Journal of General Virology (2008), 89, 3144–3149 DOI 10.1099/vir.0.2008/004820-0 Short Communication Correspondence Jun Komano ajkomano@nih.go.jp Substitution of the myristoylation signal of human Gag immunodeficiency virus type 1 Pr55 with the phospholipase C-d1 pleckstrin homology domain results in infectious pseudovirion production Emiko Urano,1,2 Toru Aoki,1 Yuko Futahashi,1 Tsutomu Murakami,1 Yuko Morikawa,2 Naoki Yamamoto1 and Jun Komano1 1AIDS Research Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan 2Kitasato Institute of Life Sciences, Kitasato University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan The matrix domain (MA) of human immunodeficiency virus type 1 Pr55Gag is covalently modified with a myristoyl group that mediates efficient viral production. However, the role of myristoylation, particularly in the viral entry process, remains uninvestigated. This study replaced the myristoylation signal of MA with a well-studied phosphatidylinositol 4,5-biphosphate-binding plasma membrane (PM) targeting motif, the phospholipase C-d1 pleckstrin homology (PH) domain. PH–Gag–Pol PM targeting and viral production efficiencies were improved compared with Gag–Pol, consistent with the estimated increases in Gag–PM affinity. Both virions were recovered in similar sucrose density-gradient fractions and had similar mature virion morphologies. Importantly, PH–Gag–Pol and Gag–Pol pseudovirions had almost identical infectivity, suggesting a dispensable role for myristoylation in the virus life cycle. PH–Gag–Pol might be useful in separating the myristoylation-dependent processes from the myristoylation- independent processes. This the first report demonstrating infectious pseudovirion production without myristoylated Pr55Gag. Received 11 June 2008 Accepted 20 August 2008 The N-terminal region [p17MA, matrix (MA) domain] of human immunodeficiency virus type 1 (HIV-1) Pr55Gag (Gag), a structural protein with multiple roles in the virus life cycle (Swanstrom & Wills, 1997), is covalently modified with a myristyol group that aids in plasma membrane (PM) targeting. Removal of this region leads to inefficient Gag targeting to the PM, resulting in dramatically reduced virus production (Bryant & Ratner, 1990; Go¨ttlinger et al., 1989; Pal et al., 1990; Zhou et al., 1994). Although viral particles can be produced by substituting MA with heterologous PM-targeting motifs, such substitution mutants show markedly reduced infectivity (Jouvenet et al., 2006; Scholz et al., 2008), probably due to an active role of MA in viral entry (Kiernan et al., 1998; Wang et al., 1993). However, direct experimental evidence of a viral entry-specific role for MA myristoylation is lacking. Such specific roles of Gag myristoylation can only be determined by separating the myristoylation-dependent PM-targeting function from other MA-associated functions. We constructed a mutant gag expression plasmid where the myristoylated region of Gag was replaced with the N- terminal pleckstrin homology (PH) domain of phospho- lipase C-d1 (PLCd1), a well-studied cellular PM-targeting 3144 motif that functions similarly to the myristoyl moiety. PLCd1 is a member of a family of inositol phospholipid- specific PLC isozymes involved in transducer-mediated intracellular responses (Berridge, 1993). The ~120 aa PH domain can bind to phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2)] and localize to the PM with high affinity and specificity (Ferguson et al., 1995b; Fiorentini et al., 2006; Harlan et al., 1994, 1995; Rhee, 2001; Yagisawa et al., 1994), and green fluorescent protein-bound PLCd1 PH domains have been used to visualize the PM in living cells (Stauffer et al., 1998; Tall et al., 2000). We used a codon-optimized HIV-1 gag–pol expression vector (pgag–pol) for genetic modification of gag, as pgag– pol increases Gag expression and facilitates protein analyses (Wagner et al., 2000). The substituted mutant retained an intact MA, with the exception of two N-terminal amino acid mutations (ATGACTG and GGCAGCG), resulting in an MGALA mutation to knock out the myristoylation signal of Gag and prevent internal translational initiation (Fig. 1a). The PLCd1 PH domain residues 1–175 (Stauffer et al., 1998) were linked to the LA–Gag N terminus by the amino acids PRAEFT, creating a PH–gag–pol expression vector (pPH-gag-pol, Fig. 1a). A control PH domain 2008/004820 G 2008 SGM Printed in Great Britain