Period using the same procedure as the virus release assay presented
Period using the same procedure as the virus release assay presented above with clarified supernatants assayed for infectivity and virion lysate samples were prepared. Infectivity assays PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27906190 were performed using the TZM-bl single-round lacZ Tat complementation assay as previously described [86]. CA present in the supernatant was measured by CA NIr immunoblot. Specific infectivity was order XAV-939 calculated dividing the virus titer by the CA band signal, expressed in blue cell-forming units per arbitrary CA fluorescence value. Heat inactivation was carried out at 70 for 20 min.Flow cytometry analysis of infected cell linesSamples were separated by SDS-PAGE electrophoresis and blotted onto PVDF-FL membranes (Millipore) using a semi-dry apparatus as previously described [84]. Blots were blocked for at least 1 h in Odyssey blocking buffer (LI-COR Biosciences) and then incubated in blocking buffer with one or two primary antiserum (a)/antibody (ies) for at least 2 h, typically overnight. Blots were washed twice with blocking buffer for 10 m then incubated with the appropriate donkey IRDye 800CW and/or IRDye680 LT fluorescently labeled secondary antibodies (LI-COR) at a 1:10,000 ratio vol/vol in blocking buffer for at least 2 h. Blots were washed five times for 10 min each time in blocking buffer, and then analyzed with an Odyssey infrared imaging system (LI-COR) using a laser intensity of between 1 and 5. Signal densities of bands were measured by the Odyssey 3.0 applicationFor each sample, medium containing 1-2 ?106 cells was centrifuged at 400 ?g for 5 min and fixed in 0.5 ml of 4 wt/vol paraformaldehyde in D-PBS (Life Technologies) for 20 min at room temperature followed by the addition of 3.5 ml permeabilization solution (PS), 0.1 (wt/vol) saponin (Sigma-Aldrich) in D-PBS, and incubation at room temperature for 10 min. Cells were then collected by centrifugation at 550 ?g for 5 min, followed by 2 washes with PS. Cells were then stained with KC57 antibody for HIV-1 CA (Beckman-Coulter, Inc.) or 2 F12 antibody for SIV CA (Quality Biological, Inc., Gaithersburg, MD) in 100 l of PS for 30 min in the dark at 4 , washed twice in 4 ml of PS and then resuspended in 200 l of PS, and analyzed immediately by an LSRII PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28381880 flow cytometer (BD Biosciences)Coren et al. Retrovirology (2015) 12:Page 12 ofData analysis was performed using FCS Express software (De Novo Software).Statistical analysis9.10.Single-round and short term infectivity data where analyzed using the Student’s t-Test function in Excel (Microsoft Inc.) with paired two-tailed parameters.Ethical approval11. 12. 13.All research was carried out under approval by the NCI at Frederick Institutional Biosafety Committee # 11-03 superseded by #14-23.Competing interests The authors declare that they have on competing interests. Authors’ contributions LVC produced the vector constructs and the immunoblots, MTT and VIA carried out flow cytometry analyses, SJ performed infectivity assays and some cell culture, GQDP contributed experimental design, CO contributed experimental design, data interpretation and manuscript assistance, DEO conceived and designed the study, produced cell lines, carried out infectivity and replication, complied data and wrote the manuscript. All authors read and approved the final manuscript. Acknowledgements We thank Theodora Hatziioannou for the African green monkey and gorilla TRIM5 genes, Hans-Peter Kleim for the Phoenix RD114 clone 22 packaging cell line, Francois-Loic Cosset for the GA.