3414 Nucleic Acids Research, 2011, Vol. 39, No. 8 heated genomic RNA dimers have slower mobility in a native gel (e.g. see 44 C lanes of NLNh in Figure 2A), which is considered to be the reason why RNA dimers are relaxed by thermal energy. In either case, ensuing Gag processing and resultant NC maturation would lead to the proper dimerization of genomic RNA and acquire infectivity. As the viral RNA from Step1.1 no longer con- tained larger components, p15 processing into p9/p6 might play a role(s) to form tight dimers. These results suggested the complexities of dimer maturation and were consistent with the recent report describing a three-step dimerization process of the HIV-1 genome (52). Some unexpected results were obtained from the stoichiometrical analysis of Gag processing and RNA di- merization (Figure 5). We ﬁrst considered the uniformity of the ‘chimeric’ virion. There could be an argument whether what we are observing is a changing proportion of two distinct populations of homo-multimeric particles or whether the particles are becoming individual, chimeric virions. Thus we showed the model of virus particle com- position here. To simplify the model, we ﬁrst imposed precondition: that one particle includes 1000 molecules of Gag and the two species of Gag (Step0 and Step1) dis- tributes evenly within the cell. The particle assembly driven by the Gag proteins follows a binomial distribution manner. Under these conditions, the calculations sug- gested that 99.9% of the particle includes 450–550 Gag of each species if the ratios of two species are 1:1. Similar results are obtained in various ratios. This outcome strongly suggests that most of the particle composition reﬂects the mixed ratio of the plasmids applied for trans- fection. Thus, we believe the data in Figure 5 represents the RNA status of a chimeric virion. It has been suggested that NC protein has RNA chap- erone activity and one NC molecule/20 nt would be sufﬁ- cient for promoting viral RNA dimerization (43). Thus, we assumed that there should be a p15 molecular concen- tration threshold imposed on the dimer transition from fragile-to-stable during Pr55 processing. It is reasonable to consider that a certain accumulation of processed Gag would trigger dimer stabilization. However, as in Figure 5, ‘intermediately-processed’ virions packaged stable and unstable dimers about equally. This means that even within the same virion protein composition, a population undergoing genome dimer stabilization encountered a sto- chastic decision process. To explain this observation, we present one working theory in Figure 9. Since NC displays both non-speciﬁc and speciﬁc RNA binding, it has been proposed that NC ﬁrst interacts with the packaging signal and then additional NC domains coat the viral RNA in a sequence-independent manner. The binding ability of NC to RNA has been suggested to be generally in the range of 1 protein molecule per 5–7 nucleotides. (53–55). Thus around 2500–4000 NCs would be required to coat the entire dimeric retroviral genome, which is approximately 9000 nucleotides per strand. Because a retroviral virion is suggested to be composed of about 2500–5000 Gag mol- ecules according to the latest reports (56,57), the quantity of NC proteins in a virion is not in excess, but in rather adequate numbers for a dimerized genome. As some reports have suggested (31,58,59), there is a possibility that additional dimer contact points on viral RNA other than the DLS, which mediate mature ribonucleoprotein complex formation within virion, may exist. Here, we assume that there are several such points on each genome (Figure 9A) and NC proteins binding to these points were simply non-speciﬁc and random, such as binding to normal nucleotides. In the fully-processed virions (Step1; Figure 9B, right), all the points are coated with sufﬁcient NC and produced the stable dimer formation. Conversely, if no points are bound with NC, the dimeric genome would remain fragile in immature par- ticles, like particles from the Step0 or PR mutants (Figure 9B, left). In the case of ‘intermediately-processed’ particles, like the virions produced from the Step0–Step1 mixtures, the dimers could be both fragile and stable. As NC (p15) numbers are not sufﬁcient in such particles, the genomes would be only partially covered by NC. If NCs covered the contact points on the genomes and properly mediated the RNA–RNA interaction, the dimer would be stable (Figure 9C, right). On the other hand, if NCs failed Figure 9. Schematic hypothetical models of virion and genome dimer- ization in various maturation conditions. (A) A diagram of a viral genome carrying one DLS and putative ‘dimer contact points (DCP)’ (B) Images of matured (right) and immature (left) virions. (C) Images of ‘intermediately-matured’ virions. Although both genomes were only partially covered with NC, the particle on left fails to form a stable genome dimer, whereas the particle on the right forms a stable dimer. The dimerization efﬁciency depends on the extent of coverage of DCP by NC. Solid lines represent viral RNA, Shaded circles represent DLS on the RNA, Open circles represent hypothetical DCP, and dotted squares represent NC (p15).