[Show abstract][Hide abstract] ABSTRACT: The glycan shield of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein serves as a barrier to antibody-mediated
neutralization and plays a critical role in transmission and infection. One of the few broadly neutralizing HIV-1 antibodies,
2G12, binds to a carbohydrate epitope consisting of an array of high-mannose glycans exposed on the surface of the gp120 subunit
of the Env protein. To produce proteins with exclusively high-mannose carbohydrates, we generated a mutant strain of Saccharomyces cerevisiae by deleting three genes in the N-glycosylation pathway, Och1, Mnn1, and Mnn4. Glycan profiling revealed that N-glycans produced by this mutant were almost exclusively Man8GlcNAc2, and four endogenous glycoproteins that were efficiently recognized by the 2G12 antibody were identified. These yeast proteins,
like HIV-1 gp120, contain a large number and high density of N-linked glycans, with glycosidase digestion abrogating 2G12
cross-reactivity. Immunization of rabbits with whole Δoch1 Δmnn1 Δmnn4 yeast cells produced sera that recognized a broad range of HIV-1 and simian immunodeficiency virus (SIV) Env glycoproteins,
despite no HIV/SIV-related proteins being used in the immunization procedure. Analyses of one of these sera on a glycan array
showed strong binding to glycans with terminal Manα1,2Man residues, and binding to gp120 was abrogated by glycosidase removal
of high-mannose glycans and terminal Manα1,2Man residues, similar to 2G12. Since S. cerevisiae is genetically pliable and can be grown easily and inexpensively, it will be possible to produce new immunogens that recapitulate
the 2G12 epitope and may make the glycan shield of HIV Env a practical target for vaccine development.
Full-text · Article · Aug 2008 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (DeltaV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds.