Fusion-associated Small Transmembrane Research Articles

Avian deltacoronaviruses encode fusion-associated small transmembrane proteins that can induce syncytia formation

Fusion-associated small transmembrane (FAST) proteins are nonstructural viral proteins that induce cell-cell fusion. FAST proteins, which previously were identified in the genomes of double-stranded RNA viruses, typically contain an acylated N-terminal ectodomain, central transmembrane domain, and C-terminal endodomain with a polybasic region. Using sequence homology and protein motif prediction, we identified accessory proteins in a subset of avian deltacoronaviruses as putative FAST proteins. Transient expression of thrush coronavirus NS7b or common moorhen coronavirus NS7a, but not night heron coronavirus NS7b, induced cell-cell fusion. Syncytia were detected in primate kidney epithelial cells or fibroblasts but not chicken embryo fibroblasts, and addition of an N-terminal FLAG peptide to the proteins ablated fusion activity. These findings suggest that multiple avian deltacoronaviruses, positive-sense RNA viruses, encode nonstructural proteins that can mediate cell-cell fusion and share features with known FAST proteins. Additional studies are needed to understand contributions of these proteins to deltacoronavirus biology.

Safe and effective in vivo delivery of DNA and RNA using proteolipid vehicles

Genetic medicines show promise for treating various diseases, yet clinical success has been limited by tolerability, scalability, and immunogenicity issues of current delivery platforms. To overcome these, we developed a proteolipid vehicle (PLV) by combining features from viral and non-viral approaches. PLVs incorporate fusion-associated small transmembrane (FAST) proteins isolated from fusogenic orthoreoviruses into a well-tolerated lipid formulation, using scalable microfluidic mixing. Screening a FAST protein library, we identified a chimeric FAST protein with enhanced membrane fusion activity that improved gene expression from an optimized lipid formulation. Systemically administered FAST-PLVs showed broad biodistribution and effective mRNA and DNA delivery in mouse and non-human primate models. FAST-PLVs show low immunogenicity and maintain activity upon repeat dosing. Systemic administration of follistatin DNA gene therapy with FAST-PLVs raised circulating follistatin levels and significantly increased muscle mass and grip strength. These results demonstrate the promising potential of FAST-PLVs for redosable gene therapies and genetic medicines.

Nonstructural protein NS17 of grass carp reovirus Honghu strain promotes virus infection by mediating cell-cell fusion and apoptosis

Fusion-associated small transmembrane (FAST) proteins can promote cell fusion, alter membrane permeability and trigger apoptosis to promote virus proliferation in orthoreoviruses. However, it is unknown whether FAST proteins perform these functions in aquareoviruses (AqRVs). Non-structural protein 17 (NS17) carried by grass carp reovirus Honghu strain (GCRV-HH196) belongs to the FAST protein family, and we preliminarily explored its relevance to virus infection. NS17 has similar domains to FAST protein NS16 of GCRV-873, comprising a transmembrane domain, a polybasic cluster, a hydrophobic patch and a polyproline motif. It was observed in the cytoplasm and the cell membrane. Overexpression of NS17 enhanced the efficiency of cell-cell fusion induced by GCRV-HH196 and promoted virus replication. Overexpression of NS17 also led to DNA fragmentation and reactive oxygen species (ROS) accumulation, and it triggered apoptosis. The findings illuminate the functions of NS17 in GCRV infection, and provide a reference for the development of novel antiviral strategies.

Development of an oncolytic mammalian orthoreovirus expressing the near-infrared fluorescent protein iRFP720

Fluorescence-guided surgery (FGS) is a useful method for removing invasive tumor tissues. For this, near-infrared (NIR) fluorescence probes are suitable for visualizing cancer cells due to their low autofluorescence, and an oncolytic mammalian orthoreovirus (MRV) expressing an NIR fluorescent protein is expected to be a novel tool for FGS. In this study, we identified the optimal insertion site of the NIR fluorescent protein gene iRFP720 (915 nt) in the MRV genome. We constructed genome plasmids for the L1, M1, and S2 segments, where a gene cassette comprising iRFP720 and T2A self-cleaving peptide was inserted in the 5′ or 3′ region of each segment. Through virus recovery, the recombinant MRV with the gene cassette at the M1 segment’s 3′ end, T3D-L(M1/3′iRFP720), was capable of replication and passaging with bright NIR fluorescence. However, the replication of T3D-L(M1/3′iRFP720) was approximately 1,000-fold lower than that of the wild-type virus. T3D-L(M1/3′iRFP720) production improved due to the transfection of a fusion-associated small transmembrane protein gene of fusogenic reovirus. Further, fluorescence signals were detected in T3D-L(M1/3′iRFP720)-infected human gastric and pancreatic cancer cells. Thus, the M1 segment’s 3′ end tolerates the expression of the long iRFP720 gene, which may propel the development of recombinant MRV vectors for FGS.

