Region Of RT Research Articles

Structure of HIV‐1 reverse transcriptase/d4TTP complex: Novel DNA cross‐linking site and pH‐dependent conformational changes

Stavudine (d4T, 2',3'-didehydro-2',3'-dideoxythymidine) was one of the first chain-terminating nucleoside analogs used to treat HIV infection. We present the first structure of the active, triphosphate form of d4T (d4TTP) bound to a catalytic complex of HIV-1 RT/dsDNA template-primer. We also present a new strategy for disulfide (S-S) chemical cross-linking between N6 of a modified adenine at the second overhang base to I63C in the fingers subdomain of RT. The cross-link site is upstream of the duplex-binding region of RT, however, the structure is very similar to published RT structures with cross-linking to Q258C in the thumb, which suggests that cross-linking at either site does not appreciably perturb the RT/DNA structures. RT has a catalytic maximum at pH 7.5. We determined the X-ray structures of the I63C-RT/dsDNA/d4TTP cross-linked complexes at pH 7, 7.5, 8, 8.5, 9, and 9.5. We found small (~0.5 Å), pH-dependent motions of the fingers subdomain that folds in to form the dNTP-binding pocket. We propose that the pH-activity profile of RT relates to this motion of the fingers. Due to side effects of neuropathy and lipodystrophy, use of d4T has been stopped in most countries, however, chemical modification of d4T might lead to the development of a new class of nucleoside analogs targeting RNA and DNA polymerases.

Penurunan Tingkat Depresi pada Lansia dengan Pendekatan Bimbingan Spiritual

Introduction: Depression is mental disorder which comes from complicated stress, it can happen for everyone including the elderly. This problem is frequently not considered as the main point of view to think because it is regarded to get good condition by its way without having serious treatment. On the reality, depression which doesn't get serious treatment to solve well, it can danger the life of the elderly. The purpose of this study was to prove the in fl uence of result in the spiritual guidance in accordance with Islamic religion for the changing score (rank) depression of the elderly who lived in the region of RT 04 Kedung Tarukan Wetan Surabaya.Method: Design used in this study was pre experimental design. Population for this study were all of the elderly who lived in the region of RT 04 Kedung Tarukan Wetan Surabaya. Total sample were 10 respondents, taken according to inclusion criteria. The independent variable was the spiritual guidance in accordance with Islamic religion where as the dependent variable was the changing score (rank) of depression. Data were collected by using structured questionnaire. Data were then analyzed using wilcoxon sign rank test with level of significance of≤ 0.05.Result: The result showed, after giving spiritual guidance in accordance with Islamic religion, the score (rank) of depression for elderly to decrease than before giving spiritual guidance in accordance with Islamic religion with significant value p = 0.005.Discussion: It can be concluded that spiritual guidance in accordance with Islamic religion having strong influence for the changing score (rank) of depression of the elderly before and after it had done. It was suggested for the competence person in RT 04 Kedung Tarukan Wetan Surabaya to do this activity continuously as one of the way to prevent and to solve depression for the elderly who lived in the region of RT 04Kedung Tarukan Wetan Surabaya.

Complete Human Immunodeficiency Virus-1 Specific T Lymphocyte Response to Chinese Human Immunodeficiency Virus-1 B/C Chronic Infectors

To characterize the human immunodeficiency virus (HIV) -specific T lymphocyte responses and identify the immunodominant regions in Chinese HIV-1 recombinant subtype B/C chronic infectors at complete genome level. Twenty-five HIV-1B/C recombinant chronic infectors were screened for their specific T lymphocyte responses to a panel of peptides corresponding to the complete HIV-1 subtype B genome by gamma interferon ELISPOT assay. Kruskal-Wallis nonparametric analysis of variance was used to test significant differences across gene regions, and Tukey pairwise analysis was used to identify differences between gene regions. Spearman rank correlation was used to assess the relation between responses. Results The order of recognized frequencies of specific T lymphocyte responses to HIV proteins was Nef>Vpr>Gag>Pol>Vpu>Env>Rev>Vif>Tat. When adjusted for protein length, Nef, Vpr, Gag, and Pol were the most intensely targeted proteins and the central region of Nef, Gag p24, Pol RT, and Vpr was most frequently recognized. No significant correlation was observed between the magnitude of IFN-gamma production of HIV-l-specific T lymphocyte responses and plasma viremia, breadth of response and CD4 counts. Conclusion The central region of Nef, Gag p24, Pol RT, and Vpr is most frequently targeted in HIV-1 B/C recombinants chronic infectors. HIV-l-specific T lymphocyte responses and plasma viremia or CD4 counts play no protective role at complete genome level in these infectors.

