Journal of Molecular Biology
Role of Residues in the Tryptophan Repeat Motif for HIV-1 Reverse Transcriptase Dimerization
Introduction
The human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) is required for the conversion of genomic RNA into double-stranded proviral DNA, catalyzed by the RNA- and DNA-dependent polymerase and ribonuclease H (RNase H) activities of the enzyme. The HIV-1 RT is an asymmetric dimer formed by the association of polypeptides p66 and p51, which are cleaved from a large Pr160GagPol precursor by the viral protease (PR) during virion assembly. p51 contains the same N-terminal sequence as p66, but lacks the C-terminal RNase H domain.1
The structure of HIV-1 RT has been elucidated by X-ray crystallography in several contexts, including the unliganded enzyme,2., 3. complexed to non-nucleoside RT inhibitors (NNRTIs),4., 5. or complexed with double-stranded DNA either with6 or without deoxynucleoside triphosphate.7., 8. Such analyses have shown that p66 can be divided structurally into the polymerase and RNase H domains, with the polymerase domain further divided into the fingers, palm, thumb and connections subdomains.8 p66 has a nucleic acid-binding cleft and a DNA polymerase active site. While p51 has the same polymerase subdomains as p66, the relative orientations of the individual subdomains differ markedly in p51. These structural analyses reveal that there are three major contacts between the p66 and p51 subunits, with most of the interaction surfaces being largely hydrophobic.9., 10.
The RT heterodimer represents the biologically relevant form of the enzyme; monomeric subunits are reported to be devoid of both polymerase and RNase H activities.11., 12. Dimerization of the p66 and p51 subunits in vitro can be achieved, though under very non-physiological conditions, and appears to occur by a two-step process.13 The first step involves a concentration-dependent reaction resulting in a dimer intermediate that can bind template/primer but lacks polymerase activity. The second step involves a slow isomerisation reaction that ultimately results in an active enzyme. Hence, formation of an active enzyme requires subunit interaction and conformational changes. The absolute requirement for dimerization of the two RT subunits to generate a fully active enzyme makes dimerization an attractive drug target. Understanding the mechanism and structural basis of dimerization is necessary for the development of strategies to interfere with this process.
The tryptophan repeat motif represents an extraordinary cluster of six tryptophan residues, five of which are separated from each other by three amino acid residues (Figure 1, Figure 6).14 The tryptophan repeat motif is found in the connection subdomain of both HIV-1 RT subunits from codons 398–414. This hydrophobic cluster is highly conserved amongst primate lentiviral reverse transcriptases.14 Interestingly, it is not present in other lentiviral RTs that are known to form heterodimers, including feline immunodeficiency virus (FIV) RT. The tryptophan repeat motif is absent from RTs from other retrovirus families, including the heterodimeric avian sarcoma leukosis virus (ASLV) and Moloney murine leukemia virus (MoMLV), which may dimerize when presented with a template.15
Previous studies suggest that the tryptophan repeat motif is important for RT dimerization. Elucidation of the kinetics of association of p66 and p51 RT subunits implicates tryptophan residues as being involved in the initial interaction of the two monomers.16 Subsequent studies have shown that a 19-mer peptide corresponding to residues 389–407 of the HIV-1 RT tryptophan repeat motif can inhibit the association between p66 and p51 when present at micromolar concentrations, but cannot induce heterodimer dissociation.17 In contrast, a peptide representing residues 403–415 had little effect on in vitro RT dimerization.17 These data suggest that the 19-mer (389–407) peptide interferes with the first step in monomer association. Moreover, a peptide corresponding to residues 395–404 could block RT dimerization in vitro and could abolish the production of viral particles in infected cells.18 The activity of this peptide was further enhanced by conjugation to carrier peptide.18
Dimerization is associated with loss of accessibility of individual residues at the HIV-1 RT dimer interface.19 Recent studies on the characterization of protein–protein interactions suggest that the free energy of binding is often distributed unevenly across the interface. These “hot spots” of binding energy tend to involve a subset of interface residues and include W, R or Y and, to a lesser extent P, L, D and K, which are occluded from the bulk solvent by energetically less important residues.20 Many of the tryptophan residues in the tryptophan repeat motif, including W402, W406 and W410 in the p66 subunit and W401 in the p51 subunit, are predicted to be buried at the dimer interface and may represent hot spots for binding.19
To facilitate studies of RT dimerization, we have developed a genetic assay based on the yeast two-hybrid (Y2H) system,21 which detects the specific interaction between the p66 and p51 RT subunits and recapitulates the effects of mutations that abrogate RT dimerization.22 The study of RT heterodimerization either in the Y2H assay or in cell-free assays does not necessarily reflect the actual process in HIV-infected cells. The HIV-1 RT is normally expressed as part of the Pr160GagPol precursor,23 and it is unclear how the RT heterodimer is formed from the Pol portion of the polyprotein. Studies have led to the hypothesis that the immediate precursor to the RT heterodimer is the p66 homodimer.10., 24. In spite of these uncertainties, examining the association of p66 and p51 subunits in yeast has provided insights into the RT dimerization process.13., 22., 25.
