HIV-1 CA-NC particles were assembled by diluting the CA-NC protein to a concentration of 0

HIV-1 CA-NC particles were assembled by diluting the CA-NC protein to a concentration of 0.3 mM in 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, and 2 mg/ml DNA oligo(TG)50. Rabbit Polyclonal to CDH23 and nondividing cells. HIV-1 viruses with capsid N57 substitutions infected dividing but not nondividing cells. Interestingly, HIV-1 viruses with N57 mutations underwent reverse Cloprostenol (sodium salt) transcription but not nuclear translocation. The mutant capsids also lost the ability to interact with Nup153 and CPSF6. The use of small molecules PF74 and BI-2 prevented the conversation of FG-containing nucleoporins (Nups), such as Nup153, with the HIV-1 core. Analysis of integration sites in HIV-1 viruses with N57 mutations revealed diminished integration into transcriptionally active genes in a manner resembling that of HIV-1 in CPSF6 knockout cells or that of HIV-1-N74D. The integration pattern of the N57 mutant HIV-1 can be explained by loss of capsid interaction with CPSF6, whereas capsid interaction with Nup153 is required for HIV-1 to infect nondividing cells. Additionally, the observed viral integration profiles suggested that integration site selection is usually a multiparameter process that depends upon nuclear factors and the state of the cellular chromatin. IMPORTANCE One of the key advantages that distinguish lentiviruses, such as HIV-1, from all other retroviruses is usually its ability to infect nondividing cells. Conversation of the HIV-1 capsid with Nup153 and CPSF6 is usually important for nuclear entry and integration; however, the contribution of each of these proteins to nuclear import and integration is not clear. Using genetics, we exhibited that these proteins contribute to different processes: Nup153 is essential for the HIV-1 nuclear import in nondividing cells, and CPSF6 is usually important for HIV-1 Cloprostenol (sodium salt) integration. In addition, nuclear factors such as CPSF6 and the state of the chromatin are known to be important for integration site selection; nevertheless, the preferential determinant influencing integration site selection is not Cloprostenol (sodium salt) known. This work demonstrates that integration site selection is usually a multiparameter process that depends upon nuclear factors and the state of the cellular chromatin. (?)151.92457.333????????(?)151.92491.487????????(?)69.646150.047????????Angle () = = 90, = 120 = Cloprostenol (sodium salt) 89.54, = 90.94, = 96.08????Resolution range (?)50C2.50 (2.54C2.50)50C1.90 (1.97C1.90)????test. Differences were considered statistically significant at a value of 0.05 (*), 0.01 (**), or 0.001 (***) or were nonsignificant. (F) PMA-treated (PMA) or untreated (Mock) U937 cells were challenged with increasing amounts of HIV-1-N57S/G208R-GFP viruses. Infection was determined by measuring the percentage of GFP-positive cells at 48 hpi. Experiments were performed at least three times, and a representative example is usually shown. To further expand the number of cell lines used to examine our viruses, we tested the myeloid cell line U937, which does not express SAMHD1. As shown in Fig. 2F, HIV-1-N57S/G208R-GFP viruses poorly infected PMA-treated U937 (nondividing) cells compared to untreated (dividing) cells. HIV-1 viruses with mutations on capsid residue N57 undergo reverse transcription but not nuclear translocation in nondividing cells. In the preceding section, we showed that mutations in the capsid residue N57 inhibit contamination of nondividing cells by HIV-1. We next tested whether HIV-1-N57S/G208R undergoes reverse transcription in nondividing cells. For this purpose, we challenged PMA-treated THP-1CSAMHD1 KO cells (nondividing cells) and measured the ability of HIV-1-N57S/G208R expressing GFP (HIV-1-N57S/G208R-GFP) as a reporter of contamination to undergo reverse transcription at 7 h postinfection (hpi). As shown in Fig. 3A, HIV-1 with the capsid mutations N57S/G208R did not infect nondividing cells (upper) but underwent reverse transcription (lower). As a control, we used nevirapine (Nev), which inhibits HIV-1 reverse transcription. These experiments suggested that HIV-1 with mutations on capsid residue N57 is usually defective only after reverse transcription in nondividing cells. Open in a separate windows FIG 3 Contamination of nondividing cells by HIV-1-N57S is usually stopped after reverse transcription but prior to nuclear translocation. (A) PMA-treated THP-1CSAMHD1 knockout (KO) cells were challenged with the indicated DNase-pretreated HIV-1-GFP viruses. (Upper) Contamination was determined by measuring the percentage of GFP-positive cells by flow cytometry at 48 hpi. (Lower) In parallel, cells from comparable infections were lysed at 7 hpi and total DNA extracted. The DNA samples collected at 7 hpi postinfection were used to determine the levels of late reverse transcripts by real-time PCR. As a control, we used 10 M the reverse transcription inhibitor nevirapine (Nev). Late reverse transcript levels were normalized to actin. (B) Similarly, HeLa cells pretreated with 0.5 g/ml aphidicolin for Cloprostenol (sodium salt) 12 h were subsequently infected by the indicated DNase-pretreated HIV-1CLuc viruses. (Upper) Contamination was determined by measuring luciferase activity at 48 hpi. In parallel, cells from comparable infections were lysed at 7 and 24 hpi, and total DNA was extracted. The DNA samples collected at 7 hpi.