Cell Biology of Virus Entry

(Env)-mediated cross-linking of their cell surface reDimiter S. Dimitrov* Laboratory of Experimental and Computational ceptors. Cholesterol depletion by methyl-b-cyclodextrin, which Biology NCI-FCRDC presumably disrupts rafts, inhibits HIV-1 infection and syncytia formation (J. Hildreth, Johns Hopkins Univ., National Institutes of Health Building 469, Room 104 Baltimore, R. Blumenthal). A limited number of specific GSL including Gb3 and GM3 promote entry of a broad Post Office Box B Miller Drive range of HIV-1 isolates (R. Blumenthal) (Hug et al., 2000). The role of rafts appears to be specific, as GSL are Frederick, Maryland 21702 required in the target but not in the Env-expressing cells for fusion. Inhibitors of GSL biosynthesis affected Envmediated fusion and infection, and this was rescued by Recent advances in our understanding of the molecular the addition of certain purified GSL. The GSL-depleted organization of cell membranes, the atomic structure cells lost the ability to induce conformational changes of viral proteins, and their interactions with receptor in Env-expressing cells but retained the property of molecules have provided novel insights into the mechaallowing complexes between CD4, coreceptors (CCR5 nisms of virus entry and raised hopes for better control and CXCR4), and gp120 of the Env to form. Thus, secby new inhibitors, vaccines, and specific delivery tools. ondary interactions between portions of gp120 and GSL These developments were discussed in a lively fashion could be required for the conformational changes in Env at the second Frederick meeting on the Cell Biology of leading to assembly of the fusion complex. GSL might Viral Entry (May 7–10, Frederick, Maryland) organized have a dual function—in addition to their role as raft by R. Blumenthal (NCI-FCRDC, Frederick) and E. Hunter components they could also specifically interact with (Univ. of Alabama, Birmingham). the Env. The role of rafts in this case could be to increase the local concentrations and arrangement of the fusion Membrane Structure: Role of Rafts in Virus Entry? complex components. The receptor for ecotropic muSphingolipids and/or glycosphingolipids (GSL) plus rine leukemia virus, CAT1, could be a “raft” protein (X. cholesterol can associate laterally to form membrane Lu and J. Silver, NIAID, Bethesda). Rafts may be also microdomains termed rafts. Such lipid rafts could assoinvolved in the fusion of Semliki Forest virus (SFV), which ciate with specific proteins while excluding others and requires the presence of cholesterol and sphingolipid may be involved in numerous cellular functions, includin the target membrane. Three regions in the SFV E1 ing membrane traffic, cell morphogenesis, and signaling subunit, all outside the fusion peptide, are involved in (Simons and Ikonen, 1997). M. Edidin (Johns Hopkins the control of cholesterol dependence (M. Kielian, Albert Univ., Baltimore) provided an overview of the field of Einstein College of Medicine, New York). Low pH trigmembrane domains and critically addressed the fundagered the E1 ectodomain association with target memmental issue of the existence and properties of rafts. branes via insertion of its fusion peptide and led to a Operational definitions of lipid domains include differenstrong E1 association with rafts, while the fusion peptide tials in lateral diffusion between related lipid analogs of influenza was not in a raft domain. and differential detergent solubility. Lipid domains are HIV-1 is a raft virus (J. Hildreth). It contains GPIsmall, mobile, unstable, and probably fluctuate in size anchored proteins (CD59 and Thy-1) and incorporates and composition. They can be disrupted by vesicle trafthe ganglioside GM1, which partitions preferentially into fic, while in turn the traffic can create dynamic yet persislipid rafts (Nguyen and Hildreth, 2000). The association tent patches of membrane proteins and lipids that are of assembling HIV-1 with rafts appears to be dependent not specific domains. Transmembrane signaling that is on the N-terminal region of Gag (A. Ono and E. Freed, initiated by immunoreceptors such as T cell receptors NIAID, Bethesda). The role of virus membrane rafts for and Fc receptors for immunoglobulins (Ig) appears to entry remains unclear. HIV-1 produced by cholesterolinvolve rafts (B. Baird, Cornell Univ., Ithaca). The IgE depleted cells was not infectious (J. Hildreth). The extent receptor is phosphorylated by Lyn tyrosine kinase only of membrane fusion activity of influenza hemagglutinin after antigen-mediated aggregation causes the recep(HA) was related to the extent of its association with tors to coalesce into rafts that contain the active kinase rafts (M. Kumar and J. Zimmerberg, NICHD, Bethesda). (and possibly exclude phosphatases). Underscoring the Other experiments, however, suggested that HA associdynamic nature of raft interactions is their regulation by ation with rafts was not needed for fusion (J. White, stimulated F-actin, which appears to cause separation Univ. of Virginia, Charlottesville). Thus, some viruses of aggregated IgE receptors and raft components, incould contain rafts in their membranes and may or may cluding Lyn, later in the sequence of cell signaling events not require rafts in the target membrane for efficient (Holowka et al., 2000). Studies similar to these would entry. Some participants felt that more convincing eviallow potential raft mechanisms to be tested for stimudence for the importance of rafts in virus entry is needed, lated viral entry that involves envelope glycoprotein but rafts were considered by many attendees as a fruitful