Inhibition of human immunodeficiency virus type 1 by triciribine involves the accessory protein nef

Abstract

Triciribine (TCN) is a tricyclic nucleoside that inhibits human immunodeficiency virus type 1 (HIV-1) replication by a unique mechanism not involving the inhibition of enzymes directly involved in viral replication. This activity requires the phosphorylation of TCN to its 5' monophosphate by intracellular adenosine kinase. New testing with a panel of HIV and simian immunodeficiency virus isolates, including low-passage-number clinical isolates and selected subgroups of HIV-1, multidrug resistant HIV-1, and HIV-2, has demonstrated that TCN has broad antiretroviral activity. It was active in cell lines chronically infected with HIV-1 in which the provirus was integrated into chromosomal DNA, thereby indicating that TCN inhibits a late process in virus replication. The selection of TCN-resistant HIV-1 isolates resulted in up to a 750-fold increase in the level of resistance to the drug. DNA sequence analysis of highly resistant isolate HIV-1(H10) found five point mutations in the HIV-1 gene nef, resulting in five different amino acid changes. DNA sequencing of the other TCN-resistant isolates identified at least one and up to three of the same mutations observed in isolate HIV-1(H10). Transfer of the mutations from TCN-resistant isolate HIV-1(H10) to wild-type virus and subsequent viral growth experiments with increasing concentrations of TCN demonstrated resistance to the drug. We conclude that TCN is a late-phase inhibitor of HIV-1 replication and that mutations in nef are necessary and sufficient for TCN resistance.

FIG. 1.

Structure of TCN.

FIG. 2.

TCN does not affect virion infectivity. H9 cells chronically infected with HIV-1_SK-1 were treated with the indicated concentrations of TCN. Following 3 days of incubation, virus replication was assayed by determination of supernatant RT and p24 expression. The supernatants were normalized for their p24 contents, and virus was assayed for infectivity by titration on HeLa-CD4-LTR-ß-Gal cells.

FIG. 3.

Mutations in nef are sufficient for TCN resistance. A portion of the HIV-1_H10 genome containing the mutations in nef was transferred to HIV-1_pNL4-3. Wild-type virus (HIV-1_IIIB and HIV-1_pNL4-3), TCN-resistant virus (HIV-1_H10), and virus engineered to have a nef mutation (HIV-1_pTAK-1) were incubated with increasing concentrations of TCN; and viral growth was assessed via determination of the supernatant reverse transcriptase activity. The data are the averages of triplicate assays for all drug concentrations.