Sensitivity of wild-type human immunodeficiency virus type 1 reverse ...

Proc. Nati. Acad. Sci. USA Vol. 91, pp. 4882-4886, May 1994 Medical Sciences

Sensitivity of wild-type human immunodeficiency virus type 1 reverse transcriptase to dideoxynucleotides depends on template length; the sensitivity of drug-resistant mutants does not PAUL L. BOYER*, CHRIS TANTILLOt, ALFREDO JACOBO-MOLINAt, RAYMOND G. NANNIt, JIANPING DINGt, EDWARD ARNOLDt, AND STEPHEN H. HUGHES*t *Advanced BioScience Laboratories-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, P.O. Box B, Building 539, Frederick, MD 21702-1201; and tCenter for Advanced Biotechnology and Medicine (CABM) and Rutgers University Chemistry Department, 679 Hoes Lane, Piscataway, NJ 08854-5638

Communicated by George F. Vande Woude, December 23, 1993 (received for review August 11, 1993)

significantly different. As a consequence, the p66/p51 heterodimer has only one DNA-binding groove and one polymerase active site, which is in the palm subdomain of p66. The corresponding region of p51 is buried in the structure and does not contact nucleic acid. The asymmetry of the two subunits of the p66/pSl heterodimer also means that any point mutation in the region of the viral genome that encodes the polymerase domain causes changes at two different positions in the HIV-1 RT heterodimer. Before the structure of HIV-1 RT was known, it was reasonable to assume that the mutations which give rise to drug resistance would cluster around the polymerase active site. Although the nonnucleoside inhibitors of HIV-1 RT appear to be structurally diverse and the mechanism by which they act is not understood, it is now clear that they bind to a hydrophobic pocket in the palm subdomain of p66 that lies near the polymerase active site in the p66 subunit (33). Mutations that confer resistance to the nonnucleoside inhibitors alter individual amino acids that form the inner surface of the hydrophobic pocket (3-5, 15, 16, 18, 19, 22, 24, 33). However, of the mutations that confer resistance to nucleoside inhibitors (6-8, 11, 12, 14, 17, 21, 25, 26), most do not lie in the immediate vicinity of the polymerase active site and are located within both the fingers subdomain and the palm subdomain (Fig. 1). Most of these mutations are in positions to interact with the template-primer. It has been suggested that the fingers subdomain of the p66 subunit is involved in the appropriate positioning of the template strand (33, 34, 36). If this suggestion is correct, the fingers subdomain could affect the precise positioning or conformation ofthe template strand, which could influence the nucleic acid/protein complex which forms the polymerase active site. This could affect the ability of the enzyme to accept or reject an incoming dNTP. Since the nucleoside-resistance mutations in the palm subdomain appear to occur at positions that could contact either the template or the primer strand, these mutations could have similar effects. We have examined the effects of varying the length of the template extension on wild-type HIV-1 RT and on two nucleoside-resistance mutations, Leu74 -+ Val (L74V) and Glu89 -+ Gly (E89G). MATERIALS AND METHODS Preparation of p66/p51 Heterodimers. We used BspMI cassette mutagenesis (37, 38) to construct the HIV-1 RT mutants L74V and E89G. These plasmids are based on the plasmid HIV-1 RT (29), which induces the expression of the 66-kDa RT in Escherichia coli. The coding regions of the mutant RTs were then modified to include codons for six consecutive histidine residues before the termination codon.

ABSTRACT Analysis of the three-dimensional structure of human Immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexed with double-stranded DNA indictes e mutations are not at the that while many nule -r putative dNTP bnding site, several are in positions to interact with the template-primer. Wild-type HIV-1 RT and two nucleodde-resistant variants, Leu74 - Val and Glues -- Gly, have been analyzed to determine the basis of resistance. The ability of the wild-type enzyme to incorporate, or reject, a 2',3'dideoxynucleoslde tripbosphate (ddNTP) is strongly affected by interactns that take place between the enzyme and the extended template strand 3-6 nt beyond the polymerase active site. Inspection of a model of the enzyme with an extended template suggests that thi interaction involves the fingers subdoin of the p66 subunit in the vinity of Leu74. These data provide direct evidence that the fingers subdomain of the p66 subunit of HIV-1 RT interacts with the template strand. The wild-type enzyme is ant to ddITP I the template extension is 3 nt or less and becomes sensitive only when the template extends more than 3 or 4 nt beyond the end of the primer strand. However, the mutant enzymes are resstant with both short and long template extensions. Taken together with the three-dimensional structure of HIV-1 RT in complex with double-stranded DNA, these data uggest that resistance to the dideoxynucleotide inhibitors results from a repositioning or change in the conformation of the template-primer that alters the ability of the enzyme to select or reject an incomig dNTP. Reverse transcriptase (RT) is the enzyme that copies the single-stranded RNA genome of retroviruses, including human immunodeficiency virus type 1 (HIV-1), into linear double-stranded DNA (1, 2). This is an essential step in the retroviral life cycle. Most anti-HIV-1 drugs that have been tested, including those approved for clinical use, inhibit RT. One of the major difficulties in using anti-HIV-1 drugs to treat AIDS is the genetic flexibility of HIV-1. Although potent nucleoside and nonnucleoside inhibitors of HIV-1 RT have been discovered, resistant mutants have been isolated for each of the drugs that has been extensively tested (3-26). HIV-1 RT is composed of two subunits, p66 and p51. The larger subunit, p66, is 560 aa long. p51 contains the first 440 aa of p66, which corresponds to the polymerase domain. The additional 120 aa of p66 comprise the RNase H domain (27-32). The polymerase domains of p66 and p51 each contain the same four subdomains, which have been called fingers, palm, thumb, and connection (33). Although the folding of the polypeptide chains within these four subdomains is similar in p66 and p51, the relative arrangement of the subdomains is

