Biomedical subjects

Vinayaka R Prasad

Publications and source records attributed to Vinayaka R Prasad.

17 recordsLinked to original sources

PubMed2006

Site-directed mutagenesis in the fingers subdomain of HIV-1 reverse transcriptase reveals a specific role for the beta3-beta4 hairpin loop in dNTP selection.

HIV-1 reverse transcriptase shares the key features of high fidelity polymerases, such as a closed architecture of the active site, but displays a level of fidelity that is intermediate to that of high fidelity, replicative polymerases and low fidelity translesion synthesis (TLS) polymerases. The beta3-beta4 loop of the HIV-1 RT fingers subdomain makes transient contacts with the dNTP and template base. To investigate the role of active site architecture in HIV-1 RT fidelity, we truncated the beta3-beta4 loop, eliminating contact between Lys65 and the gamma-phosphate of dNTP. The mutant, in a manner reminiscent of TLS polymerases, was only able to incorporate a nucleotide that was capable of base-pairing with the template nucleotide, but not a nucleotide shape-analog incapable of Watson-Crick hydrogen bonding. Unexpectedly, however, the deletion mutant differed from the TLS polymerases in that it displayed an increased fidelity. The increased fidelity was associated with reduced dNTP binding affinity as measured using the dead end complex formation. In an effort to delineate the specific amino acid residue in the deleted segment responsible for this phenotype, we examined the K65 residue. Two substitution mutants, K65R and K65A were studied. The K65A mutant behaved similarly to the deletion mutant displaying dependence on Watson-Crick hydrogen bonding, increased fidelity and reduced dNTP-binding, while the K65R was more akin to wild-type enzyme. These results underscore the key role of the K65 residue in the phenotype observed in the deletion mutant. Based on the well-known electrostatic interaction between K65 and the gamma-phosphate moiety of incoming dNTP substrate in the ternary complex structure of HIV-1 RT, we conclude that non-discriminatory interactions between beta3-beta4 loop and the dNTP in wild-type HIV-1 RT help lower dNTP selectivity. Our results show that the fidelity of dNTP insertion is influenced by protein interactions with the triphosphate moiety.

Alanine↗

PubMed2006

Structural features of mouse telomerase RNA are responsible for the lower activity of mouse telomerase versus human telomerase.

Human and mouse telomerases show a high degree of similarity in both the protein and RNA components. Human telomerase is more active and more processive than the mouse telomerase. There are two key differences between hTR [human TR (telomerase RNA)] and mTR (mouse TR) structures. First, the mouse telomerase contains only 2 nt upstream of its template region, whereas the human telomerase contains 45 nt. Secondly, the template region of human telomerase contains a 5-nt alignment domain, whereas that of mouse has only 2 nt. We hypothesize that these differences are responsible for the differential telomerase activities. Mutations were made in both the hTR and mTR, changing the template length and the length of the RNA upstream of the template, and telomerase was reconstituted in vitro using mouse telomerase reverse transcriptase generated by in vitro translation. We show that the sequences upstream of the template region, with a potential to form a double-stranded helix (the P1 helix) as in hTR, increase telomerase activity. The longer alignment domain increases telomerase activity only in the context of the P1 helix. Thus the TR contributes to regulating the level of activity of mammalian telomerases.

Animals↗

PubMed2006

Examining interactions of HIV-1 reverse transcriptase with single-stranded template nucleotides by nucleoside analog interference.

Crystallographic studies have implicated several residues of the p66 fingers subdomain of human immunodeficiency virus type-1 reverse transcriptase in contacting the single-stranded template overhang immediately ahead of the DNA polymerase catalytic center. This interaction presumably assists in inducing the appropriate geometry on the template base for efficient and accurate incorporation of the incoming dNTP. To investigate this, we introduced nucleoside analogs either individually or in tandem into the DNA template ahead of the catalytic center and investigated whether they induce pausing of the replication machinery before serving as the template base. Analogs included abasic tetrahydrofuran linkages, neutralizing methylphosphonate linkages, and conformationally locked nucleosides. In addition, several Phe-61 mutants were included in our analysis, based on previous data indicating that altering this residue affects both strand displacement synthesis and the fidelity of DNA synthesis. We demonstrate here that altering the topology of the template strand two nucleotides ahead of the catalytic center can interrupt DNA synthesis. Mutating Phe-61 to either Ala or Leu accentuates this defect, whereas replacement with an aromatic residue (Trp) allows the mutant enzyme to bypass the template analogs with relative ease.

