Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers.
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ABSTRACT:
SUBMITTER: Hendrix J
PROVIDER: S-EPMC4539982 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
| S-EPMC2887380 | biostudies-literature
Project description:During HIV-1 assembly, the retroviral structural protein Gag forms an immature capsid, containing thousands of Gag molecules, at the plasma membrane (PM). Interactions between Gag nucleocapsid (NC) and viral RNA (vRNA) are thought to drive assembly, but the exact roles of these interactions have remained poorly understood. Since previous studies have shown that Gag dimer- or trimer-forming mutants (GagZiL) lacking an NC domain can form immature capsids independent of RNA binding, it is often hypothesized that vRNA drives Gag assembly by inducing Gag to form low-ordered multimers, but is dispensable for subsequent assembly. In this study, we examined the role of vRNA in HIV-1 assembly by characterizing the distribution and mobility of Gag and Gag NC mutants at the PM using photoactivated localization microscopy (PALM) and single-particle tracking PALM (spt-PALM). We showed that both Gag and GagZiL assembly involve a similar basic assembly unit, as expected. Unexpectedly, the two proteins underwent different subsequent assembly pathways, with Gag cluster density increasing asymptotically, while GagZiL cluster density increased linearly. Additionally, the directed movement of Gag, but not GagZiL, was maintained at a constant speed, suggesting that the two proteins experience different external driving forces. Assembly was abolished when Gag was rendered monomeric by NC deletion. Collectively, these results suggest that, beyond inducing Gag to form low-ordered multimer basic assembly units, vRNA is essential in scaffolding and maintaining the stability of the subsequent assembly process. This finding should advance the current understanding of HIV-1 and, potentially, other retroviruses.
| S-EPMC6042153 | biostudies-literature
Project description:During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected in situ as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing unspliced HIV-1 RNA.
| S-EPMC5940231 | biostudies-literature
Project description:MicroRNAs (miRNAs) are small, 18-22 nt long, noncoding RNAs that act as potent negative gene regulators in a variety of physiological and pathological processes. To repress gene expression, miRNAs are packaged into RNA-induced silencing complexes (RISCs) that target mRNAs for degradation and/or translational repression in a sequence-specific manner. Recently, miRNAs have been shown to also interact with proteins outside RISCs, impacting cellular processes through mechanisms not involving gene silencing. Here, we define a previously unappreciated activity of miRNAs in inhibiting RNA-protein interactions that in the context of HIV-1 biology blocks HIV virus budding and reduces virus infectivity. This occurs by miRNA binding to the nucleocapsid domain of the Gag protein, the main structural component of HIV-1 virions. The resulting miRNA-Gag complexes interfere with viral-RNA-mediated Gag assembly and viral budding at the plasma membrane, with imperfectly assembled Gag complexes endocytosed and delivered to lysosomes. The blockade of virus production by miRNA is reversed by adding the miRNA's target mRNA and stimulated by depleting Argonaute-2, suggesting that when miRNAs are not mediating gene silencing, they can block HIV-1 production through disruption of Gag assembly on membranes. Overall, our findings have significant implications for understanding how cells modulate HIV-1 infection by miRNA expression and raise the possibility that miRNAs can function to disrupt RNA-mediated protein assembly processes in other cellular contexts.
| S-EPMC4084429 | biostudies-literature
Project description:BackgroundHuman endogenous retroviruses (HERVs), the remnants of ancient retroviral infections, constitute approximately 8% of human genomic DNA. Since HERV-K Gag expression is induced by HIV-1 Tat in T cells, induced HERV-K proteins could affect HIV-1 replication. Indeed, previously we showed that HERV-K Gag and HIV-1 Gag coassemble and that this appears to correlate with the effect of HERV-K Gag expression on HIV-1 particle release and its infectivity. We further showed that coassembly requires both MA and NC domains, which presumably serve as scaffolding for Gag via their abilities to bind membrane and RNA, respectively. Notably, however, despite possessing these abilities, MLV Gag failed to coassemble with HIV-1 Gag and did not affect assembly and infectivity of HIV-1 particles. It is unclear how the specificity of coassembly is determined.ResultsHere, we showed that coexpression of HERV-K Gag with HIV-1 Gag changed size and morphology of progeny HIV-1 particles and severely diminished infectivity of such progeny viruses. We further compared HERV-K-MLV chimeric constructs to identify molecular determinants for coassembly specificity and for inhibition of HIV-1 release efficiency and infectivity. We found that the CA N-terminal domain (NTD) of HERV-K Gag is important for the reduction of the HIV-1 release efficiency, whereas both CA-NTD and major homology region of HERV-K Gag contribute to colocalization with HIV-1 Gag. Interestingly, these regions of HERV-K Gag were not required for reduction of progeny HIV-1 infectivity.ConclusionsOur results showed that HERV-K Gag CA is important for reduction of HIV-1 release and infectivity but the different regions within CA are involved in the effects on the HIV-1 release and infectivity. Altogether, these findings revealed that HERV-K Gag interferes the HIV-1 replication by two distinct molecular mechanisms.
