We are studying the early steps in retroviral infection, particularly the fusion of viral and cell membranes mediated by viral envelope (Env) proteins and cellular receptors. Some studies focused on a normal cellular protein reported to interact with Env; other studies investigated why some antibodies that bind to Env neutralize virus, while others do not.? ? In one set of experiments we investigated the role of a cellular enzyme, protein disulfide isomerase (PDI), reported to catalyze disulfide bond rearrangements in HIV envelope protein during membrane fusion. We found that antibodies to PDI and alterations in PDI expression had little effect on cell fusion or infection mediated by HIV Env (see Ou, W., and Silver, J.: Role of protein disulfide isomerase and other thiol-reactive proteins in HIV-1 envelope protein-mediated fusion. Virol. 350: 406-417, 2006). In these experiments we over-expressed wild-type or catalytically inactive mutant PDI, intracellularly and on the cell surface, or we inhibited the synthesis of PDI by using small inhibitory RNAs (siRNA) in transiently and stably transfected cells. Our down-regulation of PDI was biologically significant in that it inhibited infection with a polyoma virus (shown in collaboration with a group at Harvard Medical School, see Gilbert, J., Ou, W., Silver, J., and Benjamin, T.: Downregulation of Protein Disulfide Isomerase Inhibits Infection by the Mouse Polyoma Virus. J Virol., In press, 2006). Nevertheless, the down-regulation had no detectable effect on fusion mediated by HIV. In contrast to the lack of effect of alterations in PDI on HIV infection, a drug (DTNB) that reacts with cell surface thiol groups blocked HIV Env-mediated cell fusion extensively. These results suggest that cell surface thiol-containing molecules other than PDI are involved in HIV Env-mediated fusion.? ? In another set of experiments, we investigated the mechanism of antibody neutralization of HIV (see Ou, W., Lu, N., Yu, S., and Silver, J.: Effect of epitope position on neutralization by anti-human immunodeficiency virus monoclonal antibody 2F5. J. Virol. 80: 2539-2547, 2006). Antibodies that neutralize multiple strains of HIV are rare, so it is important to understand how these antibodies work and how they differ from non-neutralizing antibodies. Three of five epitopes for broadly neutralizing antibodies are located in the membrane-proximal region of the TM Env protein of HIV, which has led to the idea that epitope location, especially membrane proximity, may be crucially important to the ability of some antibodies to neutralize. One of the neutralizing antibodies (2F5) has an extended hydrophobic ?finger? that may interact with the membrane. To investigate the importance of epitope location, we transferred an epitope for the 2F5 antibody to the analogous position in the TM Env protein of a murine leukemia virus (MLV), or to a different location in the MLV SU Env protein. As controls, we inserted epitopes (HA and his6) for other antibodies at the same positions. The 2F5 antibody neutralized MLV carrying the 2F5 epitope in either location, whereas antibodies to HA and his6 did not block Env-mediated fusion, despite binding as well as 2F5 to Env on the cell surface or on virus particles. This suggests that the feature important for neutralization is not epitope location, but rather a structural characteristic of the neutralizing antibody. If the structural characteristic can be identified, it might enable the engineering of monoclonal antibodies to make them neutralizing.? ? We also studied the mechanism of antibody neutralization by investigating the ability of antibodies to neutralize chimeric Env proteins. It is known that certain mutations in SU or TM can inactivate the Env function, but when combined in different molecules can result in functional Env heterotrimers, a phenomenon called ?complementation?. We put such complementing mutations in SU or TM genes, along with HA or 2F5 epitopes, to investigate whether the 2F5 antibody could neutralize when its epitope was on a non-functional monomer. The experiments showed that the 2F5 antibody neutralized even when it could only bind to Env molecules bearing inactivating mutations, i.e. it could inactivate a heterotrimer by binding to non-functional monomers within it. This result supports a controversial proposal that a single antibody molecule is sufficient to inactivate an Env trimer.
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