DNA↗

PubMed2006

Analysis of HIV-1 replication block due to substitutions at F61 residue of reverse transcriptase reveals additional defects involving the RNase H function.

We reported previously that substitutions F61L, F61W, F61Y and F61A in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase affect strand displacement synthesis [T. S. Fisher, T. Darden and V. R. Prasad (2003) J. Mol. Biol., 325, 443-459]. We have now determined the effect of these mutations on HIV replication. All mutant viruses were replication defective. Measuring replication intermediates in infected cells did not reveal a specific block as all mutants displayed reduced DNA synthesis (wild-type>F61L>F61W>F61Y>F61A). Analysis of 2-LTR circle junctions revealed that F61W and F61Y mutants generated increased aberrant circle junctions. Circle junctions corresponding to F61Y included 3'-PPT insertions suggesting ribonuclease H defect. In vitro assays mimicking PPT primer generation indicated that F61L, F61W and F61Y mutant RTs were unaffected, while F61A mutant cleaved both at PPT/U3 junction and at +6 with similar efficiencies. In assays measuring cleavage at the RNA/DNA junction to remove the PPT primer, all mutants were significantly affected with F61Y and F61A being most severely impaired. Our results show that (i) replication block of most mutants is due to more than one biochemical defect; (ii) mutations in polymerase domain can affect the function of a distal domain; and (iii) virological analyses of RT mutations can yield insight into structure-function relationship that is otherwise not obvious.

Amino Acid Substitution↗

PubMed2006

Structural determinants of slippage-mediated mutations by human immunodeficiency virus type 1 reverse transcriptase.

Single-base deletions at nucleotide runs or -1 frameshifting by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) result from template slippage during polymerization. In crystal structures of HIV-1 RT complexed with DNA-DNA template-primer, the palm subdomain in the template cleft contacts the template backbone near the proposed site of slippage via the Glu(89) side chain. We investigated the role of Glu(89) in frameshifting by perturbing this interaction. Substitutions with Asp, Gly, Ala, Val, Ser, Thr, Asn, or Lys were created in recombinant HIV RT, and frameshift frequencies of the resulting mutant RTs were measured. All substitutions led to reduced -1 frameshifting by HIV-1 RT (2-40-fold). Interestingly, the suppression of -1 frameshifting frequently coincided with an enhancement of +1 frameshifting (3-47-fold) suggesting that Glu(89) can influence the slippage of both strands. Glu(89) substitutions also led to reduced rates of dNTP misincorporation that paralleled reductions in -1 frameshifting, suggesting a common structural mechanism for both classes of RT error. Our results reveal a major influence of Glu(89) on slippage-mediated errors and dNTP incorporation fidelity. The crystal structure of HIV-1 RT reveals a salt bridge between Glu(89) and Lys(154), which may facilitate -1 frameshifting; this concept is supported by the observed reduction in -1 frameshifting for K154A and K154R mutants.

Base Sequence↗

PubMed2006

Influence of naturally occurring insertions in the fingers subdomain of human immunodeficiency virus type 1 reverse transcriptase on polymerase fidelity and mutation frequencies in vitro.

The fingers subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a hotspot for nucleoside analogue resistance mutations. Some multi-nucleoside analogue-resistant variants contain a T69S substitution along with dipeptide insertions between residues 69 and 70. This set of mutations usually co-exists with classic zidovudine-resistance mutations (e.g. M41L and T215Y) or an A62V mutation and confers resistance to multiple nucleoside analogue inhibitors. As insertions lie in the vicinity of the dNTP-binding pocket, their influence on RT fidelity was investigated. Commonly occurring insertion mutations were selected, i.e. T69S-AG, T69S-SG and T69S-SS alone, in combination with 3'-azido-2',3'-deoxythymidine-resistance mutations M41L, L210W, R211K, L214F, T215Y (LAG(AZ) and LSG(AZ)) or with an alternate set where A62V substitution replaces M41L (VAG(AZ), VSG(AZ) and VSS(AZ)). Using a lacZalpha gapped duplex substrate, the forward mutation frequencies of recombinant wild-type and mutant RTs bearing each of the above sets of mutations were measured. All of the mutants displayed significant decreases in mutation frequencies. Whereas the dipeptide insertions alone showed the least decrease (4.0- to 7.5-fold), the VAG series showed an intermediate reduction (5.0- to 11.4-fold) and the LAG set showed the largest reduction in mutation frequencies (15.3- and 16.3-fold for LAG(AZ) and LSG(AZ), respectively). Single dNTP exclusion assays for mutants LSG(AZ) and LAG(AZ) confirmed their large reduction in misincorporation efficiencies. The increased in vitro fidelity was not due to excision of the incorrect nucleotide via ATP-dependent removal. There was also no direct correlation between increased fidelity and template-primer affinity, suggesting a change in the active site that is conducive to better discrimination during dNTP insertion.

