Inhibition of replication of reactivated human immunodeficiency virus type1 (HIV-1) in latently infected U1 cells transduced with an HIV-1 long terminal repeat-driven PKR cDNA construct

Treatment of human immunodeficiency virus type 1 (HIV-1)-infected individua ls with highly active antiretroviral therapy has effectively decreased vira l load to undetectable levels. However, efforts to eliminate HIV-1 from the se individuals have been unsuccessful, due to the presence of stable, laten t viral reservoirs in resting and active CD4(+) T lymphocytes and macrophag es. These latent populations have become critical targets in the effort to eradicate HIV-1 from infected individuals. The mechanisms of HIV-1 latency have been studied by using the HIV-1-infected promonocytic cell line U1. Th e interferon-inducible double-stranded RNA-dependent p68 protein kinase (PK R), a key enzyme in the host-mediated antiviral response, is known to be do wn-regulated during HIV-1 infection. Therefore, in order to evaluate the ro le of PKR in the inhibition of replication of reactivated HIV-1 in latently infected U1 cells, we have utilized cDNA constructs containing PKR under t he transcriptional control of the HIV-1 long terminal repeat. One PKR-trans duced clone, U1/106-4:27, inhibited the tumor necrosis factor alpha (TNF-al pha)-induced replication of HIV-1 by 99% compared to control U1 cells as me asured by syncytium formation and HIV-1 p24 antigen enzyme-linked immunosor bent assay. Western blot analysis showed an increase in PKR expression thro ugh 96 h postinduction in the U1/106-4:27 clone, concomitant with maximal i ncreases in phosphorylation of the or subunit of eukaryotic initiation fact or 2 and NF-kappa B activity at 72 h postinduction. These results demonstra te that overexpression of PKR can inhibit the replication of reactivated HI V-1 in latently infected cells and confirm the involvement of PKR in the in terferon-associated antiviral pathway against HIV-1 infection. Additionally , treatment of the PKR-transduced U1/1064:27 clone with the protease inhibi tor saquinavir (250 nM) completely inhibited TNF-alpha-induced HIV-1 replic ation.