Multifaceted Sequence-Dependent and -Independent Roles for Reovirus FAST Protein Cytoplasmic Tails in Fusion Pore Formation and Syncytiogenesis

Fusogenic reoviruses utilize the FAST proteins, a novel family of nonstructural viral membrane fusion proteins, to induce cell-cell fusion and syncytium formation. Unlike the paradigmatic enveloped virus fusion proteins, the FAST proteins position the majority of their mass within and internal to the membrane in which they reside, resulting in extended C-terminal cytoplasmic tails (CTs). Using tail truncations, we demonstrate that the last 8 residues of the 36-residue CT of the avian reovirus p10 FAST protein and the last 20 residues of the 68-residue CT of the reptilian reovirus p14 FAST protein enhance, but are not required for, pore expansion and syncytium formation. Further truncations indicate that the membrane-distal 12 residues of the p10 and 47 residues of the p14 CTs are essential for pore formation and that a residual tail of 21 to 24 residues that includes a conserved, membrane-proximal polybasic region present in all FAST proteins is insufficient to maintain FAST protein fusion activity. Unexpectedly, a reextension of the tail-truncated, nonfusogenic p10 and p14 constructs with scrambled versions of the deleted sequences restored pore formation and syncytiogenesis, while reextensions with heterologous sequences partially restored pore formation but failed to rescue syncytiogenesis. The membrane-distal regions of the FAST protein CTs therefore exert multiple effects on the membrane fusion reaction, serving in both sequence-dependent and sequence-independent manners as positive effectors of pore formation, pore expansion, and syncytiogenesis.The only examples of nonenveloped viruses that induce cell-cell fusion and syncytium formation occur within the family Orthoreoviridae, an extremely diverse group of viruses containing segmented double-stranded RNA genomes (9). In recent years, the viral proteins responsible for the syncytiogenic phenotype of the fusogenic orthoreoviruses and aquareoviruses have been identified and characterized (14, 18, 41, 46). These fusion-associated small transmembrane (FAST) proteins define a new family of viral fusogens with several unique biological and biophysical properties. Unlike the well-characterized enveloped virus fusion proteins, reovirus FAST proteins are nonstructural viral proteins and are therefore not involved in mediating virus-cell fusion and virus entry (18, 21, 46). The FAST proteins are instead dedicated to inducing cell-cell fusion and syncytium formation following their expression and trafficking to the plasma membrane of virus-infected or transfected cells (14, 17, 46). Data from previously reported studies also suggest that the FAST proteins serve as virulence factors for the fusogenic reoviruses, promoting virus dissemination and increased tissue destruction (6, 43). How this atypical family of viral fusogens functions to mediate cell-cell membrane fusion remains unclear.The unusual biological role of the FAST proteins as nonstructural, virus-encoded, “cellular” fusogens is embodied in structural features that clearly distinguish the FAST proteins from the membrane fusion proteins of enveloped viruses. There are currently four distinct members of the FAST protein family, named according to their molecular masses: the homologous p10 proteins of avian reovirus (ARV) and Nelson Bay reovirus and the unrelated p14, p15, and p22 proteins of reptilian reovirus (RRV), baboon reovirus, and Atlantic salmon aquareovirus, respectively (14, 18, 41, 46). These proteins are the smallest known fusogens, ranging from 95 to 198 amino acids in size, and assume an asymmetric topology in the plasma membrane, with a single transmembrane domain that separates small N-terminal ectodomains of ∼20 to 41 residues from equal-sized or considerably larger C-terminal endodomains of ∼36 to 141 residues (Fig. ​(Fig.1A).1A). A number of structural motifs in both the ecto- and endodomains of the FAST proteins have been identified, including sites of acylation, hydrophobic patches, a membrane-proximal polybasic region, and regions rich in proline, cysteine, or arginine, proline, and histidine. Each of the FAST proteins has its own signature repertoire and arrangement of these motifs. Determining how these various motifs contribute to the fusogenic activity of the FAST proteins remains an area of active investigation.Open in a separate windowFIG. 1.ARV p10 and RRV p14 FAST protein topologies and tail truncations. (A) Diagrammatic representation of the p10 and p14 FAST proteins showing their topology in the plasma membrane. Both are single-pass transmembrane proteins with N-terminal ectodomains on the surface of cells and C-terminal endodomains in the cytoplasm. Structural motifs include hydrophobic patches (HP), polybasic motifs (PB), fatty acid modifications (indicated by squiggly lines) that are either the N-terminal myristoylation or palmitoylation of a dicysteine motif (CC), and a polyproline motif (PP). The total number of residues in each protein is indicated by the numbers. (B) The amino acid sequences of the p10 and p14 endodomains are shown, along with the motifs described above. Progressive truncations of the CTs were constructed (arrows), with the numbers indicating the last amino acid present in the full-length proteins or each truncation.Numerous studies of diverse fusion processes define five general steps of the pathway for membrane fusion and syncytium formation: membrane binding, close membrane apposition, hemifusion (i.e., the mixing of the outer leaflets of the two bilayers), stable pore formation, and pore expansion (12, 13, 44). The well-characterized enveloped virus fusion proteins utilize extensive structural rearrangement of their complex ectodomains to provide mechanical energy to draw membranes into close proximity and promote membrane merger (21, 53). The limited size of the FAST protein ectodomains precludes such a mechanical model for membrane fusion, necessitating the development of alternate models to explain how the diminutive FAST proteins breach the thermodynamic barriers that prevent the spontaneous merger of biological membranes. The FAST proteins are both necessary and sufficient to mediate membrane fusion (51). However, data from recent studies indicate that for maximal cell fusion activity, the FAST proteins rely on surrogate adhesins to mediate close membrane apposition (42). Data from recent studies also indicate that a small percentage of the p14 FAST protein expressed in virus-infected or transfected cells is proteolytically processed to generate a bioactive, soluble endodomain that recruits cellular pathways to drive the expansion of stable fusion pores into the extended fusion apertures needed for syncytium formation (50). The FAST proteins therefore utilize accessory proteins to mediate the prefusion (membrane binding and apposition) and postfusion (pore expansion) stages of syncytiogenesis, retaining within their rudimentary structures all that is required to mediate the actual process of membrane merger. This subdivision of the multistep process of syncytium formation is reflected in, and is perfectly suited to, the evolution of the FAST proteins as virus-encoded cellular fusogens.The small size of the FAST protein ectodomains and their donor membrane-focused topology contrast markedly with enveloped virus fusion proteins that position the majority of their mass external to the membrane. While the complex ectodomains of the enveloped virus fusion proteins clearly play an essential role in the fusion reaction, the involvement of their cytoplasmic tails (CTs) is far less certain, and no consistent picture of the role of these C-terminal tails has emerged. The CTs of many enveloped viral fusion proteins, including baculovirus (31), severe acute respiratory syndrome coronavirus (5), vesicular stomatitis virus (36), parainfluenza virus type 2 (56), and influenza A virus subtype H3 (10), play no role in the membrane fusion reaction. Of the fusion protein tails that do modulate the fusion reaction, the majority serve inhibitory roles, including the F proteins of measles virus and parainfluenza virus type 5 SER (7, 45, 52), glycoprotein B from several herpesviruses (22, 24, 28), and the fusion proteins of numerous retroviruses (1, 8, 30, 32, 34, 47, 48). These inhibitory cytoplasmic domains alter the conformation of the fusion protein ectodomains, thereby coupling virion maturation to fusion competence (1, 2, 35, 52, 54). In the few cases where extensive tail truncations adversely affect fusion, these truncations generally decrease but do not eliminate syncytiogenesis, and it is the membrane-proximal portion of the tail that promotes pore formation or pore expansion (20, 25, 26, 32).Since the FAST proteins are nonstructural viral proteins, their CTs (also referred to as endodomains) are not required to suppress fusion activity until after virus particle assembly. At the same time, the disproportionate size of their endodomains strongly suggests that these CTs play an important role in membrane fusion activity. Although one such role of the p14 CT is the generation of a soluble endodomain that recruits cellular factors involved in pore expansion, the majority of p14 is not proteolytically processed, suggesting that FAST protein CTs may serve additional roles as components of the intact protein (50). We now show that C-terminal truncations of the p10 and p14 FAST proteins reduced and eventually eliminated cell-cell fusion. Fluorescence-based pore formation assays coupled with tail reextension studies further revealed that FAST protein CTs drive fusion pore formation and expansion in both sequence-dependent and sequence-independent manners. The membrane-distal regions of FAST protein CTs therefore exert multiple effects on the mechanism of membrane fusion.