Shayman et al. have studied large vessel reactivity and pathology in this model

e of HIV-1 nuclear entry has been mapped to the HIV-1 CA. These results also suggest a functional link between the HIV-1 CA and nuclear entry. We previously generated simian-tropic HIV-1 that replicates efficiently in cynomolgus monkey cells. This virus encodes a CA with SIVmac239-derived loops between helices 4 and 5 and between -helices 6 and 7, along with the entire SIVmac239 vif. These SIVmac239-derived sequences allow HIV-1 to escape from restriction factors in monkey cells, including cyclophilin A, TRIM5, and ApoB mRNA editing catalytic subunit 3G. However, the replicative capability of this virus in human cells was severely impaired. By long-term cultivation of human CEM-SS cells infected with NL-4/5S6/7SvifS, we succeeded in partially rescuing the replicative capability of this virus in human cells. This adapted virus encoded a G-to-E substitution at the 116th position of the CA. Interestingly, this G116E mutation also occurred after adaptation in rhesus monkey cells. In the work presented here, we examined the mechanism by which the replicative capability of NL-4/5S6/7SvifS was severely impaired in human cells. Program), the BssHII to ApaI fragment was replaced with the corresponding fragment of NL-4/5S6/7SvifS 2173565 or NL-4/5SG116E6/7SvifS. Viral titers were measured with the RETROtek antigen ELISA kit. Viral infections CEM-SS cells were infected with HIV-1 derivatives at titers equivalent to 20 ng of p24 per reaction. Culture supernatants were collected periodically, and p24 levels were measured using an ELISA kit. Real-time PCR analysis CEM-SS cells were infected with DNase I-pretreated HIV-1 derivatives at titers equivalent to 80 ng of p24 per reaction. DNase I pretreatment consisted of incubation with DNase I for 30 min at 7685384 room temperature. After 2 hr on ice, infected cells were washed with PBS, resuspended in medium, and returned to 37 C until harvesting at the indicated time point post-infection. Genomic DNA was extracted by using the QIAamp DNA Blood Mini kit. After digestion with 1 unit/l DpnI for 4 hr at 37 C, 30 ng of DNA was analyzed for U5/gag, 2-LTR, and AluHIV by real-time PCR using published primers and TaqMan probes in an Applied Biosystems 7500 Real-Time PCR System. In situ uncoating assay The in situ uncoating assay was conducted as previously described. Briefly, the labeled virus was generated by cotransfecting 9 g NL-Nh CA mutant 221244-14-0 proviral plasmid, 4 g S15-dTomato-expressing plasmid, 4 g vesicular stomatitis virus G protein -expressing plasmid, and 1 g GFPVpr-expressing plasmid into 10-cm plates of 293T cells using PEI. HeLa cells were spinoculated with the labeled virus for 2 hr at 16 C in the presence or absence of bafilomycin A . Virus-containing supernatant then was removed and replaced with 37 C medium in the presence or absence of BafA, shifted to 37 C, and fixed with 3.7% formaldehyde in 0.1M PIPES buffer at the indicated time point post-infection. The fixed HeLa cells were permeabilized with blocking solution for 5 min at room temperature, stained with anti-p24 mAb AG3.0 is one of the most important molecular mechanisms that regulate almost every aspect of cellular processes in eukaryotes. Common PTMs, such as phosphorylation, ubiquitylation, SUMOylation, acetylation, and glycosylation, alter protein conformation, surface charge, stability, and molecular interactions and thereby, profoundly impact protein function. As one of the most important PTMs, reversible phosphorylation is highly dynamic and plays a