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The mechanism by which cells expressing HIV envelope glycoproteins progress from binding CD4+ cells to syncytia formation is not entirely understood. The purpose of these investigations was to use physical and biochemical tools (temperature shifts, soluble CD4, protease inhibitors, and a battery of anti-CD4 monoclonal antibodies) to isolate discrete steps during syncytia formation. Previously (Fu et al., J Virol 1993;67:3818), we found that preincubation of cells stably expressing HIV-1 gp 160 (TF228.1.16) with CD4+ SupT1 cells at 16 degrees C, a temperature that is nonpermissive for syncytia formation, resulted in an increased rate of syncytia formation when the cocultures were shifted to the syncytia-permissive temperature of 37 degrees C. We have since found that syncytia formation is further enhanced by shifting the cocultures from 16 to 4 degrees C prior to incubation at 37 degrees C. Together, these data suggest that two discrete states, which we term the first and second activation intermediates (FAI and SAI), are involved in syncytia formation. We have found that acquisition of the FAI (by preincubation at 16 degree C) is sensitive to some serine protease inhibitors (PI), soluble CD4 (sCD4), shedding of gp120, and anti-CD4 monoclonal antibodies (MAb) directed toward the CDR-1/2 and CDR-3 regions of domain 1 on CD4. Expression of the FAI (formation of syncytia by shifting from 16 to 37 degrees C) remains sensitive to sCD4, shedding of gp120, and MAb directed toward CDR-1/2 but is less sensitive to MAb that bind CDR-3 and is insensitive to PI. Similarly, acquisition of the SAI (shifting cocultures from 16 to 4 degrees C), is sensitive to sCD4, shedding of gp120, and MAb directed toward CDR-1/2. In contrast, expression of the SAI (shifting cocultures from 16 to 4 to 37 degrees C) is sensitive only to MAb directed toward CDR-1/2 and cannot be blocked by sCD4, shedding of gp120, or PI. These data allow us to propose that syncytia formation, mediated by HIV-1 envelope glycoproteins, proceeds by a multistep cascade.

Assembly and budding of retroviruses is believed to involve a complex interaction of envelope and capsid proteins at the host cell membrane. The nature of these interactions is, however, incompletely understood. Studies of the topography of the surface of HIV-1 have shown that the envelope glycoprotein projections (knobs) are arranged in a T = 7 levo rotational symmetry. Similarly, an icosahedral structure has been suggested for the p17 matrix of HIV-1. In an effort to investigate whether there is a structural interaction between these molecules, virions whose maturation was blocked by an inhibitor of HIV protease were studied using cytochemistry, morphometry, and 2D fast Fourier transform image enhancement. Analysis of the relationship between core morphology and the topographic distribution of envelope glycoprotein projections on HIV-1 provided structural evidence of an interaction between Env and Gag proteins. Furthermore, image enhancement revealed a periodic substructure in the Pr55gag plaque. Taken together, the data suggest an interaction between Pr55gag and the gp120-gp41 complex during assembly and budding of HIV-1. This interaction may, in part, contribute to determining the amount of Env glycoprotein that will be incorporated into a virion, and therefore play a role in the biology of HIV-1.

The mechanism of CD4-mediated fusion via activated human immunodeficiency virus type 1 (HIV-1) gp41 and the biological significance of soluble CD4 (sCD4)-induced shedding of gp120 are poorly understood. The purpose of these investigations was to determine whether shedding of gp120 led to fusion activation or inactivation. BJAB cells (TF228.1.16) stably expressing HIV-1 envelope glycoproteins (the gp120-gp41 complex) were used to examine the effects of pH and temperature on sCD4-induced shedding of gp120 and on cell-to-cell fusion (syncytium formation) with CD4+ SupT1 cells. sCD4-induced shedding of gp120 was maximal at pH 4.5 to 5.5 and did not occur at pH 8.5. At physiologic pH, sCD4-induced shedding of gp120 occurred at 22, 37, and 40 degrees C but neither at 16 nor 4 degrees C. In contrast, syncytia formed at pH 8.5 (maximally at pH 7.5) but not at pH 4.5 to 5.5. At pH 7.5, syncytia formed at 37 and 40 degrees C but not at 22, 16, or 4 degrees C. Preincubation of cocultures of TF228.1.16 and SupT1 cells at 4, 16, or 22 degrees C before the shift to 37 degrees C resulted in similar, increased, or decreased syncytium formation, respectively, compared with the control. Furthermore, an activated intermediate of CD4-gp120-gp41 ternary complex may form at 16 degrees C; this intermediate rapidly executes fusion upon a shift to 37 degrees C but readily decays upon a shift to the shedding-permissive but fusion-nonpermissive temperature of 22 degrees C. These physicochemical data indicate that shedding of HIV-1 gp120 is not an integral step in the fusion cascade and that CD4 may inactivate the fusion complex in a process analogous to sCD4-induced shedding of gp120.

The surface of HIV-1, like that of other retroviruses, is studied with virally encoded glycoproteins which appear ultrastructurally as electron-dense spikes or knobs. The glycoprotein that forms the spike structure, gp120, is non-covalently bound to the transmembrane glycoprotein gp41. Mature HIV-1 virions do not have as many spikes as the genetically related retroviruses HIV-2 and SIV. gp120 is lost from HIV-1 during viral morphogenesis and after incubation of the virus with the soluble form of cellular receptor CD4. In this study we used ultrastructural cytochemistry and morphometry to quantitate the distribution of envelope glycoprotein spikes on budding and mature HIV-1 virions and to look for alternatives to the laborious and somewhat subjective spike-counting technique for envelope spike analysis on HIV-1. HIV-1, strain HTLV-IIIB, was examined after staining of envelope glycoproteins with either tannic acid, immunogold staining for gp120 (gp120-immunogold), or lectin-gold staining with concanavalin A for mannose residues (ConA-HRP-gold) and frequency distributions of spikes or gold particles per micron HIV-1 membrane generated. Envelope spikes were normally distributed on membranes of budding and mature HIV-1. However, the density of spikes per micron viral membrane on mature HIV-1 virions was approximately 50% of that observed on budding virions. ConA-HRP-gold and gp120-immunogold did not efficiently label budding virions. The shape of the frequency distribution for ConA-HRP-gold particles on mature virions was similar to that for envelope spikes and could be used to quantitate envelope glycoproteins on HIV-1. In addition, ConA-HRP-gold staining was able to detect the loss of envelope proteins after treatment of virus with soluble CD4. gp120-immunogold labeling was patchy and many virions were unlabeled. ConA-HRP-gold staining proved to be a rapid, reliable, and easily quantifiable method for estimation of envelope glycoprotein density on mature HIV-1. However, the loss of spike structures throughout the life cycle of HIV-1 can effectively be determined only by direct spike counting.

Synthetic peptide analog inhibitors of human immunodeficiency virus type 1 (HIV-1) protease were used to study the effects of inhibition of polyprotein processing on the assembly, structure, and infectivity of virions released from a T-cell line chronically infected with HIV-1. Inhibition of proteolytic processing of both Pr55gag and Pr160gag-pol was observed in purified virions from infected T cells after treatment. Protease inhibition was evident by the accumulation of precursors and processing intermediates of Pr55gag and by corresponding decreases in mature protein products. Electron microscopy revealed that the majority of the virion particles released from inhibitor-treated cells after a 24-h treatment had an immature or aberrant capsid morphology. This morphological change correlated with the inhibition of polyprotein processing and a loss of infectivity. The infectivity of virion particles purified from these chronically infected cell cultures was assessed following treatment with the inhibitor for 1 to 3 days. Virions purified from cultures treated with inhibitor for 1 or 2 days demonstrated a 95- to 100-fold reduction in virus titers, and treatment for 3 days resulted in complete loss of detectable infectivity. The fact that virions from treated cultures were unable to establish infection over the 7- to 10-day incubation period in the titration experiments strongly suggests that particles produced by inhibitor-treated cells were unable to reactivate to an infectious form when they were purified away from exogenous protease inhibitor. Thus, a block of HIV-1 protease processing of viral polyproteins by specific inhibitors results in a potent antiviral effect characterized by the production of noninfectious virions with altered protein structures and immature morphologies.

Human immunodeficiency virus (HIV) infects cells after binding of the viral envelope glycoprotein gp120 to the cell surface recognition marker CD4. gp120 is noncovalently associated with the HIV transmembrane envelope glycoprotein gp41, and this complex is believed responsible for the initial stages of HIV infection and cytopathic events in infected cells. Soluble constructs of CD4 that contain the gp120 binding site inhibit HIV infection in vitro. This is believed to occur by competitive inhibition of viral binding to cellular CD4. Here we suggest an alternative mechanism of viral inhibition by soluble CD4 proteins. We demonstrate biochemically and morphologically that following binding, the soluble CD4 proteins sT4, V1V2,DT, and V1[106] (amino acids 1-369, 1-183, and -2 to 106 of mature CD4) induced the release of gp120 from HIV-1 and HIV-1-infected cells. gp120 release was concentration-, time-, and temperature-dependent. The reaction was biphasic at 37 degrees C and did not take place at 4 degrees C, indicating that binding of soluble CD4 was not sufficient to release gp120. The appearance of free gp120 in the medium after incubation with sT4 correlated with a decrease in envelope glycoprotein spikes on virions and exposure of a previously cryptic epitope near the amino terminus of gp41 on virions and infected cells. The concentration of soluble CD4 proteins needed to induce the release of gp120 from virally infected cells also correlated with those required to inhibit HIV-mediated syncytium formation. These results suggest that soluble CD4 constructs may inactivate HIV by inducing the release of gp120. We propose that HIV envelope-mediated fusion is initiated following rearrangement and/or dissociation of gp120 from the gp120-gp41 complex upon binding to cellular CD4, thus exposing the fusion domain of gp41.