E. Urano and others The intracellular distribution of Gag, PH–Gag and PH4A– Gag was analysed by immunofluorescence microscopy of transfected 293T cells (Fig. 1c). Transfected cells were grown for 24 h, fixed (4 % formaldehyde), permeabilized (0.1 % Triton X-100 for 5–30 min) and incubated with mouse anti-p24CA and goat anti-mouse antibodies (GE Healthcare Bio-Sciences) conjugated to streptavidin–Alexa Fluor 555 (Invitrogen). Cells were stained with Hoechst 33258, mounted and analysed using confocal microscopy as described previously (Futahashi et al., 2007). Gag was found to be distributed throughout the cytoplasm and at the cell periphery. In contrast, PH–Gag signals were mostly detected at the cell periphery and PH4A–Gag was distributed homogeneously in the cytoplasm. These data clearly showed that PH–Gag targeted the PM more efficiently than Gag, consistent with the Western blot analysis (Fig. 1b). These results also suggested that the efficient PM targeting of PH–Gag depends on the ability of the PH domain to bind PI(4,5)P(2). Similar observations were made in NP2 and COS7 cells. VLP production was also examined. Tissue culture super- natants of pgag-pol-, pPH-gag-pol- or pPH4A-gag-pol- transfected 293T cells were passed through nitrocellulose filters (0.45 mm) and the virions were collected by centrifugation (541 000 g for 1 h). Viral antigens, except for PH4A, were detected with anti-p24CA and anti-p17MA antibodies (Fig. 1b). Gag and PH–Gag were further processed by the viral proteases in the virions compared with the cell lysates, as indicated by the increased signals for CA and MA relative to Gag. Interestingly, approxi- mately one-fifth of the PH–Gag in the virion was cleaved close to the PH–MA junction. Presumably, the amino acid sequence at the C end of the PH domain ELQN/FLKE (aa 164–171, where the protease cleaves at the N–F junction) served as a viral protease recognition site as it matched the substrate consensus sequence and resembled the NC–p1 junction, RQAN/FLGK (de Oliveira et al., 2003; Swanstrom & Wills, 1997). Alternatively, the N terminus of LA–Gag (EFTL/AADS) might be targeted by the viral protease. Thus, the MA released from PH–MA, designated MA*, possibly has 10 aa attached to its N terminus. The VLP production efficiency was quantified as the concentration of CA in transfected 293T cell culture supernatants relative to that in cell lysates using a p24 ELISA (Zeptometrics). When the CA concentrations of the virion fractions were normalized to those of the cell lysates, the pPH-gag-pol viral production efficiency was 3.2-fold higher than that of pgag-pol (3.2±2.0-fold, n514, P,0.001 by Wilcoxon’s matched pairs rank test; repres- entative experiments are shown in Table 1). In contrast, pPH4A-gag-pol produced viral particles less efficiently than pgag-pol (0.09±0.07-fold, n56, P,0.05 by Wilcoxon’s matched pairs rank test; representative experiments are shown in Table 1). These data were consistent with the Western blot analysis (Fig. 1b). To characterize the physical properties of PH–Gag–Pol VLPs, we measured the specific density of virions and examined the virion morphology. Firstly, the VLPs were subjected to 20–70 % (w/w) equilibrium sucrose gradient centrifugation (120 000 g for 16 h) and fractions were recovered from the bottom to the top. The peak fraction containing the viral CA antigen was determined by p24 ELISA. Gag–Pol and PH–Gag–Pol VLPs were detected in fractions with densities of 1.15±0.01 (n55) and 1.16±0.01 (n54) g ml21, respectively (not statistically significant; representative experiments are shown in Fig. 2a). Secondly, ultrathin sections of fixed 293T cells (2 % glutaraldehyde, 2 % osmium tetroxide) transfected with pgag-pol or pPH-gag-pol were imaged by transmission electron microscopy (JEM1200EX at 80 kV or JEM2000EX at 100 kV; JEOL). The PH–Gag–Pol and Gag–Pol VLP Table 1. Efficiency of virus production from 293T cells transfected with pgag-pol, pPH-gag-pol or pPH4A-gag-pol expression vector Experiment Plasmid p24CA (ng per well)* Virus production efficiency (B/A) Fold increase relative to pgag-pol Cell lysate (A) Culture supernatant (B) pgag-pol pPH-gag-pol pPH4A-gag-pol pgag-pol pPH-gag-pol pPH4A-gag-pol pgag-pol pPH-gag-pol pPH4A-gag-pol pgag-pol pPH-gag-pol pPH4A-gag-pol 4 043 1 989 3 175 3 521 2 638 5 913 3 359 1 454 4 226 9 666 4 699 5 527 4 869 8 363 103 3 887 7 688 1 125 4 160 5 172 75 8 996 18 273 534 1 2 3 4 3146 *Cells grown in six-well plates were transfected using Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen). 1.204 4.206 0.033 1.104 2.914 0.190 1.239 3.558 0.018 0.931 3.889 0.097 – 3.49 0.03 – 2.64 0.17 – 2.87 0.01 – 4.18 0.10 Journal of General Virology 89