A novel Vpr peptide interactor fused to integrase (IN) restores integration activity to IN-defective HIV-1 virions

A novel approach to complement human immunodeficiency virus type I (HIV-1) integrase (IN)-defective virions has been identified. The approach involves fusion of a 23-amino-acid stretch to the N-terminus of wild-type IN and co expression of this chimera with the IN-defective proviral template in virus producing cells. The 23-amino-acid peptide represents a Vpr "interactor," referred to as the the WxxF or WF domain, which apparently leads to docking of the domain along with the fusion partner onto HIV-1 Vpr, thus permittin g virion incorporation of the chimeric protein when expressed, in trans, wi th other viral products. Transfection of the WF-IN expression plasmid along with HIV-1 viral clones that produce Vpr, but bear an IN mutation, results in the release of a proportion of viral particles that are competent for i ntegration. The extent of complementation was assessed using the MAGI cell assay, where integration of viral DNA results in the eventual appearance of easily visible multinucleated blue syncytia. The efficiency of dWF-IN (dou ble copy of WF domain) complementation is not improved markedly by incorpor ation of a HIV-1 protease cleavage site (PR) between the dWF domain and IN (dWF-PR-IN), unlike that observed with Vpr fusions to IN. Furthermore, the ability of Vpr-PR-IN and dWF-PR-IN to complement IN-defective proviral clon es, both of which bear an intervening protease cleavage site, appear compar able. Western blotting analyses using virions isolated through sucrose cush ions demonstrate clearly the incorporation of the dWF-IN fusion protein int o Vpr containing HIV-1 particles but not in Vpr-deficient virions. Addition al Western blotting analyses indicate that all Vpr-IN and dWF-IN chimeras, with or without a PR site, are packaged into virions. The efficiency of vir ion incorporation of Vpr-IN and dWF-IN chimeras appears approximately compa rable by Western blotting analysis. The ability of dWF-IN to complement IN- defective proviruses with efficiency similar to that of Vpr-PR-IN and dWF-P R-IN indicates that dWF-IN retains the full complement of functions necessa ry for integration of proviral DNA and is likely due to the benign nature o f this small domain at the amino-terminus of IN. (C) 1999 Academic Press.