The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ), a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.

The alphaviruses and flaviviruses include many important human pathogens, such as the dengue, West Nile, and Chikungunya viruses. These enveloped viruses infect cells by a membrane fusion reaction triggered by the low pH in endosomes. Fusion is mediated by viral membrane proteins through their acid-dependent conversion from a dimer on the virus surface to a homotrimer inserted in the host cell membrane. Here we review recent studies on the regulatory mechanisms that silence these fusion proteins during virus exit, and that sense low pH and mediate protein refolding during virus entry. We discuss results using truncated proteins to dissect the fusion reaction, and future research directions including the development of antiviral therapies against these medically important viruses.

The alphavirus Semliki Forest virus (SFV) uses a membrane fusion reaction to infect host cells. Fusion of the virus and cell membranes is triggered by low pH in the endosome and is mediated by the viral membrane protein E1. During fusion, E1 inserts into the target membrane, trimerizes, and refolds into a hairpin conformation. Formation of the E1 homotrimer is critical to membrane fusion, but the mechanism of trimerization is not understood. The crystal structure of the postfusion E1 trimer shows that an aspartate residue, D188, is positioned in the central core trimer interface. D188 is conserved in all reported alphavirus E1 sequences. We tested the contribution of this amino acid to trimerization and fusion by replacing D188 with alanine (D188A) or lysine (D188K) in an SFV infectious clone. These mutations were predicted to disrupt specific interactions at this position and/or change their pH dependence. Our results indicated that the D188K mutation blocked SFV fusion and infection. At low pH, D188K E1 inserted into target membranes but was trapped as a target membrane-inserted monomer that did not efficiently form the stable core trimer. In contrast, the D188A mutant was infectious, although trimerization and fusion required a lower pH. While there are extensive contacts between E1 subunits in the homotrimer, the D188K mutant identifies an important "hot spot" for protein-protein interactions within the core trimer.In an aqueous environment, phospholipid bilayers are stable structures that do not spontaneously fuse. Fusion is inhibited by high energy barriers that oppose both the initial mixing of the outer membrane leaflets and the subsequent merger of the inner leaflets that forms a stable fusion pore joining the two membrane compartments (18). Enveloped viruses have developed efficient strategies to promote fusion between the virus and cellular membranes to deliver the viral genome into the host cell (reviewed in references 19 and 40). Such virus-membrane fusion reactions are mediated by transmembrane fusion proteins in the virus envelope. These proteins rearrange from a metastable prefusion conformation to a stable target membrane-inserted postfusion conformation. The energy released by these conformational changes acts to induce local bending of the membranes, destabilizing the bilayers and lowering the activation energy for fusion (8,18). The refolding from the prefusion to the postfusion form is initiated by specific triggering mechanisms, such as receptor and/or coreceptor interactions or exposure to low pH in the endocytic pathway.Virus membrane fusion proteins have been grouped into several classes based on shared structural features (23, 24). The postfusion structures of class I fusion proteins, exemplified by the influenza virus hemagglutinin and human immunodeficiency virus type 1 gp41, contain a central ␣-helical coiled-coil domain. The class II fusion proteins, exemplified by alphavirus E1 and flavivirus E, contain primarily ␤-sheet structures. The class III proteins, such as vesicular stomati...

