Phenotyping of Cytomegalovirus Drug Resistance Mutations by Using Recombinant Viruses Incorporating a Reporter Gene

ABSTRACT A new recombinant phenotyping method was developed for the analysis of drug resistance mutations in human cytomegalovirus (CMV). CMV strain T2211 was derived from strain AD169 by inserting unique restriction sites and a secreted alkaline phosphatase (SEAP) reporter gene for rapid viral quantitation. Specific viral UL97 and pol gene mutations were transferred by recombination into T2211, and their drug resistance phenotypes (for ganciclovir, foscarnet, or cidofovir) were determined by the drug concentrations required to reduce supernatant SEAP activity by 50% (IC50). Changes in the IC50 conferred by the mutations tested (UL97 M460V, C592G, A594V, and L595S and pol del981-2) were similar to those previously reported in marker transfer and conventional plaque reduction assays. The combination of UL97 C592G and pol del981-2 conferred much higher ganciclovir resistance than either mutation alone. The UL97 polymorphism D605E had no measurable effect on ganciclovir susceptibility, alone or in combination with common UL97 resistance mutations. Transfer into strain T2211 facilitates the phenotyping of newly observed mutations, combinations of mutations, and clinical CMV sequences without an accompanying viral isolate.

Lovastatin selectively inhibits ras activation of the 12-O-tetradecanoylphorbol-13-acetate response element in mammalian cells

To evaluate ras-mediated signal transduction, an alkaline phosphatase gene (SEAP) was placed under the control of the ras-inducible phorbol ester response element (TRE) in murine fibroblasts (TRE-SEAP cells). The Kirsten ras gene was placed under the control of the glucocorticoid-inducible mouse mammary tumor virus promoter and introduced into the TRE-SEAP cells. Dexamethasone increased ras expression in the TRE-SEAP cells carrying the Kirsten ras gene and stimulated SEAP activity 25-fold. Lavostatin blocked dexamethasone induction of SEAP activity (50% inhibitory concentration, 0.5 microM) but did not affect phorbol ester-induced SEAP activity in the same cells. Lovastatin also did not block forskolin induction of SEAP activity in cells expressing SEAP under the control of the cyclic AMP response element.

Lovastatin selectively inhibits ras activation of the 12-O-tetradecanoylphorbol-13-acetate response element in mammalian cells.

To evaluate ras-mediated signal transduction, an alkaline phosphatase gene (SEAP) was placed under the control of the ras-inducible phorbol ester response element (TRE) in murine fibroblasts (TRE-SEAP cells). The Kirsten ras gene was placed under the control of the glucocorticoid-inducible mouse mammary tumor virus promoter and introduced into the TRE-SEAP cells. Dexamethasone increased ras expression in the TRE-SEAP cells carrying the Kirsten ras gene and stimulated SEAP activity 25-fold. Lavostatin blocked dexamethasone induction of SEAP activity (50% inhibitory concentration, 0.5 microM) but did not affect phorbol ester-induced SEAP activity in the same cells. Lovastatin also did not block forskolin induction of SEAP activity in cells expressing SEAP under the control of the cyclic AMP response element.