We are studying the interaction between retroviral envelope and receptor proteins, which mediate viral entry. Some studies involve mutating the receptor, or the envelope, to see what portions of these proteins are involved in various steps in the fusion process. Other approaches include modifying the surface charge or lipid composition of the membranes involved in fusion. In one set of experiments we engineered cells to express wild-type HIV or murine leukemia virus (MLV) envelope proteins along with a portion of these proteins that is known to form a homo-trimer (the N-heptad repeat region). The heptad repeat regions were linked to green fluorescent protein to allow visualization and antibody detection. The heptad repeat chimeric proteins formed mixed heterotrimers with wild-type envelope proteins. Unexpectedly, we found that this very efficiently blocked cleavage of the wild-type envelope precursor protein into the mature form of envelope protein required for membrane fusion. Since the envelope proteins of many viruses in addition to retroviruses (e.g. influenza, SARS) go through similar steps of trimerization and cleavage, this work suggests a potentially general mechanism to inhibit formation of infectious virus via molecules that interact with envelope proteins during their synthesis. In another set of experiments, we investigated the role of a cellular enzyme, protein disulfide isomerase (PDI), reportedly required on the cell surface for disulfide bond rearrangements in HIV envelope protein postulated to occur during membrane fusion. We found that over-expressing wild-type or mutant PDI, intracellularly or on the cell surface, or inhibiting the synthesis of PDI via small inhibitory RNAs (siRNA), had no effect on fusion mediated by HIV envelope in our experimental system, whereas inhibition of PDI with our constructs did reduce susceptibility to infection by polyoma virus in experiments performed by our collaborators at Harvard Medical School. PDI is believed to be required for disulfide bond rearrangements in the polyoma virus capsid protein during ?uncoating? of this virus in the endoplasmic reticulum. Thus, our experiments cast doubt on the value of PDI as a target for anti-HIV therapies. In another line of experiments, we investigated HIV-inhibitory effects of polyanions concentrated at the cell surface; these results are summarized in the Scientific Advances section of this report. Finally, we began studies of the role of the peri-membrane region of HIV and MLV envelope protein, i.e. the part of the TM envelope protein that lies within about 20 amino acids of the virus membrane. In different viruses this region is thought to be involved in forming a ?clamp? with an upstream region of envelope protein (N-heptad repeat) that brings virus and cell membranes together, or in destabilizing virus or cell membranes during membrane fusion. In HIV, this region is one of a few rare sites where antibodies that can neutralize virus bind. We exchanged peri-membrane regions from different viruses (MLV, HIV, VSV) and tested to see if the chimeric envelope proteins were functional, as might be expected if the peri-membrane region provides a ?generic? function such as ?membrane destabilization?, or non-functional, as might be expected if the peri-membrane region interacts with other portions of the envelope protein. We also made point mutations and substituted various antigenic epitopes for the HIV neutralizing epitope to investigate why antibodies binding to this region of HIV are neutralizing. These studies are described further under Future Plans.
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