RNA aptamers directed to human immunodeficiency virus type 1 Gag polyprotein bind to the matrix and nucleocapsid domains and inhibit virus production.
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.
Ramalingam
Dhivya
D
Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Duclair
Sonald
S
Datta
Siddhartha A K
SA
Ellington
Andrew
A
Rein
Alan
A
Prasad
Vinayaka R
VR
D052157:Aptamers, Nucleotide / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D015683:Gene Products, gag / Q000737:chemistry / Q000378:metabolism*; D015488:HIV Antigens / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D019065:Virus Assembly / Q000187:drug effects; D014779:Virus Replication / Q000187:drug effects; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism*
D052157:Aptamers, Nucleotide; D015683:Gene Products, gag; D015488:HIV Antigens; D019590:Nucleocapsid Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1