Impact of template overhang-binding region of HIV-1 RT on the binding and orientation of the duplex region of the template-primer

Fingers domain of HIV-1 RT is one of the constituents of the dNTP-binding pocket that is involved in binding of both dNTP and the template-primer. In the ternary complex of HIV-1 RT, two residues Trp-24 and Phe-61 located on the beta1 and beta3, respectively, are seen interacting with N + 1 to N + 3 nucleotides in the template overhang. We generated nonconservative and conservative mutant derivatives of these residues and examined their impact on the template-primer binding and polymerase function of the enzyme. We noted that W24A, F61A, and F61Y and the double mutant (W24A/F61A) were significantly affected in their ability to bind template-primer and also to catalyze the polymerase reaction while W24F remained unaffected. Using a specially designed template-primer with photoactivatable bromo-dU base in the duplex region at the penultimate position to the primer terminus, we demonstrated that F61A, W24A, F61Y as well as the double mutant were also affected in their cross-linking ability with the duplex region of the template-primer. We also isolated the E-TP covalent complexes of these mutants and examined their ability to catalyze single dNTP incorporation onto the immobilized primer terminus. The E-TP covalent complexes from W24F mutant displayed wild-type activity while those from W24A, F61A, F61Y, and the double mutant (W24A/F61A) were significantly impaired in their ability to catalyze dNTP incorporation onto the immobilized primer terminus. This unusual observation indicated that amino acid residues involved in the positioning of the template overhang may also influence the binding and orientation of the duplex region of the template-primer. Molecular modeling studies based on our biochemical results suggested that conformation of both W24 and F61 are interdependent on their interactions with each other, which together are required for proper positioning of the +1 template nucleotide in the binary and ternary complexes.

Comparative Molecular Field Analysis (CoMFA) for Thiotetrazole Alkynylacetanilides, a Non‐Nucleoside Inhibitor of HIV‐1 Double Mutant K103N/Y181C Reverse Transcriptase

Non‐nucleoside reverse transcriptase inhibitors (NNRTIs) are allosteric inhibitors of the HIV‐1 reverse transcriptase. HIV‐1 resistance to nucleoside RT inhibitors such as AZT can arise through mutations in the coding region of RT. Recently a series of thioterazolyl acetanilides were reported as potent inhibitors of HIV‐1 wild type and double mutant K103N/Y181C reverse transcriptase. In the present study 3D quantitative structure activity relationship (3D QSAR) studies involving comparative molecular field analysis (CoFMA) were performed on 28 thiotetrazole alkynylacetanilides. CoMFA was performed using sibyl software 6.7v. The molecules were divided into training set and test set. The developed model based on training set containing 20 molecules gave Q2 value of 0.587 and non cross validation R2 value of 0.961 and standard error of estimate 0.098 for CoMFA. The steric and electrostatic contributions are 42.7% and 57.3% The cross validation on test set gave R2 value of 0.74. The CoMFA model provided the most significant correlation of steric and electrostatic fields with biological activities. The information rendered by 3D QSAR model may afford valuable clues to optimize the lead and design new potential inhibitors

Highly diversified multiply drug-resistant HIV-1 quasispecies in PBMCs: a case report

BackgroundAlthough drug resistance is a major challenge in HIV therapy, the effect of drug resistance mutations on HIV evolution in vivo is not well understood. We have now investigated genetic heterogeneity in HIV-1 by performing drug resistance genotyping of the PR-RT regions of viruses derived from plasma and peripheral blood mononuclear cells (PBMCs) of a single patient who had failed multiple regimens of anti-retroviral therapy.ResultsPatterns of drug resistance mutations showed that the viral populations in PBMCs were more heterogeneous than in plasma. Extensive analysis of HIV from infected PBMCs in this patient showed that high-level diversity existed among 109 cloned PR-RT sequences and that the majority of mutations were related to drug resistance. Moreover, the PBMCs included archival species that reflected the treatment history of the patient while those in plasma were mainly related to the most recent treatment. Some of the proviral clones contained single or multiple mutations in various combinations. Approximately eighteen percent of the proviral clones derived from infected PBMCs were defective, i.e. 5.5% contained single nucleotide deletions (frameshift mutations) and 12.8% encoded in-frame stop codons (nonsense mutations). Amino acid substitutions in PR and the polymerase region of RT occurred in 12–15% of cases but were much less frequent in the RNase H region of RT, which might not have been under drug selection pressure.ConclusionSelective drug pressure can yield multiple drug-resistant quasispecies that include archival and replication-incompetent species in PBMC reservoirs.

Role of Integrase in Reverse Transcription of the Saccharomyces cerevisiae Retrotransposon Ty1

Reverse transcriptase (RT) with its associated RNase H (RH) domain and integrase (IN) are key enzymes encoded by retroviruses and retrotransposons. Several studies have implied a functional role of the interaction between IN and RT during the replication of retroviral and retrotransposon genomes. In this study, IN deletion mutants were used to investigate the role of IN on the RT activity of the yeast Saccharomyces cerevisiae retrotransposon Ty1. We have identified two domains of Ty1 integrase which have effects on RT activity in vivo. The deletion of a domain spanning amino acid residues 233 to 520 of IN increases the exogenous specific activity of RT up to 20-fold, whereas the removal of a region rich in acidic amino acid residues between residues 521 and 607 decreases its activity. The last result complements our observation that an active recombinant RT protein can be obtained if a small acidic tail mimicking the acidic domain of IN is fused to the RT-RH domain. We suggest that interaction between these acidic amino acid residues of IN and a basic region of RT could be critical for the correct folding of RT and for the formation of an active conformation of the enzyme.

