Complementation of integrase function in HIV-1 virions.
Int Conf AIDS 1996 Jul 7-12; 11:217 (abstract no. Th.A.381) Fletcher TM 3d, Soares MA, Wu X, McPherson S, Muesing MA, Boeke JD, Kappes JC, Hahn BH; Univ. of Alabama at Birmingham, Birmingham, AL.
We have previously demonstrated that HIV/SIV virion associated accessory proteins (Vpr, Vpx, Vif) can be utilized to target biologically active fusion proteins to the virus particle. In this paper, we examined whether coexpression of Vpr-integrase fusion proteins can restore the biological activity of int-mutant HIV-1 proviruses in a single round virus infectivity assay (MAGI). Vpr-int fusion (and control) genes were cloned into a eukaryotic expression vector and cotransfected with wild-type and int-mutant HIV-1 proviruses previously shown to be defective in the MAGI cell assay (H12A, H16A, E69A/K71A, K244A/E246A). Vpr-int fusion genes with and without a natural protease cleavage site (R-PC-IN and R-IN), as well as a cleavage site mutant (R-deltaPC-IN), were tested along with control constructs containing the vpr (R) or int (IN) gene alone. Western blot analysis of transfected cell supernatants showed that all Vpr-int fusion constructs expressed appropriately sized proteins which were packaged into HIV-1 virions. However, analysis in the MAGI cell assay demonstrated that only R-PC-IN, and not R-IN or R-deltaPC-IN, could complement the integrase defect in the mutant HIV-1 proviruses. This ability correlated with protease processing (R-IN and R-deltaPC-IN were NOT cleaved by the viral protease). No packaging or restoration of function was observed with the IN control construct. These results indicate that (i) HIV-1 integrase can be packaged into virions independent of the Gag/Pol precursor when expressed as a Vpr fusion protein, (ii) this Vpr-mediated packaging restores the biological activity of int-mutant HIV-1 proviruses in the MAGI cell assay, and (iii) the natural N-terminus of the integrase is required for successful complementation. To our knowledge this is the first time that in trans complementation of integrase function in the context of virions has been achieved.
Keywords: AEGIS, Gene Products, vpr, Virion, HIV-1, HIV Integrase, HIV Protease, Proviruses, Integrase Inhibitors, HIV Antibodies, HIV Protease Inhibitors, HIV Core Protein p24, HIV Antigens, HIV-1 Reverse Transcriptase, Blotting, Western, HIV, Anti-HIV Agents, HIV Infections, ICA11