Amino Acid Substitution↗

PubMed2005

HIV-1 reverse transcriptase mutations that confer decreased in vitro susceptibility to anti-RT DNA aptamer RT1t49 confer cross resistance to other anti-RT aptamers but not to standard RT inhibitors.

RNA and DNA aptamers specific for HIV-1 reverse transcriptase (RT) can inhibit reverse transcription in vitro. RNA aptamers have been shown to potently block HIV-1 replication in culture. We previously reported mutants of HIV-1 RT with substitutions N255D or N265D that display resistance to the DNA aptamer RT1t49. Variant viruses bearing these mutations singly or in combination were compromised for replication. In order to address the wider applicability of such aptamers, HIV-1 RT variants containing the N255D, N265D or both (Dbl) were tested for the extent of their cross-resistance to other DNA/RNA aptamers as well as to other RT inhibitors. Both N265D and Dbl RTs were resistant to most aptamers tested. N255D mutant displayed mild resistance to two of the DNA aptamers, little change in sensitivity to three and hypersensitivity to one. Although all mutants displayed wild type-like ribonuclease H activity, their activity was compromised under conditions that prevent re-binding. This suggests that the processivity defect caused by these mutations can also affect RNase H function thus contributing further to the replication defect in mutant viruses. These results indicate that mutants conferring resistance to anti-RT aptamers significantly affect many HIV-1 RT enzymatic activities, which could contribute to preventing the development of resistance in vivo. If such mutations were to arise in vivo, our results suggest that variant viruses should remain susceptible to many existing anti-RT inhibitors. This result was tempered by the observation that NRTI-resistance mutations such as K65R can confer resistance to some anti-RT aptamers.

Journal Article↗

PubMed2005

Human telomerase RNA template sequence is a determinant of telomere repeat extension rate.

Human telomerase is a specialized reverse transcriptase that utilizes an integral RNA subunit to template the synthesis of telomeres. In the present study, we demonstrate that the human telomerase template sequence not only determines the composition, but also the rate of synthesis, of telomere repeats. Mutagenesis of the template sequence identified variants that reconstitute enzymes with repeat extension rates that were either faster or slower than wild type template. Changes in extension rate could not be attributed solely to altered heteroduplex melting, strongly suggesting that specific interactions between telomerase template, protein, and products contribute significantly in determining repeat extension rate. Furthermore, some substitutions that had no effect on extension rate led to striking increases in repeat processivity, indicating that processivity and extension rates can be regulated independently of each other. Our results suggest that telomerase RNA template sequence is a key determinant of the contribution of telomerase to telomere length regulation.

Base Sequence↗

PubMed2005

Aptamers directed to HIV-1 reverse transcriptase display greater efficacy over small hairpin RNAs targeted to viral RNA in blocking HIV-1 replication.

RNA molecules can be powerful inhibitors of HIV-1 replication. To determine the relative efficacy of siRNAs and RNA aptamers, a direct comparison of three anti-HIV reverse transcriptase aptamers and three shRNAs targeted to HIV-1(R3b) was made. U6 promoter-driven anti-HIV genes were delivered into CEMx174 cells via a retroviral vector, and transduced cells were sorted out via green fluorescent protein function and challenged with HIV. The results show that, at low virus input, shRNAs can block HIV as efficiently as aptamers. When expressed in target cells, both classes of inhibitors blocked early events of reverse transcription, suggesting they are both able to access intracellular reverse transcription complexes. However, at higher multiplicities of infection (m.o.i. of 50), while the aptamers could efficiently inhibit HIV replication, shRNAs did not. RNase protection assays indicated similar steady-state levels or nucleocytoplasmic distribution showing that the differential efficacy was not a reflection of intracellular concentration. The higher potency of anti-RT aptamers could be due to their ability to inhibit two successive rounds of reverse transcription owing to their unique ability to be encapsidated into virion particles. Furthermore, anti-RT aptamers expressed in T cells afforded protection against high-dose infection by chimeric RT-SHIV viruses.

