E. Urano and others gp120 (anti-gp120 antibodies from Santa Cruz Biotechnology; Fig. 2c). This was presumably because PH interfered with the MA–Env interaction. Alternatively, PH may actively incorporate cellular proteins that block efficient Env incorporation into virions. We were unable to evaluate the entry efficacy of PH–Gag–Pol virions pseudotyped by HIV-1 Env because of the limit of detection. PH–Gag–Pol virion infectivity was re-examined by virions pseudotyped with vesicular stomatitis virus G glycoprotein (VSV-G). The incorporation efficiencies of VSV-G into Gag–Pol and PH–Gag–Pol virions were similar (anti-VSV-G antibody from Sigma; Fig. 2c). The lentiviral vector system was used to test this, as HIV-1 provirus gene modifications often fail to produce infectious virions, probably due to viral gene dysregulation. 293T cells were transfected with expression plasmids for Gag–Pol, VSV-G (Komano et al., 2004), Rev and Vpu (a generous gift from Dr H. Go¨ttlinger, University of Massachusetts Medical School, MA, USA), and with a packaging vector encoding a luciferase expression cassette. The HIV-1-based vector expressing firefly luciferase upon infection was recovered 2 days post-transfection. 293T cells were exposed to virus- containing culture supernatants with similar CA concen- trations, and luciferase activities were measured at 2–3 days post-infection. When viral preparations with similar p24 concentrations were used, the luciferase activities of PH– Gag–Pol and Gag–Pol virus-infected cells were almost identical to each other (Fig. 2d, left graph). Luciferase expression was blocked by the non-nucleoside reverse transcriptase inhibitor nevirapine (NVP; Boehringer Ingelheim) but not by the CCR5 inhibitor (TAK-779; NIH AIDS Research and Reference Reagent Program), suggesting that gene transduction was mediated by viral infection (Fig. 2d, right panel). Similar results were obtained in several independent experiments. Thus, the PLCd1 PH domain can functionally replace the HIV-1 Gag myristoylation signal to support both viral production and entry processes, and this myristoylation is dispensable for MA function in the early phase of the virus life cycle. This is the first report describing an infectious pseudovirion without myristoylated Gag. Given that PH–Gag can enhance virus production, HIV-1 with PH–Gag might have been expected to be selected in nature. This is not the case, presumably because the addition of PH to the HIV-1 genome would increase its genome size close to the upper limit that can be incorporated into the retroviral particle, leading to a decrease in genome uptake efficiency, which is clearly a growth disadvantage, despite the enhanced virus production with PH-Gag. More importantly, PH–Gag is unable to incorporate HIV-1 Env efficiently enough to support the production of fully infectious virions. Our data point to the selective advantage of myristoylated Gag in viral evolution. The myristoylation-dependent Gag–PM association [max- imal dissociation constant (Kd) of ~0.5–1.061025 M] is presumably important for Gag multimerization at the PM (Provitera et al., 2006). After the first contact of Gag with 3148 the PM, the membrane binding of Gag is assumed to be stabilized by the Gag–PI(4,5)P(2) interaction (Ono et al., 2004; Saad et al., 2006). The multimerization of Gag appears to induce a conformational change in MA to expose myristoyl groups to enhance the PM targeting of Gag. The higher-order Gag multimerization is probably facilitated by the increased local concentrations of Gag at the PM. Although Gag and PH–Gag are similar to the extent that PI(4,5)P(2) is involved in their PM association, Gag binds to one of the acyl chains of PI(4,5)P(2), as modelled previously (Saad et al., 2006), whilst the PH domain binds the phosphorylated inositol group (Lemmon et al., 1995). The Kd of binding between the PLCd1 PH domain and PI(4,5)P(2) (~1–261026 M; Lemmon et al., 1995) suggests that the primary force driving PH–Gag to the PM is at least 2.5-fold stronger than that of myristoylation-mediated PM targeting of Gag. This might be one reason why PH–Gag–Pol was 3.2-fold more efficient at virion production than Gag–Pol. Our data suggest that the myristoyl group-dependent Gag–PM affinity is not a prerequisite for efficient Gag assembly at the PM or for viral production. The MA has multiple functions throughout the virus life cycle (reviewed by Bukrinskaya, 2007; Fiorentini et al., 2006; Hearps & Jans, 2007; Klein et al., 2007). In the PH– Gag–Pol virion, approximately one-fifth of the PH–MA was unanchored from the PM as MA* (Fig. 1b), which might accompany the pre-integration complex to support nuclear targeting. Using PH–Gag–Pol might enable separation of myristoylation-dependent and -independent MA functions, particularly during the entry phase. PH– Gag–Pol might also be useful for producing high-titre lentiviral vectors or for studying Gag trafficking in cells that poorly support PM targeting of myristoylated Gag, such as rodent cells. Furthermore, functional assays comparing the virus production of Gag–Pol and PH– Gag–Pol might enable the identification of chemical inhibitors or cellular factors specifically targeting myris- toylated Gag. Acknowledgements We thank Dr H. Go¨ttlinger for critically reading the manuscript. This work was supported by the Japan Health Science Foundation, the Japanese Ministry of Health, Labor and Welfare (H18-AIDS-W-003) and the Japanese Ministry of Education, Culture, Sports, Science and Technology (18689014 and 18659136). References Berridge, M. J. (1993). Inositol trisphosphate and calcium signalling. Nature 361, 315–325. Bryant, M. & Ratner, L. (1990). Myristoylation-dependent replication and assembly of human immunodeficiency virus 1. Proc Natl Acad Sci U S A 87, 523–527. Bukrinskaya, A. (2007). HIV-1 matrix protein: a mysterious regulator of the viral life cycle. Virus Res 124, 1–11. Journal of General Virology 89