Substitution of conserved hydrophobic residues in motifs B and C of HIV-1 RT alters the geometry of its catalytic pocket.

Recent crystallographic data suggest that a number of hydrophobic residues seen clustered between the structurally conserved alphabetabetaalpha motif of the palm subdomain and at the junction of palm and fingers subdomains of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) provide an optimal geometry to the alphabeta sandwich of the palm subdomain, which harbors the catalytic site and the primer-binding grip region. This region has also been implicated in binding to the non-nucleoside RT inhibitors. We have evaluated the impact of conserved and nonconserved amino acid substitutions at four hydrophobic positions in this region of HIV-1 RT, in the context of their biochemical characteristics. The residues that have been analyzed include Ile-167, Leu-187, and Val-189 which are located within the alphabetabetaalpha motif, while Trp-153 lies next to the conserved LPQG motif, at the juncture of the palm and fingers subdomains. Our results show that all substitutions at I167 with the exception of I167T were deleterious to enzyme function in contrast to substitutions at V189 which enhanced the enzymatic activity. Ala substitution at residues W153 and L187 also substantially hindered the polymerase function of the enzyme. Further analysis revealed that the defective mutant derivatives of I167 were substantially impaired in their apparent dNTP binding abilities, thereby impacting the geometry of the dNTP binding pocket. The extent of misinsertion and misincorporation was higher in the case of RT variants of W153 and V189, specifically on a DNA template. Interestingly, none of the mutant derivatives of these residues were resistant to nucleoside inhibitors. A salient finding was that all nonconserved mutants of these residues exhibited hypersensitivity to nevirapine. We have analyzed these findings and their significance in the context of the HIV-1 RT structure and propose that these residues exert their effect via their indirect interactions with the template-primer through residues in their vicinity.

Mutational Analysis of the Fingers and Palm Subdomains of Human Immunodeficiency Virus Type-1 (HIV-1) Reverse Transcriptase

We have analyzed the human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) polymerase domain between amino acids 91 and 157 by site-directed mutagenesis. We have constructed a series of amino acid substitutions using BspMI cassettes, and have assayed the RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and Rnase H activities of the mutant HIV-1 RTs. The regions of HIV-1 RT between amino acids 91 and 119 between amino acids 151 and 157 lie within the palm subdomain and include part of the polymerase active site. A number of amino acids within these regions have been identified as being directly or indirectly involved with polymerization, since amino acid substitutions at these residues decrease the polymerase activity without affecting Rnase H activity. The region of HIV-1 RT between amino acids 120 and 150 lies within the fingers subdomain of the HIV-1 polymerase. We believe that the fingers subdomain plays a role in positioning the template. Many amino acid substitutions in this region decrease or abolish both the polymerase and the Rnase H functions.

The 'helix clamp' in HIV-1 reverse transcriptase: a new nucleic acid binding motif common in nucleic acid polymerases.

Amino acid sequences homologous to 259KLVGKL (X)16KLLR284 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) are conserved in several nucleotide polymerizing enzymes. This amino acid motif has been identified in the crystal structure model as an element of the enzyme's nucleic acid binding apparatus. It is part of the helix-turn-helix structure, alpha H-turn-alpha I, within the 'thumb' region of HIV-1 RT. The motif grasps the complexed nucleic acid at one side. Molecular modeling studies on HIV-1 RT in complex with a nucleic acid fragment suggest that the motif has binding function in the p66 subunit as well as in the p51 subunit, acting as a kind of 'helix clamp'. Given its wide distribution within the nucleic acid polymerases, the helix clamp motif is assumed to be a structure of general significance for nucleic acid binding.

Kinetic theory of granular shear flow: Constitutive relations for the hard-disk model

A kinetic theory for the constitutive Theological relations of rapid granular shear flow of hard circular disks, characterized by a coefficient of restitutione and a surface roughness coefficientβ, is formulated. From a set of general constitutive equations for single-particle dynamical variables, the approximate expressions for the limit of small and large dimensionless dissipative parameterR t are obtained. HereR t is defined as the ratio σγ/〈ν〉, where ν is the fluctuation of translational velocity from the mean flow velocity, σ is the diameter of a disk, and γ is the shear rate. At smallR t the theoretical predictions can be compared with “exact” computer simulation results of granular dynamics that are also reported. The agreement between theory and simulation is better than expected; the present theory is accurate up to high packing density in this region ofR t .