Proc. Nadl. Acad. Sci. USA Vol. 87, pp. 523-527, January 1990 Biochemistry Myristoylation-dependent replication and assembly of human immunodeficiency virus 1 (AIDS/virus assembly/gag precursor) MARTIN BRYANT*t AND LEE RATNERtf§ Departments of *Pediatrics, tMedicine, and §Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO 63110 Communicated by Stuart Kornfeld, September 25, 1989 (receivedfor review May 23, 1989) ABSTRACT Covalent linkage of myristic acid to the N- virus types I and II, human immunodeficiency virus 1 (HIV-1), terminal glycine residue ofPr55gag, the precursor ofthe major and simian immunodeficiency virus type I] has recently been structural proteins of human immunodeficiency virus 1 (HIV- described (1, 18-23). In cells infected by MoMLV (22) or 1), facilitates an essential step in virus assembly and propaga- MPMV (23), myristoylation of the gag polypeptide is required tion. Substitution of the myristoyl-acceptor glycine with ala- for normal virion assembly and infectious particle production. nine, in a functional clone of HIV-1, eliminates virus replica- HIV, like other mammalian retroviruses, encodes a gag tion. Complementation of this defect, in trans, restores precursor, Pr55gag, which is normally processed by the viral infectious particle production. The nonmyristoylated (myr-) protease to the major structural proteins found in the mature gag precursor accumulates in infected cells and is not processed extracellular virus particle. Both Pr55gag and the matrix into the mature capsid components of the intact virion. How- protein (p17), which is proteolytically cleaved from the ever, myr- Pr55gag can be processed by purified HIV protease N-terminal end ofPr55gag, are myristoylated (see Fig. 1) (24, in vitro, demonstrating that the myristoyl moiety is not required 25). Analysis of the amino acid sequence of these proteins for cleavage by the protease. Myristoylation of Pr55gag is not from different HIV-1 isolates shows that the myristoylation necessary for localization but is required for stable membrane acceptor is always an N-terminal glycine residue (26-28). association and assembly of HIV-1. Furthermore, the Prl80gag-pol precursor, which contains gag proteins as well as the viral replicative enzymes [prote- The covalent attachment of long-chain fatty acids to select ase, reverse transcriptase (RT), and integrase], is probably subsets of eukaryotic cellular proteins and viral polypeptides also myristoylated. The current study examines the role of confers a change in hydrophobicity that may be important in myristoylation of the structural gag polyprotein precursor defining protein quaternary structure, directing intracellular Pr55gag of HIV-1 in virus replication. Our results show that trafficking and signal transduction, and/or in specific pro- addition of the myristate moiety is required for stable mem- tein-protein or protein-lipid interactions at the plasma mem- brane association, proteolytic processing, and assembly of brane. The specific fatty acid and its distinct mode of Pr55gag into infectious extracellular particles of HIV-1. attachment probably influence the unique biochemical func- tion served by each acylated protein (1-5). MATERIALS AND METHODS The most thoroughly studied acyl proteins have been Cell Lines. T-lymphoid cell lines H9 and Jurkat were virus-related polypeptides because of their high level of provided by J. Hoxie and R. C. Gallo (National Cancer expression in infected cells. Schmidt and Schlesinger (6) were Institute). COS-1 and HeLa cell lines were obtained from the first to describe the attachment of fatty acid to the envelope American Type Culture Collection. glycoproteins of vesicular stomatitis and Sindbis viruses and DNA Clones. pGG1 contains a functional clone of HIV-1 to suggest an association of this type of protein modification and was previously designated pHXB2gpt2 (29). Plasmid with virus assembly and propagation (7). Subsequently, the pAenv is a derivative of pGG1 constructed by digestion with structural proteins of nonenveloped viruses have also been the restriction endonucleases Sal I and Xho I, followed by shown to contain covalently bound fatty acid. The capsid treatment with T4 DNA ligase thus deleting the entire enve- protein VP4 and its precursors in picornaviruses are myris- lope gene of HIV-1 (nucleotides 5366-8474). toylated (8-11). The position of the myristate moiety at the Mutagenesis. The glycine to alanine substitution was made interface between protein subunits in poliovirus suggests that with the 20-mer oligonucleotide GCTCTCGCAGCCATCTC- it may play a role in virus capsid assembly (12). The structural TCT using oligonucleotide-directed mutagenesis as de- proteins ofpolyomavirus (10) and