Effects of mutations in the polymerase domain on the polymerase, RNase H and strand transfer activities of human immunodeficiency virus type 1 reverse transcriptase 1 1Edited By J.Karn

The 2,3-dideoxy-3-thiacytidine drug-resistant M184I HIV-1 reverse transcriptase (RT) has been shown to synthesize DNA with decreased processivity compared with the wild-type RT. M184A displays an even more severe processivity defect. However, the basis of this decreased processivity has been unclear, and both primer-template binding and dNTP interaction defects have been proposed to account for it. In this study, we show that the altered properties of the M184I and M184A RT mutants that we have measured, including decreased processivity, a slower rate of primer extension, and increased strand transfer activity, can all be explained by a defect in dNTP utilization. These alterations are observed only at low dNTP concentration and vanish as the dNTP concentration is raised. The mutant RTs exhibit a normal dissociation rate from a DNA primer-RNA template while paused during synthesis. Slower than normal synthesis at physiological dNTP concentration, coupled with normal dissociation from the primertemplate, results in the lowered processivity. The mutant RTs exhibit normal DNA 3-end-directed and RNA 5-end-directed ribonuclease H activity. The reduced rate of DNA synthesis causes an increase in the ratio of ribonuclease H to polymerase activity thereby promoting increased strand transfer. These latter results are consistent with an observed higher rate of recombination by HIV-1 strains with Met-184 mutations. HIV-13 reverse transcriptase (RT) is a heterodimer consisting of a 66-kDa subunit (p66) and a 51-kDa subunit (p51). The p51 and p66 subunits share a common N terminus; the p51 subunit lacks 120 amino acids from the C terminus of the p66 subunit. Active sites for polymerase and ribonuclease H (RNase H) reside in p66. The highly conserved motif YXDD in the polymerase active site of HIV-1 and other retroviral RTs is essential for polymerase activity (1). In HIV-1 RT, Met-184 is the second amino acid in this conserved YXDD motif (2). Two mutations, M184I and M184V, arise in HIV-1 RT after treatment with the RT inhibitor 2Ј,3Ј-dideoxy-3Ј-thiacytidine, or 3TC (3-5), and confer high level resistance to 3TC. 3TC is a nucleoside analog that lacks the 3-OH of the ribose ring; incorporation of 3TC blocks viral DNA synthesis. It is the L-pseudo sugar version of 3TC that is used to treat patients. The M184I/V mutations cause steric hindrance between the oxathiolane ring of 3TCTP and the ␤-branched side chain of valine or isoleucine, which hinders incorporation of 3TCTP (6 -8). Wild-type MLV RT has valine in the second position of the YXDD motif (Val-223), and wild-type MLV is resistant to 3TC (9). Replacing the valine at position X in the YXDD motif of the WT MLV RT with methionine (V223M) makes the virus more susceptible to 3TC, and the MLV RT more sensitive to 3TCTP (10). Both HIV-1 and MLV RTs with an alanine substitution at the X position are moderately resistant to 3TC in vitro, even though alanine is not a ␤-branched amino acid (10, 11). This suggests that other factors, such as altered positioning of the template prim...

Section: Methodsmentioning

confidence: 99%

“…Processivity is the quantitative parameter of processive synthesis, indicating the average number of nucleotides added each time the RT engages the primer (12). The M184V mutant of HIV-1 RT has, relative to the wild-type enzyme, reduced processivity (13)(14)(15)(16). The M184I RT mutant is even less processive (15)(16)(17).…”

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confidence: 99%

Apparent Defects in Processive DNA Synthesis, Strand Transfer, and Primer Elongation of Met-184 Mutants of HIV-1 Reverse Transcriptase Derive Solely from a dNTP Utilization Defect

Gao¹,

Hanson²,

Balakrishnan³

et al. 2008

“…However, experimental evidence from HIV-1 (13)(14)(15) 2 and murine leukemia virus (MuLV) (9,16,17) model systems demonstrates that cleavages within the PPT can occur. The MuLV PPT can also be internally cleaved by the isolated RNase H domain from MuLV RT; furthermore, the specificity of cleavage at the 3Ј-end of the PPT is lost when the polymerase domain is removed (18,19).…”

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confidence: 99%

“…A detailed investigation of mutants with substitutions other than alanine for Trp 266 indicated that there is a strong correlation between buried apolar side-chain surface area and quantitative changes in P/T binding; it was suggested that both hydrophobic interactions and hydrogen bonding contribute to the stability of the RT-DNA complex (48). Other studies have shown that mutations in the thumb subdomain can have a dramatic effect on the polymerase, RNase H, and minus-strand DNA transfer activities of HIV-1 RT (15). RNase H assays with HIV-1 PPT-containing substrates consisting of a short DNA primer annealed to a longer RNA template revealed that the cleavage activities of wild-type (WT) RT and two ␣H mutants, W266T and G262A, differ, indicating that mutations in the thumb subdomain can affect the efficiency and specificity of RNase H cleavage (15).…”

