We demonstrate that the S4 genome segment of baboon reovirus (BRV) contains two sequential partially overlapping open reading frames (ORFs), both of which are functional in vitro and in virus-infected cells. The 15-kDa gene product (p15) of the 5-proximal ORF induces efficient cell-cell fusion when expressed by itself in transfected cells, suggesting that p15 is the only viral protein required for induction of syncytium formation by BRV. The p15 protein is a small, hydrophobic, basic, integral membrane protein, properties shared with the p10 fusion-associated small transmembrane (FAST) proteins encoded by avian reovirus and Nelson Bay reovirus. As with p10, the BRV p15 protein is also a nonstructural protein and, therefore, is not involved in virus entry. Sequence analysis indicates that p15 shares no significant sequence similarity with the p10 FAST proteins and contains a unique repertoire and arrangement of sequence-predicted structural and functional motifs. These motifs include a functional N-terminal myristylation consensus sequence, an N-proximal prolinerich motif, two potential transmembrane domains, and an intervening polybasic region. The unique structural properties of p15 suggest that this protein is a novel member of the new family of FAST proteins.Membrane fusion is an essential cellular event, the precise mechanism of which remains poorly understood. Much of our understanding of membrane fusion is derived from studies of enveloped virus fusion proteins. Structure-function analysis of numerous enveloped virus fusion proteins has led to a generalized model of protein-mediated membrane fusion. This model suggests that triggered conformational changes in the metastable viral fusion proteins expose a previously concealed hydrophobic fusion peptide for insertion into, and destabilization of, the target lipid bilayer (16,43,50,51). Further extensive conformational changes are predicted to be involved in supplying the energy required to pull the donor and target bilayers together and allow the fusion reaction to proceed 1 (6, 50, 51). Based on the nature of the structural rearrangements, the enveloped virus fusion proteins fall into two general classes. Class I proteins, such as the influenza virus hemagglutinin, human immunodeficiency virus gp41, and Ebola virus GP2, use coiled-coil rearrangements to drive the fusion process while class II proteins, such as those of the alphaviruses, flaviviruses, and rhabdoviruses, undergo extensive multimer rearrangements in the absence of apparent coiled-coil interactions (19,34,42,43,44).Our recent characterization of a fusion-associated small transmembrane (FAST) protein encoded by certain fusogenic reoviruses (39) suggests that extensive conformational changes in large, complex, multimeric viral fusion proteins may not be a universal requirement for protein-mediated membrane fusion. The only examples of nonenveloped viruses that induce syncytium formation are all members of the family Reoviridae, a diverse group of nonenveloped viruses with double-stranded RNA geno...

Previous studies of the avian reovirus strain S1133 (ARV-S1133) S1 genome segment revealed that the open reading frame (ORF) encoding the C viral cell attachment protein initiates over 600 nucleotides distal from the 5 end of the S1 mRNA and is preceded by two predicted small nonoverlapping ORFs. To more clearly define the translational properties of this unusual polycistronic RNA, we pursued a comparative analysis of the S1 genome segment of the related Nelson Bay reovirus (NBV). Sequence analysis indicated that the 3-proximal ORF present on the NBV S1 genome segment also encodes a C homolog, as evidenced by the presence of an extended N-terminal heptad repeat characteristic of the coiled-coil region common to the cell attachment proteins of reoviruses. Most importantly, the NBV S1 genome segment contains two conserved ORFs upstream of the C coding region that are extended relative to the predicted ORFs of ARV-S1133 and are arranged in a sequential, partially overlapping fashion. Sequence analysis of the S1 genome segments of two additional strains of ARV indicated a similar overlapping tricistronic gene arrangement as predicted for the NBV S1 genome segment. Expression analysis of the ARV S1 genome segment indicated that all three ORFs are functional in vitro and in virus-infected cells. In addition to the previously described p10 and C gene products, the S1 genome segment encodes from the central ORF a 17-kDa basic protein (p17) of no known function. Optimizing the translation start site of the ARV p10 ORF lead to an approximately 15-fold increase in p10 expression with little or no effect on translation of the downstream C ORF. These results suggest that translation initiation complexes can bypass over 600 nucleotides and two functional overlapping upstream ORFs in order to access the distal C start site.The orthoreoviruses consist of a number of diverse virus species, all of which have a similar capsid structure and a genome composed of 10 double-stranded RNA (dsRNA) segments (38). For the most part, the genome segments of reoviruses are monocistronic, each encoding a single unique polypeptide. The mammalian reovirus (MRV) S1 genome segment, however, is bicistronic and encodes two unrelated polypeptides from overlapping open reading frames (ORFs). The 1 viral cell attachment protein is encoded by a large ORF that originates near the 5Ј end of the mRNA and spans almost the complete length of the S1 mRNA (12, 37). A second small ORF, beginning at nucleotide position 71, is nested within the 1 ORF in a ϩ1 reading frame and encodes the small nonstructural 1NS protein of MRV (14,23).Since the translation start site for 1 is in a nonpreferred context (a pyrimidine at position Ϫ3), leaky scanning by the preinitiation complex most likely accounts for translation of the downstream 1NS ORF from the MRV S1 mRNA (28). In addition to leaky scanning, there is evidence suggesting that translation of the 1 protein from the bicistronic S1 mRNA is modulated by ribosomal pausing at the downstream 1NS start site (7, 15).As wit...

