Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly

Sardo, Luca; Hatch, Steven C.; Chen, Jianbo; Nikolaitchik, Olga A.; Burdick, Ryan C.; Chen, De; Westlake, Christopher J.; Lockett, Stephen; Pathak, Vinay K.; Hu, Wei-Shau

doi:10.1128/jvi.01146-15

Cited by 37 publications

(54 citation statements)

References 35 publications

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“…The dynamic interactions between HIV-1 Gag and RNA genome on plasma membrane have been described (29)(30)(31). We have previously studied the population dynamics of HIV-1 RNA on the plasma membrane and found that the presence of Gag protein significantly extends the time that HIV-1 RNA resides near the plasma membrane, indicating that most viral RNAs on the plasma membrane are in Gag-RNA complexes (31).…”

Section: Discussionmentioning

confidence: 99%

“…We have previously studied the population dynamics of HIV-1 RNA on the plasma membrane and found that the presence of Gag protein significantly extends the time that HIV-1 RNA resides near the plasma membrane, indicating that most viral RNAs on the plasma membrane are in Gag-RNA complexes (31). We have also examined HIV-1 RNA dimerization using live-cell total internal reflection fluorescence microscopy and observed that HIV-1 RNA molecules interact with each other dynamically on the plasma membrane, often when RNAs are associated with Gag signals.…”

Section: Discussionmentioning

confidence: 99%

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Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly

Dilley

Nikolaitchik

Galli

et al. 2017

J Virol

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Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly.IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly. Understanding the mechanism used by HIV-1 to ensure genome packaging provides significant insights into viral assembly and replication.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly

Dilley

Nikolaitchik

Galli

et al. 2017

J Virol

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“…We emphasize that under native conditions, it is almost certain that Gag plays the dominant role in defining the preferred site of assembly by tethering gRNAs to the PM through the activity of its N-terminal matrix domain (13,40). However, we point out that our data do not rule out potential contributions from one or more cellular RNA binding proteins in addition to Gag that, like the MS2 targeting proteins, may modulate transient Gag/gRNA interactions with membranes or other cellular machineries.…”

Section: Discussionmentioning

confidence: 99%

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly

Becker

Sherer

2017

J Virol

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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 fulllength 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, RNAdependent 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.KEYWORDS Gag, MS2, packaging, Psi, RNA trafficking, RRE, genomes, human immunodeficiency virus, translation, virus assembly T he spatial distribution of mRNAs within the cytoplasm is a core determinant of mRNA turnover, cytoplasmic utilization, and the formation of functional macromolecular complexes (1-3). Viruses face severe challenges in this regard during the p...

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“…Our results also shed light on the time frame in which RNA dimerization occurs during virus assembly. In the presence of sufficient Gag proteins for virus assembly, it is likely that most HIV-1 RNAs are associated with some Gag proteins, because the overall dynamics of the viral RNA is altered (25). Thus, the RNA dimerization process is likely to be the merging of two Gag-RNA complexes, regardless of whether the Gag signal is detected.…”

Section: /Mkatementioning

confidence: 99%

HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein

Chen

Rahman

Nikolaitchik

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA-Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA-Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms.retrovirus | RNA genome | Gag-RNA complex | virus assembly | RNA-binding protein

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Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly

Cited by 37 publications

References 35 publications

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly

Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly

HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein

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