Base Pairing↗

PubMed2004

Nucleoside-analog resistance mutations in HIV-1 reverse transcriptase and their influence on polymerase fidelity and viral mutation rates.

Nucleoside-analog inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) were the first drugs used against the virus. It is long known that monotherapy with these and other drugs leads to the rapid development of viral resistance and it is being increasingly appreciated that a significant percentage of individuals receiving highly active antiretroviral therapy (HAART) also develop resistance. Considering the fact that RT is responsible both for optimal rate of replication and an accurate copying of the viral genome, the consequence of drug-resistance mutations in RT to the biochemistry of this enzyme and to the biology of the virus are critically important. The biochemistry of HIV-1 reverse transcriptase variants harboring nucleoside-analog resistance mutations has been studied extensively. In this review, we describe a number of studies into the polymerase fidelity of nucleoside-analog resistant HIV-1 reverse transcriptase as well as the mutation rate of HIV-1 harboring these mutations.

Antiretroviral Therapy, Highly Active↗

PubMed2004

Interaction between human immunodeficiency virus type 1 reverse transcriptase and integrase proteins.

Reverse transcriptase (RT) and integrase (IN) are two key catalytic enzymes encoded by all retroviruses. It has been shown that a specific interaction occurs between the human immunodeficiency virus type 1 (HIV-1) RT and IN proteins (X. Wu, H. Liu, H. Xiao, J. A. Conway, E. Hehl, G. V. Kalpana, V. R. Prasad, and J. C. Kappes, J. Virol. 73:2126-2135, 1999). We have now further examined this interaction to map the binding domains and to determine the effects of interaction on enzyme function. Using recombinant purified proteins, we have found that both a HIV-1 RT heterodimer (p66/p51) and its individual subunits, p51 and p66, are able to bind to HIV-1 IN. An oligomerization-defective mutant of IN, V260E, retained the ability to bind to RT, showing that IN oligomerization may not be required for interaction. Furthermore, we report that the C-terminal domain of IN, but not the N-terminal zinc-binding domain or the catalytic core domain, was able to bind to heterodimeric RT. Deletion analysis to map the IN-binding domain on RT revealed two separate IN-interacting domains: the fingers-palm domain and the carboxy-terminal half of the connection subdomain. The carboxy-terminal domain of IN alone retained its interaction with both the fingers-palm and the connection-RNase H fragments of RT, but not with the half connection-RNase H fragment. This interaction was not bridged by nucleic acids, as shown by micrococcal nuclease treatment of the proteins prior to the binding reaction. The influences of IN and RT on each other's activities were investigated by performing RT processivity and IN-mediated 3' processing and joining reactions in the presence of both proteins. Our results suggest that, while IN had no influence on RT processivity, RT stimulated the IN-mediated strand transfer reaction in a dose-dependent manner up to 155-fold. Thus, a functional interaction between these two viral enzymes may occur during viral replication.

Binding Sites↗

PubMed2004

Tat protein of human immunodeficiency virus type 1 subtype C strains is a defective chemokine.

Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is correlated with increased monocyte migration to the brain, and the incidence of HAD among otherwise asymptomatic subjects appears to be lower in India than in the United States and Europe (1 to 2% versus 15 to 30%). Because of the genetic differences between HIV-1 strains circulating in these regions, we sought to identify viral determinants associated with this difference. We targeted Tat protein for these studies in view of its association with monocyte chemotactic function. Analyses of Tat sequences representing nine subtypes revealed that at least six amino acid residues are differentially conserved in subtype C Tat (C-Tat). Of these, cysteine (at position 31) was highly (>99%) conserved in non-subtype C viruses and more than 90% of subtype C viruses encoded a serine. We hypothesized a compromised chemotactic function of C-Tat due to the disruption of CC motif and tested it with the wild type C-Tat (CS) and its two isogenic variants (CC and SC) derived by site-directed mutagenesis. We found that the CS natural variant was defective for monocyte chemotactic activity without a loss in the transactivation property. While the CC mutant is functionally competent for both the functions, in contrast, the SC mutant was defective in both. Therefore, the loss of the C-Tat chemotactic property may underlie the reduced incidence of HAD; although not presenting conclusive evidence, this study provides the first evidence for a potential epidemiologic phenomenon associated with biological differences in the subtype C viruses.

