title: Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins.
abstract: In virus-infected cells, the envelope glycoprotein (Env) precursor, gp160, of human immunodeficiency virus type 1 is cleaved by cellular proteases into a fusion-competent gp120-gp41 heterodimer in which the two subunits are noncovalently associated. However, cleavage can be inefficient when recombinant Env is expressed at high levels, either as a full-length gp160 or as a soluble gp140 truncated immediately N-terminal to the transmembrane domain. We have explored several methods for obtaining fully cleaved Env for use as a vaccine antigen. We tested whether purified Env could be enzymatically digested with purified protease in vitro. Plasmin efficiently cleaved the Env precursor but also cut at a second site in gp120, most probably the V3 loop. In contrast, a soluble form of furin was specific for the gp120-gp41 cleavage site but cleaved inefficiently. Coexpression of Env with the full-length or soluble form of furin enhanced Env cleavage but also reduced Env expression. When the Env cleavage site (REKR) was mutated in order to see if its use by cellular proteases could be enhanced, several mutants were found to be processed more efficiently than the wild-type protein. The optimal cleavage site sequences were RRRRRR, RRRRKR, and RRRKKR. These mutations did not significantly alter the capacity of the Env protein to mediate fusion, so they have not radically perturbed Env structure. Furthermore, unlike that of wild-type Env, expression of the cleavage site mutants was not significantly reduced by furin coexpression. Coexpression of Env cleavage site mutants and furin is therefore a useful method for obtaining high-level expression of processed Env.
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, USA. jbinley@scripps.edu
authors.forename: James M
authors.initials: JM
authors.lastname: Binley
authors.forename: Rogier W
authors.initials: RW
authors.lastname: Sanders
authors.forename: Aditi
authors.initials: A
authors.lastname: Master
authors.forename: Charmagne S
authors.initials: CS
authors.lastname: Cayanan
authors.forename: Cheryl L
authors.initials: CL
authors.lastname: Wiley
authors.forename: Linnea
authors.initials: L
authors.lastname: Schiffner
authors.forename: Bruce
authors.initials: B
authors.lastname: Travis
authors.forename: Shawn
authors.initials: S
authors.lastname: Kuhmann
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Burton
authors.forename: Shiu-Lok
authors.initials: SL
authors.lastname: Hu
authors.forename: William C
authors.initials: WC
authors.lastname: Olson
authors.forename: John P
authors.initials: JP
authors.lastname: Moore
mesh_terms: D010450:Endopeptidases / Q000378:metabolism*; D045683:Furin; D015686:Gene Products, env / Q000378:metabolism*; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000378:metabolism; D019302:HIV Envelope Protein gp160 / Q000235:genetics / Q000378:metabolism; D015700:HIV Envelope Protein gp41 / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D006367:HeLa Cells; D006801:Humans; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*
chemical_list: D015686:Gene Products, env; D015699:HIV Envelope Protein gp120; D019302:HIV Envelope Protein gp160; D015700:HIV Envelope Protein gp41; D011498:Protein Precursors; D011994:Recombinant Proteins; D010450:Endopeptidases; D013381:Subtilisins; D045683:Furin