Inhibition of HIV type 1 replication using lentiviral-mediated delivery of mutant tRNA(Lys3)A58U.
In previous studies, we showed that residue A58 of cellular tRNALys3 is necessary for appropriate termination of viral plus-strand strong-stop DNA (+SS DNA), and therefore plays a critical role in the life cycle of HIV-1. We also performed proof-of-principle studies that established that a mutant form of this tRNA primer (tRNA(Lys3)A58U, which lacks the M1A58 residue necessary for +SS DNA termination) could inhibit HIV-1 replication. In the present work, we examined whether a third generation lentiviral vector (SIN) could be used to deliver tRNA(Lys3)A58U to CEM cells. Using both viral kinetic studies and limiting dilution assays (LDA), we observed significant impairment of HIV-1 replication, up to 3 logs in the LDA, in CEM sublines expressing mutant tRNA(Lys3)A58U. No inhibition occurred in cells that either expressed wild-type tRNA(Lys3) or were transduced with empty SIN vector. Further, we observed impairment of viral replication using primary isolates of both HIV-1 and HIV-2 in sublines containing tRNA(Lys3)A58U. We also detected "breakthrough" HIV-1 replication in some tRNA(Lys3)A58U-expressing cultures. Interestingly, analyzed breakthrough viruses appeared to be both genetically and phenotypically wild type. One possible explanation for virological breakthrough is that it reflects the gradual accumulation of HIV-1 within the infected cell culture, to a level that ultimately exceeds the containment "threshold" conferred by tRNA(Lys3)A58U. The fact that HIV-1 does not appear to acquire heritable resistance to tRNA(Lys3)A58U-mediated blockade differentiates this antiviral modality from other therapeutic interventions. It also suggests that tRNA-mediated inhibition of viral replication might be a valuable adjunct to other antiviral approaches.
Renda
Matthew J
MJ
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Bradel-Tretheway
Birgit
B
Planelles
Vicente
V
Bambara
Robert A
RA
Dewhurst
Stephen
S
D002460:Cell Line; D004279:DNA, Viral / Q000096:biosynthesis; D005822:Genetic Vectors; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014779:Virus Replication / Q000187:drug effects*
D004279:DNA, Viral; D012346:RNA, Transfer, Amino Acyl; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase