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PubMed1996

Targeting HIV reverse transcriptase for anti-AIDS drug design: structural and biological considerations for chemotherapeutic strategies.

The reverse transcriptase of HIV is a key target for the antiviral treatment of AIDS. Numerous potent inhibitors of RT have been described including all of the drugs that have been currently licensed for the treatment of AIDS, but their efficacy has been limited by the emergence of drug-resistant HIV variants. Extensive biochemical, genetic, and clinical data about HIV RT enzymatic mechanisms, inhibition, and drug resistance have been reported. This information, taken together with structural data from crystallographic studies of HIV-1 RT, has set the stage for structure-based design of improved inhibitors of this essential viral enzyme. Comparisons of the different crystal structures of HIV-1 RT shows that the enzyme has great conformational flexibility, providing additional possibilities for drug targeting. Recent clinical and virological data suggest that HIV-1 RT enzymes that carry drug-resistance mutations can be substantially impaired and that combinations of RT inhibitors can produce significant clinical benefit in the treatment of AIDS. An immediate goal is to use the available information to design specific inhibitors or combination therapies that will select for relatively less fit HIV variants.

Binding Sites↗
PubMed1999

Mutations in the primer grip region of HIV reverse transcriptase can increase replication fidelity.

Mutations in the primer grip region of human immunodeficiency virus reverse transcriptase (HIV-RT) affect its replication fidelity. The primer grip region (residues 227-235) correctly positions the 3'-ends of primers. Point mutations were created by alanine substitution at positions 224-235. Error frequencies were measured by extension of a dG:dA primer-template mismatch. Mutants E224A, P225A, P226A, L228A, and E233A were approximately equal to the wild type in their ability to extend the mismatch. Mutants F227A, W229A, M230A, G231A, and Y232A extended 40, 66, 54, 72, and 76% less efficiently past a dG:dA mismatch compared with the wild type. We also examined the misinsertion rates of dG, dC, or dA across from a DNA template dA using RT mutants F227A and W229A. Mutant W229A exhibited high fidelity and did not produce a dG:dA or dC:dA mismatch. Interestingly, mutant F227A displayed high fidelity for dG:dA and dC:dA mismatches but low fidelity for dA:dA misinsertions. This indicates that F227A discriminates against particular base substitutions. However, a primer extension assay with three dNTPs showed that F227A generally displays higher fidelity than the wild type RT. Clearly, primer grip mutations can improve or worsen either the overall or base-specific fidelity of HIV-RT. We hypothesize that wild type RT has evolved to a fidelity that allows genetic variation without compromising yield of viable viruses.

Base Pair Mismatch↗
PubMed1996

Strand transfer is enhanced by mismatched nucleotides at the 3' primer terminus: a possible link between HIV reverse transcriptase fidelity and recombination.

Strand transfer catalyzed by HIV reverse transcriptase (RT) was examined. The system consisted of a 142 nt RNA (donor) to which a 50 nt DNA primer was hybridized. The primer bound such that its 3' terminal nucleotide hybridized to the 12th nt from the 5' end of the donor. The 3' terminal nucleotide on the primer was either a G, A or T residue. Since the corresponding nucleotide of the donor was a C, the G formed a matched terminus and the A or T a mismatched terminus. The efficiency with which DNA bound to the donor transferred to a second RNA, termed acceptor, was monitored. The acceptor was homologous to the donor for all but the last 9 nt at the 5' end of the donor. Therefore, homologous strand transfer could occur at any point prior to the DNA being extended into the nonhomologous region on the donor. Strand transfer occurred approximately twice as efficiently with the mismatched versus matched substrates. The mismatched nucleotide was fixed into transfer products indicating that excision of the mismatch was not required for RT extension or transfer. Results suggest that base misincorporations by RT may promote recombination by enhancing strand transfer.

Base Sequence↗
PubMed1997

Mutations in HIV reverse transcriptase which alter RNase H activity and decrease strand transfer efficiency are suppressed by HIV nucleocapsid protein.

Structural studies of authentic HIV reverse transcriptase (RT) suggest a role for the p51 carboxyl terminus in forming an active RNase H conformation [Rodgers, D. W., Gamblin, S. J., Harris, B. A., Ray, S., Culp, J. S., Hellmig, B., Woolf, D. J., Debouck, C. & Harrison, S. C. (1995) Proc. Natl. Acad. Sci. USA 92, 1222-1226]. We have purified mutant RT heterodimers containing deletion of 5, 9, or 13 amino acids from the p51 carboxyl terminus. These "selectively deleted" heterodimers have been analyzed for changes in RNA-dependent DNA polymerase activity, RNase H activity, and the ability to catalyze DNA strand transfer. As deletions extended into the p51 subunit, a decrease in the stability of the RT-DNA complex was apparent. The largest effect was observed for p66/p51Delta13 RT, which showed a 3-fold decrease relative to wild-type RT. RNase H activity was measured by digestion of the RNA in a 5' 32P-labeled RNA/DNA hybrid. Deletion of 5 or 9 amino acids from p51 had little effect on synthesis-dependent and synthesis-independent RNase H activities. In contrast, deletion of 13 amino acids from p51 increased the length of the hydrolysis products of both RNase H activities by 8-10 bp, thus changing the spatial relationship between the polymerase and RNase H active sites from a distance of 17-18 bp to 26-27 bp. The Delta13 derivative was also incapable of efficient DNA strand transfer. This defect in strand transfer could be suppressed by the 71-amino acid form of HIV nucleocapsid protein (NC) but not by the 55-amino acid form (NC55) or by equine infectious anemia virus NC. These results provide evidence for the existence of a specific complex between RT and NC and are discussed in terms of the role of this complex in proviral DNA synthesis.

Base Sequence↗
PubMed2005

Non-nucleoside HIV reverse transcriptase inhibitors, Part 6[1]: synthesis and anti-HIV activity of novel 2-[(arylcarbonylmethyl)thio]-6-arylthio DABO analogues.

2-(Arylcarbonylmethyl)thio-6alpha-naphthylmethyl derivatives of dihydro-alkoxy-benzyl-oxopyrimidines (DABO) were newly found to exhibit activity against both HIV-1 and HIV-2. To further explore their structure-activity relationship, the modified S-DABO analogues (5a-g and 6e-f) with a 1-naphthylthio or phenylthio group at the C-6 position were synthesized. S-Alkylation of 5-ethyl-2-thiouracil with substituted 2-bromo-acetophenones provided crude 2-[(arylcarbonylmethyl)thio]-5-ethyl-(3H)-uracil 2a-e, which was directly subjected to toluenesulfonylation with TsCl to afford disulfonate 4a-e. Substitution of 4a-e with arylthiol afforded the desired S-DABO analogues 5a-g and 6e-f. The compounds were evaluated for their in vitro anti-HIV activity in MT-4 cells. The IC(50) values for anti-HIV-1 activity fall into the range 0.37-29.50 microM, and the IC(50) values for anti-HIV-2 activity fall into the range 23.11-181.07 microM. The results indicated that these compounds are moderately active against HIV-1 and HIV-2.

Anti-HIV Agents↗
PubMed2005

HIV reverse transcriptase structures: designing new inhibitors and understanding mechanisms of drug resistance.

HIV reverse transcriptase (RT) is one of the main targets for the action of anti-AIDS drugs. The selection of drug-resistant HIV is a key problem in the continued treatment of the infection and thus new drugs are required. A significant body of information consisting of HIV-1 RT crystal structures with bound inhibitors has become available during the past several years, and, increasingly, such data will be of use in developing novel inhibitors. Two examples of crystal structures of HIV-1 RT with bound inhibitors have been published recently, one with the non-nucleoside CP94707 and the second with the nucleotide analogue drug tenofovir. Such structures will help the design of new drugs and improve our understanding of the mechanisms of resistance.

Drug Design↗
PubMed2003

Interaction of HIV reverse transcriptase with structures mimicking recombination intermediates.

Interactions between human immunodeficiency virus (HIV) reverse transcriptase (RT) and structures mimicking intermediates proposed to occur during recombination (strand transfer) were investigated. One mechanism proposed for strand transfer is strand exchange in which a homologous RNA (acceptor) "invades" a donor RNA.DNA duplex (replication intermediate) on which DNA synthesis is occurring. The acceptor displaces the donor of the duplex and binds to the DNA. During exchange a transient trimeric structure forms. A model structure was designed with a replication intermediate to which an acceptor RNA was bound. The acceptor was bound to the 5'-end of the DNA over a 54-base region, whereas the donor associated with the DNA 3'-end over a 28-base region. The dimeric constituents of the trimer (acceptor RNA.DNA and donor RNA.DNA) were also constructed. The acceptor RNA.DNA formed a branched structure in this case. Results showed that RT could cleave the RNA portion of all the structures examined. Association with junction substrates was less stable as determined by off-rates. On the trimer, RT cleaved both RNAs but showed a clear preference for cleaving the donor RNA region. This preference was accentuated by HIV nucleocapsid protein (NC). Results suggest that during recombination RT generally associates with the donor-RNA portion of the trimer and the acceptor RNA is protected but not immune from cleavage. The partial protection likely allows the acceptor RNA to more easily complete strand exchange and shield this RNA to provide a means to salvage replication if the DNA were to dissociate from the cleaved donor RNA.

DNA Replication↗
PubMed1995

Human T helper cells specific for HIV reverse transcriptase: possible role in intrastructural help for HIV envelope-specific antibodies.

Cooperation between B cells specific for an antigen exposed on a viral structure and T helper (Th) cells specific for an internal antigen, as demonstrated with influenza, hepatitis B and rabies viruses, has been termed intrastructural help. Th cells specific for internal proteins of HIV, which are much less mutated than its exposed antigens, may be valuable in vaccine design against this virus. We investigated the human Th repertoire specific for the core HIV antigen reverse transcriptase (p66), and determined whether these cells could be candidate intrastructural T helpers. CD4+ T lines and clones were generated from non-immune individuals by stimulation with p66-pulsed antigen-presenting cells (APC). Specific lines were obtained with p66 from 19 out of 21 (90%) of these individuals, vs. 7 out of 29 (24%) with gp120. Diverse epitopes were recognized by different individuals, and various V beta genes were used by these clones. Clones using the same V beta genes were of diverse origin, according to VDJ region sequence. Of these lines 45% responded to p66 in the context of HIV virions. Moreover, p66-specific clones could respond to APC that had internalized HIV complexed with envelope-specific monoclonal antibodies, suggesting that p66-specific Th cells may participate in intrastructural help. These studies indicate that p66-specific Th cells are detectable in vitro in most naive individuals and exhibit clonal heterogeneity, and that the majority recognize an HIV conserved antigen. They respond to p66 following processing of whole virions and are clearly candidates for intrastructural help. If confirmed in vivo, p66 should be included among vaccine candidates investigated to optimize the anti-HIV Th response.

Amino Acid Sequence↗
PubMed1999

New human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) mutants with increased fidelity of DNA synthesis. Accuracy, template binding, and processivity.

Infidelity of DNA synthesis by human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) is a presumptive determinant of HIV-1 hypervariability and is incompletely understood at the mechanistic and structural levels. Amino acid substitution at only three residues, including Asp-76 (Kim, B., Hathaway, T. R., and Loeb, L. A. (1996) Biochemistry 37, 5831-5839), is known to increase fidelity. We report here that substitution at Arg-78 can also increase accuracy. Mutant R78A RT showed reduced primer extension in misincorporation assays lacking a complementary dNTP and exhibited a 9-fold decrease in mutation frequency in the M13mp2 lacZ forward mutation assay. Previous structural studies indicate that Arg-78 and Asp-76 lie in a region that interacts with template nucleotides. Interestingly, R78A RT exhibited 6- to 8-fold decreases in binding affinity (K(d)) for RNA and DNA templates relative to wild type RT. In contrast, D76V RT, which also increases fidelity (Kim et al., 1996), showed a 6- to 7-fold increased affinity. The processivity of R78A RT on both RNA and DNA templates was substantially reduced relative to wild type RT, whereas the processivity of D76V RT was increased. We discuss relationships of fidelity, template binding, and processivity in these and other HIV RT mutants.

Amino Acid Substitution↗
PubMed1989

Stepwise mechanism of HIV reverse transcriptase: primer function of phosphorothioate oligodeoxynucleotide.

Primer recognition by purified HIV reverse transcriptase has been investigated. Earlier we found that the reaction pathway for DNA synthesis is ordered, with template-primer and free enzyme combining to form the first complex in the reaction sequence (Majumdar et al., 1988). We now find that d(C)28 is a linear competitive inhibitor of DNA synthesis against poly[r(A)].oligo[d(T)] as template.primer, indicating that d(C)28 and the template.primer combine with the same form of the enzyme in the reaction scheme, i.e., the free enzyme. The phosphorothioate oligodeoxynucleotide Sd(C)28 also is a linear competitive inhibitor against template.primer. However, the Ki for inhibition (approximately 2.8 nM) is approximately 200-fold lower than the Ki for inhibition by d(C)28. Since the inhibition is linear competitive, the dissociation constant is equal to the Ki for inhibition. Filter binding assays confirmed high-affinity binding between Sd(C)28 and the enzyme and yielded a KD similar to the Ki for inhibition. Substrate kinetic studies of DNA synthesis using Sd(C)28 as primer, and poly[r(I)] as template, revealed that the Km for Sd(C)28 is 24 nM. The Km for this primer is, therefore, 8-fold higher than the KD for enzyme-primer binding (2.8 nM). These results enable calculation of real time rate values for the enzyme-primer association (kon = 5.7 x 10(8) M-1 s-1) and dissociation (koff = 1.6 s-1).

Binding, Competitive↗
PubMed1989

Structural characterization of HIV reverse transcriptase: a target for the design of specific virus inhibitors.

The reverse transcriptase (RT) of HIV is an important target for chemotherapy as demonstrated by the effective treatment of AIDS patients with zidovudine, a potent inhibitor of RT. Structural studies of HIV RT were therefore undertaken with a view to designing more effective inhibitors. To obtain sufficient quantities of enzyme for these studies the reverse transcriptase gene of HIV was cloned into a high level expression plasmid yielding reverse transcriptase at a level of 10% of the total Escherichia coli proteins. Monoclonal antibodies to RT were raised in mice and have been used to purify the enzyme by immunoaffinity chromatography. Crystallization of the enzyme has been achieved and studies are underway to determine its three-dimensional structure. In addition, carboxy-terminal truncated mutants were prepared by inserting stop codons into the gene at appropriate sites. The proteins expressed were analysed for RT and RNase H activity and used for mapping RT epitopes. This, together with previous data on site-directed mutagenesis of conserved regions of HIV RT has helped to map some of the structural and functional regions of the enzyme.

Animals↗
PubMed2004

Taking aim at a moving target: designing drugs to inhibit drug-resistant HIV-1 reverse transcriptases.

HIV undergoes rapid genetic variation; this variation is caused primarily by the enormous number of viruses produced daily in an infected individual. Because of this variation, HIV presents a moving target for drug and vaccine development. The variation within individuals has led to the generation of diverse HIV-1 subtypes, which further complicates the development of effective drugs and vaccines. In general, it is more difficult to hit a moving target than a stationary target. Two broad strategies for hitting a moving target (in this case, HIV replication) are to understand the movement and to aim at the portions that move the least. In the case of anti-HIV drug development, the first option can be addressed by understanding the mechanism(s) of drug resistance and developing drugs that effectively inhibit mutant viruses. The second can be addressed by designing drugs that interact with portions of the viral machinery that are evolutionarily conserved, such as enzyme active sites.

Binding Sites↗
PubMed2006

Molecular dynamics model of unliganded HIV-1 reverse transcriptase.

HIV-1 RT is one of the most important antiviral targets in the treatment of acquired immunodeficiency syndrome (AIDS). Several crystallographic structures are available for this enzyme, mostly with bound inhibitors. Despite their importance for structure based drug design towards new anti-HIV retrovirals, the X-ray structures of the unliganded enzyme could only be obtained incomplete, with a low resolution and until recently even the conformation of the p66 thumb was controversial. In this work we have aligned different X-ray RT structures, and built up a computational model of RT using homology modeling, which was afterwards refined and validated through MD simulations with explicit solvent. The model enzyme was structurally stable through the whole MD simulation, showing a RMSD of 2 Angstrom from the starting geometry. The Ramanchandram plot has improved along the simulation. Both intra-domain and interdomain movements were observed. The thumb kept its closed conformation through the whole simulation. A contact map, hydration sites study and a detailed analysis of the solvation of the nucleotide binding site are also presented.

Binding Sites↗
PubMed2001

Physical mapping of HIV reverse transcriptase to the 5' end of RNA primers.

Enzymatic analysis of RNA cleavage products has suggested that human immunodeficiency virus (HIV) reverse transcriptase (RT) binds to the 5' end of RNAs that are recessed on a longer DNA template (RNA primers) yet binds to the 3' end of DNA primers. One concern is that RT molecules bound at the 3' end of RNA would not be easily detected because RT may not catalyze substantial RNA extension or cleavage when bound to the 3' end. We used physical mapping to show that RT binds preferentially to the 5' end of RNA primers. An HIV-RT that lacked RNase H activity (HIV-RT(E478Q)) was incubated with the RNA-DNA hybrid followed by the addition of Escherichia coli RNase H. RT protected a approximately 23-base region at the 5' end of the RNA and 4 additional bases on the DNA strand. This footprint correlated well with the crystal structure of HIV-RT. No protection of the RNA 3' end was observed, although when dNTPs were included, low levels of extension occurred, indicating that RT can bind this end. Wild-type HIV-RT cleaved the RNA and then extended a small portion of the cleaved fragments, suggesting that very small RNAs may be bound similar to DNA primers.

3' Untranslated Regions↗
PubMed2000

Intranasal immunization with HIV reverse transcriptase: effect of dose in the induction of helper T cell type 1 and 2 immunity.

HIV reverse transcriptase (RT) is immunologically recognized by the host after natural infection with HIV. To study the immune response to this nonenvelope protein and the effect of the antigen dose and the immunization route on the differential induction of cellular or humoral responses, CB6F1 mice were immunized intranasally with various doses of recombinant RT (0.025-25 microg/dose) in the presence of the mucosal adjuvant cholera toxin (CT). An antigen-specific, dose-dependent, in vitro proliferative response was observed in splenic cells from all mouse groups immunized with RT and CT. Proliferative responses in salivary gland-associated lymph node (SGALN) cells from the same mice were detected only with higher antigen dose immunizations (2.5, 25 microg/dose). IFN-gamma, a Th1-type cytokine, was detected in RT-stimulated culture supernatants from splenic and SGALN cells from all groups of mice immunized with RT and CT in a dose-dependent fashion. IL-4, a Th2-type cytokine, was detected in RT-stimulated culture supernatants from splenic and SGALN cells from mice immunized with higher doses of RT and CT. RT-specific IgG2a, a Th1-type-related antibody, was detected consistently in sera from all animals immunized with RT and CT and was predominant in mice immunized with lower antigen doses (0.025, 0.25 microg/dose). RT-specific IgG1, a Th2-type-related antibody, was detected consistently in mice immunized with higher antigen doses and was predominant in these groups. These studies demonstrate the immunogenicity of recombinant RT and the effect of the antigen dose in the induction of Th1-type and Th2-type immune responses after mucosal immunization.

AIDS Vaccines↗
PubMed1998

Sulfoquinovosyldiacylglycerol, KM043, a new potent inhibitor of eukaryotic DNA polymerases and HIV-reverse transcriptase type 1 from a marine red alga, Gigartina tenella.

A new sulfolipid, KM043, which belongs to the 6-sulfo-alpha-D-quinovopyranosyl-(1-->3')-1',2'-diacylglycerol (SQDG) class of compounds, has been isolated from a marine red alga, Gigartina tenella, as a potent inhibitor of eukaryotic DNA polymerases and HIV-reverse transcriptase type 1. Its structure was determined on the basis of spectroscopic and gas chromatographic analyses. The inhibition was dose-dependent, and complete (more than 90%) inhibition of DNA polymerase alpha (pol. alpha), DNA polymerase beta (pol. beta) and HIV-reverse transcriptase type 1 (HIV-RT) was observed at concentrations of 5, 10, and 30 microM, respectively.

Chromatography, Gas↗
PubMed1991

HIV reverse transcriptase inhibiting antibodies detected by a new technique: relation to p24 and gp41 antibodies, HIV antigenemia and clinical variables.

A new assay for HIV reverse transcriptase activity inhibiting antibodies (RTI-ab) was used for the analysis of a large collection of sera sampled before and after confirmation of HIV infection. In this assay HIV-RT was preincubated with diluted serum, after which residual RT activity was determined by a technique using a template coupled to macrobeads and 125I-lodo-deoxyuridine-triphosphate as the tracer-substrate. Of the 936 sera analysed, 818 were found positive for RTI-ab, and 824 were positive in Western blot (Wb). The prevalence of RTI-ab compared to Wb was therefore 99.3%. The corresponding figure for 930 sera analysed for envelope-ab, i.e., gp41-ab, was 823 positive, and of these 930 sera 815 were Wb positive, giving a comparative prevalence of 101%. In contrast, only 678 samples of 993 analyzed for core ab, i.e., p24, were positive, giving a prevalence of 77.0% as 880 of these samples were Wb positive. Thus, RTI-ab was as prevalent as gp41-ab, and although the analyses of RTI-ab amounts in different stages showed decreasing levels in stage IV compared to stages II or III, all of the sera except 1 were found positive in stages III and IV. Further, it was found that both the few RTI-ab negative samples in stage II and the few RTI-ab positive samples among Wb negative sera were sampled in connection with seroconversion. The specificity of the RTI-ab assay was 100% in a test of 200 serum samples from HIV negative blood donors. It was concluded that RTI-ab analyses can be made highly sensitive and specific and useful for studies of HIV infection.

AIDS Serodiagnosis↗
PubMed1994

Inhibitory Effects of Polyphenolic Catechins from Chinese Green Tea on HIV Reverse Transcriptase Activity.

Three polyphenolic catechins, epigallocatechin (1), epicatechin-3-O-gallate (2) and epigallocatechin-3-O-gallate (3), were isolated from Chinese green tea, Ti-Kaun-Yin (Camellia sinensis) and demonstrated as a new class of human immunodeficiency virus-reverse transcriptase (HIV-RT) inhibitor. The concentrations required for 50% inhibition for the compounds (1), (2) and (3) were 7.80, 0.32 and 0.68 &mgr;M, respectively. The polyphenolic catechins with a galloyl group at the 3 position were potent inhibitors of HIV-RT. Kinetic analysis indicated that the polyphenolic catechins were competitive inhibitors with respect to the template-primer (rA)(n)(dT)(12-18) and noncompetitive inhibitors to dTTP. Copyright 1994 S. Karger AG, Basel

Journal Article↗