Figure 5

Phosphatase treatment does not affect in vitro RT activity. (A) Recombinant M-MuLV RT was assayed directly (RT1 = 500 milliUnits [mU], RT2 = 100 mU, RT3 = 20 mU, RT4 = 4 mU) or was incubated for 60 mins at 37°C in PBS or CIAP buffer +/- CIAP enzyme prior to exogenous RT activity assay, overnight at 37°C using DIG-UTP and colourimetric detection of incorporated DIG. (B) H3B and Hut-78 cells were co-cultured and lysed at 40 min post-cell mixing and lysates were subjected to 0–60% linear sucrose equilibrium gradient sedimentation. Fractions (1 ml) were collected from the top of the gradient and viral Gag DNA analysed by real time PCR. Fractions 7–8, containing HIV DNA and sedimenting at 1.19–1.25 g/ml sucrose was immunoprecipitated with AIDS patient sera and represented the RTCs used subsequently in the in vitro RT activity assay. (C) Samples from virions, cell lysates and RTCs were incubated for 60 mins at 37°C in PBS or CIAP buffer +/- CIAP enzyme prior to exogenous RT activity assay, overnight at 37°C using DIG-UTP and colourimetric detection of incorporated DIG. Results were normalized against the RT activity observed in the absence of CIAP and represents data from 3 independent dephosphorylation and RT activity assays and from 2 independent RTC preparations.