Select members of the Reoviridae are the only nonenveloped viruses known to induce syncytium formation. The fusogenic orthoreoviruses accomplish cell-cell fusion through a distinct class of membrane fusioninducing proteins referred to as the fusion-associated small transmembrane (FAST) proteins. The p15 membrane fusion protein of baboon reovirus is unique among the FAST proteins in that it contains two hydrophobic regions (H1 and H2) recognized as potential transmembrane (TM) domains, suggesting a polytopic topology. However, detailed topological analysis of p15 indicated only the H1 domain is membrane spanning. In the absence of an N-terminal signal peptide, the H1 TM domain serves as a reverse signal-anchor to direct p15 membrane insertion and a bitopic N exoplasmic /C cytoplasmic topology. This topology results in the translocation of the smallest ectodomain (ϳ20 residues) of any known viral fusion protein, with the majority of p15 positioned on the cytosolic side of the membrane. Mutagenic analysis indicated the unusual presence of an N-terminal myristic acid on the small p15 ectodomain is essential to the fusion process. Furthermore, the only other hydrophobic region (H2) present in p15, aside from the TM domain, is located within the endodomain. Consequently, the p15 ectodomain is devoid of a fusion peptide motif, a hallmark feature of membrane fusion proteins. The exceedingly small, myristoylated ectodomain and the unusual topological distribution of structural motifs in this nonenveloped virus membrane fusion protein necessitate alternate models of proteinmediated membrane fusion.The baboon reovirus (BRV) p15 protein is a novel member of the recently described fusion-associated small transmembrane (FAST) protein family (9,11,45). The FAST proteins are unusual membrane fusion proteins encoded by the fusogenic subgroup of orthoreoviruses, one of the few examples of nonenveloped viruses that induce cell-cell fusion and syncytium formation (15,18). At 10 to 15 kDa, the reovirus FAST proteins are the smallest known viral membrane fusion proteins and are unlikely to undergo the types of extensive structural rearrangements required for enveloped virus fusion protein activity (27,48). The FAST proteins are also the only examples of nonstructural viral proteins that induce membrane fusion (11, 45). As a result of their nonstructural nature, the FAST proteins play no role in reovirus entry. Their sole purpose appears to reflect enhanced dissemination of the infection via syncytium formation, following FAST protein expression in reovirus-infected cells (16,17). The unusual structural features of the FAST proteins and their unique role in the virus replication cycle suggest the mechanism of FAST-mediated membrane fusion is unlikely to adhere to the existing paradigm, which is derived from studies of the enveloped virus fusion proteins (5,48,52).In addition to BRV p15, FAST proteins have been recently characterized from avian reovirus (ARV), Nelson Bay reovirus (NBV), and reptilian reovirus (RRV) (9, 45). Although th...

Background:The p15 FAST protein mediates cell-cell fusion using a 19-residue ectodomain. Results: The p15 ectodomain requires both an N-terminal myristate and a polyproline type II helix for membrane fusion activity. Conclusion:The p15 ectodomain functions as a novel fusion peptide motif. Significance: This novel fusion peptide provides new insights into the essential biological process of protein-mediated membrane fusion.

The S1 genome segments of avian and Nelson Bay reovirus encode tricistronic mRNAs containing three sequential partially overlapping open reading frames (ORFs). The translation start site of the 3 -proximal ORF encoding the C protein lies downstream of two ORFs encoding the unrelated p10 and p17 proteins and more than 600 nucleotides distal from the 5 -end of the mRNA. It is unclear how translation of this remarkable tricistronic mRNA is regulated. We now show that the p10 and p17 ORFs are coordinately expressed by leaky scanning. Translation initiation events at these 5 -proximal ORFs, however, have little to no effect on translation of the 3 -proximal C ORF. Northern blotting, insertion of upstream stop codons or optimized translation start sites, 5 -truncation analysis, and poliovirus 2A protease-mediated cleavage of eIF4G indicated C translation derives from a full-length tricistronic mRNA using a mechanism that is eIF4G-dependent but leaky scanning-and translation reinitiation-independent. Further analysis of artificial bicistronic mRNAs failed to provide any evidence that C translation derives from an internal ribosome entry site. Additional features of the S1 mRNA and the mechanism of C translation also differ from current models of ribosomal shunting. Translation of the tricistronic reovirus S1 mRNA, therefore, is dependent both on leaky scanning and on a novel scanning-independent mechanism that allows translation initiation complexes to efficiently bypass two functional upstream ORFs.The scanning model of translation initiation accounts for the vast majority of translation events occurring in eukaryotic cells (1). Recruitment of the 40 S ribosomal subunit is facilitated by the presence of a 5Ј-terminal m 7 Gppp cap and canonical eukaryotic initiation factors (eIFs) 4 involved in cap binding and creation of the 43 S and 48 S preinitiation complexes. Translation initiation generally occurs at the 5Ј-proximal AUG start codon after scanning of the 5Ј-untranslated region (UTR) by the preinitiation complex and recruitment of the 60 S subunit.