AIDS Dementia Complex↗

PubMed2003

Substitutions at Phe61 in the beta3-beta4 hairpin of HIV-1 reverse transcriptase reveal a role for the Fingers subdomain in strand displacement DNA synthesis.

Unlike most DNA polymerases, retroviral reverse transcriptases (RTs) are capable of strand displacement DNA synthesis in vitro, unassisted by other proteins. While human immunodeficiency virus type 1 (HIV-1) RT has been shown to possess this rare ability, the structural determinants responsible are unknown. X-Ray crystallographic and biochemical studies have indicated that the beta3-beta4 hairpin of the fingers subdomain of HIV-1 RT contains key contacts for the incoming template strand. In order to assess the possible role of the fingers subdomain in strand displacement synthesis, a set of substitutions was created at the highly conserved Phe61 residue, which is thought to contact the template strand immediately ahead of the dNTP-binding site. Purified heterodimeric RTs containing Phe61 substitutions displayed altered degrees of strand displacement synthesis on nicked and gapped duplex DNA templates with the relative order being: F61Y > or = F61L > wild-type = F61A > F61W. In order to verify that the effects on strand displacement synthesis were not an indirect effect of alterations in processivity, all Phe61 mutants were tested for processive polymerization. While the strand displacement activity of F61W RT variant was affected severely, it displayed a wild-type-like processivity. In contrast, both F61L and F61Y substitutions, despite showing enhanced strand displacement synthesis, displayed reduced processivity. In contrast, the processivity of F61A mutant, which had displayed nearly wild-type-like strand displacement synthesis, was affected most. These results showed that the effects of Phe61 substitutions on strand displacement are not due to global changes in polymerase processivity. Analysis of pause sites during DNA polymerization on double-stranded templates revealed that the wild-type and the Phe61 mutant RTs interact with the template quite differently. Modeling a 5 nt duplex DNA ahead of the dNTP-binding site of HIV-1 RT suggested a correlation between the ability of the side-chain of the amino acid residue at position 61 to stabilize the first base-pair of the DNA duplex to be melted and the degree of strand displacement synthesis. Our results confirm a role for F61 residue in processive synthesis and indicate that the fingers subdomain harbors a structural determinant of strand displacement synthesis by HIV-1 RT.

Amino Acid Substitution↗

PubMed2003

Mutations proximal to the minor groove-binding track of human immunodeficiency virus type 1 reverse transcriptase differentially affect utilization of RNA versus DNA as template.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), like all retroviral RTs, is a versatile DNA polymerase that can copy both RNA and DNA templates. In spite of extensive investigations into the structure-function of this enzyme, the structural basis for this dual template specificity is poorly understood. Biochemical studies with two mutations in HIV-1 RT that affect residues contacting the template-primer now provide some insight into this specialized property. The mutations are N255D and N265D, both adjoining the minor groove-binding track, in the thumb region. The N265D substitution led to a loss of processive polymerization on DNA but not on RNA, whereas N255D drastically reduced processive synthesis on both templates. This differential template usage was accompanied by a rapid dissociation of the N265D variant on DNA but not RNA templates, whereas the N255D variant rapidly dissociated from both templates. Molecular dynamics modeling suggested that N265D leads to a loss of template strand-specific hydrogen bonding, indicating that this is a key determinant of the differential template affinity. The N255D substitution caused local changes in conformation and a consequent loss of interaction with the primer, leading to a loss of processive synthesis with both templates. We conclude that N265 is part of a subset of template-enzyme contacts that enable RT to utilize DNA templates in addition to RNA templates and that such residues play an important role in facilitating processive DNA synthesis on both RNA and DNA templates.

Binding Sites↗

PubMed2002

Substitutions of Phe61 located in the vicinity of template 5'-overhang influence polymerase fidelity and nucleoside analog sensitivity of HIV-1 reverse transcriptase.