The enveloped alphaviruses infect cells via a low-pH-triggered membrane fusion reaction mediated by the viral transmembrane protein E1. During fusion, E1 inserts into the target membrane and refolds to a hairpin-like postfusion conformation in which domain III (DIII) and the juxtamembrane stem pack against a central core trimer. Although zinc has previously been shown to cause a striking block in alphavirus fusion with liposome target membranes, the mechanism of zinc's effect on the E1 fusion protein is not understood. Here we developed a cell culture system to study zinc inhibition of fusion and infection of the alphavirus Semliki Forest virus (SFV). Inclusion of 2 mM ZnCl 2 in the pH 5.75 fusion buffer caused a decrease of ϳ5 logs in SFV fusion at the plasma membrane. Fusion was also inhibited by nickel, a chemically related transition metal. Selection for SFV zinc resistance identified a key histidine residue, H333 on E1 DIII, while other conserved E1 histidine residues were not involved. An H333N mutation conferred resistance to both zinc and nickel, with properties in keeping with the known pH-dependent chelation of these metals by histidine. Biochemical studies demonstrated that zinc strongly inhibits formation of the postfusion E1 trimer in wild-type SFV but not in an H333 mutant. Together our results suggest that zinc acts by blocking the fold-back of DIII via its interaction with H333. E nveloped viruses infect cells by membrane fusion reactions that are triggered at the plasma membrane or in the endocytic pathway (reviewed in references 16, 28, and 43). Virus membrane fusion is mediated by conformational changes in specialized transmembrane proteins on the virus surface. During fusion, these viral proteins interact with the target cell membrane via a hydrophobic fusion peptide and refold to a hairpin-like conformation that brings the fusion peptide and transmembrane anchor into close proximity. Inhibition of these conformational changes blocks virus fusion and infection and is a potent antiviral strategy (11,29). In some cases, peptides or domains of the fusion protein can act as dominant negative inhibitors of the refolding reaction (10,20,23,36). In other cases, small molecules that block fusion protein conformational changes have been identified by in silico studies or screens for inhibitors (6,12,35). Definition of the detailed molecular mechanism of inhibition is critical to improve such inhibitors and to develop them into usable antiviral therapies.Alphaviruses are small enveloped plus-sense RNA viruses that include important human pathogens such as Chikungunya virus and eastern equine encephalitis virus and the well-characterized Semliki Forest virus (SFV) (reviewed in reference 18). Alphaviruses infect cells by endocytic uptake and a membrane fusion reaction triggered by endosomal low pH (reviewed in reference 17). Fusion is mediated by the transmembrane E1 protein, an elongated molecule containing three domains (DI to DIII), arranged with the central DI connecting on one side to DII and on the...

Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells by a low-pH-triggered membrane fusion reaction mediated by the viral E1 protein. E1 inserts into target membranes and refolds to a hairpin-likehomotrimer containing a central core trimer and an outer layer composed of domain III and the juxtamembrane stem region. The key residues involved in mediating E1 trimerization are not well understood. We recently showed that aspartate 188 in the interface of the core trimer plays a critical role. Substitution with lysine (D188K) blocks formation of the core trimer and E1 trimerization and strongly inhibits virus fusion and infection. Here, we have isolated and characterized revertants that rescued the fusion and growth defects of D188K. These revertants included pseudorevertants containing acidic or polar neutral residues at E1 position 188 and a second-site revertant containing an E1 K176T mutation. Computational analysis using multiconformation continuum electrostatics revealed an important interaction bridging D188 of one chain with K176 of the adjacent chain in the core trimer. E1 K176 is completely conserved among the alphaviruses, and mutations of K176 to threonine (K176T) or isoleucine (K176I) produced similar fusion phenotypes as D188 mutants. Together, our data support a model in which a ring of three salt bridges formed by D188 and K176 stabilize the core trimer, a key intermediate of the alphavirus fusion protein.Enveloped viruses contain a phospholipid bilayer that surrounds and protects the viral genome until fusion of the virus and host membranes delivers the genome into the cytoplasm. Fusion is mediated by transmembrane fusion proteins in the virus envelope. Viruses have evolved specific mechanisms to trigger membrane fusion upon interaction with the host cell (15, 42). For example, the fusion protein of the human immunodeficiency virus is triggered by receptor and coreceptor binding, while alphaviruses such as Semliki Forest virus (SFV) and flaviviruses such as dengue virus are triggered by exposure to acidic pH. The fusion trigger initiates the conversion of the fusion protein from the metastable prefusion state to the more energetically stable postfusion state (14, 15). The energy released during the refolding of the membrane fusion protein drives the merger of the viral and host membranes.Alphaviruses take advantage of the low-pH environment of the endocytic pathway to trigger membrane fusion during entry (37). E1 is the fusion protein and forms heterodimers with the E2 protein on the virus surface. These heterodimers are organized into trimers (E2/E1) 3 to form the icosahedral glycoprotein shell (21,30,43). Alphaviruses bind to cell surface receptors and are internalized by clathrin-mediated endocytosis and delivered to endosomes (16). Here, low pH induces E1/E2 heterodimer dissociation, E1 insertion into endosomal membranes, and the refolding of E1 to the final postfusion homotrimer conformation (16, 37). The resultant membrane fusion releases the viral RNA genome into the cytop...