Soluble CD4 and CD4 Immunoglobulin-Selected HIV-1 Variants: A Phenotypic Characterization

Abstract

The selection of HIV-1 resistance to neutralization by both monovalent and bivalent forms of soluble CD4 was demonstrated under various conditions. Phenotypic traits of the neutralization-resistant variants were systematically explored in order to gain insight into which aspects of the interactions with CD4 are most expendable to HIV-1 replication. The size of the nonneutralized fraction after treatment of preparations of the HIV-1 isolate IIIB and a molecular clone derived from it (HX10), with either monovalent soluble CD4 (sCD4) or bivalent CD4-Ig, was determined. These fractions were greater for the polyclonal IIIB than for the viral clone, and greater after treatment with sCD4 than with CD4-Ig. The virus in the nonneutralized fractions exhibited 2- to 20-fold lower sensitivity to the neutralizing agents than did unselected virus. In addition, clonal HIV-1 (HX10) was cultured in the presence of sCD4 or CD4-Ig for 12 weeks, so as to allow for accumulation of mutations that would confer stronger resistance to the selecting agent. Variants were obtained with up to 100-fold increased resistance to sCD4 or CD4-Ig. Detergent-solubilized gp120 from sCD4- and CD4-Ig-selected virus showed decreases in affinity for sCD4 and CD4-Ig. The monoclonal antibodies 6H10, to the gp120-binding site in domain 1 of CD4, and 5A8, to domain 2 of CD4, inhibited the induction by the viral escape variants of syncytium formation of C8166 cells. In general, the concentration of antibody 6H10 that inhibited the escape variants was lower than the concentration that inhibited the wild type, whereas there was no significant difference for the domain 2 antibody 5A8. We interpret this as a weaker attachment of the escape variants than of the wild-type virus to cellular CD4, but as an intact dependence of the variants on CD4 interactions for gaining entry into cells.