Previous work with recombinant soluble CD4 and CD4-Ig hybrid proteins demonstrated viral inactivation at low levels of CD4-Ig binding. We have extended these studies, using flow cytometry to measure CD4-Ig binding to envelope glycoprotein gp120 on the surface of HIV-1 infected cells. The fluorescent signal increased as a function of CD4-Ig concentration, reaching saturation at 20 nM CD4-Ig, and decreased when subsaturating amounts of CD4-Ig were competed with soluble recombinant gp120, reaching 50% inhibition at <20 nM gp120. Virus survival was measured in a sensitive plaque forming assay over the full range of CD4- Ig binding, between 0 and 99% of maximal binding. Virus survival was essentially 0 whenever CD4-Ig binding was over 20% of saturating. Virus survival reached 37% (1/e) when just 2.7% of available gp120 sites bound CD4-Ig; i.e., when one gp120 molecule out of 36 was bound by CD4-Ig. At each level of average CD4-Ig binding, the fraction of virus surviving equaled the fraction of viruses binding less than this critical value. These results suggest that low level CD4-Ig binding may trigger a conformational change in the virus, converting it to an inactive state. They are consistent with observations that much greater levels of CD4 binding can trigger gp120 shedding from the virus. The recent discovery of fresh isolates, which bind CD4-Ig normally but resist inactivation suggests that these isolates may have lost the triggering mechanism. We are currently studying these isolates for ways to restore triggering.
