Effect of HIV constructs containing protease-reverse transcriptase fusion proteins on viral replication


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Abstract

Objective:To determine whether disruption of the cleavage site between protease and reverse transcriptase (RT) or the HIV-1 frameshift site could yield trans-dominant negative HIV-1 variants that interfere with wild-type viral replication.Design:Residues at the cleavage site between the HIV-1 protease and RT coding regions were mutagenized to produce a protease-RT (PR-RT) fusion protein that was expressed in the context of a full-length provirus. The PR-RT cleavage site mutation was also combined with a read-through mutation at the frameshift site in order to overexpress the mutant Gag-Pol polyproteins.Methods:COS-7 cells were transiently transfected with the mutant constructs to produce viruses harbouring the PR-RT fusion protein. In addition, we performed cotransfection studies in various cell types to analyze the inhibition of wild-type replication by the mutant constructs.Results:Immunoblot analysis revealed that this novel mutation prevented cleavage between the two proteins and that both existed as a PR-RT fusion protein in each of cellular and viral lysates. While both the protease and RT components of this fusion protein remained functionally active, viruses containing the cleavage site mutation were less infectious in tissue culture than wild-type viruses produced by COS-7 cells. This defect was further pronounced when the cleavage site mutation between protease and RT was overexpressed as a consequence of an additional mutation that interfered with frameshifting. Cotransfection of COS-7 cells with the mutant constructs and wild-type HIV-1 interfered with the replication of the latter and reduced the infectiousness of the virus particles produced.

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