To evaluate the genome RNA dimer stability of these single cleavage site mutants, genomic RNA isolated from mutant virions were incubated at various temperatures and their thermostability was examined with native northern blot analysis (Figure 2A). Genomic RNA was mainly observed as a dimer at 25 C in all mutant virions except for the PR mutant (Figure 2C). The amounts of monomeric RNA for the p2/NC and NC/p1 mutants at 25 C were slightly higher than WT (NLNh) (Figure 2C and D). On the other hand, the amounts of monomers for the other mutants (MA/CA, CA/p2 and p1/p6) were similar to that of WT from 25 C to 44 C. These results indicated that both cleavage sites at the NC termini are important for genomic RNA dimerization. It is also possible that insufﬁcient Gag-cleavage of the p2/NC mutant might contribute to the relatively low stability of its dimeric genome. The mutants to mimic cleavage intermediates of Pr55 It was previously demonstrated that the processing rates of the ﬁve Pr55 cleavage sites were not equal (38). The processing rate of the cleavage site between p2 and NC (p2/NC) is the fastest, while p1/p6 and MA/CA are second and the third, respectively. The processing rates of the two remaining sites are much slower than those of the three sites mentioned above. Although the results from single cleavage site mutants (p2/NC and NC/p1) showed the importance of NC in RNA dimerization (Figure 2), such composition of intermediate proteins would hardly appear during native virion maturation. As virion maturation is suggested to occur during or immediately after virion budding (39,40), it is very difﬁcult to observe this matur- ation process in a time series. Therefore, four sequential HIV-1 Gag mutants were constructed (Figure 3A) in an attempt to generate a sequential series of Gag maturation intermediates and to help study the RNA dimer transition from fragile to stable, which occurs in virions during mat- uration. Step0 carries all cleavage site mutations and its Pr55 would not be processed by PR. The mutants Step1, 1.1 and 2 carried four, three and two cleavage site muta- tions, respectively. Thus, these sequential mutants were expected to mimic the maturation process of Gag in a step-wise manner. All mutations were conﬁrmed to yield expected fusion proteins, respectively by western blotting analysis (Figure 3B). Step0 mostly expressed unprocessed Pr55. Step1 contained p41 and p15 fusion proteins, Step1.1 contained p41 and p9 fusion proteins, and Step2 contained p25 and p9 fusion proteins. When genomic RNA isolated from the sequential mutants was examined by native northern blotting, the sequential mutants contained a higher rate of monomeric genomes than the WT (Figure 3C and D). The genomic RNA from Step0 was exclusively monomeric, similar to the PR. The monomer rate of Step1, where Gag was cleaved only at p2/NC, was dramatically reduced (<40%) in comparison to that of Step0, and was compar- able to the rate of Step1.1. Viral genomes from immature particles were observed as monomers on a native gel, and were proposed to be unstable and fragile dimers (13). These results suggested that the initial cleavage of Gag, Nucleic Acids Research, 2011, Vol. 39, No. 8 3409 which processes p2/NC, was critical for genomic RNA dimer stabilization in HIV-1. The monomer rate of the Step2 was gradually reduced to 25%, but still higher than that of WT. Finally, the CA/p2 mutant showed a similar level of monomer content to WT. Taken together, viral genome dimerization showed triphasic states during virion protein maturation. Consecutive Gag processing is required for genome dimer maturation As shown in Figure 3, our data indicated that the propor- tions of genomic RNA dimers in the Step1 and 1.1 mutants were similar, but we did notice that their RNA proﬁles were not completely identical. To compare the RNA proﬁles of these samples more precisely, we scanned the RNA signals on the blot and densitometric- ally analyzed their distribution (Figure 4). We ﬁrst focused on the peak position of RNA dimer in each mutant (Figure 4B). The mobility of the dimer in Step1 was con- siderably lower than that of WT, suggesting its apparent molecular mass to be relatively high. The peak mobility of Step1.1 and 2 were slightly lower than the mobility of WT, while the dimer mobility of CA/p2 and NC/p1 mutants were very similar to WT. We then examined the RNA signal distribution pattern of the mutants on the blots (Figure 4C) When genomic RNA were analyzed on the gel under native conditions (35 C), an intense signal of monomeric RNA from PR mutant formed a sharp peak, although it was preceded by a smaller peak at the dimer position. The RNA signal of Step0 formed the highest peak at the same position as PR and overall signal distributions looked very similar. Interestingly, Step1 formed a broader RNA signal range in comparison to WT. Although the peak signal was detected at the dimer position, the mobility was shorter and signal inten- sity was relatively weak. Additional signals were observed at larger molecule areas than the normal dimer and at a monomer position. The RNA signal of Step1.1 formed the highest peak at the dimer position, with a modest intensity at the monomer area, which was also observed for Step1. The signal pattern of Step2 was similar to that of Step1.1, but with reduced signals at the monomer area. The signal of CA/p2 mutant was almost the same shape as that of WT, whereas that of NC/p1 was more aberrant and analo- gous to that of Step1, but the peak signal of dimer was more concentrated. These results suggested that p2/NC processing alone was not sufﬁcient to complete the dimer- ization of genomic RNA at WT levels and the ensuing cleavages were also required to change the genome into uniform dimers. Stoichiometric analysis of Gag processing and RNA dimerization The results presented in Figure 3 revealed that p2/NC processing was the most important step for stable dimeric RNA formation. This suggested that the progres- sion of the initial Pr55 cleavage would stimulate the genomic RNA maturation. To investigate the extent of the required processing, and whether a threshold for the RNA dimerization switch from fragile to stable exists, we