High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis

Abstract

A long-standing question in retrovirus biology is how RNA genomes are distributed among virions. In the studies presented in this report, we addressed this issue by directly examining HIV-1 RNAs in virions using a modified HIV-1 genome that contained recognition sites for BglG, an antitermination protein in the Escherichia coli bgl operon, which was coexpressed with a fragment of BglG RNA binding protein fused to a fluorescent protein. Our results demonstrate that the majority of virions (>90%) contain viral RNAs. We also coexpressed HIV-1 genomes containing binding sites for BglG or the bacteriophage MS2 coat protein along with 2 fluorescent protein-tagged RNA binding proteins. This method allows simultaneously labeling and discrimination of 2 different RNAs at single-RNA-detection sensitivity. Using this strategy, we obtained physical evidence that virions contain RNAs derived from different parental viruses (heterozygous virion) at ratios expected from a random distribution, and we found that this ratio can be altered by changing the dimerization sequences. Our studies of heterozygous virions also support a generally accepted but unproven assumption that most particles contain 1 dimer. This study provides answers to long-standing questions in HIV-1 biology and illustrates the power and sensitivity of the 2-RNA labeling method, which can also be adapted to analyze various issues of RNA biogenesis including the detection of different RNAs in live cell imaging.Bgl ͉ MS2 ͉ dimerization ͉ DIS ͉ fluorescent protein

Nuclear import of APOBEC3F-labeled HIV-1 preintegration complexes

“…The supernatant containing virus was collected 18 h after transfection, filtered (0.45 μm), and concentrated by ultracentrifugation (100,000 × g, 1.5 h, 4°C) through a 20% sucrose cushion, and resuspended in either PBS or culture medium. For the production of noninfectious, Gag-CeFP-labeled viruses, the same plasmids described above were used, except pHDV-EGFP was replaced with a mixture of pGagCeFP-BglSL (5 μg) and pGag-BglSL (5 μg) (11).…”

“…Single-virion analysis was performed as previously described (11,47), with minor modification. Briefly, a small amount of concentrated virus (0.4 μL) was diluted in PBS and centrifuged onto IbiTreated μ-slides (Ibidi) at 1200 × g for 1 h. Epifluorescence microscopy was performed with an inverted Nikon Eclipse Ti microscope and a 100× N.A.-1.40 oil objective, using an X-Cite 120 system (EXFO Photonic Solution) for illumination.…”

Cell biology of retroviral RNA packaging

“…Since complete virions are known to contain dimeric genomes, 70,[85][86][87][88] this suggests that the genomes are recruited as a preformed dimer, rather than as individual molecules that subsequently dimerize during or after packaging. Consistent with this notion, it was reported that MLV gRNA dimerization is coupled to splicing and transcription processes, suggesting that MLV gRNA exits the nucleus as dimeric forms [89][90][91][92] (Fig.…”

“…By coupling protein cross-linking experiments with membrane flotation approaches, it was shown that Gag forms recognized by MS2 fused to GFP. Detection of MS2-GFP-bound RNA in VLPs collected from cell culture supernatant, either by microscopy 61,70,71 or by protein gel blotting analysis, 68 indicated that the modified genomes were efficiently packaged into virions. Importantly, this encapsidation was dependent on the RNA packaging signal.…”

Properties and functions of the nucleocapsid protein in virus assembly

“…Given that about 1,500 NC molecules are present per viral core, and thought to extensively coat the genome, each molecule, if evenly distributed, should bind 11-12 nucleotide residues on the average. 14,15 As a molecular replicative machine, the nucleocapsid contains about 50 copies of the viral enzymes RT, protease (PR) and integrase (IN) in dimeric forms. In addition, about 100 to 200 molecules of the viral protein R (VPR) are being incorporated into the nucleocapsid (reviewed in refs.…”