MRadionucleotides, (methyl- 3H)dTTP and [a-32 P]dTTP were purchased from
MRadionucleotides, (methyl- 3H)dTTP and [a-32 P]dTTP were purchased from Perkin Elmer, (Shelton, CT); poly (rC)-poly(dG)12-18 was purchased from Amersham Pharmacia Biotech, (Piscataway, NJ); and Polynucleotide poly (rA) and primer oligo(dT) 12-18 were purchased from Boehringer Mannheim (IN). The oligonucleotides used for mutagenesis were synthesized and high pressure liquid chromatography purified by Diversified Biopharma Solutions Inc. (Loma Linda, CA). Complete Dulbecco’s Modified Eagles Medium (DMEM) containing 10 heat inactivated fetal bovine serum (FBS) and penicillin/streptomycin was used to grow 293T cells. Complete PubMed ID: RPMI medium containing 20 FBS, 26 IU of IL-2, penicillin/streptomycin and glutamine was used to culture Peripheral blood mononuclear (PBM) cells. MT2 cells were grown in RPMI containing 10 FBS, penicillin/streptomycin and glutamine.Cells and virusPBM cells were prepared from Buffy coats received from commercial vendors (Red Cross and LifeSouth Community Blood Center, Atlanta, GA) using Ficoll gradients. Primary human embryonic kidney cells 293T, indicator cell line HeLa-CD4-LTR-b-galactosidase and proviral clone pNL4-3 [9,10] were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institute of Health.Site-specific mutagenesis and generation of mutant virusesVarious single point mutants were created in the background of proviral clone pNL4-3 by using pALTER -1 mutagenesis system of Promega (Madison, WI) according to manufacturer’s guidelines and our previously described PM01183 site protocols [11,12]. Mutagenic oligonucleotide pNL74I 5′-GAAATCTACTATTT TTCTCCAT-3′ was used to create L74I mutation in the background of NL4-3 (wild type) and NL4-3 containing K65R mutation. Mutants K65R, L74V, and K65R+L74V that have been previously analyzed for replication capacity and in vitro RT processivity were used as controls [12,13]. Viruses were produced using SuperFect R reagentChunduri et al. Virology Journal 2011, 8:33 3 of(Quiagen, Valencia, CA) and manufacturer’s guidelines. Cells (293T) were split into 60 ?10 mm dishes 24 h-48 h prior to transfection. To generate virus the complex containing 10 g of DNA in 150 l of PubMed ID: serum-free medium and 30 l of SuperFect reagent was incubated at room temperature for 10 min. One ml of complete DMEM was added drop by drop onto 293 cells that were washed once with phosphate buffer saline (PBS). Cells were incubated at 37 in the presence of 5 CO2 for 3 h. The remaining medium-complex was removed and the cells were washed with 4 ml of PBS. Four ml of complete DMEM was added and dishes were incubated for 72 h-96 h. Culture supernatants were collected and centrifuged for 5 min at 833g (g = 1.2) to pellet any debris. Culture supernatants were filtered (0.22 m) and saved in aliquots of 0.5 ml and 1 ml at -80 . Viral RNA was isolated by QiAamp?viral RNA mini kit (Qiagen Sciences, Valencia, CA). RT PCR was performed using SuperscriptTM III one-step RT PCR system (Invitrogen, Carlsbad, CA). All the stock viruses were confirmed by sequencing viral RNA using primer 74F, 5′-GTAGGACCTACACCTGTCAAC-3′ [14].Quantification of virusthen 1 ml of DMEM with 10 calf serum was added to each well. After 48 h, the medium was removed and the cells were fixed at room temperature with 2 ml of phosphate-buffered saline (PBS) containing 1 formaldehyde and 0.2 glutaraldehyde for 5 min. The cells w.