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confidence: 99%

Residues in the αH and αI Helices of the HIV-1 Reverse Transcriptase Thumb Subdomain Required for the Specificity of RNase H-catalyzed Removal of the Polypurine Tract Primer

Powell¹,

Beard²,

Bębenek³

et al. 1999

During retrovirus replication, reverse transcriptase (RT) must specifically interact with the polypurine tract (PPT) to generate and subsequently remove the RNA primer for plus-strand DNA synthesis. We have investigated the role that human immunodeficiency virus-1 RT residues in the ␣H and ␣I helices in the thumb subdomain play in specific RNase H cleavage at the 3-end of the PPT; an in vitro assay modeling the primer removal step was used. Analysis of alanine-scanning mutants revealed that a subgroup exhibits an unusual phenotype in which the PPT is cleaved up to seven bases from its 3-end. Further analysis of ␣H mutants (G262A, K263A, N265A, and W266A) with changes in residues in or near a structural motif known as the minor groove binding track showed that the RNase H activity of these mutants is more dramatically affected with PPT substrates than with non-PPT substrates. Vertical scan mutants at position 266 were all defective in specific RNase H cleavage, consistent with conservation of tryptophan at this position among lentiviral RTs. Our results indicate that residues in the thumb subdomain and the minor groove binding track in particular, are crucial for unique interactions between RT and the PPT required for correct positioning and precise RNase H cleavage.The virus-encoded enzyme reverse transcriptase (RT) 1 of human immunodeficiency virus type 1 (HIV-1) and other retroviruses catalyzes the conversion of genomic RNA to a doublestranded DNA replicative intermediate. As minus-strand DNA is synthesized, the RNA template is degraded by the RNase H activity of RT, which cleaves the RNA strand in an RNA-DNA hybrid (Refs. 1 and 2; for reviews, see . This results in the production of many small RNA fragments, any one of which could potentially serve as a primer to initiate synthesis of plus-strand DNA (6). However, a short, purine-rich sequence known as the polypurine tract (PPT) is almost exclusively used as the primer for plus-strand initiation (Refs. 7-12, 61; for a review, see Ref. 6). Why the PPT sequence is selected from all of the other available primers has been the subject of much speculation.One possibility is that the PPT sequence is intrinsically resistant to RNase H degradation and therefore survives as the sole RNA primer available for plus-strand initiation. However, experimental evidence from HIV-1 (13-15) 2 and murine leukemia virus (MuLV) (9, 16, 17) model systems demonstrates that cleavages within the PPT can occur. The MuLV PPT can also be internally cleaved by the isolated RNase H domain from MuLV RT; furthermore, the specificity of cleavage at the 3Ј-end of the PPT is lost when the polymerase domain is removed (18,19). Finally, Escherichia coli RNase H catalyzes cleavages within the MuLV PPT (9,16,17,19) as well as within the HIV-1 PPT. 3A second possibility to explain selection of the PPT primer is related to its unique helical structure (12,20) and the shape and width of the major groove, which is wider than that of other RNA-DNA hybrids (20, 21). These structural factors could cause bi...

“…Two lines of evidence support this suggestion. First, biochemical analyses of mutant RTs and RDRPs have identified a number of amino acid replacements in motif B that severely compromise polymerase activity (7)(8)(9)(10)(11)(12)(13)(14)(15). Second, the primary sequence of motif B is strictly conserved in independent virus isolates; in human immunodeficiency virus type-1 (HIV-1), this region is nearly invariant (16).…”

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confidence: 99%

Purifying Selection Masks the Mutational Flexibility of HIV-1 Reverse Transcriptase

Smith

Anderson

Preston

2004

DNA and RNA polymerases share a core architecture composed of three structurally conserved motifs: A, B, and C. Although the amino acid sequences of these motifs are highly conserved between closely related organisms, variation across broader evolutionary distances suggests that only a few residues in each motif are indispensable for polymerase function. To test this, we constructed libraries of human immunodeficiency virus type-1 (HIV-1) containing random single amino acid replacements in motif B of reverse transcriptase (RT), and we used selection in culture to assess RT function. Despite the nearly absolute constancy of motif B in vivo, virus replicating in culture tolerated a range of conservative and nonconservative substitutions at 10 of the 11 amino acid positions examined. These included residues that are invariant across all retroviral subfamilies and highly conversed in diverse retroelements. Several mutants retained wild type infectivity, and serial passage experiments revealed replacements that were neutral or even beneficial to viral fitness. In addition, a number of the selected variants exhibited altered susceptibility to the nucleoside analog inhibitors AZT and 3TC. Taken together, these data indicate that HIV-1 tolerates a range of substitutions at conserved RT residues and that selection against slightly deleterious mutations (purifying selection) in vivo masks a large repertoire of viable phenotypic variants. This mutational flexibility likely contributes to HIV-1 evolution in response to changing selection pressures in infected individuals.