Hiv type 1 p24 antigen elisa

The VSV-G pseudotyped HIV-1-ΔVif luciferase reporter viruses with A3G or its mutants were produced by co-transfection of pNL4-3/ΔenvΔvif-Luc and pVSV-G with the A3G expression vector (pcDNA3/HA-A3G) in HEK293T cells as previously described (Shindo et al., 2003 (link); Kobayashi et al., 2004 (link)). The target M8166 cells were infected with reporter viruses normalized by p24 amount, which was measured by HIV Type 1 p24 Antigen ELISA (ZeptoMetrix). The infectivity was measured by luciferase activity and values were normalized to the infectivity of the virus produced in the absence of A3G expression.

The plasmids pcDNA3.1-APOBEC3G-HA and pNL4-3 were transfected into 293T cells and cultured with or without hop-8. The cells were collected and the expression of A3G-HA, p55, p24, Vif, and beta-actin was analyzed by western blot. The supernatant was collected and centrifuged at 2000 g and the cell debris discarded. To normalize viral input, the levels of p24 were determined using HIV Type 1 p24 Antigen ELISA (ZeptoMetrix Corporation, Buffalo, NY, USA). TZM-bl cells were infected with supernatant containing 20 ng of HIV-1 p24, and the residual infection was determined using relative luciferase activity. To determine the incorporation of A3G in progeny virion, plasmids pcDNA3.1-APOBEC3G-HA and pNL4-3 were transfected into 293T cells and cultured with or without hop-8 for 48 h. The supernatant was collected and centrifuged at 2000× g. The cell debris was discarded. The viral particles were lysed with 0.5% Triton-X100. A3G and p24 levels in the supernatant were determined by western blot.

The pseudotyped HIV-1 Gag-iFRET labeled virus was produced by co-transfecting HEK293T cells with pHIV-1 Gag-iFRETΔEnv and pNL4-3ΔEnv parental plasmids at three different ratios as described in section “Cell Cultures and Virus Production,” together with the HIV-1 envelope expression plasmid, pSVIII-92HT593.1. The pSVIII-92HT593.1 construct was obtained from Dr. Beatrice Hahn through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 92HT593.1 gp160 Expression Vector (cat# 3077) (Gao et al., 1996 (link)). HIV-1 Gag-iFRETΔPro labeled virus was produced by co-transfecting HEK293T cells with pHIV-1 Gag-iFRETΔPro and pNL4-3ΔPro parental plasmid at the same ratios as HIV-1 Gag-iFRET. The viral titer was measured by HIV Type 1 p24 Antigen ELISA (ZeptoMetrix). The following day after 5 × 10³ TZM-bl cells seeded in 96 well plates, an equal amount of virus (total of 5 ng HIV-1 p24) was added to the TZM-bl target cells, and then cultured at 37°C for 48 h in a CO₂ incubator. Luciferase activity in the infected cells was measured with the Luciferase Assay System (Promega) on a 2030 ARVO X3 plate reader (Perkin Elmer) to quantify virus infectivity.