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Related Experiment Videos

Gammaherpesviruses encode functional dihydrofolate reductase activity.

Gábor Gáspár1, Erik De Clercq, Johan Neyts

  • 1Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.

Biochemical and Biophysical Research Communications
|October 3, 2002
PubMed
Summary
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Gammaherpesvirus dihydrofolate reductase (DHFR) enzymes from HHV-8, HVS, and RRV were overexpressed and purified. These viral DHFR enzymes exhibit distinct kinetic properties and are less sensitive to folate antagonists compared to human DHFR.

Area of Science:

  • Biochemistry
  • Virology
  • Enzymology

Background:

  • Gammaherpesviruses, including human herpesvirus 8 (HHV-8), herpesvirus saimiri (HVS), and rhesus rhadinovirus (RRV), are DNA viruses that can cause disease in various hosts.
  • Dihydrofolate reductase (DHFR) is a crucial enzyme in folate metabolism, essential for DNA synthesis and cell proliferation.
  • Viral DHFR enzymes are potential targets for antiviral therapies, although their biochemical properties and susceptibility to inhibitors can differ from human DHFR.

Purpose of the Study:

  • To overexpress and purify the dihydrofolate reductase (DHFR) enzymes from three gammaherpesviruses: HHV-8, HVS, and RRV.
  • To characterize the kinetic properties (K(m) values for dihydrofolate and NADPH) of these viral DHFR enzymes.
  • To investigate the inhibitory effects of known folate antagonists on the catalytic activity of the viral DHFR enzymes.

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Main Methods:

  • Overexpression and purification of recombinant DHFR enzymes from HHV-8, HVS, and RRV in Escherichia coli.
  • Enzymatic assays to determine Michaelis-Menten constants (K(m)) for dihydrofolate (DHF) and NADPH.
  • Enzyme inhibition studies using various folate antagonists, including methotrexate, aminopterin, trimethoprim, pyrimethamine, and PT523.

Main Results:

  • All three gammaherpesvirus DHFR enzymes were successfully overexpressed, purified, and found to be catalytically active.
  • The K(m) values for dihydrofolate for HHV-8, HVS, and RRV DHFR were approximately 5-15-fold higher than that of human DHFR.
  • The K(m) values for NADPH were similar or slightly higher for the viral DHFR enzymes compared to human DHFR.
  • Folate antagonists competitively inhibited viral DHFR activity with respect to DHF but were markedly less inhibitory than against human DHFR.

Conclusions:

  • The DHFR enzymes from HHV-8, HVS, and RRV possess distinct kinetic parameters compared to human DHFR, particularly regarding their affinity for dihydrofolate.
  • The reduced sensitivity of viral DHFR to common folate antagonists suggests potential for developing selective antiviral agents.
  • These findings provide a biochemical basis for understanding gammaherpesvirus replication and offer insights for targeted antiviral drug design.