RNA-protein interactions govern antiviral specificity and encapsidation of broad spectrum anti-HIV reverse transcriptase aptamers.
RNA aptamers that bind HIV-1 reverse transcriptase (RT) inhibit HIV-1 replication, but little is known about potential aptamer-specific viral resistance. During replication, RT interacts with diverse nucleic acids. Thus, the genetic threshold for eliciting resistance may be high for aptamers that make numerous contacts with RT. To evaluate the impact of RT-aptamer binding specificity on replication, we engineered proviral plasmids encoding diverse RTs within the backbone of HIV-1 strain NL4-3. Viruses inhibited by pseudoknot aptamers were rendered insensitive by a naturally occurring R277K variant, providing the first demonstration of aptamer-specific resistance in cell culture. Naturally occurring, pseudoknot-insensitive viruses were rendered sensitive by the inverse K277R mutation, establishing RT as the genetic locus for aptamer-mediated HIV-1 inhibition. Non-pseudoknot RNA aptamers exhibited broad-spectrum inhibition. Inhibition was observed only when virus was produced in aptamer-expressing cells, indicating that encapsidation is required. HIV-1 suppression magnitude correlated with the number of encapsidated aptamer transcripts per virion, with saturation occurring around 1:1 stoichiometry with packaged RT. Encapsidation specificity suggests that aptamers may encounter dimerized GagPol in the cytosol during viral assembly. This study provides new insights into HIV-1's capacity to escape aptamer-mediated inhibition, the potential utility of broad-spectrum aptamers to overcome resistance, and molecular interactions that occur during viral assembly.
Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, MO 65211, USA.|Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.
Margaret J
MJ
Lange
Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.|Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA.
Phuong D M
PDM
Nguyen
Department of Biological Engineering, University of Missouri, Columbia, MO 65211, USA.
Mackenzie K
MK
Callaway
Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, MO 65211, USA.|Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.
Marc C
MC
Johnson
Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, MO 65211, USA.|Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.|Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA.|Department of Biological Engineering, University of Missouri, Columbia, MO 65211, USA.
Donald H
DH
Burke
D052157:Aptamers, Nucleotide / Q000378:metabolism / Q000494:pharmacology*; D002213:Capsid / Q000378:metabolism; D057809:HEK293 Cells; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D006801:Humans; D020125:Mutation, Missense; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011533:Proviruses / Q000201:enzymology / Q000648:ultrastructure; D012367:RNA, Viral / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D018894:Reverse Transcriptase Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection; D014779:Virus Replication / Q000187:drug effects
D052157:Aptamers, Nucleotide; D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase