| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1996: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Integration is an essential step to establish the proviral state of retroviruses including human immunodeficiency virus type 1 (HIV-1). In this study, the extent of integration dependency for viral gene expression and possible function (s) of the integrase (IN) of HIV-1 in vivo were addressed. Towards these ends, we made several IN mutants by introducing single or double amino acid substitution into the highly conserved HHCC (zinc-finger domain) or D,D35E (catalytic site) motif of HIV-1 IN.In addition, the attachment (att) site mutants were also made, in which the IN was kept intact but the sequences of the U3 or U5 teminal region was altered or deleted. The effect of each mutation was examined by using single-step infection system with emvelope psuedotype virus. The relative integration efficiency of each mutant was estimated by examining the stability of each de novo synthesized viral DNA with quantitative PCR method. The efficiency of the catalytic site and the att site deletion mutants were estimated to be as low as 0.5 % of WT level. The gene expression level of each mutant measured by using highly sensitive luciferase assay was shown to well correlate with each integration efficiency, demonstrating that integration is obligate step for HIV-1 gene expression. Meanwhile, we also found that all the three zinc-finger mutants examined here, were sevearly defective in the de novo synthesis of viral DNA after the infection, suggesting impairment of these mutants before or in the reverse transcription (RT) process. Finally from mutational analysis of att site indicated that terminal 11 bp is minimum cis element required for spesific IN interaction, and that IN recognize each att site indipendently, suggesting importance of IN multimerization for conserted integration in vivo.
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