To create stable APOBEC3G expressing cell lines, the plas mid DNA of pcDNA3. 1 Apo E/Hygromycin, Pim signaling pcDNA3. one Apo U/Hygromycin, pcDNA3. one Apo M/ Hygromycin was transfected into HEK293 cells. HEK293 cells that stably develop a substantial degree of APOBEC3G, APOBEC3G UBA2 or APOBEC3G UBA2 had been 1st estab lished by collection of hygromycin resistant cells for 2 weeks and verified from the Western blot analyses. To produce infectious viral particles, HEK293 was inoculated into six very well plate one particular day ahead of pNL4 3 plasmid transfection to make certain sufficient cell confluency. The full length molecular clone pNL4 three and pNL4 three ?Vif plasmid was then transfected into HEK293 cells to produce wild form Vif or mutant Vif HIV one viruses. Forty eight hours p. t, HIV 1 viral particles have been harvested through the supernatants of HEK293 cells by centrifugation at 1,000 rpm/min for 5 min.
The isolated viral particles have been split into 2 ml aliquots and stored in 80 C. To make sure equal amounts of viral infection, the viral stocks were normalized by identifying ranges of p24 anti gens in every single viral stock. The level of p24 antigen was established through the use of a business p24 antigen kit from ZeptoMetrix Co. following the manufac tures instructions. Viral infections To assess the suppressive effect of APOBEC3G, APOBEC3G UBA2 and APOBEC3G UBA2 on viral repli cation in proliferating CD4 T lymphocytes, CEM SS cells that stably express a plasmid management, APOBEC3G, APOBEC3G UBA2 and APOBEC3G UBA2 were established. 1 106 CEM SS cells were either mock contaminated or infected with 3000 TCID50 of HIV 1NL4 3 and HIV 1NL4 3?Vif.
The viral replication was measured by p24 antigenemia. MAGI assay MAGI assay was employed to find out the viral infectivity as previously described. Briefly, MAGI CCR5 cells have been cultured in 6 nicely plates one particular day before infection to ensure that cells can attain around 40 50% confluency over the day of infection. The medium of every very well was removed ahead of the viral supernatant was extra to infect cells in the total volume of 300l of finish DMEM with 20g/ml of DEAE dextran. Cells in virus containing medium have been incubated at 37 C in a 5% CO2 incubator. Just after two hrs incubation, 1. five ml full DMEM medium was additional to each very well. The cells were further incubated beneath the same problem for 48 hrs after incubation. The media were eliminated and two ml fixing remedy was extra to every properly.
Cells were washed twice with PBS after which fixed for 5 min. with 600l of staining answer. Cells were then incubated for two hrs at 37 C in the non CO2 incubator. Staining was stopped by removing the staining solution and washed twice with PBS. Blue dots had been counted as infected cells as described previously under the microscope. The viral infectivity was deter mined by evaluating the complete variety of infected cells with uninfected cells in each effectively.