Determining the Frequency and Mechanisms of HIV-1 and HIV-2 RNA Copackaging by Single-Virion Analysis

Dilley, Kari A.; Ni, N.; Nikolaitchik, Olga A.; Chen, Jianbo; Galli, Andrea; Hu, Wei-Shau

doi:10.1128/jvi.05147-11

Cited by 22 publications

(28 citation statements)

References 34 publications

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“…4E). These results are consistent with our previous observation that HIV-1 and HIV-2 RNAs can copackage at a low efficiency (21).…”

Section: /Mkatesupporting

confidence: 93%

“…Construct 1-AAG-BSLMSL is similar to 1-Gag-BSLMSL except that the Gag translational start codon was changed from AUG to AAG to abolish the translation of functional Gag. The previously described construct HIV-2-noGag-BglSL (21) is referred to as 2-noGag-BSL for clarity in the present report; this HIV-2-based construct contains two stop-codon mutations in the gag gene-one at codon 17 of Gag in MA and one at codon 171 of CA-to abolish Gag expression (21). Construct globin-MSL was modified from r-Cfp-betaglobin (27), which encodes a β-globin gene under the control of the tet promoter.…”

Section: Methodsmentioning

confidence: 99%

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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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“…4E). These results are consistent with our previous observation that HIV-1 and HIV-2 RNAs can copackage at a low efficiency (21).…”

Section: /Mkatesupporting

confidence: 93%

Section: Methodsmentioning

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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“…Recent studies using electron tomography showed the viral RNPs incorporated in the budding virions possess different lengths; however, only a few virus particles were analyzed, and the segments with similar length could not be differentiated by this method (14,22). The development of single-particle approaches has allowed the characterization of virus particles and events in the virus life cycle that might have been missed in the observations of the entire virus population (23)(24)(25). Therefore, to study the genome packaging of influenza viruses, methods that allow stoichiometric analysis of viral RNAs within single-virus particles with individual RNA sensitivity are required.…”

mentioning

confidence: 99%

One influenza virus particle packages eight unique viral RNAs as shown by FISH analysis

Chou¹,

Vafabakhsh

Doğanay

et al. 2012

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

160

141

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Influenza A virus possesses a segmented genome of eight negativesense, single-stranded RNAs. The eight segments have been shown to be represented in approximately equal molar ratios in a virus population; however, the exact copy number of each viral RNA segment per individual virus particles has not been determined. We have established an experimental approach based on multicolor single-molecule fluorescent in situ hybridization (FISH) to study the composition of viral RNAs at single-virus particle resolution. Colocalization analysis showed that a high percentage of virus particles package all eight different segments of viral RNAs. To determine the copy number of each RNA segment within individual virus particles, we measured the photobleaching steps of individual virus particles hybridized with fluorescent probes targeting a specific viral RNA. By comparing the photobleaching profiles of probes against the HA RNA segment for the wild-type influenza A/Puerto Rico/8/34 (PR8) and a recombinant PR8 virus carrying two copies of the HA segment, we concluded that only one copy of HA segment is packaged into a wild type virus particle. Our results showed similar photobleaching behaviors for other RNA segments, suggesting that for the majority of the virus particles, only one copy of each RNA segment is packaged into one virus particle. Together, our results support that the packaging of influenza viral genome is a selective process.

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“…Interestingly, mutation of core lentiviral cis and trans elements did not improve the efficiency of this technology. Reverse transcriptase mutants V148I and Q151N and complementary DIS mutations did not increase full-length proviral reconstitution, despite reports that they increase the rate of template-switching25262728. This may have been due to impaired infectivity with these variants33 and reduced efficiency of provirus synthesis, despite any increase in recombination frequency.…”

Section: Discussionmentioning

confidence: 69%

Lentiviral vectors can be used for full-length dystrophin gene therapy

Counsell

Asgarian

Meng

et al. 2017

Sci Rep

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Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a ‘template-switching’ lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.

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Determining the Frequency and Mechanisms of HIV-1 and HIV-2 RNA Copackaging by Single-Virion Analysis

Cited by 22 publications

References 34 publications

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

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

One influenza virus particle packages eight unique viral RNAs as shown by FISH analysis

Lentiviral vectors can be used for full-length dystrophin gene therapy

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