Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins - PubMed (original) (raw)

. 2002 Mar;76(6):2606-16.

doi: 10.1128/jvi.76.6.2606-2616.2002.

Rogier W Sanders, Aditi Master, Charmagne S Cayanan, Cheryl L Wiley, Linnea Schiffner, Bruce Travis, Shawn Kuhmann, Dennis R Burton, Shiu-Lok Hu, William C Olson, John P Moore

Affiliations

Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins

James M Binley et al. J Virol. 2002 Mar.

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.

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Figures

FIG. 1.

FIG. 1.

In vitro enzymatic cleavage of Env with plasmin or soluble furin. Eight micrograms of partially (∼50%) cleaved, purified JR-FL gp140SOS or gp120 was incubated with a protease for various times at 37°C. The Env proteins were then analyzed by reducing SDS-PAGE and Western blotting. (A) gp140SOS proteins from a 2- or 16-h plasmin (20 μg) digest. The Western blot was probed with the anti-gp41 MAb 2F5. (B) gp140SOS and gp120 samples from a 16-h plasmin (20 μg) digest. The Western blot was probed with the anti-gp120 MAb B12. (C) gp140SOS was incubated at pH 7.5 with or without plasmin or soluble furin for 16 h at 37°C. The Western blot was probed with MAb B12. (D) JR-FL gp140SOS was incubated with or without furin for 16 h at the pH indicated. The Western blot was probed with MAb B12. The percent cleavage achieved by soluble furin was calculated as described in Materials and Methods and was expressed with reference to the control in the last lane (no furin; 0% cleavage).

FIG. 2.

FIG. 2.

Cleavage and expression of Env proteins with or without furin. Culture supernatants containing 35S-labeled Env proteins were immunoprecipitated with either HIVIG or SIVIG as appropriate and then analyzed by reducing SDS-PAGE. Results shown are representative of three repeats. In each panel, the percent cleavage was calculated as described in Materials and Methods. Additionally, the relative expression of the gp120-plus-gp140 or gp120-plus-gp160 bands was calculated and expressed as a ratio relative to a standard (expression defined as 1.00) in each gel. (A) Soluble gp140 proteins were expressed in 293T cells transfected with pPPI4-based plasmids. (B) The JR-FL gp140SOS and gp140WT proteins were expressed as in panel A but in the presence of variable amounts of coexpressed, full-length furin. (C) BSC40 cells were infected with vaccinia viruses v-VS4 (expressing SIVmne Gag-Pol) and/or v-VSE5 (expressing SIVmne Env) at an MOI of 5, as indicated. Some of the cells were coinfected, also at an MOI of 5, with the vaccinia virus vv:hfur expressing full-length furin (second and fourth lanes). (D) JR-FL gp140WT and gp140UNC proteins were expressed in 293T cells transfected with pPPI4-based plasmids, with or without cotransfection of full-length furin. (E) JR-FL gp140WT and gp140SOS proteins were expressed in 293T cells transfected with pPPI4-based plasmids, alone or with cotransfection of either full-length furin (FL) or truncated, soluble furin (∂TC), as indicated.

FIG. 3.

FIG. 3.

Altering the cleavage sequence can increase Env processing by cellular proteases. (A) JR-FL gp140SOS with the wild-type REKR (first and second lanes) or the mutant RRRKKR (third and fourth lanes) or RRRRRR (fifth and sixth lanes) cleavage site sequence was expressed in the absence (first, third, and fifth lanes) or presence (second, fourth, and sixth lanes) of cotransfected full-length furin. (B) JR-FL gp140SOS RRRRRR was expressed with 0, 0.1, 1, or 10 μg of cotransfected full-length furin. (C) DU151 gp140SOS (first and second lanes) and its RRRRRR mutant (third and fourth lanes) were expressed in the absence (−) or presence (+) of cotransfected full-length furin. All samples were labeled and immunoprecipitated as described in the legend to Fig. 2. The percent cleavage and relative expression of Env were calculated as for Fig. 2.

FIG. 4.

FIG. 4.

Env cleavage site mutants are functional for infection and fusion. (A) The infectivities for HeLa-CD4-CCR5 cells of pNL-luc viruses pseudotyped with wild-type JR-FL gp160WT, the JR-FL gp160RRRRRR mutant, or VSV-G were measured by use of a single-round infection assay with a luciferase readout. Normalized luciferase values for negative-control viruses lacking envelope (derived from pNL-luc transfection supernatants) were <1 U. (B) Cell-cell fusion mediated by the gp160WT or gp160RRRRRR protein was analyzed in a dye transfer assay.

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