Abbreviations: HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase; dsDNA, double-stranded DNA; AZTTP, 3'-

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azido-3'-deoxythymidine 5'-triphosphate. tTo whom reprint requests should be addressed. 4882

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Proc. Natl. Acad. Sci. USA 91 (1994)

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FIG. 1. Stereodiagram showing the locations of HIV-1 RT nucleoside drug-resistance mutations relative to the polymerase active site and to the template-primer. The coordinates of HIV-1 RT and the template-primer duplex region are derived from the 3.0-A resolution structure of an HIV-1 RT/dsDNA complex (34, 35). The 10-base poly(dA) template extension has been modeled. The polypeptide backbones of the HIV-1 fingers and palm subdomains are represented as cyan and red Ca tracings, respectively. The Ca positions of the three essential aspartic residues at the polymerase active site (Aspl0, Asp185, and Asp186) are indicated in white. The Ca positions of nucleoside-resistance mutations are numbered and their corresponding structural elements are highlighted as ribbons. The fingers subdomain contains position 41 (in helix aA), positions 67, 69, and 70 (in the P3-,4 loop), and position 74 (in strand p4). The palm subdomain contains positions 89 (in p5a), 184 (in the 89-810 hairpin), 215 (in 81la), and 219 (in Pl3lb). The template and primer strands are white and green, respectively, and the nucleotides in the template overhang have been numbered in agreement with Figs. 2 and 3. An orange dTTP has been docked at the putative dNTP binding site. The nucleoside-resistance mutations are not located in the immediate vicinity of the putative dNTP binding site; instead, most are in positions to interact with the template-primer. This model suggests that many nucleoside drug-resistance mutations govern selection of a dNTP substrate by altering the position of the template-primer relative to the wild-type enzyme. The change in HIV-1 RT interactions with the template-primer alters the structure at the active site, permitting the enzyme to discriminate between dNTP and ddNTP substrates.

The coding regions for wild-type, L74V, and E89G RT were cloned into plasmids similar in concept to p6HRTPROT (39). The constructs are based on the vector pT5m and are grown in E. coli BL21 (40, 41). After induction with isopropyl P-D-thiogalactopyranoside, the plasmids simultaneously express the HIV-1 p66 RT subunit and the HIV-1 protease. Approximately 50%6 of the overexpressed p66 is processed to p51 by the protease, and p66/pSi heterodimers accumulate in the bacteria. The p66/pSi heterodimers were

purified by metal chelate chromatography (39, 42). RT Assays. DNADNA template-primers were based on a synthetic oligonucleotide (pGTCCCTGTTCGGGCGCCA) and its complement. This sequence matches the duplex region of the template-primer present in the crystal structure (34, 35). The oligonucleotides were resuspended at 5.0 A2w/ml in double-distilled water and hybridized to yield the DNA-DNA substrates. The polymerase activity of the p66/ p5l heterodimers was assayed in 100 id of 25 mM Tris, pH 8.0/75 mM KCl/8.0 mM MgCl2/2.0 mM dithiothreitol/10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) containing acetylated bovine serum albumin at 100 pg/ml. The reaction mixture was supplemented with the appropriate labeled dNTP and unlabeled dNTPs (10 ,uM). Each RT reaction mixture contained 0.005 A260 unit of the DNA-DNA template-primer. ddNTPs were added as indicated in the figures. After incubation at 37°C for 1 hr, the assay was stopped by the addition of 100 pI of a 10-mg/ml solution of sheared and denatured salmon sperm DNA,

