Nucleic Acids Research, 2011, Vol. 39, No. 8 3413 Figure 8. The infectivity of the mutants into T-cell lines. (A) Overall infectivity into M8166/H1Luc cells. (B) Viral DNA production measured by real-time PCR assay within infected cells (MT-4) 18 h post-infection. (C) Viral DNA production within TRIM5alpha+/ Jurkat cells. In each ﬁgure, the value of WT is set as one. The data are the average of at least three independent experiments, and the error bars represent SEM. regardless of PR cleavage (46). Thus we think that PR/ Step0 RNA in native condition partially forms fragile dimers, and it gradually dissociate and is dissected in frag- ments to be smeared signals as the temperature rises. The blot also indicated that the main transition of the RNA from a monomer to a dimer state occurred between the Step0 and Step1 mutants (Figure 3). Pr55 Gag is ini- tially cleaved by PR into intermediate proteins p41 (MA-CA-p2) and p15 (NC-p1-p6). Previous reports showed that p15, as well as NC of HIV-1, interacted with in vitro synthesized viral RNA, and GST-fused re- combinant p15 protein efﬁciently induced dimerization of viral RNA fragments (47,48). Our results showed that p15 also promoted genomic RNA dimerization in a nascent virion. A recent report showed the nucleotide binding ability of Pr55 Gag was 10-fold higher than NC proteins (p15, p9 and NC), while its destabilizing effect on double-stranded nucleotides was much lower than the NC proteins (49). Taken together with our results, we assumed that the primary processing of Pr55, which is anchored to the viral inner membrane during virion assembly, would release p15 within the virion. Then the tight, non-speciﬁc binding between nucleotide and Pr55 is relieved, enabling the NC region of p15 to interact with the proper sites on genomic RNAs to initiate dimer maturation. The melting temperature (Tm) of RNA from Step2 seems similar to those of Step1/1.1, although the monomer contents are different. We regard that the thermal stability of ‘already dimerized’ RNA in the mutants are similar regardless of dimer formation efﬁciency. Thus the thermal dissociation curve of the mutants would be converged to present similar Tm. The molecular mass of dimerized genomic RNA isolated from the Step1 mutant included larger and smaller components than WT (Figure 4). We propose two hypotheses on this observation. First, non-viral RNAs may interact with the detected genomic RNA dimer. Previous reports have suggested that in addition to viral genomes, non-selective RNAs derived from the host cell are packaged in virions (50,51). It is possible that p15 from the Step1 mutant is somewhat different from p9 or WT NC in its ability to recognize its proper viral RNA target. Thus, the slower-migrating complex detected in the Step1 mutant may be composed of genomic RNAs and other non-viral RNAs. Second, genomic RNA dimers in the Step1 mutant may be in a relaxed conformation. It has been often observed that