Designed Viruses and Designed Host Cells

T-20 or enfuvirtide was rationally designed to interfere with fusion of the viral envelope and the plasma membrane by inhibiting a required conformational change in the transmembrane protein gp41. In clinical trials, the emergence of escape mutants was observed within 14 days of monotherapy with T-20. The method to assay field isolates for the evolution of resistance to this drug required a more complex approach: HeLa cells were stably transfected with LTR-driven reporter and with CD4 and CCR5 receptors, and the reporter signal was shown to decrease in a T-20 dose-dependent fashion in cells challenged with HIV. To demonstrate that indeed viral entry is the target ofT-20, the viral envelope proteins from laboratory HIV particles were replaced with envelope proteins from the escape mutants, and these pseudotyped viruses were used in the screen in the presence of escalating doses of T-20. As expected, virus replication - and thus reporter gene activity - was stronger for a given T-20 concentration in pseudotyped viruses when compared to laboratory strain reference [75], demonstrating that T-20 drives HIV into selection of envelope-protein escape mutants. T-20 therefore should be a good candidate for combination therapy with drugs that target other aspects of the viral life cycle, such as reverse transcription or morphogenesis via the viral protease.

Another approach using recombinant viruses on designed cells was performed to search for inhibitors of transactivation and to characterize the mechanism of inhibition. This approach is instructive, as this principle should be applicable to other screens where a specific trans-activator and promoter interaction is to be targeted by a drug: a replication-deficient adenovirus (a DNA virus vector completely unrelated to retroviruses) containing the LTR-reporter cassette was used as tester on HeLa cells that had been engineered to constitutively express the Tat protein. Following infection with the adenoviral vector, the inhibitor was added [82] and the reduction of reporter expression was measured. Using an adenoviral vector to transduce the reporter allows better efficiency and consistency compared to transfection.

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