attempted to snapshot the initial virion maturation process of HIV-1 through co-transfection. The ratios of Step1 to Step0 were changed from 10:0 to 0:10 successively by mixing two plasmids for co-transfection and the series of resultant viral particles were harvested. The monomeric viral genome content from various particles were visualized and analyzed by northern blot (Figure 5A). The graph represents the relationship between monomeric genome content and proportion of processed Gag (Step1) within the virion (Figure 5B), and the data were ﬁt to a linear regression model (R2 & 0.956). These results indicated that the increase of cleavage products may not have a threshold for the transition from monomeric to dimeric RNA genomes. Virion morphology and genome dimerization It is well-known that HIV-1 virion morphology dramatic- ally changes during Pr55 processing. To investigate whether the morphological changes of the virion are correlated with genomic RNA dimerization, virion mor- phological features of the four sequential step mutants and NC/p1 mutant were observed with electron micros- copy (Figure 6). Virion morphologies were classiﬁed into four main groups (Figure 6A) and morphological classiﬁ- cations were based on viral core and membrane conditions (Table 1). The morphology of all PR virions were typical immature particles (Group A) as demonstrated by many previous reports [e.g. (41)]. As expected, virions from the Step0 mutant were very similar to those of PR, Figure 5. Stoichiometric analysis of Gag processing and RNA dimer- ization. PR mutant plasmid and the mixtures of Step0 and 1 plasmids were applied for transfection, and a series of resultant viral particles were harvested. The ratios of Step 1 to Step 0 was changed successively from 10:0 to 0:10. (A) Virion RNA proﬁles were detected by northern blotting in a native agarose gel as in Figure 2. (B) The monomer content of each RNA in native condition was calculated and repre- sented by a scatter diagram. The data are the average of three inde- pendent experiments and the error bars represent SEM. The formula and R-squared value of the straight-line approximation are shown. Nucleic Acids Research, 2011, Vol. 39, No. 8 3411 containing a thick electron-dense ring and devoid of a conical-shaped core, termed a doughnut-shaped morph- ology. The morphologies of the WT, as well as the NC/ p1 virions, were classiﬁed into two major groups. One was the typical mature particle (Group D), with a conical core surrounded by an envelope (Figure 6A). The second was an enveloped particle containing some amorphous struc- tures rather than an obvious core (Group C). Most of the virions from Step1 and 1.1 were immature (Group B), but did not have an apparent electron-dense ring like Group A particles, with envelopes that were thicker than those of Group C. The virions from Step2 contained both Group B Figure 6. Virion morphology of the mutants. (A) Classiﬁcation of virion morphology judged by electron microscopy analysis. The criter- ion for judgment was summarized in Table 1. (B) The distribution chart of virion morphology in each mutant. A minimum of one hundred particles were surveyed and classiﬁed for each mutant. Table 1. Classiﬁcation of virion morphology based on electron-microscopic observation Group A B C D Core Ring-shaped Amorphous Amorphous or non-conical Conical Envelope Thick Thick Thin Thin