Genetic labeling of HIV-1 proteins and its impact on virus infectivity.

Abstract

<p>(A) Diagram of the HIV-1 <i>gag</i> (red), <i>pol</i> (brown) and <i>env</i> (blue) open reading frames and the positions where the tetracysteine (TC) tag motif was inserted. The majority of the TC motifs were inserted five amino acids downstream (-N tags) or upstream (-C tags) of the protease cleavage site and the first or last five amino acids of the viral protein coding sequence were repeated to prevent alterations in the minimal recognition site for the viral protease. The left insert shows the crystal structure of the RT enzyme and the positions in the “thumb” region where the TC tag was inserted are shown in blue. The right insert shows the crystal structure of the Env gp120 and the positions in the V1/V2 region where the TC tag was inserted are shown in blue. (B) Virion protein processing profiles of 293T cell-derived HIV^MA-C, HIV^CA-N, HIV^CA-C, HIV^NC-N, HIV^NC-C, HIV^PR-C, HIV^RT-N, HIV^RT-C, HIV^RN-N, HIV^RN-C and HIV^IN-N (lanes 1-12, respectively). HIV-1 proteins were detected by Western Blot using pooled HIV-positive patient sera. Data are representative of 2 independent experiments. (C) The infectivity of HIV^MA-C, HIV^CA-N, HIV^CA-C, HIV^NC-N, HIV^NC-C, HIV^PR-C, HIV^RT-N, HIV^RN-N, HIV^RN-C and HIV^IN-N was assessed by comparing their replication kinetics in peripheral blood mononuclear cells with HIV^wt. Samples were collected at 3, 7, 11 and 14 days post infection, and the levels of virus replication were monitored using an <i>in vitro</i> reverse transcriptase assay that is specific for HIV-1 enzymatic activity. Data are representative of 2-3 independent donors. Error bars represent technical replicates. (D) The capacity of several HIV^TC to enter target cells was assessed by measuring β-lactamase activity in MT-2 cells that have been infected with HIV^TC containing a β-lactamase-Vpr fusion protein. Error bars, s.d. are based on the averages of 2-3 independent experiments. (E) The capacity of HIV^TC to infect target cells was assessed by measuring luciferase activity in the indicator TZM-bl cells that have been infected with the indicated viruses. Error bars, s.d. are based on the averages of 2-3 independent experiments.</p