Proc. Natl. Acad. Sci. USA Vol. 96, pp. 1492–1497, February 1999 Genetics Lethal mutagenesis of HIV with mutagenic nucleoside analogs LAWRENCE A. LOEB*†,JOHN M. ESSIGMANN‡,FARHAD KAZAZI*§,JUE ZHANG*, KARL D. ROSE*¶, i AND JAMES I. MULLINS *Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, and iDepartment of Microbiology, University of Washington, Seattle, WA 98195; and ‡Department of Chemistry and Division of Toxicology, Massachusetts Institute of Technology, Cambridge, MA 02139 Communicated by Manfred Eigen, Max Planck Institute for Biophysical Chemistry, Go¨ttingen, Germany, December 14, 1998 (received for review November 1, 1998) ABSTRACT The human immunodeficiency virus (HIV) structure of the population. Within the quasispecies are drug- replicates its genome and mutates at exceptionally high rates. resistant viruses that are present early in infection before As a result, the virus is able to evade immunological and exposure to a drug, as well as mutant viruses that can easily chemical antiviral agents. We tested the hypothesis that a acquire additional mutations that render them drug resistant further increase in the mutation rate by promutagenic nucle- (10). oside analogs would abolish viral replication. We evaluated The exceptionally high rate of mutagenesis of RNA viruses deoxynucleoside analogs for lack of toxicity to human cells, (11, 12), coupled with the finding that most HIV virions in the incorporation by HIV reverse transcriptase, resistance to blood appear to be nonviable (13), suggest that the HIV repair when incorporated into the DNA strand of an genome is unable to tolerate many additional mutations RNAzDNA hybrid, and mispairing at high frequency. Among without a loss of viability. Thus, even a small increase in the candidates tested, 5-hydroxydeoxycytidine (5-OH-dC) ful- mutation rate might result in the virus population reaching an filled these criteria. In seven of nine experiments, the presence error threshold beyond which the population cannot be sus- of this analog resulted in the loss of viral replicative potential tained because of a loss of viral replication capacity and after 9–24 sequential passages of HIV in human CEM cells. infectivity. Here, we examined the capacity of mutagenic In contrast, loss of viral replication was not observed in 28 deoxynucleoside analogs to modify the fidelity of replication of control cultures passaged in the absence of the nucleoside HIV during sequential passages in culture and to induce lethal analog, nor with other analogs tested. Sequence analysis of a mutagenesis. portion of the HIV reverse transcriptase gene demonstrated a disproportionate increase in G 3 A substitutions, mutations MATERIALS AND METHODS predicted to result from misincorporation of 5-OH-dC into the cDNA during reverse transcription. Thus, ‘‘lethal mutagene- Serial Transfer Experiments. Stock preparations of HIV- 6 y sis’’ driven by the class of deoxynucleoside analogs repre- 1LAI containing approximately 10 infectious units ml were sented by 5-OH-dC could provide a new approach to treating titered by syncytium induction (14). HIV was added at a 3 HIV infections and, potentially, other viral infections. multiplicity of 0.01 to 1-ml aliquots of medium containing 2 105 CEM cells that were previously incubated for 1 hr with or without different deoxynucleoside analogs. After 4 hr at 37°C, Current therapies for the treatment of HIV infection include 1 the infected cells were washed twice with PBS (without Mg2 combinations of inhibitors of the viral reverse transcriptase 1 or Ca2 ) and resuspended in 1 ml of medium with or without (RT) and protease. Drugs that target the viral RT either are the analog in a 48-well plate. Half the volume of the medium nucleosides that terminate viral DNA synthesis, such as with fresh analog was replenished at 2 days. After 4–6 days, the zidovudine (ZDV), dideoxyinosine (ddI), and dideoxycytidine indicated amount of supernatant was transferred to fresh cells (ddC), or are nonnucleoside analogs that bind to a hydropho- that were preincubated with or without analog for 1 hr. This bic cavity adjacent to the polymerase active site, such as procedure was iterated for the indicated number of cycles; nevirapine (1). Unfortunately, the rapid evolution of HIV in aliquots of both cells and supernatants were frozen at each vivo results in the emergence of viruses resistant to each of passage. Virus production was monitored by measuring HIV these agents. Combination therapy involving RT and protease p24 core antigen in culture supernatants with the Abbott inhibitors has been very successful in reducing viral loads and, Antigen ELISA Kit. All experiments were carried out with a in principle, should reduce the outgrowth of resistant viruses. double-blind protocol. Even this approach, however, is limited by drug availability, Insertions of dNTPs by HIV RT. A59-32P-end-labeled patient compliance, and the likelihood that