3408 Nucleic Acids Research, 2011, Vol. 39, No. 8 Electron microscopy 293T cells were transfected with appropriate plasmids. Supernatants were collected 36 h post-transfection and ultracentrifuged for 30 min at 4 C and 49 000 g through a 20% sucrose cushion. Pellets were washed twice with 0.15 M phosphate buffered saline (PBS, pH 7.2) and ﬁxed with 2% glutaraldehyde in PBS at 4 C for 120 min. The ﬁxed pellets were washed ﬁve times with PBS. The ﬁxed pellets were further ﬁxed in 1% osmium tetroxide in the same buffer at 4 C for 60 min, washed in the buffer, dehydrated in a graded ethanol series and embedded in epoxy resin (Plain Resin; Nisshin-EM, Tokyo, Japan). Ultrathin sections were cut using a Porter Blum ultramicrotome (Sorvall MT-5000; Du Pont, Newton, CT, USA) and mounted on a copper grid (300 mesh) sup- ported by a carbon-coated collodion ﬁlm. Finally, the sections were double stained with uranyl acetate and lead citrate. All the sections were observed under a transmission electron microscope (H-7650; Hitachi, Tokyo, Japan) with an accelerating voltage of 80 kV. Electron micrographs were obtained at 10 000-fold magniﬁcation. Endogenous RT assay Puriﬁed virions were suspended with PBS( ) and endogen- ous RT assay was performed essentially as previously described (32). Aliquots of virus suspensions were incubated in 30 ml of endogenous reverse transcription reaction mixture (0.01% Triton X-100, 50 mM NaCl, 50 mM Tris–HCl [pH 8.0], 10 mM dithiothreitol, 5 mM MgCl2, 100 mM each dATP, dCTP, dGTP and dTTP). As a negative control, reactions without dTTP were performed in parallel. After 2 h of incubation at 37 C, the reaction was terminated by addition of 120 ml of stop mix (50 mg of proteinase K per ml, 20 mg of tRNA per ml, 1.5 mM EDTA [pH 8.0]). After incubation at 60 C for 1 h, the proteinase K was heat-inactivated by incubation at 95 C for 15 min. 4 ml aliquots of the reaction mixtures were subjected to real-time PCR analysis. A portion of virion suspension with PBS ( ) was dispensed and viral RNA was puriﬁed directly by High Pure Viral RNA Kit (Roche Applied Science, Mannheim, Germany). Viral RNA was applied for quantitation with real-time PCR. Infection assay The infection of env-pseudotyped virus into human T-cell lines were performed essentially as previously described (25). M8166/H1Luc cells (33), containing HIV-1 LTR- luciferase vector, were used for single-round infection ex- periments to determine overall infectivity of virus by Tat expression. Luciferase activity of cells was determined by the Bright-Glo luciferase assay system (Promega, Madison, WI, USA). TRIM5-KD/Luc-KD Jurkat cells (kind gifts from Dr Jeremy Luban) and MT-4 cells (34) were used for viral infection and viral DNA quantitation by real-time PCR assay. 1 106 cells per sample were infected with puriﬁed virus and total cellular DNA was extracted with GenElute mammalian genomic DNA miniprep kit (Sigma-Aldrich, St Louis, MO, USA) at 18 h post infection. Equal amount of total DNA was applied for real-time PCR analysis. Real-time PCR Real-time PCR analysis was performed essentially as pre- viously described (35). For detection of viral RNA from puriﬁed virion, Gag primers (TM-GagF: GCAGCCATG CAAATGTTAAAAGAG, and TM-GagR: TCCCCTTG GTTCTCTCATCTGG) were applied with One Step SYBR PrimeScript PLUS RT-PCR kit (Takara Bio Inc.). For detection of viral DNA from MT-4 cells, primers and Taqman probes were selected according to criteria described elsewhere (36). For detection of viral DNA from TRIM5-KD/Luc-KD Jurkat cells, Gag primers and a probe (NL1359F: AGTGGGGGGACAT CAAGCAGCCATGCAAAT, NL1500R: TGCTATGTC AGTTCCCCTTGGTTCTCT, and NLgag1400TqmnFB: FAM-ATCAATGAGGAAGCTGCAGAATGGGA -BHQ-1) were applied RESULTS Maturation of NC is important for the completion of genome dimerization To investigate which cleavage site(s) of HIV-1 Gag protein is responsible for genomic RNA dimerization, ﬁve single Gag-cleavage site mutants, MA/CA, CA/p2, p2/NC, NC/ p1 and p1/p6 (Figure 1B) were constructed. Protein proﬁles of the mutants released from 293T cells were assessed by Western blotting, using antibodies against MA, CA, NC and p6 (Figure 1C). The mutant MA/CA yielded an MA–CA fusion product (p41) and the mutant CA/p2 yielded a CA-p2 fusion product (p25), as expected. Only a faint band corresponding to the p2-NC fusion product (p9) was observed in the p2/NC mutant and a strong signal was observed as the p32 or p33 intermediate, which was consistent with a previous report (17). This can be considered as an aberrant product comprised by CA-p2-NC fusion protein and may be due to the block of the primary cleavage at p2/NC of Gag, resulting in an aberrant exhibition of other cleavage sites to PR leading to insufﬁcient processing. However, there was also a clear CA protein signal on anti-CA blot in the p2/NC mutant (Figure 1C). As the CA/p2 junction must be cleaved for CA release, it is reasonable to assume that a certain amount of p2-NC protein existed in the mutant. The faint signal from the p9 on the anti-NC blot would suggest that the antibody recognition epitope of NC is somehow masked on the p2-NC fusion protein. The Asn to Thr substitution (amino acid #432) in the mutant NC/ p1 was located just proximal to the heptameric slippery sequence, UUUUUUA, where the programmed riboso- mal frame shifting for Gag-Pol precursor translation occurs (37). Nonetheless the heptameric sequence itself was conserved completely, the mutation introduced was expected to have little effect on frame shifting. The mutant NC/p1 actually yielded p9 properly. The mutant p1/p6 yielded a p1–p6 fusion product (p8) and the substi- tution did not affect the peptide sequence of Pol protein. Taken together, all the mutations expressed as expected.