Berkhout Lab - Ben Berkhout - HIV AIDS research Laboratory

The HIV-1 Envelope glycoprotein

Folding, Structure & Function
The envelope glycoproteins gp120 and gp41 are embedded in the viral membrane and mediate fusion of the viral membrane with the membrane of the target T cell. The viral Env gene encodes a gp160 precursor protein. Subsequent maturation steps of the gp160 precursor protein in the endoplasmatic reticulum and Golgi apparatus involve protein folding, glycosylation, formation of disulfide bridges, oligomerization, signal sequence cleavage and cleavage into gp120 and gp41. In the functional trimeric gp120-gp41 complex, gp120 is non-covalently bound to gp41, which anchors the envelope glycoptoteins in the viral membrane. Fusion of viral and cellular membranes involves interactions of gp120 with the receptor (CD4) and one of the co-receptors (mainly CCR5 and CXCR4) and subsequent conformational changes that expose the fusion peptides in gp41.
In collaboration with the group of Ineke Braakman (University of Utrecht), we study the folding process of the HIV-1 envelope proteins gp120 and gp41. Biochemical and molecular-virological techniques and molecular modeling are used to dissect the role and importance of the intramolecular disulfide bridges. Forced-evolution experiments with defective viruses containing mutations in the cysteines yield revertant viruses with compensatory mutations. These compensatory mutations elucidate important structural features of this protein.

Antigen & Immunogen
We have used the knowledge on the folding, structure and function of the envelope glycoproteins to design improved versions of envelope-based subunit vaccines. The structure of the envelope glycoproteins provides the virus with various possibilities to evade the immune system. Variable loops and extensive glycosylation shield the conserved regions that are vulnerable to neutralizing antibodies. The CD4 binding site is recessed, thus making it less accessible to antibodies. The co-receptor binding site only becomes exposed upon CD4 binding, thereby limiting time and space for antibody interference. Furthermore, incomplete gp160 precursor cleavage and dissociation of gp120 from gp41 result in non-functional proteins that may act as decoy for the immune system.
In collaboration with James Binley and John Moore (Cornell University, New York) we try to improve the antigenicity an possibly immunogenicity of envelope subunit vaccines. In particular, we focus on a novel antigen, the SOS protein, which has the gp120 and gp41 subunits covalently linked by an intermolecular disulfide bridge. We are employing molecular and virological techniques to improve the folding, cleavage, oligomerization and antigenicity of potential subunit vaccines.

Sanders RW, Busser E, Moore JP, Lu M, Berkhout B. Evolutionary repair of HIV type 1 gp41 with a kink in the N-terminal helix leads to restoration of the six-helix bundle structure. AIDS Res Hum Retroviruses. 2004 Jul;20(7):742-9.

Sanders RW, Dankers MM, Busser E, Caffrey M, Moore JP, Berkhout B. Evolution of the HIV-1 envelope glycoproteins with a disulfide bond between gp120 and gp41. Retrovirology. 2004 Mar 9;1(1):3.

Sanders RW, Vesanen M, Schuelke N, Master A, Schiffner L, Kalyanaraman R, Paluch M, Berkhout B, Olson WC, Lu M, Moore JP. 2002. Stabilization of the soluble, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1. J Virol. 2002 Sep;76(17):8875-89.

Sanders RW, Venturi M, Schiffner L, Kalyanaraman R, Katinger H, Lloyd KO, Kwong PD, Moore JP. The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120. J Virol. 2002 Jul;76(14):7293-305.

Schulke N, Vesanen MS, Sanders RW, Zhu P, Lu M, Anselma DJ, Villa AR, Parren PW, Binley JM, Roux KH, Maddon PJ, Moore JP, Olson WC. Oligomeric and conformational properties of a proteolytically mature, disulfide-stabilized human immunodeficiency virus type 1 gp140 envelope glycoprotein. J Virol. 2002 Aug;76(15):7760-76.

Binley JM, Sanders RW Aditi Master, Charmagne S. Cayanan, Cheryl L. Wiley, Linnea Schiffner, Bruce Travis, Shawn Kuhmann, Dennis R. Burton, Shiu-Lok Hu, William Olson, John P. Moore.. 2002. Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins. J Virol 2002 Mar;76(6):2606-16

Das AT, Land A, Braakman I, Klaver B, Berkhout B. 1999. HIV-1 evolves into a nonsyncytium-inducing virus upon prolonged culture in vitro. Virology 263: 55-69.

Binley JM, Sanders RW, Clas B, Schuelke N, Master A, Guo Y, Kajumo F, Anselma DH, Maddon PJ, Olson WC, Moore JP. 2000. A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associtated structure. J. Virol. 74: 627-643.

Sanders RW, Schiffner L, Master A, Kajumo F, Guo Y, Dragic T, Moore JP, Binley JM. 2000. Variable loop-deleted variants of the human immunodeficiency virus type 1 envelope glycoprotein can be stabilized by the introduction of an intermolecular disulfide bridge between gp120 and the gp41 subunits. J. Virol. 74: 5091-5100.