to cover the points correctly, the dimer formation would remain fragile (Figure 9C, left). Electron microscopy analysis of the sequential cleavage site mutants showed successive morphological changes during Pr55 Gag maturation (Figure 6). Virions classiﬁed into Group A were <10% from Step0 to Step1, and most of the remaining virions changed to Group B, another immature particle classiﬁcation. As the change from Group A to B was characterized by disappearance of an electron dense ring inside the viral membrane, the observed ring could be composed by the p15 region within Pr55. After the cleavage at p2-NC position, the ordered array of the p15 region would be lost and p15 would be released from perimembrane. This transition was synchronous with initial dimeric RNA stabilization, suggesting the release of genome RNA from a perimembrane localization to a space inside the virion where p15 would trigger dimer maturation. The cleavage at p1–p6 (from Step 1 to 1.1) did not affect virion morph- ology, although this process contributed to a uniformly dimerized genome (Figure 4). The thin-membraned virions (Group C) became abundant after the third pro- teolytic processing at the MA/CA region, represented by the Step2 mutant. A certain portion of Group C particles could easily contain the conical cores of Group D, which are meant to be matured virions, but cut transversely the morphology can appear different. Nonetheless half of Step2 mutant particles were classiﬁed into Group B and thus showing unique morphology. Viral membranes of Group B may appear to be thick because of Pr55 CA region fusion to MA, which lines the inner membrane. It has been speculated that viral dimeric RNA could mature in the absence of the viral core (44). Our study not only conﬁrmed this hypothesis, but additionally revealed that RNA maturation would begin and approach completion before core formation in the virion. In addition, although morphological proportion of the virions produced from the NC/p1 mutant was almost similar to WT (Figure 6B), genome dimerization in the NC/p1 mutant was less efﬁcient than in WT (Figure 2). This suggests that proper core formation alone may be insufﬁcient to fully accomplish RNA maturation. Taken together, particle morphology dynamics during viral maturation did not clearly synchronize with the transition of dimeric RNA status. The processing speed of the NC-p1 cleavage site in vitro has been suggested to be very low and nearly equal to that of CA-p2 (38). This observation leads to the idea that an intermediary such as the NC/p1 mutant could be generated as much as the CA/p2 mutant during the ﬁnal phase of virion maturation. However, the data from viral RNA proﬁles (Figures 2 and 4) indicated that NC/p1 mutant contained aberrant RNA and could not support infection. In contrast, the RNA proﬁles or infectivity of CA/p2 mutant were very similar to WT. In addition, CA/ p2 mutant has been reported to contain tightly condensed internal RNP cores with a size and morphology similar to those of WT particles (16). These observations imply that CA-p2 cleavage, but not NC-p1 cleavage, would occur during the ﬁnal phase of virion maturation. This insight might be explained by the ﬁndings of the recent paper (60) Nucleic Acids Research, 2011, Vol. 39, No. 8 3415 which presented the stimulation of p15 (NC-p1-p6) cleavage by PR in the presence of ssRNA/DNA. Due to a simple biochemical assay which ignored the existence of ssRNA genome interacting with most of Pr55 molecules, the processing rate of NC-p1 site within the virions may be underestimated. The infectivity assay of the mutants unveiled very intriguing results. The endogenous RT activity of viral RNA in the virion was fully detected following the initial cleavage of Pr55, whereas the production of viral DNA in the infected cell was not observed until comple- tion of NC maturation. These results suggested that although viral RNA was fully active as the template for viral reverse transcriptase just after initial cleavage of the precursor, much more virion maturation processes were required for the RT of viral RNA within cells. We form two possibilities to explain these results. One is the exist- ence of cellular restriction system to eliminate immature viral RNA just after the penetration into the cytoplasm. As the restriction activity was not mediated by human T5a (Figure 8C), we assume it could be cytoplasmic RNases or some other enzymes to corrupt RNP of virus. The second possibility is a defect in penetration into the host cell by the mutant. As the most of the mutants failed to form proper virion structure, there may be some defects in the release of RNP into the cells somewhere during viral fusion and uncoating. However, when we examined the infectivity of NC/p1 mutant, we found that NC/p1 produced only <10% early viral DNA in infected cell in comparison to WT (data not shown). As virion morph- ology of the NC/p1 mutant is nearly similar to the WT (Figure 6B), the uncoating defects of the mutants alone may not fully explain this difference. In the in vitro assay, p9 or p15 proteins, which are immature NC intermediates, were suggested to obtain in- sufﬁcient RNA chaperone activity required for strand transfer during reverse transcription (49). Although our results of the endogenous RT assay were not consistent with the study, different experimental conditions can offer partial explanation to this discrepancy. As the samples applied for the endogenous RT assay are puriﬁed virions, many viral/host factors are present within the assay conditions and would participate in the reaction. In summary, we suggested here that mature NC or the intermediate proteins containing NC region are involved in genomic RNA dimerization within the HIV-1 particle. Dimer stabilization begins during the primary cleavage (p2-NC) of Pr55 Gag. However, the primary cleavage alone is not sufﬁcient for completion of dimerization and ensuing cleavages are required to generate a stable and uniform dimer. The majority of this process does not require a mature viral core. In contrast, the primary cleavage is more than enough to initiate endogenous RT activity of viral RNA, whereas overall viral infectivity, as well as intracellular viral DNA production, required the near-completion of viral maturation. Other retroviruses also have NC and dimeric RNA genomes, suggesting that RNA dimerization could occur in the same manner as HIV-1. Future studies with HIV-1 or other retroviruses will contribute to clarify the general