In one version of our Y2H system, the p66 RT subunit is fused to the C terminus of the LexA DNA-binding domain to generate the bait fusion, while the p51 RT subunit is fused to the C terminus of the Gal4 activation domain (Gal4AD), which is referred to as the prey fusion.22., 25. Specific interaction between the bait and prey results in the transactivation of the Lac Z reporter gene in yeast. Therefore, the interaction can be quantified accurately by measuring β-galactosidase (β-gal) activity in the yeast reporter strain. One of the advantages of the Y2H system is that we can select and screen for suppressor mutations that restore dimerization to an assembly-defective mutant.22 These studies can provide insight into the structural basis of mutations that interfere with RT dimerization and shed light on amino acid contacts that are energetically important for subunit association. In this study, we performed a systematic mutagenesis of each of the tryptophan residues in the tryptophan repeat motif to assess their individual affects on RT dimerization. We identified suppressors that restore dimerization to an assembly-defective mutant and identified a region in p66 that appears to make important contacts with p51.
Section snippets
Effect of mutations in the tryptophan repeat motif on RT heterodimerization in the Y2H system
The tryptophan repeat motif is present in the connection subdomain of both subunits of the HIV-1 RT and consists of six tryptophan residues at codons 398, 401, 402, 406, 410 and 414 that are highly conserved amongst primate lentiviral RTs (Figure 1).14 Furthermore, a highly conserved tyrosine residue is located at codon 405 (Figure 1). To determine the contribution of each of these conserved residues to HIV-1 RT dimerization we mutagenized the individual tryptophan and tyrosine residues to
Discussion
In this study, we have shown using the Y2H assay that mutagenesis of W401 and W414 in the p66 subunit impairs dimerization with p51 significantly, whereas mutagenesis of other tryptophan residues and Y405 to leucine does not result in detectable effects on RT dimerization. These data were confirmed by studies on RT expressed in bacteria. While W401 and W414 in the p66 subunit are near the heterodimer interface, they do not appear to make direct contacts with amino acid residues in p51,
Bacterial and yeast strains
Saccharomyces cerevisiae strain CTY10-5d (MATa ade2 trp1-901 leu2-3,112 his3-200 gal4−gal80− URA3∷lexA-lacZ) contains an integrated GAL1-lacZ gene with the lexA operator (a gift from Stanley Fields, State University of New York, Stony Brook). Escherichia coli mutator strain XL1-Red (Stratagene, La Jolla, CA) was used for random mutagenesis whereas XL1-Blue (Stratagene) was used to amplify the mutated library and for cloning into expression vectors pQE-70 (Qiagen, Chatsworth, CA) and
Acknowledgements
We thank Drs Wayne A. Hendrickson and Marianna Orlova for helpful discussions, Dr Vinayaka Prasad for providing the pRT6H/NB-PROT construct, Drs Clyde A. Hutchison III, John Wrobel and Michael Conrad for providing pE66MK331A, and Dr Dag Helland for providing RT monoclonal antibodies. G.T. was supported, in part, by C. J. Martin Fellowship 977375 awarded by the National Health and Medical Research Council of Australia. S.P.G. is an Investigator of the Howard Hughes Medical Institute. H.-E.G.A.
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Present addresses: G. Tachedjian, AIDS Molecular Biology Unit, Macfarlane Burnet Institute for Medical Research and Public Health, Commercial Road, Melbourne, Victoria, Australia 3004. GPO Box 2284; H.-E. G. Aronson, Columbia Genome Center, Russ Berrie Medical Science Pavilion, 1150 St. Nicholas Avenue, New York, NY 10032, USA; J. M. McCoy, Biogen Inc. 14 Cambridge Center, Cambridge, MA 02142, USA.