followed by 3 ml of ice-cold 10%6 (wt/vol) trichloroacetic acid. The labeled polymer was collected on Whatman GF/C filters by suction filtration and counted. Modeling of Extended Template Strand. Modeling was based on the structure of the ternary complex of HIV-1 RT, a 19-mer/18-mer double-stranded DNA (dsDNA) templateprimer, and a monoclonal Fab fragment (refs. 34 and 35; Brookhaven Protein Databank entry 1HMI). Model building was performed with INSIGHT version 2.0 (Biosym, Technologies, San Diego) on a Silicon Graphics 4D/240 GTX computer. Positioning of the template extension was based only on the dsDNA structure, and potential contacts with the protein were not used as a guide. The phosphate positions of the duplex region near the polymerase active site were used to anchor the terminus of an A-form helical poly(dA) fragment. Although single-stranded polynucleotides are flexible, structural studies have indicated that single-stranded nucleic acids can adopt helical conformations (43) and we have, in the absence of structural data, modeled the extended template as a helix. Only the 10-base template extension was modeled; the sequence and structure of the 18-bp duplex region corresponds to the structure of the DNA in the crystalline complex (34, 35).

RESULTS We examined the effects of template extension length on the wild-type HIV-1 RT heterodimer and on two nucleosideresistance mutations, L74V and E89G. L74V was found in

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clinical isolates of HIV-1 and confers resistance to 2',3'dideoxyinosine (ddl) (21).'E89G, which was selected in an E. coli expression system, confers resistance to ddGTP (17, 25). It has been reported (14) that some HIV-1 RTs bearing nucleoside-resistance mutations do not show resistance in in vitro assays. We tested the recombinant L74V and E89G RT heterodimers for resistance to ddNTP inhibitors. The L74V heterodimer had considerable resistance to inhibition by ddITP relative to the wild-type enzyme in an in vitro reaction using either poly(rC)oligo(dG) or a DNADNA templateprimer (data not shown). It has been suggested that ddITP is converted to ddATP in vivo and that ddATP is the metabolite that blocks HIV-1 replication (44). In an in vitro reaction, the L74V mutant showed significant resistance to ddATP using a DNA-DNA template-primer (data not shown). The L74V heterodimer also showed cross resistance to ddCTP and ddGTP using DNA*DNA template-primers (data not shown). In these in vitro assays, we did not see significant resistance to 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP) or ddTTP. We also tested the resistance of the E89G mutant to various dideoxy compounds. In agreement with Prasad et al. (17), we found that E89G showed resistance to ddITP, ddGTP, and ddATP (data not shown). Leu74 is located on the ,84 strand of the HIV-1 RT fingers subdomain. In the structure of the complex containing RT and the 19/18-mer template-primer, Leu74 of the p66 subunit lies beyond the end of the 1-nt template extension (34, 36). Leu74 does not appear to be in a position to interact directly with the incoming dNTP or ddNTP (Fig. 1). Models with extended template strands were built based on the crystal structure. In these models, the sugar-phosphate backbone of the extended single-stranded template makes contact with the p66 Leu74 2-3 nt beyond the end of the primer strand (Fig. 1). This suggests that the incorporation of ddNTPs might depend on the length of the template strand. To test this possibility, a series of DNA-DNA template-primers were prepared in which the duplex regions of the template-primers matched the sequence of the duplex region of the templateprimer in the crystal structure (34-36). This sequence, pGTCCCTGTTCGGGCGCCA, is a DNA-DNA version of the 18-base RNA-RNA duplex formed between the primer binding site in the RNA genome of HIV-1 and the 3' end of the tRNALYs-3 primer. The various template-primers differed only in the length (1-10 nt) and composition of the extended single-stranded portion of the template. In vitro polymerization reactions were done. Reactions using a series of template-primers with a variable number of cytosines in the template extension were performed in the absence of an inhibitor and in the presence of 5 p&M ddITP. Reactions were allowed to go to completion. Under these conditions the assay measures the ability of the unlabeled ddNTP to compete with the labeled dNTP for incorporation; incorporation of a ddNTP blocks any further incorporation of dNTPs. Parallel reactions were done with the wild-type HIV-1 RT heterodimers and the L74V mutant heterodimers and the incorporation of radioactively labeled dGTP was monitored. When the template strands were short (1-3 nt beyond the duplex region), both the wild-type and mutant heterodimers were relatively resistant to ddITP. With longer templates, the wild-type enzyme became sensitive (incorporated the inhibitor). The mutant enzyme showed some sensitivity to the drug with longer templates; however, it was always more resistant than the wild-type enzyme (Fig. 2A). The experiment was repeated with a similar series of duplex oligonucleotides with thymidine extensions of 1-8 nt and ddATP as the inhibitor. The outcome of these experiments was similar. Both the wild-type and the mutant enzyme showed considerable resistance when the templates extended