Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thymidylate kinases are essential enzymes with roles in DNA synthesis and repair and have been the target of drug development for antimalarials, antifungals, HIV treatment, and cancer therapeutics. Human thymidylate kinase (hTMPK) conversion of the anti-HIV prodrug 3'-azido-3'-deoxythymidine (AZT or zidovudine) monophosphate to diphosphate is the rate-limiting step in the activation of AZT. A point mutant (F105Y) has been previously reported with significantly increased activity for the monophosphate form of the drug [3'-azidothymidine-5'-monophosphate (AZTMP)]. Using solution nuclear magnetic resonance (NMR) techniques, we show that while the wild-type (WT) and F105Y hTMPK adopt the same structure in solution, significant changes in dynamics may explain their different activities toward TMP and AZTMP. 13C spin-relaxation measurements show that there is little change in dynamics on the ps to ns time scale. In contrast, methyl 1H relaxation dispersion shows that AZTMP alters adenosine nucleotide handling in the WT protein but not in the mutant. Additionally, the F105Y mutant has reduced conformational flexibility, leading to an increase in affinity for the product ADP and a slower rate of phosphorylation of TMP. The dynamics at the catalytic center for F105Y bound to AZTMP are tuned to the same frequency as WT bound to TMP, which may explain the mutant's catalytic efficiency toward the prodrug.
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PMID:Stabilization of Active Site Dynamics Leads to Increased Activity with 3'-Azido-3'-deoxythymidine Monophosphate for F105Y Mutant Human Thymidylate Kinase. 3206 97

There has been resurgence in determining the role of host metabolism in viral infection yet deciphering how the metabolic state of single cells affects viral entry and fusion remains unknown. Here, we have developed a novel assay multiplexing genetically-encoded biosensors with single virus tracking (SVT) to evaluate the influence of global metabolic processes on the success rate of virus entry in single cells. We found that cells with a lower ATP:ADP ratio prior to virus addition were less permissive to virus fusion and infection. These results indicated a relationship between host metabolic state and the likelihood for virus-cell fusion to occur. SVT revealed that HIV-1 virions were arrested at hemifusion in glycolytically-inactive cells. Interestingly, cells acutely treated with glycolysis inhibitor 2-deoxyglucose (2-DG) become resistant to virus infection and also display less surface membrane cholesterol. Addition of cholesterol in these in glycolytically-inactive cells rescued the virus entry block at hemifusion and enabled completion of HIV-1 fusion. Further investigation with FRET-based membrane tension and membrane order reporters revealed a link between host cell glycolytic activity and host membrane order and tension. Indeed, cells treated with 2-DG possessed lower plasma membrane lipid order and higher tension values, respectively. Our novel imaging approach that combines lifetime imaging (FLIM) and SVT revealed not only changes in plasma membrane tension at the point of viral fusion, but also that HIV is less likely to enter cells at areas of higher membrane tension. We therefore have identified a connection between host cell glycolytic activity and membrane tension that influences HIV-1 fusion in real-time at the single-virus fusion level in live cells.
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PMID:Single-cell glycolytic activity regulates membrane tension and HIV-1 fusion. 3208 46


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