Analysis of HIV-1 integrase structure and function in viralcDNA synthesis

The human immunodeficiency virus (HIV) integrase (IN) protein is required for the integration of the reverse transcribed viral genome (cDNA) into the host cell DNA. In addition, the IN protein augments viral cDNA synthesis in infected cells by an unknown mechanism. To investigate how IN augmented viral cDNA synthesis, we used a cDNA synthesis defective HIIV-1/HIV-2 chimeric (SG3 IN2) virus in a gain of function study. Long-term culturing of SG3 IN2 virus resulted in the acquisition of mutations throughout the viral genome that facilitated viral cDNA synthesis and infectivity. The IN region was demonstrated to contain mutations that were primarily responsible for the improvements in viral infectivity. Further analysis revealed that three IN mutations (Q96H, K127E, and V204I) were responsible for much of the increase in viral cDNA synthesis.; To investigate whether the IN region that contained the 204 residues (the central domain alpha6 helix) is important for viral cDNA synthesis, we introduced mutations into this region. Virions containing IN mutated at alpha6 helix were found to have maturation, cDNA synthesis, and infectivity defects, but were not defective in IN enzymatic function. The mutant IN protein present within these virions had aberrant folding and multimerization. This suggested that a6 helix is necessary for correct IN multimerization and viral cDNA synthesis.; The HIV-1 IN central domain has a structural similarity to the double-stranded RNA binding domain (dsRBD). The IN protein was shown to incorporate into virions in a viral RNA genome (vRNA) dependent manner, and mutations within the IN dsRBD-like region caused a loss of IN incorporation. Virions containing IN with dsRBD mutations had reduced viral cDNA synthesis and infectivity and exhibited virion morphology defects. Trans-complementation analysis indicated that this IN motif is important for virion morphology and cDNA synthesis when IN is incorporated as part of Pr160Gag-Pol.; Together these findings show that alpha6 helix and the dsRBD-like region in IN are determinants required for normal maturation and efficient viral cDNA synthesis.