been suggested that Gag precursor, as well as viral NC protein, have RNA chaperone activity and are required for the proper formation of dimeric RNA in the virion (11,12). A defect in its capability to stably dimerize genomic RNA was found in a PR virus (13,14), which led to a hypothesis that one or more Gag cleavage products help form or stabilize genomic RNA dimers. There are ﬁve cleavage sites in the HIV-1 Gag protein and the sequential processing of Gag by PR has been discussed so far (15). Some preceding studies suggested that speciﬁc Gag cleavage sites or protein regions contribute to viral genome dimerization (16–20). In light of these ﬁndings, we constructed two sets of Gag mutants which could rep- resent cleavage intermediates, effectively snapshooting the process of virion maturation in this study. To systematic- ally clarify the dynamic correlation between viral RNA and protein maturation in viral life cycle, virion protein, genomic RNA, virion morphology and infectivity of these mutants were examined comprehensively. We found that NC maturation is critical for the accomplishment of RNA dimerization and viral infectivity, but unnecessary for proper reverse transcription of viral RNA and virion maturation. The mutual relationship between viral protein and RNA maturation was discussed for a further under- standing of the retroviral life cycle. MATERIALS AND METHODS Constructs The plasmid pNLNh (21), which contains a 4-base inser- tion mutation to abrogate Env protein expression on the env gene of HIV-1 proviral clone pNL4-3 (22), was used as the wild-type (WT) and as the progenitor for the mutant constructs described. The amino acid sequence of the Gag cleavage sites and the constitution of the mutants are summarized in Figure 1. The Gag cleavage site mutants MA/CA, CA/p2, p2/NC, NC/p1 and p1/p6, were con- structed according to the previous reports (16,23) using PCR stitch mutagenesis (24). The double cleavage site mutant Step2, containing CA/p2 and NC/p1 mutations, was constructed by introducing N432T amino acid substi- tution to the CA/p2 mutant. The triple cleavage site mutant Step1.1, containing MA/CA, CA/p2 and NC/p1 mutations, was constructed by introducing Y132I amino acid substitution to the Step2 mutant. The quadruple cleavage site mutant Step1, containing all four cleavage site mutations except p2/NC, was constructed by intro- ducing F448S amino acid substitution to the Step1.1 mutant. The quintuple cleavage site mutant Step0, con- taining all Gag cleavage sites mutations, was constructed by introducing M377V amino acid substitution to the Step1 mutant. The HIV-1 proviral mutant MS172, which lacks PR activity, was previously described (25). Cell culture and transfection 293T cells (26) were maintained in Dulbecco’s modiﬁed Eagle’s medium supplemented with 10% fetal calf serum, penicillin and streptomycin. The cells were trans- fected with the calcium phosphate method (27). To recover virus particles, 5 mg of plasmid DNA was used Nucleic Acids Research, 2011, Vol. 39, No. 8 3405 Figure 1. Gag-cleavage site mutations of HIV-1. (A) Summary of the mutations used in this study. Upper ﬁgure represents HIV-1 Gag and its cleavage site sequences. The slashes represent the position of cleavage and the numbers beneath the letters designate amino acid positions in Pr55. The correlation between the mutation and the mutants constructed are summarized in the table. Details on the mu- tations Y132I, M377V, N432T and F448S have been reported (23), as well as the L363I and M367I mutations (16). The M367I mutation was introduced to block an HIV-1 PR-mediated cryptic cleavage site within the p2 protein because this cryptic cleavage has been reported to occur when proteolytic cleavage between CA and p2 was inhibited by L363I mutation. (B) Schematic representations of the single Gag-cleavage site mutants. Shaded boxes represent fusion proteins which resulted from the mutations. (C) Detection of HIV-1 protein produced in pelleted virions by western blotting with various anti-Gag antibodies. Positions of Gag proteins and precursors were indicated. The asterisks indicate aberrant proteins (‘Results’ section). to transfect the cells in a 100 mm dish. Production of par- ticles containing a mixture of quadruple and quintuple Gag cleavage sites mutants was achieved by co- transfection of Step1 and 0 plasmids in various ratios. Virus puriﬁcation Supernatants of transfected cells were collected 72 h post-transfection and treated with DNase I for elimination