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

• DOI: 10.1073/pnas.0906822106
• Publication Date: 2009-07-24T01:36:29Z
• Authors: Jianbo Chen; Olga Nikolaitchik; Jatinder Singh; Andrew Wright; Craig E. Bencsics; John M. Coffin; Na Ni; Stephen Lockett; Vinay K. Pathak; Wei-Shau Hu
• Keywords: 0301 basic medicine; Heterozygote; Base Sequence; Staining and Labeling; Virus Assembly; Homozygote; Molecular Sequence Data; Virion; RNA-Binding Proteins; Genome, Viral; Fluorescence; Cell Line; 3. Good health; 03 medical and health sciences; Bacterial Proteins; Operon; HIV-1; Humans; Nucleic Acid Conformation; RNA, Viral; Capsid Proteins; Base Pairing; Levivirus

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.