simian virus 40 (11), as well scribed by Kunkel (30). The resultant plasmid was designated as the PreS1 protein ofhepadnaviruses (i.e., hepatitis B virus) pGA1 and the mutation was confirmed by the dideoxynucle- (13), are also modified by attachment of myristic acid and otide chain-termination method for sequencing DNA (31). may influence nucleocapsid assembly and/or infectivity. Transfection. Cells were transfected by the calcium phos- Myristoylation of p6Ov-src of Rous sarcoma virus is re- phate precipitation technique for gene transfer (32). Stable quired for its stable association with cellular membranes and transfectants were isolated by cotransfection of HeLa cells morphological transformation of cells (14, 15). Similarly, with either pGA1 or pGG1 and SFneo [similar to pSV2neo myristoylation has been proposed to be an important factor in with the addition of the spleen focus-forming virus long transformation by gag-onc fusion proteins with kinase activity terminal repeat (33)]. Cell clones resistant to G418 (400 (16, 17). In addition, fatty acid modification of the gag pre- ,ug/ml, GIBCO) and positive by HIV-1 p24 ELISA (DuPont) cursor to the internal structural proteins of many mammalian were selected for protein studies. retroviruses [Moloney murine leukemia virus (MoMLV), Ma- son-Pfizer monkey virus (MPMV), human T-cell leukemia Abbreviations: MoMLV, Moloney murine leukemia virus; MPMV, Mason-Pfizer monkey virus; HIV, human immunodeficiency virus; RT, reverse transcriptase. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Washington payment. This article must therefore be hereby marked "advertisement" University School of Medicine, Clinical Science Building Box 8125, in accordance with 18 U.S.C. §1734 solely to indicate this fact. 660 South Euclid Avenue, Saint Louis, MO 63110. 523 Downloaded by guest on September 26, 2021 524 Biochemistry: Bryant and Ratner Proc. Natl. Acad. Sci. USA 87 (1990) Virus Assays. Cell supernatants were tested on various Biological Effect of Myristoylation Minus Mutation. The days posttransfection for virus-specific antigen using the p24 pGA1 mutant and the parent plasmid pGG1 were transfected ELISA (sensitivity of 0.03 ng/ml). Both the major structural into CD4- HeLa (Fig. 2) or COS-1 cells (not shown) using nucleocapsid protein of HIV-1, p24, and the p24-containing calcium phosphate coprecipitation (32). On day 4 posttrans- Pr55gag can be detected by this assay (unpublished obser- fection, CD4' human lymphoid cells (H9) were added to vation). Virions were concentrated 10:1 from culture super- allow propagation and amplification of infectious virus re- natants by polyethylene glycol precipitation (30% in 150 mM leased from the transfected cells. The nonadherent H9 cells NaCl/0.1 mM phenylmethylsulfonyl fluoride), solubilized, were removed from the adherent HeLa cells on day 8 and and virus-associated RT activity was measured by [32P]TTP were maintained in culture for an additional 2 weeks. Virus- incorporation using a poly(rA)-oligo(dT) template (34). specific antigen production, as measured by p24 antigen Immunoprecipitation and Immunoblotting. HIV-1-specific ELISA, was detected in supernatant solutions from both antigens in lysates from 4 x 106 cells or cell-free supernatants pGG1 and pGA1 transfected cultures prior to the addition of were characterized by immunoblot techniques (35) or were the H9 cells (Fig. 2). However, neither virus antigen nor RT immunoprecipitated after labeling with [35S]methionine/ activity could be detected in the H9 cultures after cocultiva- cysteine (specific activity, 1031 Ci/mmol; 1 Ci = 37 GBq) or tion with pGA1 transfected HeLa (Fig. 2) or COS-1 cells (not [3H]myristic acid (39.3 Ci/mmol). Cells labeled with myris- shown). In contrast, production of infectious HIV by the tate were grown in 5% delipidated fetal calf serum. Pooled pGG1 transfected cells was readily identified by syncytia AIDS patients' sera containing a high titer of antibody to HIV formation among the H9 cells, p24 soluble antigen produc- antigens were used in both procedures. Cell pellets were tion, and RT activity (Fig. 2). The pGA1 defect could, how- lysed in RIPA buffer [50 mM Tris-HCI, pH 7.4/0.5% Triton ever, be complemented in trans, by the gag-pol expression X-100/100 mM NaCl/aprotinin (100 units/ml)/0.1 mM phe- plasmid pAenv (Fig. 2). Similar results were obtained after nylmethylsulfonyl fluoride] and used directly for immunoblot direct transfection of the CD4+ Jurkat cell line (data not analysis. The HIV-1 and cellular proteins were separated by shown). These results show that the glycine to alanine sub- SDS/PAGE (36) and transferred by electroblotting to a stitution at the N terminus of the gag polyprotein Pr55gag