Cytoplasmic HIV-1 RNA is mainly transported by diffusion in the presence or absence of Gag protein

Chen, Jianbo; Grünwald, David; Sardo, Luca; Galli, Andrea; Plisov, Sergey; Nikolaitchik, Olga A.; Chen, De; Lockett, Stephen; Larson, Daniel R.; Pathak, Vinay K.; Hu, Wei-Shau

doi:10.1073/pnas.1413169111

Cited by 55 publications

(91 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is possible that once the viral RNA leaves the TIRF observation volume, it rarely or never reenters this region; in this scenario, the length of time an RNA molecule can be tracked, i.e., ϳ3 s, is the amount of time the RNA stays near this region. However, we observed that most HIV-1 RNAs move in a nondirectional, random-walking manner in the cytoplasm (27). Thus, it is highly likely that a large portion of the RNAs may leave and reenter the region near the plasma membrane; in this scenario, the time that the RNAs stay near the plasma membrane may be longer than indicated by the track lengths of the viral RNAs.…”

mentioning

confidence: 79%

“…Construct 1-GagBFP-BSL was derived from 1-GagCeFP-BSL (3) by replacing the cerulean fluorescent protein (CeFP) gene with the blue fluorescent protein (BFP) gene (29). Constructs 1-Gag⌬CTDBFP-BSL and 1-Gag⌬CTD-BSL were derived from 1-GagBFP-BSL and 1-Gag-BSL (27), respectively, by deleting the region encoding the last 83 amino acids of the capsid. Plasmid 1-GagLZCeFP-BSL was constructed by replacing a portion of the gag sequence of 1-GagCeFP-BSL (3) with the counterpart from 1-GagLZmCherry-BSL (27), which encodes a leucine zipper motif in place of the NC domain.…”

Section: Methodsmentioning

confidence: 99%

“…Constructs 1-Gag⌬CTDBFP-BSL and 1-Gag⌬CTD-BSL were derived from 1-GagBFP-BSL and 1-Gag-BSL (27), respectively, by deleting the region encoding the last 83 amino acids of the capsid. Plasmid 1-GagLZCeFP-BSL was constructed by replacing a portion of the gag sequence of 1-GagCeFP-BSL (3) with the counterpart from 1-GagLZmCherry-BSL (27), which encodes a leucine zipper motif in place of the NC domain. Plasmid Bgl-Eos was derived from Bgl-YFP (27) by replacing the yellow fluorescent protein (YFP) gene with the d2EosFP (Eos) gene, which encodes a dimeric Eos fluorescent protein (28).…”

Section: Methodsmentioning

confidence: 99%

“…Plasmid 1-GagLZCeFP-BSL was constructed by replacing a portion of the gag sequence of 1-GagCeFP-BSL (3) with the counterpart from 1-GagLZmCherry-BSL (27), which encodes a leucine zipper motif in place of the NC domain. Plasmid Bgl-Eos was derived from Bgl-YFP (27) by replacing the yellow fluorescent protein (YFP) gene with the d2EosFP (Eos) gene, which encodes a dimeric Eos fluorescent protein (28). HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin (50 U/ml), and streptomycin (50 U/ml).…”

Section: Methodsmentioning

confidence: 99%

“…We sought to address these unanswered questions regarding Gag-RNA interaction during virus assembly. Previously, we showed that cytoplasmic HIV-1 RNA has dynamic movement and is transported mainly by diffusion (27). We reasoned that because TIRF observations are limited to a very shallow volume, an RNA molecule may move in and out of the observation volume during the observation time due to its dynamic movement.…”

mentioning

confidence: 99%

See 4 more Smart Citations

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

Sardo

Hatch

Chen

et al. 2015

J Virol

Self Cite

View full text Add to dashboard Cite

To increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we observed that only a portion, ϳ13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions. IMPORTANCENascent HIV-1 particles assemble on plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we labeled and monitored the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stayed near the plasma membrane for only a few minutes in the absence of Gag, whereas most HIV-1 RNAs stayed at the plasma membrane for 15 to 60 min in the presence of Gag. Our results also demonstrated that only a small proportion of the HIV-1 RNAs, approximately 1/10 to 1/3 of the RNAs that reached the plasma membrane, was incorporated into viral protein complexes. These studies determined the dynamics of HIV-1 RNA on the plasma membrane and obtained temporal information on RNA-Gag interactions that lead to RNA encapsidation.

show abstract

mentioning

confidence: 79%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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

Sardo

Hatch

Chen

et al. 2015

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

A structurally plastic ribonucleoprotein complex mediates post‐transcriptional gene regulation in HIV‐1

2016

View full text Add to dashboard Cite

HIV replication requires the nuclear export of essential, intron-containing viral RNAs. To facilitate export, HIV encodes the viral accessory protein Rev which binds unspliced and partially spliced viral RNAs and creates a ribonucleoprotein complex that recruits the cellular Chromosome maintenance factor 1 export machinery. Exporting RNAs in this manner bypasses the necessity for complete splicing as a prerequisite for mRNA export, and allows intron-containing RNAs to reach the cytoplasm intact for translation and virus packaging. Recent structural studies have revealed that this entire complex exhibits remarkable plasticity at many levels of organization, including RNA folding, protein–RNA recognition, multimer formation, and host factor recruitment. In this review, we explore each aspect of plasticity from structural, functional, and possible therapeutic viewpoints.

show abstract