The resulting viruses were then incubated for 2 h at 37 C in a buffer containing 10 mM MgCl2 and 50 Uml of RNase free DNase I. Virus particles were further concentrated by centrifugation through a 30% sucrose cushion in PBS at 24,000 RPM in a Beckman SW 28 rotor for 2 h at 4 C. Virus pellets were resus pended either in RPMI medium containing 20 mM HEPES pH 7. 4 or in PBS for RNA isola tion and selleck chem Calcitriol Western blot analysis. For infection viral titers were normalized to 0. 01 or 0. 1 pg of p24CA per cell, using a p24 ELISA kit. Infection of Sup T1 cells was performed in 12 well plates by spinoculation at 1000g for 2 h at 18 C, according to the published protocol. The cells were washed twice with PBS at room temperature and incubated in culture medium at 37 C for 0 48 h.

For infection with nevirapine, the cells were pre treated overnight with 10 uM nevirapine and then cul tivated for Inhibitors,Modulators,Libraries 24 h after infection in the fresh culture media containing 10 uM nevirapine. Western blot analysis The suspensions of virus particles Inhibitors,Modulators,Libraries in PBS were mixed with equal volumes of Laemmli Sample Buffer, heated in boiling water for 2 min and then subjected to SDS PAGE. Proteins were transferred to PVDF mem branes, and detected using anti HIV 1 p24 or anti HIV 1 integrase mouse monoclonal antibo dies from NIH AIDS Research Reference Reagent Program, or anti HIV 1 RT monoclonal anti body from Abcam. The HIV 1 GagPol polyprotein was identified using human HIV immunoglobulin also from NIH AIDS Research Reference Reagent Program. Specific bands were visualized by ECL.

Quantification of the Western blotting results was performed using ImageJ software. Endogenous reverse transcription in viral particles Preparations of viral particles containing 100 ng of p24CA were used for ERT assay. The virus particles were incubated with or Inhibitors,Modulators,Libraries without dNTP mixture for 1. 5, 2, 3, and 5 h at 37 C in ERT buffer as previously described. Samples were collected and DNA was purified with 25 ug of glycogen using Iso Quick DNA Extraction Kit. RT products were analyzed by real time PCR with primer sets specific for strong stop viral DNA as described below. RNA purification and RT reaction RNA was purified from suspensions of virus particles containing 250 ng of p24CA using RNA STAT 50LS RNA isolation solution according to manufacturers Inhibitors,Modulators,Libraries protocol. Reverse transcrip tion of isolated RNA to cDNA for subsequent quantita tive real time PCR analysis was Inhibitors,Modulators,Libraries performed using GeneAmp RNA PCR Kit components and the oligo dT primer accord ing to manufacturers protocol. Reverse transcription complex isolation etc and purification of DNA from RTCs and cell lysates Approximately 5106 infected Sup T1 cells were col lected and washed twice with 40 ml cold PBS.