Julie D. Turner

CD4-independent infection by HIV-2 is mediated by Fusin/CXCR4

Several members of the chemokine receptor family have been shown to function in association with CD4 to permit HIV-1 entry and infection. However, the mechanism by which these molecules serve as CD4-associated cofactors is unclear. In the present report, we show that one member of this family, termed Fusin/ CXCR4, is able to function as an alternative receptor for some isolates of HIV-2 in the absence of CD4. This conclusion is supported by the finding that (1) CD4-independent infection by these viruses is inhibited by an anti-Fusin monoclonal antibody, (2) Fusin expression renders human and nonhuman CD4-negative cell lines sensitive to HIV-2-induced syncytium induction and/or infection, and (3) Fusin is selectively down-regulated from the cell surface following HIV-2 infection. The finding that one chemokine receptor can function as a primary viral receptor strongly suggests that the HIV envelope glycoprotein contains a binding site for these proteins and that differences in the affinity and/or the availability of this site can extend the host range of these viruses to include a number of CD4-negative cell types.

CD4-independent utilization of the CXCR4 chemokine receptor by HIV-1 and HIV-2

HIV entry is mediated by an interaction between CD4 and members of the chemokine receptor family of proteins. It is likely that CD4 induces conformational changes in the viral envelope glycoproteins that facilitate a subsequent interaction with the chemokine receptor. To understand these events, variants of HIV-2 and HIV-1 have been derived that are able to interact directly with CXCR4 in the absence of CD4. One HIV-2 variant. termed HIV-2/vcp, has an expanded host range that includes CXCR4+/CD4- lymphoid and nonlymphoid cell lines. In contrast to T-tropic isolates of HIV-1, HIV-2/vcp was shown to induce > 95% downregulation of CXCR4 on chronically infected cells and was able to superinfect HIV-1-infected cells. A variant of HIV-1/IIIB termed HIV-1/IIIBx was also derived that is both replication competent and fusogenic for a CD4-negative subclone of SupT1 cells, termed BC7. Infection of BC7 cells by HIV-1/IIIBx was resistant to anti-CD4 monoclonal antibodies but inhibited by the anti-CXCR4 mAb, 12G5. HIV-1/IIIBx was highly fusogenic on 3T3 cells expressing CXCR4 in the absence of CD4. In contrast to HIV-2/vcp, the host range of HIV-1/IIIBx was highly restricted and replication in several CD4+/CXCR4+ lymphoid cell lines was reduced compared to HIV-1/IIIB. In addition, HIV-1/IIIBx failed to downregulate CXCR4 on chronically infected cells. These studies indicate that HIV-1 and HIV-2 variants can be derived in vitro that utilize CXCR4 in the absence of CD4. Although the mechanism(s) for these changes remain unclear, possibilities include an increased avidity of the viral envelope glycoprotein for CXCR4 and/or the increased exposure of the chemokine receptor binding site. Further biochemical and molecular analysis of the envelope glycoproteins from these viruses should be helpful in addressing these and other possibilities.

Efficient Autoproteolytic Processing of the MHV-A59 3C-like Proteinase from the Flanking Hydrophobic Domains Requires Membranes

Abstract The replicase gene of the coronavirus MHV-A59 encodes a serine-like proteinase similar to the 3C proteinases of picornaviruses. This proteinase domain is flanked on both sides by hydrophobic, potentially membrane-spanning, regions. Cell-free expression of a plasmid encoding only the 3C-like proteinase (3CLpro) resulted in the synthesis of a 29-kDa protein that was specifically recognized by an antibody directed against the carboxy-terminal region of the proteinase. A protein of identical mobility was detected in MHV-A59-infected cell lysates.In vitroexpression of a plasmid encoding the 3CLpro and portions of the two flanking hydrophobic regions resulted in inefficient processing of the 29-kDa protein. However, the efficiency of this processing event was enhanced by the addition of canine pancreatic microsomes to the translation reaction, or removal of one of the flanking hydrophobic domains. Proteolysis was inhibited in the presence ofN-ethylmaleimide (NEM) or by mutagenesis of the catalytic cysteine residue of the proteinase, indicating that the 3CLpro is responsible for its autoproteolytic cleavage from the flanking domains. Microsomal membranes were unable to enhance thetransprocessing of a precursor containing the inactive proteinase domain and both hydrophobic regions by a recombinant 3CLpro expressed fromEscherichia coli.Membrane association assays demonstrated that the 29-kDa 3CLpro was present in the soluble fraction of the reticulocyte lysates, while polypeptides containing the hydrophobic domains associated with the membrane pellets. With the help of a viral epitope tag, we identified a 22-kDa membrane-associated polypeptide as the proteolytic product containing the amino-terminal hydrophobic domain.

Small-Molecule Intramimics of Formin Autoinhibition: A New Strategy to Target the Cytoskeletal Remodeling Machinery in Cancer Cells

Although the cancer cell cytoskeleton is a clinically validated target, few new strategies have emerged for selectively targeting cell division by modulating the cytoskeletal structure, particularly ways that could avoid the cardiotoxic and neurotoxic effects of current agents such as taxanes. We address this gap by describing a novel class of small-molecule agonists of the mammalian Diaphanous (mDia)-related formins, which act downstream of Rho GTPases to assemble actin filaments, and their organization with microfilaments to establish and maintain cell polarity during migration and asymmetric division. GTP-bound Rho activates mDia family members by disrupting the interaction between the DID and DAD autoregulatory domains, which releases the FH2 domain to modulate actin and microtubule dynamics. In screening for DID-DAD disruptors that activate mDia, we identified two molecules called intramimics (IMM-01 and -02) that were sufficient to trigger actin assembly and microtubule stabilization, serum response factor-mediated gene expression, cell-cycle arrest, and apoptosis. In vivo analysis of IMM-01 and -02 established their ability to slow tumor growth in a mouse xenograft model of colon cancer. Taken together, our work establishes the use of intramimics and mDia-related formins as a new general strategy for therapeutic targeting of the cytoskeletal remodeling machinery of cancer cells.