Biomedical subjects

Vinay K Pathak

Publications and source records attributed to Vinay K Pathak.

22 records · Page 2Linked to original sources

PubMed2003

Retroviral mutation rates and reverse transcriptase fidelity.

Genetic variation in retroviral populations provides a mechanism for retroviruses to escape host immune responses and develop resistance to all known antiretroviral drugs. Retroviruses, like all RNA viruses, exhibit a high mutation rate. Polymerization errors during DNA synthesis by reverse transcriptase, which lacks a proofreading activity, is a major mechanism for generating genetic variation within retroviral populations. In this review, we summarize our current understanding of the processes that contribute to the generation of mutations in retroviruses. An overview of in vivo and in vitro studies of retroviral mutation rates determined by various fidelity assays is provided. Extensive mutational analyses of RTs are beginning to elucidate the relationship between structural determinants of RTs and fidelity of DNA synthesis. Recently, it was observed that the Y586F mutation in MLV RT results in a dramatic increase in the mutation rate in the vicinity of adenine-thymie tracts (AAAA, TTTT, and AATT), which are associated with bends in DNA. These results indicate that the template-primer duplex is a component of the polymerase active site and its structure can influence nucleotide selectivity and the mutation rate. Additionally, the results also suggest that the Y586 residue and the RNase H primer grip are structural determinants of RT that have evolved to attenuate the effects of unusual conformations of the template-primer duplex, such as bends in DNA, on fidelity of DNA synthesis.

Animals↗

PubMed2002

Structure activity of 3-aryl-1,3-diketo-containing compounds as HIV-1 integrase inhibitors.

The 4-aryl-2-hydroxy-4-oxo-2-butenoic acids and their isosteric tetrazoles are among an emerging class of aryl beta-diketo (ADK)-based agents which exhibit potent inhibition of HIV-1 integrase (IN)-catalyzed strand transfer (ST) processes, while having much reduced potencies against 3'-processing (3'-P) reactions. In the current study, L-708,906 (10e) and 5CITEP (13b), which are two examples of ADK inhibitors that have been reported by Merck and Shionogi pharmaceutical companies, served as model ADK leads. Structural variations to both the "left" and "right" sides of these molecules were made in order to examine effects on HIV-1 integrase inhibitory potencies. It was found that a variety of groups could be introduced onto the left side aryl ring with maintenance of good ST inhibitory potency. However, introduction of carboxylic acid-containing substituents onto the left side aryl ring enhanced 3'-P inhibitory potency and reduced selectivity toward ST reactions. Although both L-708,906 and 5CITEP show potent inhibition of IN in biochemical assays, there is a disparity of antiviral activity in cellular assays using HIV-1-infected cells. Neither 5CITEP nor any other of the indolyl-containing inhibitors exhibit significant antiviral effects in cellular systems. Alternatively, consistent with literature reports, L-708,906 does provide antiviral protection at low micromolar concentrations. Interestingly, several analogues of L-708,906 with varied substituents on the left side aryl ring, while having good inhibitory potencies against IN in extracellular assays, are not antiviral in whole-cell systems.

Acetoacetates↗

PubMed2002

Y586F mutation in murine leukemia virus reverse transcriptase decreases fidelity of DNA synthesis in regions associated with adenine-thymine tracts.

Using in vivo fidelity assays in which bacterial beta-galactosidase or green fluorescent protein genes served as reporters of mutations, we have identified a murine leukemia virus (MLV) RNase H mutant (Y586F) that exhibited an increase in the retroviral mutation rate approximately 5-fold in a single replication cycle. DNA-sequencing analysis indicated that the Y586F mutation increased the frequency of substitution mutations 17-fold within 18 nt of adenine-thymine tracts (AAAA, TTTT, or AATT), which are known to induce DNA bending. Sequence alignments indicate that MLV Y586 is equivalent to HIV-1 Y501, a component of the recently described RNase H primer grip domain, which contacts and positions the DNA primer strand near the RNase H active site. The results suggest that wild-type reverse transcriptase (RT) facilitates a specific conformation of the template-primer duplex at the polymerase active site that is important for accuracy of DNA synthesis; when an adenine-thymine tract is within 18 nt of the polymerase active site, the Y586F mutant RT cannot facilitate this specific template-primer conformation, leading to an increase in the frequency of substitution mutations. These findings indicate that the RNase H primer grip can affect the template-primer conformation at the polymerase active site and that the MLV Y586 residue and template-primer conformation are important determinants of RT fidelity.

Amino Acid Sequence↗

PubMed2002

Zinc finger domain of murine leukemia virus nucleocapsid protein enhances the rate of viral DNA synthesis in vivo.

In vitro studies have indicated that retroviral nucleocapsid (NC) protein facilitates both DNA synthesis by reverse transcriptase (RT) and annealing of the nascent DNA with acceptor template. Increasing the rate of DNA synthesis is expected to reduce the frequency of RT template switching, whereas annealing the nascent DNA with acceptor template promotes template switching. We performed a mutational analysis of the murine leukemia virus (MLV) NC zinc finger domain to study its effect on RT template switching in vivo and to explore the role of NC during reverse transcription. The effects of NC mutations on RT template switching were determined by using a previously described in vivo direct-repeat deletion assay. A trans-complementation assay was also developed in which replication-defective NC mutants were rescued by coexpression of replication-defective RT mutants that provided wild-type NC in trans. We found that mutations in the MLV NC zinc finger domain increased the frequency of template switching approximately twofold. When a predicted stem-loop RNA secondary structure was introduced into the template RNA, the template-switching frequency increased 5-fold for wild-type NC and further increased up to an additional 6-fold for NC zinc finger domain mutants, resulting in an overall increase of as much as 30-fold. Thus, wild-type NC increased the efficiency with which RT was able to reverse transcribe through regions of RNA secondary structure that might serve as RT pause sites. These results provide the first in vivo evidence that NC enhances the rate of DNA synthesis by RT in regions of the template possessing stable RNA secondary structure.

Amino Acid Motifs↗