The present results have implications for the mechanism of SIV virus-mediated membrane fusion, and the relationship of structural changes in the CT to exposure of receptor binding sites on fusogenic or nonfusogenic forms of Env proteins. The data indicate that the sequence, the α-helical content, and the length of the CT domain have effects on the stability of the trimeric structure of the Env protein and play different roles in cell–cell fusion and infectivity. Enveloped viruses enter cells by fusion of the viral envelope with a cellular membrane, which is mediated by metastable fusion-mediating glycoproteins that use the energy released from irreversible protein refolding for the work of membrane fusion.9,35,46
The proteins then undergo a cascade of tightly regulated conformational changes, releasing the fusion proteins to a lower energy state. We found that fusion activation is suppressed by stabilizing the CT structure of Env proteins. An increase in the α-helical content in the CT domain was found to inhibit fusion activity. Previous results demonstrated that an elongated CT of a paramyxovirus F protein interferes with membrane fusion in a sequence-dependent manner.47
The glycoprotein 3HBaa (CT of 45 aa) with the lowest trimer stability exhibited the highest fusion activity among chimeric mutant Envs, and the glycoprotein 3HBii with the most stable trimeric structure exhibited the lowest level of fusion activity. It was previously shown29
that both full-length and truncated TM subunits of the SIVmac239 envelope protein form stable oligomers that are expressed on cell surfaces. The Env-T17 protein with a CT domain of 17 aa had optimal fusion activity and was able to form syncytia in an extended range of cells, whereas Envs with shorter (3 aa) or longer CT sequences demonstrated reduced fusion activity.29
In the present study, we observed that Env-T17 demonstrated stable TM trimer species at 95°C and exhibited similar cell–cell fusion activity compared to 3HBii in mouse fibroblast 3T3T4R5 cells.
We did not observe a correlation between the level of incorporation of Env proteins, fusion activity, and infectivity. Previous studies indicated that the level of incorporation into virions of the Env protein with a full-length cytoplasmic domain was lower than that of Env with truncated CT into SIV or HIV virions.48
It was also shown that sequence changes in the SU subunit and truncation of gp41 can significantly increase the level of Env incorporation into SIV virions, but SIV with truncated Env exhibits a defect in infectivity.11,31,32
Despite the apparent importance of fusion activity for early steps of infection, we observed that cytoplasmic domain modifications had differential effects on fusion activity and infectivity, and these effects were dependent on the content and length of the CT. Several mutant Env proteins were incorporated very effectively into pseudotyped virions, which exhibited high specific infectivity. The 3HBii Env with a more stable trimeric structure exhibited low infectivity and fusion activity. Pseudotyped virions with Env-T45 were more infectious than pseudotyped virions with the 3HBai or 3HBaa Env but less fusogenic than 3HBaa. Previously, we observed that replication of SIV with full-length Env was cell type dependent and occurred only in monkey cell lines; in contrast, SIV with truncated Env produced infectious particles in all types of monkey and human cell lines tested.11
Here, we found that transfected human epithelial 293T cells produced lower levels of infectious virions pseudotyped with full-length or 3HBii Env than with other truncated Env mutants.
Several studies indicate that known coreceptors are not absolutely required for CD4 binding.49–51
It is possible that HIV-1 strains become CD4 dependent as a mechanism to shield the conserved coreceptor binding site from neutralizing antibody.52
We observed that mutant 3HBai Env and 3HBii with three or seven isoleucine residues in GCN4-related sequences added to truncated cytoplasmic tails showed reduced binding activity to CD4 molecules as well as differences in fusion activity and infectivity. 3HBaa with intermediate fusion activity and infectivity, and Env-T45 with the highest infectivity, exhibited the highest CD4 binding activity. We also studied interactions with CCR5 by using a blocking assay with a CCR5-specific (2D7) monoclonal antibody. 2D7 may inhibit HIV-1 binding by steric hindrance rather than by direct interruption of the binding site.42
These results may indicate that the Env proteins that escape the antibody blocking effect could have higher affinity for CCR5.
Our studies have focused on effects of the cytoplasmic domain on Env structure, trimer stability, and their relation to fusion activity and infectivity. The gp41 ectodomain undergoes a series of conformational changes to form a fusion-active state during virus–cell membrane fusion.53,54
The differences in reactivity of Envs with CD4 and CCR5 may reflect changes in gp41 ectodomain conformations due to modifications of the CT domain of the prefusogenic forms. We observed that defined modifications of the CT modulate infectivity and cause changes in the stability of the trimeric structure that promote fusion activity. The addition of helical CT structures stabilizes Env oligomers and results in changes in Env incorporation, infectivity, and cell fusion activity. Recent work has shown differences between the mechanisms of cell-to-cell fusion and virus entry.55
Cell–cell fusion occurs at the cell surface and the role of cortical actin is consistent with this event.56
Infection, however, occurs after HIV enters cells via endosomes and excludes cell surface proteins from this process.55
Properties of incoming virus particles including the level of incorporation of Env molecules, their association with lipid rafts, or association with actin filaments may be important for this process.