Enhanced Fusion Pore Expansion Mediated by the Trans-Acting Endodomain of the Reovirus FAST Proteins

The reovirus fusion-associated small transmembrane (FAST) proteins are virus-encoded membrane fusion proteins that function as dedicated cell–cell fusogens. The topology of these small, single-pass membrane proteins orients the majority of the protein on the distal side of the membrane (i.e., inside the cell). We now show that ectopic expression of the endodomains of the p10, p14, and p15 FAST proteins enhances syncytiogenesis induced by the full-length FAST proteins, both homotypically and heterotypically. Results further indicate that the 68-residue cytoplasmic endodomain of the p14 FAST protein (1) is endogenously generated from full-length p14 protein expressed in virus-infected or transfected cells; (2) enhances syncytiogenesis subsequent to stable pore formation; (3) increases the syncytiogenic activity of heterologous fusion proteins, including the differentiation-dependent fusion of murine myoblasts; (4) exerts its enhancing activity from the cytosol, independent of direct interactions with either the fusogen or the membranes being fused; and (5) contains several regions with protein–protein interaction motifs that influence enhancing activity. We propose that the unique evolution of the FAST proteins as virus-encoded cellular fusogens has allowed them to generate a trans-acting, soluble endodomain peptide to harness a cellular pathway or process involved in the poorly understood process that facilitates the transition from microfusion pores to macrofusion and syncytiogenesis

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

Heart and Skeletal Muscle Inflammation of Farmed Salmon Is Associated with Infection with a Novel Reovirus

Atlantic salmon (Salmo salar L.) mariculture has been associated with epidemics of infectious diseases that threaten not only local production, but also wild fish coming into close proximity to marine pens and fish escaping from them. Heart and skeletal muscle inflammation (HSMI) is a frequently fatal disease of farmed Atlantic salmon. First recognized in one farm in Norway in 1999[1], HSMI was subsequently implicated in outbreaks in other farms in Norway and the United Kingdom[2]. Although pathology and disease transmission studies indicated an infectious basis, efforts to identify an agent were unsuccessful. Here we provide evidence that HSMI is associated with infection with piscine reovirus (PRV). PRV is a novel reovirus identified by unbiased high throughput DNA sequencing and a bioinformatics program focused on nucleotide frequency as well as sequence alignment and motif analyses. Formal implication of PRV in HSMI will require isolation in cell culture and fulfillment of Koch's postulates, or prevention or modification of disease through use of specific drugs or vaccines. Nonetheless, as our data indicate that a causal relationship is plausible, measures must be taken to control PRV not only because it threatens domestic salmon production but also due to the potential for transmission to wild salmon populations

Turbot reovirus (SMReV) genome encoding a FAST protein with a non-AUG start site

<p>Abstract</p> <p>Background</p> <p>A virus was isolated from diseased turbot <it>Scophthalmus maximus </it>in China. Biophysical and biochemical assays, electron microscopy, and genome electrophoresis revealed that the virus belonged to the genus <it>Aquareovirus</it>, and was named <it>Scophthalmus maximus </it>reovirus (SMReV). To the best of our knowledge, no complete sequence of an aquareovirus from marine fish has been determined. Therefore, the complete characterization and analysis of the genome of this novel aquareovirus will facilitate further understanding of the taxonomic distribution of aquareovirus species and the molecular mechanism of its pathogenesis.</p> <p>Results</p> <p>The full-length genome sequences of SMReV were determined. It comprises eleven dsRNA segments covering 24,042 base pairs and has the largest S4 genome segment in the sequenced aquareoviruses. Sequence analysis showed that all of the segments contained six conserved nucleotides at the 5' end and five conserved nucleotides at the 3' end (5'-GUUUUA ---- UCAUC-3'). The encoded amino acid sequences share the highest sequence identities with the respective proteins of aquareoviruses in species group <it>Aquareovirus </it>A. Phylogenetic analysis based on the major outer capsid protein VP7 and RNA-dependent RNA polymerase were performed. Members in <it>Aquareovirus </it>were clustered in two groups, one from fresh water fish and the other from marine fish. Furthermore, a fusion associated small transmembrane (FAST) protein NS22, which is translated from a non-AUG start site, was identified in the S7 segment.</p> <p>Conclusions</p> <p>This study has provided the complete genome sequence of a novel isolated aquareovirus from marine fish. Amino acids comparison and phylogenetic analysis suggested that SMReV was a new aquareovirus in the species group <it>Aquareovirus </it>A. Phylogenetic analysis among aquareoviruses revealed that VP7 could be used as a reference to divide the aquareovirus from hosts in fresh water or marine. In addition, a FAST protein with a non-AUG start site was identified, which partially contributed to the cytopathic effect caused by the virus infection. These results provide new insights into the virus-host and virus-environment interactions.</p

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

Membrane metabolism mediated by Sec14 family members influences Arf GTPase activating protein activity for transport from the trans-Golgi

The budding yeast Saccharomyces cerevisiae contains a family of Arf (ADP-ribosylation factor) GTPase activating protein (GAP) proteins with the Gcs1 + Age2 ArfGAP pair providing essential overlapping function for the movement of transport vesicles from the trans-Golgi network. We have generated a temperature-sensitive but stable version of the Gcs1 protein that is impaired only for trans-Golgi transport and find that deleterious effects of this enfeebled Gcs1-4 mutant protein are relieved by increased gene dosage of the gcs1-4 mutant gene itself or by the SFH2 gene (also called CSR1), encoding a phosphatidylinositol transfer protein (PITP). This effect was not seen for the SEC14 gene, encoding the founding member of the yeast PITP protein family, even though the Gcs1 and Age2 ArfGAPs are known to be downstream effectors of Sec14-mediated activity for trans-Golgi transport. Sfh2-mediated suppression of inadequate Gcs1-4 function depended on phospholipase D, whereas inadequate Gcs1-4 activity was relieved by increasing levels of diacylglycerol (DAG). Recombinant Gcs1 protein was found to bind certain phospholipids but not DAG. Our findings favor a model of Gcs1 localization through binding to specific phospholipids and activation of ArfGAP activity by DAG-mediated membrane curvature as the transport vesicle is formed. Thus, ArfGAPs are subject to both temporal and spatial regulation that is facilitated by Sfh2-mediated modulation of the lipid environment