High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis
| Autor: | Stephen J. Lockett, Jianbo Chen, John M. Coffin, Andrew Wright, Craig E. Bencsics, Wei-Shau Hu, Olga A. Nikolaitchik, Vinay K. Pathak, Jatinder Singh, Na Ni |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Heterozygote
Base pair viruses Molecular Sequence Data RNA-binding protein Genome Viral Biology Fluorescence Cell Line Retrovirus Bacterial Proteins Operon Bacteriophage MS2 Humans Small nucleolar RNA Binding site Base Pairing Levivirus Genetics Multidisciplinary Base Sequence Staining and Labeling Virus Assembly Homozygote Virion RNA-Binding Proteins RNA Biological Sciences biology.organism_classification Cell biology Antitermination HIV-1 Nucleic Acid Conformation RNA Viral Capsid Proteins |
| Zdroj: | Proceedings of the National Academy of Sciences. 106:13535-13540 |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|