3412 Nucleic Acids Research, 2011, Vol. 39, No. 8 and C particles. We classiﬁed the virions from each step into groups and drew a chart to represent the morpho- logical transition of the HIV-1 virion during maturation (Figure 6B). These results indicated that dimer stabiliza- tion of the HIV-1 genome, occurring at the timing of p2-NC cleavage, was consistent with the start of virion morphological change from Group A to B, but not with viral core formation. Endogenous RT activity, infectivity and maturation process Although the maturation process is required for virion infectivity, the sequential steps to acquire infectivity, remains unclear. To identify the maturation step which prepares viral RNA ready for reverse transcription, the endogenous RT activity of the mutant virions were assessed. The virions were puriﬁed from transfectant supernatants, re-suspended in PBS ( ) then incubated with detergent and dNTPs. The amount of viral DNA was determined by real-time PCR with two sets of primers, R/U5 (early products) and 2ndTfr (late products) (Figure 7). The RT product of Step0 was reduced by one order of magnitude when compared to WT for the early products, and was attenuated by more than two orders for the late products. Step1 RT products were increased by 2- to 3-fold in both at early and late RT stages. A gradual reduction in the amount of viral DNA was observed, which eventually reached WT levels by the fourth cleavage event (CA/p2 mutant). These data indicated that the initial Pr55 cleavage prepares the viral RNA for RT within virion. The enzymatic activity of viral reverse transcriptase was conﬁrmed by exogenous RT assay. All mutants, with the exception of PR, showed similar RT activity as the WT (data not shown). We ﬁnally compared the infectivity of the mutants in T-cell lines. Single-round virus infectivity assays using marker cells (M8166/H1Luc) revealed that only the CA/ p2 mutant showed similar infectivity to WT (Figure 8A). We performed real-time PCR analysis of viral DNA within infected cells (MT-4) to determine when the defect occurred during virion maturation (Step1, 1.1 and 2), and located the defect at a step before and/or early-RT (Figure 8B). The TRIM5alpha protein (T5a) restricts retrovirus infection during the uncoating process prior to the initiation of RT (42). Although human T5a is sug- gested to have little anti-HIV-1 effect, immature virions, may be affected by it. To determine the effect of human T5a on the mutants, T5a knock-down (KD) and control cells were applied for infection assay (Figure 8C). The viral DNA production in both cells was very similar, sug- gesting no participation of T5a restriction with immature virions. DISCUSSION We have described a sequential mutational analysis series of the HIV-1 Gag cleavage sites to clarify the relationship between virion maturation and genome RNA dimeriza- tion, adding further mechanical insight into previously reported observations. Figure 7. The endogenous RT activity in the virions of mutants. The original values are calculated by dividing the viral DNA amount by the viral RNA amount in virions. Relative amount to WT of early (R-U5: A) and late (2nd Tfer :B) products of RT are shown. The data are the average of at least three independent experiments, and the error bars represent SEM. The statistical signiﬁcance was evaluated by a Student’s t-test. *P < 0.05; **P < 0.01. The results from the ﬁve single cleavage site mutants in Figures 1 and 2 showed that NC/p1 site as well as p2/NC is involved in genomic RNA dimerization, which is con- sistent with previous studies (17,18). NC maturation, indicated by the release of NC from p2 and p1, would be a critical factor for genomic RNA dimerization. NC has been reported to convert fragile RNA dimers into stable dimers in a cell-free system, suggesting that NC is an important contributor to RNA dimer formation (43). The partially processed NCs, p2-NC and NC-p1, may have attenuated RNA dimerization properties, indicated by the failure to form normal RNA dimers and leaving them in an unstable state. The dissociation temperature of the RNA dimer in our experiments, 45 C, was relatively low in comparison to those in other studies (18,44,45). This might be due to the subtle differences in assay con- ditions, such as the presence of formamide in RNA buffer. The examination of genomic RNAs derived from four sequential mutants showed many interesting results (Figure 3). The RNA from Step0 mutant was very similar to that from PR. This result clearly shows that Pr55 cleavage, and not PR activity itself, is required for RNA maturation. Although PR and Step0 mutant show immature RNA phenotype, the blots change with increasing temperature. The RNA in the PR have been suggested to form fragile dimers (13). In addition, the retroviral RNA in the virion contains many nicks