Functional replacement of HIV Gag PM targeting signal Fig. 2. Physical and biological properties of PH–Gag–Pol VLPs. (a) The virions produced by the pgag-pol (WT) and pPH-gag- pol (PH) expression vectors were analysed by equilibrium sucrose density-gradient centrifugation. The virion-containing fraction was determined by an ELISA detecting p24CA. Representative data from four to five independent experiments are shown. In this experiment, WT (filled circles) and PH (open circles) VLPs migrated in the 1.152 or 1.162 g ml”1 density fractions, respectively. (b) Transmission electron microscopy images of 293T cells transfected with pgag-pol (WT) or pPH-gag-pol (PH). Representative images are shown. (c) Incorporation of HIV-1 Env (upper panel) and VSV-G (lower panel) into Gag–Pol (WT) and PH–Gag–Pol (PH) virions. The virion fractions were subjected to Western blot analysis detecting gp120, VSV-G and p24CA. (d) The early phase of the HIV-1 life cycle is supported by PH–Gag–Pol. 293T cells were exposed to virus-containing culture supernatants with similar CA concentrations (270 and 220 ng ml”1 for Gag–Pol and PH–Gag–Pol, respectively), and luciferase activities were measured at 2–3 days post-infection as relative light units (RLU). The luciferase activities of PH–Gag– Pol (PH) and Gag–Pol (WT) virus-infected cells were almost identical (left graph). The luciferase transduction by WT (bars 1 and 2) and PH (bars 3 and 4) pseudovirions was performed in the presence of nevirapine (NVP, bars 1 and 3) or TAK-779 (bars 2 and 4). The luciferase signals decreased in the presence of NVP for both WT (bar 1) and PH (bar 3) but not in the presence of TAK-779 for both WT (bar 2) and PH (bar 4), respectively. Representative data from several independent experiments are shown. Asterisks indicate statistical significance (P,0.01, n53, Student’s t-test). diameters were almost identical (Fig. 2b). Virion budding structures showed that the electron-dense layer, which represented multimerized Gag, of the PH–Gag–Pol VLP was slightly separated from the viral envelope compared with that of the Gag–Pol VLP (Fig. 2b). This indicated that the PH domain was positioned between the viral envelope and the electron-dense layer. In contrast, the morphologies of the mature PH–Gag–Pol and Gag–Pol virions were similar, suggesting that myristoylation is dispensable for http://vir.sgmjournals.org mature virion morphology and that the PH–Gag–Pol virion may be infectious. We examined HIV-1 Env incorporation into the PH–Gag– Pol virion. To do this, we used codon-optimized gp160 (p96ZM651gp160-opt; NIH AIDS Research and Reference Reagent Program). The PH–Gag–Pol virion incorporated HIV-1 Env less efficiently than the Gag–Pol virion as demonstrated by Western blot analysis detecting CA and 3147