INVESTIGATION OF THE MECHANISM OF GRIFFITHSIN (GRFT): A POTENT HIV ENTRY INHIBITOR

Griffithsin (GRFT) is a potent protein lectin that has been shown to inhibit HIV infection by binding to high mannose glycans on the surface of gp120. However, important biochemical details on the antiviral mechanism of GRFT action remain unexplored. In chapter 2, we investigate the role of individual carbohydrate binding sites in the function of GRFT. Individual point mutants of GRFT showed a small loss of binding to gp120, but a significant loss of activity to inhibit viral infection. The disparity between HIV-1 gp120 binding ability and HIV inhibitory potency for these GRFT variants indicates that gp120 binding and virus neutralization do not necessarily correlate, and suggests a mechanism that is not based on simple gp120 binding. In chapter 3 we investigated the role of the dimer in GRFT's anti-HIV activity. An obligate dimer of GRFT was designed, as well as a "one-armed" obligate dimer which has one carbohydrate binding arm mutated while the other subunit remained intact. While GRFT only needs one functional arm to tightly bind to gp120, it needs both arms to potently inhibit HIV entry. Furthermore, our experiments showed that GRFT needs both arms to alter the structure of gp120 by exposing the CD4 binding site as well as to cross-link two separate gp120 subunits. Evidence is provided that the dimer form of Grft is critical to the function of this protein in HIV inhibition. In chapter 4 we studied a chemokine analog vMIP-II (viral macrophage inflammatory protein-II) that inhibits multiple HIV strains by binding to both CCR5 and CXCR4 co-receptors. We have made a vMIP-II variant, "5P12-vMIP-II" in which the N-terminus of vMIP-II has been replaced by the N-terminus of 5P12-RANTES that have been shown to greatly enhance the anti-HIV potency of the chemokine RANTES for R5 HIV strains. Both vMIP-II and 5P12-vMIP-II showed the ability to inhibit multiple strains of HIV. While the 5P12 N-terminus did not improve the potency of the protein, our results provide evidence about the interaction of vMIP-II with CCR5, indicating that vMIP-II does not bind CCR5 in the same way as human chemokines