A Virus-Encoded Cell–Cell Fusion Machine Dependent on Surrogate Adhesins

The reovirus fusion-associated small transmembrane (FAST) proteins function as virus-encoded cellular fusogens, mediating efficient cell–cell rather than virus–cell membrane fusion. With ectodomains of only ∼20–40 residues, it is unclear how such diminutive viral fusion proteins mediate the initial stages (i.e. membrane contact and close membrane apposition) of the fusion reaction that precede actual membrane merger. We now show that the FAST proteins lack specific receptor-binding activity, and in their natural biological context of promoting cell–cell fusion, rely on cadherins to promote close membrane apposition. The FAST proteins, however, are not specifically reliant on cadherin engagement to mediate membrane apposition as indicated by their ability to efficiently utilize other adhesins in the fusion reaction. Results further indicate that surrogate adhesion proteins that bridge membranes as close as 13 nm apart enhance FAST protein-induced cell–cell fusion, but active actin remodelling is required for maximal fusion activity. The FAST proteins are the first example of membrane fusion proteins that have specifically evolved to function as opportunistic fusogens, designed to exploit and convert naturally occurring adhesion sites into fusion sites. The capacity of surrogate, non-cognate adhesins and active actin remodelling to enhance the cell–cell fusion activity of the FAST proteins are features perfectly suited to the structural and functional evolution of these fusogens as the minimal fusion component of a virus-encoded cellular fusion machine. These results also provide a basis for reconciling the rudimentary structure of the FAST proteins with their capacity to fuse cellular membranes

HAP2(GCS1)-Dependent Gamete Fusion Requires a Positively Charged Carboxy-Terminal Domain

HAP2(GCS1) is a deeply conserved sperm protein that is essential for gamete fusion. Here we use complementation assays to define major functional regions of the Arabidopsis thaliana ortholog using HAP2(GCS1) variants with modifications to regions amino(N) and carboxy(C) to its single transmembrane domain. These quantitative in vivo complementation studies show that the N-terminal region tolerates exchange with a closely related sequence, but not with a more distantly related plant sequence. In contrast, a distantly related C-terminus is functional in Arabidopsis, indicating that the primary sequence of the C-terminus is not critical. However, mutations that neutralized the charge of the C-terminus impair HAP2(GCS1)-dependent gamete fusion. Our results provide data identifying the essential functional features of this highly conserved sperm fusion protein. They suggest that the N-terminus functions by interacting with female gamete-expressed proteins and that the positively charged C-terminus may function through electrostatic interactions with the sperm plasma membrane

Liposome reconstitution of a minimal protein-mediated membrane fusion machine

Biological membrane fusion is dependent on protein catalysts to mediate localized restructuring of lipid bilayers. A central theme in current models of protein-mediated membrane fusion involves the sequential refolding of complex homomeric or heteromeric protein fusion machines. The structural features of a new family of fusion-associated small transmembrane (FAST) proteins appear incompatible with existing models of membrane fusion protein function. While the FAST proteins function to induce efficient cell–cell fusion when expressed in transfected cells, it was unclear whether they function on their own to mediate membrane fusion or are dependent on cellular protein cofactors. Using proteoliposomes containing the purified p14 FAST protein of reptilian reovirus, we now show via liposome–cell and liposome–liposome fusion assays that p14 is both necessary and sufficient for membrane fusion. Stoichiometric and kinetic analyses suggest that the relative efficiency of p14-mediated membrane fusion rivals that of the more complex cellular and viral fusion proteins, making the FAST proteins the simplest known membrane fusion machines

The P2 capsid protein of the nonenveloped rice dwarf phytoreovirus induces membrane fusion in insect host cells

Insect transmission is an essential process of infection for numerous plant and animal viruses. How an insect-transmissible plant virus enters an insect cell to initiate the infection cycle is poorly understood, especially for nonenveloped plant and animal viruses. The capsid protein P2 of rice dwarf virus (RDV), which is nonenveloped, is necessary for insect transmission. Here, we present evidence that P2 shares structural features with membrane-fusogenic proteins encoded by enveloped animal viruses. When RDV P2 was ectopically expressed and displayed on the surface of insect Spodoptera frugiperda cells, it induced membrane fusion characterized by syncytium formation at low pH. Mutational analyses identified the N-terminal and a heptad repeat as being critical for the membrane fusion-inducing activity. These results are corroborated with results from RDV-infected cells of the insect vector leafhopper. We propose that the RDV P2-induced membrane fusion plays a critical role in viral entry into insect cells. Our report that a plant viral protein can induce membrane fusion has broad significance in studying the mechanisms of virus entry into insect cells and insect transmission of nonenveloped plant and animal viruses

Avian Reovirus Major μ-Class Outer Capsid Protein Influences Efficiency of Productive Macrophage Infection in a Virus Strain-Specific Manner

We determined that the highly pathogenic avian reovirus strain 176 (ARV-176) possesses an enhanced ability to establish productive infections in HD-11 avian macrophages compared to avian fibroblasts. Conversely, the weakly pathogenic strain ARV-138 shows no such macrophagotropic tendency. The macrophage infection capability of the two viruses did not reflect differences in the ability to either induce or inhibit nitric oxide production. Moderate increases in the ARV-138 multiplicity of infection resulted in a concomitant increase in macrophage infection, and under such conditions the kinetics and extent of the ARV-138 replication cycle were equivalent to those of the highly infectious ARV-176 strain. These results indicated that both viruses are apparently equally capable of replicating in an infected macrophage, but they differ in the ability to establish productive infections in these cells. Using a genetic reassortant approach, we determined that the macrophagotropic property of ARV-176 reflects a post-receptor-binding step in the virus replication cycle and that the ARV-176 M2 genome segment is required for efficient infection of HD-11 cells. The M2 genome segment encodes the major μ-class outer capsid protein (μB) of the virus, which is involved in virus entry and transcriptase activation, suggesting that a host-specific influence on ARV entry and/or uncoating may affect the likelihood of the virus establishing a productive infection in a macrophage cell