Lethal mutagenesis of HIV with mutagenic nucleoside analogs

Abstract

The human immunodeficiency virus (HIV) replicates its genome and mutates at exceptionally high rates. As a result, the virus is able to evade immunological and chemical antiviral agents. We tested the hypothesis that a further increase in the mutation rate by promutagenic nucleoside analogs would abolish viral replication. We evaluated deoxynucleoside analogs for lack of toxicity to human cells, incorporation by HIV reverse transcriptase, resistance to repair when incorporated into the DNA strand of an RNA.DNA hybrid, and mispairing at high frequency. Among the candidates tested, 5-hydroxydeoxycytidine (5-OH-dC) fulfilled these criteria. In seven of nine experiments, the presence of this analog resulted in the loss of viral replicative potential after 9-24 sequential passages of HIV in human CEM cells. In contrast, loss of viral replication was not observed in 28 control cultures passaged in the absence of the nucleoside analog, nor with other analogs tested. Sequence analysis of a portion of the HIV reverse transcriptase gene demonstrated a disproportionate increase in G --> A substitutions, mutations predicted to result from misincorporation of 5-OH-dC into the cDNA during reverse transcription. Thus, "lethal mutagenesis" driven by the class of deoxynucleoside analogs represented by 5-OH-dC could provide a new approach to treating HIV infections and, potentially, other viral infections.

Figure 1

Scheme for the progressive accumulation of mutations in HIV RNA as a result of repetitive infection of host cells. dY and dYTP refer to nucleoside and nucleoside triphosphate analogs. Y and M are analogs and mutations, respectively, present in the HIV genome. The wavy lines represent viral RNA and the solid lines represent viral DNA.

Figure 2

Production of HIV-1_LAI in the course of sequential passage of cell culture supernatants in the absence or presence of 5-OH-dC. Cultures containing 2 × 10⁵ CEM cells were maintained for 88 hr; thereafter, supernatant containing the same amount of p24 was transferred to fresh cells. Three replicates are shown.

Figure 3

Sequential passage of HIV. Experiments I and II each contain data on three sets of control and 5-OH-dC-treated cultures. The arrows indicate the passage number for which aliquots were obtained for the preparation of DNA for control (C), 5-OH-dC-treated (L), and breakthrough (B) viruses. Results are expressed as the amount of p24 in culture supernatants. In Exp. I, cultures were maintained for 29 passages, and 5 μl of the supernatant was transferred at each step irrespective of the p24 titer. In experiment II, cultures were maintained for 24 passages, and 1 μl of the supernatant was transferred.