| S-EPMC5406883 | biostudies-literature
Project description:Understanding how amyloid-? peptide interacts with living cells on a molecular level is critical to development of targeted treatments for Alzheimer's disease. Evidence that oligomeric A? interacts with neuronal cell membranes has been provided, but the mechanism by which membrane binding occurs and the exact stoichiometry of the neurotoxic aggregates remain elusive. Physiologically relevant experimentation is hindered by the high A? concentrations required for most biochemical analyses, the metastable nature of A? aggregates, and the complex variety of A? species present under physiological conditions. Here we use single molecule microscopy to overcome these challenges, presenting direct optical evidence that small A?(1-40) oligomers bind to living neuroblastoma cells at physiological A? concentrations. Single particle fluorescence intensity measurements indicate that cell-bound A? species range in size from monomers to hexamers and greater, with the majority of bound oligomers falling in the dimer-to-tetramer range. Furthermore, while low-molecular weight oligomeric species do form in solution, the membrane-bound oligomer size distribution is shifted towards larger aggregates, indicating either that bound A? oligomers can rapidly increase in size or that these oligomers cluster at specific sites on the membrane. Calcium indicator studies demonstrate that small oligomer binding at physiological concentrations induces only mild, sporadic calcium leakage. These findings support the hypothesis that small oligomers are the primary A? species that interact with neurons at physiological concentrations.
| S-EPMC3162019 | biostudies-literature
Project description:In the area of artificial genetics the development of non-enzymatic self-organization of synthetic building blocks is critical for both providing biopolymers with extended functions and understanding prebiotic processes. While reversibility is believed to have played a major role in early functional genetic materials, we previously reported an efficient DNA-templated ligation of suitably designed 5'-end boronic acid and 3'-end ribonucleosidic half-sequences. Here, we report the enzyme-free and activation-free DNA- and RNA-templated assembly of bifunctional hexamers. The reversible assembly was found to be regio- and sequence specific and the stabilities of the resulting duplexes were compared to their nicked counterparts. To go further with our understanding of this unprecedented process we also examined an auto-templated duplex self-assembly representing a key step toward the evolution of sequence-defined synthetic polymers.
| S-EPMC5588539 | biostudies-literature
Project description:We report a computational model for the assembly of HIV-1 Gag into immature viral particles at the plasma membrane. To reproduce experimental structural and kinetic properties of assembly, a process occurring on the order of minutes, a coarse-grained representation consisting of a single particle per Gag molecule is developed. The model uses information relating the functional interfaces implicated in Gag assembly, results from cryo electron-tomography, and biophysical measurements from fluorescence microscopy, such as the dynamics of Gag assembly at single virions. These experimental constraints eliminated many classes of potential interactions, and narrowed the model to a single interaction scheme with two non-equivalent interfaces acting to form Gags into a hexamer, and a third interface acting to link hexamers together. This model was able to form into a hexameric structure with correct lattice spacing and reproduced biologically relevant growth rates. We explored the effect of genomic RNA seeding punctum growth, finding that RNA may be a factor in locally concentrating Gags to initiate assembly. The simulation results infer that completion of assembly cannot be governed simply by Gag binding kinetics. However the addition of membrane curvature suggests that budding of the virion from the plasma membrane could factor into slowing incorporation of Gag at an assembly site resulting in virions of the same size and number of Gag molecules independent of Gag concentration or the time taken to complete assembly. To corroborate the results of our simulation model, we developed an analytic model for Gag assembly finding good agreement with the simulation results.
| S-EPMC5909904 | biostudies-literature
Project description:Human immunodeficiency virus type 1 (HIV-1) Gag-RNA interactions are required for virus assembly. However, our prior study found that a defect in particle production exhibited by an HIV-1 proviral mutant with a severe deletion in the RNA-binding nucleocapsid (NC) region of Gag, NX, could be reversed by eliminating its protease activity. While our follow-up study indicated that a secondary RNA-binding site in Gag can also provide the required RNA-binding function, how protease activity inhibits NX virion production is still unclear. Therefore, we tested three possible mechanisms: NX virions are unstable and fall apart after budding; NX Gag assembly is slowed, allowing protease processing to start before particle formation; or the protease region within NX Gag-Pol becomes activated prematurely and processes the assembling Gag. We found that NX particles were as stable as wild-type virions. Furthermore, even a modest slowing of protease activity could rescue NX. Pulse-chase analysis revealed that the initial particle production by NC-deleted Gag was delayed compared to that of wild type Gag, but once started, the rate of production was similar, revealing a defect in the initiation of assembly. Wild-type Gag particle production was not eliminated or decreased in the presence of excess NX Gag-Pol, inconsistent with a premature activation of protease. Overall, these results indicate that the particle formation defect of NX is due to delayed initiation of assembly caused by the absence of NC in Gag, making it vulnerable to protease processing before budding can occur. Therefore, NC plays an important initiating role in Gag assembly.
| S-EPMC2708646 | biostudies-literature
Project description:Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements: the 5' packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM.IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV-1) virus particles at the plasma membrane (PM). Artificially tethering viral mRNAs encoding Gag capsid proteins (gag-pol mRNAs) to distinct non-PM subcellular locales, such as cytoplasmic vesicles or the actin cytoskeleton, markedly alters Gag subcellular distribution, relocates sites of assembly, and reduces net virus particle production. These observations support a model for native HIV-1 assembly wherein HIV-1 gag-pol mRNA localization helps to confine interactions between Gag, viral RNAs, and host determinants in order to ensure virion production at the right place and right time. Direct perturbation of HIV-1 mRNA subcellular localization may represent a novel antiviral strategy.
| S-EPMC5331792 | biostudies-literature