virus populations 15-mer DNA primer was hybridized to the 39 end of a 46-mer harbored by treated individuals will eventually develop drug DNA template containing either dG or dA at position 16 from resistance. its 39 end. The reaction mixture contained 25 mM TriszHCl The development of HIV resistance to host immunity or y (pH 8.0), 10 mM MgCl2,40mMKCl,2mMDTT,0.1mg ml chemotherapy results both from the high replication rate of the BSA, 50 nM template-primer, and the stated amount of either virus and from the infidelity of the HIV RT. HIV-1-infected dCTP or its 5-hydroxy analog 5-OH-dCTP. The concentration 10 individuals produce approximately 10 virions per day (2), and of HIV RT was adjusted such that the reaction was linear with the HIV RT produces one error per 2,000–5,000 nucleotides time and ,20% of the primer was extended. After incubation polymerized (3–7). As a result, HIV genomes within an at 37°C, the reactions were terminated by the addition of an infected individual do not exist as a homogeneous nucleotide sequence, but rather as a ‘‘quasispecies’’ (8, 9), an ensemble of Abbreviations: RT, reverse transcriptase; 5-OH-dCTP, 5-hy- related genomes in which selection operates at the level of the droxydeoxycytidine triphosphate; 5-OH-dC, 5-hydroxydeoxycytidine. †To whom reprint requests should be addressed at: University of The publication costs of this article were defrayed in part by page charge Washington, Department of Pathology, Box 357705, Seattle, WA 98195-7705. e-mail: [email protected]. payment. This article must therefore be hereby marked ‘‘advertisement’’ in §Present address: AMGEN, Inc., Thousand Oaks, CA 91320-1789. accordance with 18 U.S.C. §1734 solely to indicate this fact. ¶Present address: University of Maryland at Baltimore Dental School, PNAS is available online at www.pnas.org. Baltimore, MD 21201. 1492 Downloaded by guest on September 26, 2021 Genetics: Loeb et al. Proc. Natl. Acad. Sci. USA 96 (1999) 1493 equal volume of denaturing sample buffer. The samples were sisted of 30 cycles. A 1-ml aliquot of each reaction mixture was boiled and the 16-mer product was resolved from the 15-mer diluted into a second 50-ml PCR mixture containing inner substrate by electrophoresis through a 14% polyacrylamidey primers corresponding to nucleotides 2281–2318 and 2881– urea gel. The amount of product generated was quantitated by 2916 and amplified for an additional 20 cycles. The amplified PhosphorImager (Molecular Dynamics) analysis and the ki- DNA was ligated into the PCR II-TOPO TA cloning vector netic constants were calculated from Hanes–Woolf plots (15). (Invitrogen). The nucleotide sequence of the HIV inserts was Nucleoside Analogs. The nucleoside analogs initially tested determined by sequencing both strands, using forward and included the following compounds. 5-Hydroxydeoxycytidine reverse primers complementary to the adjacent M13 DNA. (5-OH-dC) is formed in DNA on exposure to reactive oxygen species (16). 5-OH-dCTP is incorporated into DNA by DNA polymerases and HIV RT (17, 18) and yields predominantly RESULTS z 3 z G C A T substitutions, although other mispairings have Lethal mutagenesis results from a progressive accumulation of been observed. When present in double-stranded DNA, 5-OH- mutations in the HIV genome because of the incorporation of dC is excised by Escherichia coli endonuclease III (Nth protein) mutagenic deoxynucleoside analogs during each round of viral or formamidopyrimidine-DNA glycosidase (19, 20) or by replication (Fig. 1). The analog is taken up by cells as a homologous enzymes in eukaryotic cells (21). In contrast, nucleoside and is phosphorylated by cellular kinases to the z when present in an RNA DNA hybrid, 5-OH-dC is resistant to corresponding deoxynucleoside triphosphate. The mutagenic 4 digestion (22). O -Methyl-dTTP is incorporated efficiently nucleoside triphosphate then can be incorporated during HIV into DNA by a number of DNA polymerases, including RTs RNA template-directed synthesis of the minus DNA strand. (23, 24). Incorporation into HIV cDNA followed by methyl- 3 We postulate that mutagenesis would occur more frequently in transferase-mediated DNA repair would yield G A substi- the HIV genome because of the incorporation of a mutagenic tutions (25). Even though there is evidence for repair of z 4 analog into an RNA DNA hybrid for the following reasons. O -methyl-dT by several pathways (26, 27), the rate of removal First, reverse transcription occurs in the cytoplasm, whereas from rat liver DNA (t of 20–60 hr) is very slow. O6- 1/2 repair of cellular DNA is a nuclear process. Second, DNA Methyl-dG is produced in DNA by alkylating agents and repair enzymes have evolved to utilize double-stranded DNA base-pairs with thymidine at high frequency (28–30), and the that is present in a B-type structure, whereas RNAzDNA triphosphate is incorporated into DNA by HIV RT as well as hybrids are in an A-type structure (38).