RNA Aptamers Directed to Human Immunodeficiency Virus Type 1 Gag Polyprotein Bind to the Matrix and Nucleocapsid Domains and Inhibit Virus Production
| Autor: | Sonald Duclair, Vinayaka R. Prasad, Siddhartha A. K. Datta, Alan Rein, Dhivya Ramalingam, Andrew D. Ellington |
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| Rok vydání: | 2011 |
| Předmět: |
Riboswitch
HIV Antigens viruses Aptamer Immunology Gene Products gag Biology Virus Replication gag Gene Products Human Immunodeficiency Virus Microbiology Cell Line Transcription (biology) Virology Humans Viral matrix protein Structure and Assembly Virus Assembly RNA Aptamers Nucleotide Nucleocapsid Proteins Group-specific antigen Molecular biology Viral replication Capsid Insect Science HIV-1 Protein Binding |
| Zdroj: | Journal of Virology. 85:305-314 |
| ISSN: | 1098-5514 0022-538X |
| Popis: | Gag orchestrates the assembly and release of human immunodeficiency virus type 1 (HIV-1) particles. We explored here the potential of anti-Gag RNA aptamers to inhibit HIV-1 replication. In vitro , RNA aptamers raised against an HIV-1 Gag protein, lacking the N-terminal myristate and the C-terminal p6 (DP6-Gag), could bind to matrix protein (MA), nucleocapsid protein (NC), or entire DP6-Gag protein. Upon cotransfection with pNL4-3.Luc molecular clone into 293T cells, six of the aptamers caused mild inhibition (2- to 3-fold) in the extracellular capsid levels, and one aptamer displayed 20-fold inhibition. The reduction was not due to a release defect but reflected Gag mRNA levels. We hypothesized that the aptamers influence genomic RNA levels via perturbation of specific Gag-genomic RNA interactions. Binding studies revealed that the “NC-binders” specifically compete with the packaging signal (ψ) of HIV-1 for binding to DP6-Gag. Therefore, we tested the ability of two NC-binders to inhibit viruses containing ψ-region deletions (ΔSL1 or ΔSL3) and found that the NC-binders were no longer able to inhibit Gag synthesis. The inability of these aptamers to inhibit ψ-deleted viruses correlated with the absence of competition with the corresponding ψ transcripts lacking SL1 or SL3 for binding DP6-Gag in vitro . These results indicate that the NC-binding aptamers disrupt Gag-genomic RNA interaction and negatively affect genomic RNA transcription, processing, or stability. Our results reveal an essential interaction between HIV-1 Gag and the ψ-region that may be distinct from that which occurs during the encapsidation of genomic RNA. Thus, anti-Gag aptamers can be an effective tool to perturb Gag-genomic RNA interactions. |
| Databáze: | OpenAIRE |
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