Human immunodeficiency virus type 1 reverse transcriptase (RT) is an error-prone DNA polymerase. Structural determinants of its fidelity are incompletely understood. RT/template primer contacts have been shown to influence its fidelity and sensitivity to nucleoside analog inhibitors. The Phe(61) residue, located within the beta 3 sheet of the finger subdomain, is highly conserved among retroviral RTs. The crystal structure of a ternary complex revealed that Phe(61) contacts the first and second bases of the 5'-template overhang. To determine whether such contacts influence the dNTP-binding pocket, we performed a limited vertical scanning mutagenesis (Phe --> Ala, Leu, Trp, or Tyr) at Phe(61). The F61A mutant displayed the highest increase in fidelity, followed by the F61L and F61W variants, which had intermediate phenotypes. F61Y RT had a minimal effect. The increase in fidelity of the F61A mutant was corroborated by a 12-fold decrease in its forward mutation rate. The Phe(61) mutant RTs also displayed large reductions in sensitivity to 2',3'-dideoxythymidine triphosphate and 2',3'-dideoxy,2'3'-didehydrothymidine triphosphate. Mutants displaying the largest increase in fidelity (F61A and F61L) were also the most resistant. These results suggest that contacts between the finger subdomain of human immunodeficiency virus type 1 RT and the template 5'-overhang are important determinants of the geometry of the dNTP-binding pocket.

Adenosine Triphosphate↗

PubMed2002

Potent inhibition of human immunodeficiency virus type 1 replication by template analog reverse transcriptase inhibitors derived by SELEX (systematic evolution of ligands by exponential enrichment).

RNA aptamers derived by SELEX (systematic evolution of ligands by exponential enrichment) and specific for human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) bind at the template-primer cleft with high affinity and inhibit its activity. In order to determine the potential of such template analog RT inhibitors (TRTIs) to inhibit HIV-1 replication, 10 aptamers were expressed with flanking, self-cleaving ribozymes to generate aptamer RNA transcripts with minimal flanking sequences. From these, six aptamers (70.8,13, 70.15, 80.55,65, 70.28, 70.28t34, and 1.1) were selected based on binding constants (K(d)) and the degree of inhibition of RT in vitro (50% inhibitory concentration [IC(50)]). These six aptamers were each stably expressed in 293T cells followed by transfection of a molecular clone of HIV(R3B). Analysis of the virion particles revealed that the aptamers were encapsidated into the virions released and that the packaging of the viral genomic RNA or the cognate primer, tRNA(Lys)(3), was apparently unaffected. Infectivity of virions produced from 293T cell lines expressing the aptamers, as measured by infecting LuSIV reporter cells, was reduced by 90 to 99.5% compared to virions released from cells not expressing any aptamers. PCR analysis of newly made viral DNA upon infection with virions containing any of the three aptamers with the strongest binding affinities (70.8,13, 70.15, and 80.55,65) showed that all three were able to form the minus-strand strong-stop DNA. However, virions with the aptamers 70.8 and 70.15 were defective for first-strand transfer, suggesting an early block in viral reverse transcription. Jurkat T cells expressing each of the three aptamers, when infected with HIV(R3B), completely blocked the spread of HIV in culture. We found that the replication of nucleoside analog RT inhibitor-, nonnucleoside analog RT inhibitor-, and protease inhibitor-resistant viruses was strongly suppressed by the three aptamers. In addition, some of the HIV subtypes were severely inhibited (subtypes A, B, D, E, and F), while others were either moderately inhibited (subtypes C and O) or were naturally resistant to inhibition (chimeric A/D subtype). As virion-encapsidated TRTIs can predispose virions for inhibition immediately upon entry, they should prove to be efficacious agents in gene therapy approaches for AIDS.

Anti-HIV Agents↗

PubMed2002

Mutations that confer resistance to template-analog inhibitors of human immunodeficiency virus (HIV) type 1 reverse transcriptase lead to severe defects in HIV replication.

We isolated two template analog reverse transcriptase (RT) inhibitor-resistant mutants of human immunodeficiency virus (HIV) type 1 RT by using the DNA aptamer, RT1t49. The mutations associated, N255D or N265D, displayed low-level resistance to RT1t49, while high-level resistance could be observed when both mutations were present (Dbl). Molecular clones of HIV that contained the mutations produced replication-defective virions. All three RT mutants displayed severe processivity defects. Thus, while biochemical resistance to the DNA aptamer RT1t49 can be generated in vitro via multiple mutations, the overlap between the aptamer- and template-primer-binding pockets favors mutations that also affect the RT-template-primer interaction. Therefore, viruses with such mutations are replication defective. Potent inhibition and a built-in mechanism to render aptamer-resistant viruses replication defective make this an attractive class of inhibitors.

Anti-HIV Agents↗