High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis.
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.
Chen
Jianbo
J
HIV Drug Resistance Program, National Cancer Institute, Frederick, MD 21702, USA.
Nikolaitchik
Olga
O
Singh
Jatinder
J
Wright
Andrew
A
Bencsics
Craig E
CE
Coffin
John M
JM
Ni
Na
N
Lockett
Stephen
S
Pathak
Vinay K
VK
Hu
Wei-Shau
WS
D001426:Bacterial Proteins / Q000235:genetics; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D036022:Capsid Proteins / Q000378:metabolism; D002460:Cell Line; D005453:Fluorescence; D016679:Genome, Viral / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology*; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D017909:Levivirus / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009876:Operon / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics; D013194:Staining and Labeling; D014771:Virion / Q000235:genetics*; D019065:Virus Assembly / Q000235:genetics*
D001426:Bacterial Proteins; D036022:Capsid Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; C064872:antiterminator proteins, Bacteria