Biomedical subjects

Prem L Sharma

Publications and source records attributed to Prem L Sharma.

5 recordsLinked to original sources

PubMed2005

Quantifying mixed populations of drug-resistant human immunodeficiency virus type 1.

In order to survive prolonged treatment with antiretroviral nucleoside analogs, the human immunodeficiency virus type 1 (HIV-1) is selectively forced to acquire mutations in the reverse transcriptase (RT) gene. Some of these mutations are more common than others and have become markers for antiretroviral resistance. For the early detection of these markers, a novel MultiCode-RTx one-step testing system to rapidly and simultaneously characterize mixtures of HIV-1 targets was designed. For cDNA, nucleotide polymorphisms for codon M184V (ATG to GTG) and K65R (AAA to AGA) could be differentiated and quantified even when the population mixture varied as much as 1 to 10,000. Standard mixed-population curves using 1 to 100% of the mutant or wild type generated over 4 logs of total viral particle input did not affect the overall curves, making the method robust. The system was also applied to a small set of samples extracted from infected individuals on nucleoside reverse transcriptase inhibitor therapy. Of 13 samples tested, all were positive for HIV and 10 of the 13 genotypes determined were concordant with the line probe assay. MultiCode-RTx could be applied to other drug-selected mutations in the viral genome or for applications where single-base changes in DNA or RNA occur at frequencies reaching 0.01% to 1%, respectively.

Anti-HIV Agents↗

PubMed2005

Retrovirus reverse transcriptases containing a modified YXDD motif.

The YXDD motif, where X is a variable amino acid, is highly conserved among various viral RNA-dependent DNA polymerases. Mutations in the YXDD motif can abolish enzymatic activity, alter the processivity and fidelity of enzymes and decrease virus infectivity. This review provides a summary of the significant documented studies on the YXDD motif of HIV-1, simian immunodeficiency virus, feline immunodeficiency virus and murine leukaemia virus and the impact of mutation that this motif has had on viral pathogenesis and drug treatment.

Amino Acid Motifs↗

PubMed2004

Replication-dependent 65R-->K reversion in human immunodeficiency virus type 1 reverse transcriptase double mutant K65R + L74V.

Understanding of the mechanisms of interaction among nucleoside reverse transcriptase inhibitor (NRTI)-selected mutations in the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) coding sequence is essential for the design of newer drugs and for enhancing our vision of the structure function relationship among amino acids of the polymerase domain of HIV-1. Although several nucleoside reverse transcriptase inhibitors select RT mutations K65R and L74V, the combination of 65R + 74V is rare in clinics. A novel NRTI (-)-beta-d-dioxolane-guanosine (DXG) is known to select in vitro either the 65R or 74V mutant virus. These mutations were not selected together during repeated passaging of the HIV-1 in the presence of this drug. To analyze the impact of these RT mutations on viral replication, a double mutant containing K65R + L74V was created by site-directed mutagenesis in a pNL4-3 background. Replication kinetic assays revealed that the mutant K65R + L74V is unstable, and 65R-->K reversion occurs during replication of virus in phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication kinetic assays in MT-2 cells demonstrated that double mutant 65R + 74V is highly attenuated for replication and the initiation of reversion is related to the increase in RT activity. Additionally, the suppression of viral replication in the presence of DXG or under suboptimal human recombinant interleukin-2 leads to minimal or no 65R-->K reversion. These observations provide evidence that 65R-->K reversion in the double mutant 65R + 74V is dependent on a specific rate of viral replication in a pNL4-3 background. A similar phenomenon may occur in vivo, which may have implications for treatment management strategies.

Arginine↗

PubMed2004

Nucleoside inhibitors of human immunodeficiency virus type 1 reverse transcriptase.

The development of novel compounds that can effectively inhibit both wild type and the most consensus resistant strains of human immunodeficiency virus type 1 (HIV-1) is the primary focus in HIV disease management. Combination therapy, comprising at least three classes of drugs, has become the standard of care for acquired immunodeficiency syndrome (AIDS) or HIV-infected individuals. The drug cocktail can comprise all three classes of HIV inhibitors, including nucleoside reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI). Due to their competitive mode of inhibition and requirement for metabolic activation, almost all NRTI drugs lack the virological potency of NNRTI or PI drugs. However, data from clinical trials indicate that sustained viral suppression could not be achieved with NRTI, NNRTI or PIs alone. Therefore, the NRTIs will remain essential components of highly active antiretroviral therapy (HAART) for the foreseeable future, because they enhance the virological potency of the regimen, they do not bind excessively to protein and most regimens are small pills/tablets given once a day. It has become apparent in recent years that the prolonged use of certain NRTIs exhibits adverse events as a class, limiting the length of time for which they can be safely used. Of major clinical concern is their association with the potentially fatal lactic acidaemia and hepatic steatosis. These class events, as well as individual drug effects, such as peripheral neuropathy, are linked to delayed mitochondrial destruction. In addition to toxicity, the development of resistance-conferring mutations against exposure to nucleoside analogs currently in use influences long-term therapeutic benefits. Of critical importance for the evaluation of new NRTIs are recent studies showing that the efficiency of discrimination or excision by pyrophosphorolysis in the presence of nucleotides of a given NRTI is a key determinant in the emergence of one or the other resistance pathway.

Anti-HIV Agents↗

PubMed2003

Fluorescent dye terminator sequencing methods for quantitative determination of replication fitness of human immunodeficiency virus type 1 containing the codon 74 and 184 mutations in reverse transcriptase.

The fluorescent dye-labeled dideoxynucleotide automated DNA sequencing system has been routinely used for monitoring the development of resistance mutations in human immunodeficiency virus type 1 reverse transcriptase (RT) and protease genes during therapy. This system has provided information regarding the presence of mixtures of nucleotides in the clinical samples but has not previously been validated for the quantitative determination between peak heights and relative DNA concentration. We evaluated this system by using various ratios of wild-type and mutated DNA fragments and by performing sequencing reactions at actual melting temperatures of specific primers. Several different ratios of purified DNA fragments containing mixtures of L74/V74 and M184/V184 were sequenced, and peak heights were measured. Regression analysis between ratios of peak heights and DNA concentration demonstrated a statistically significant linear correlation, suggesting that the quantification of two different species of DNA in a mixture could be achieved with the fluorescent dye-labeled dideoxynucleotide system. These strategies have broader implications for the quantification of replication fitness of viruses, particularly those containing RT mutations at codons 74 and 184.

Codon↗