The nonstructural p17 protein of a fusogenic bat-borne reovirus regulates viral replication in virus species- and host-specific manners

Nelson Bay orthoreovirus (NBV), a member of the family Reoviridae, genus Orthoreovirus, is a bat-borne virus that causes respiratory diseases in humans. NBV encodes two unique nonstructural proteins, fusion-associated small transmembrane (FAST) protein and p17 protein, in the S1 gene segment. FAST induces cell–cell fusion between infected cells and neighboring cells and the fusogenic activity is required for efficient viral replication. However, the function of p17 in the virus cycle is not fully understood. Here, various p17 mutant viruses including p17-deficient viruses were generated by a reverse genetics system for NBV. The results demonstrated that p17 is not essential for viral replication and does not play an important role in viral pathogenesis. On the other hand, NBV p17 regulated viral replication in a bat cell line but not in other human and animal cell lines. Nuclear localization of p17 is associated with the regulation of NBV replication in bat cells. We also found that p17 dramatically enhances the cell–cell fusion activity of NBV FAST protein for efficient replication in bat cells. Furthermore, we found that a protein homologue of NBV p17 from another bat-borne orthoreovirus, but not those of avian orthoreovirus or baboon orthoreovirus, also supported efficient viral replication in bat cells using a p17-deficient virus-based complementation approach. These results provide critical insights into the functioning of the unique replication machinery of bat-borne viruses in their natural hosts.

Homology-based classification of accessory proteins in coronavirus genomes uncovers extremely dynamic evolution of gene content.

Coronaviruses (CoVs) have complex genomes that encode a fixed array of structural and nonstructural components, as well as a variety of accessory proteins that differ even among closely related viruses. Accessory proteins often play a role in the suppression of immune responses and may represent virulence factors. Despite their relevance for CoV phenotypic variability, information on accessory proteins is fragmentary. We applied a systematic approach based on homology detection to create a comprehensive catalogue of accessory proteins encoded by CoVs. Our analyses grouped accessory proteins into 379 orthogroups and 12 super‐groups. No orthogroup was shared by the four CoV genera and very few were present in all or most viruses in the same genus, reflecting the dynamic evolution of CoV genomes. We observed differences in the distribution of accessory proteins in CoV genera. Alphacoronaviruses harboured the largest diversity of accessory open reading frames (ORFs), deltacoronaviruses the smallest. However, the average number of accessory proteins per genome was highest in betacoronaviruses. Analysis of the evolutionary history of some orthogroups indicated that the different CoV genera adopted similar evolutionary strategies. Thus, alphacoronaviruses and betacoronaviruses acquired phosphodiesterases and spike‐like accessory proteins independently, whereas horizontal gene transfer from reoviruses endowed betacoronaviruses and deltacoronaviruses with fusion‐associated small transmembrane (FAST) proteins. Finally, analysis of accessory ORFs in annotated CoV genomes indicated ambiguity in their naming. This complicates cross‐communication among researchers and hinders automated searches of large data sets (e.g., PubMed, GenBank). We suggest that orthogroup membership is used together with a naming system to provide information on protein function.

Isolation and Genomic Characterization of Avian Reovirus From Wild Birds in South Korea.

Avian reoviruses (ARVs) cause severe arthritis, tenosynovitis, pericarditis, and depressed growth in chickens, and these conditions have become increasingly frequent in recent years. Studies on the role of wild birds in the epidemiology of ARVs are insufficient. This study provides information about currently circulating ARVs in wild birds by gene detection using diagnostic RT-PCR, virus isolation, and genomic characterization. In this study, we isolated and identified 10 ARV isolates from 7,390 wild birds' fecal samples, including migratory bird species (bean goose, Eurasian teal, Indian spot-billed duck, and mallard duck) from 2015 to 2019 in South Korea. On comparing the amino acid sequences of the σC-encoding gene, most isolates, except A18-13, shared higher sequence similarity with the commercial vaccine isolate S1133 and Chinese isolates. However, the A18-13 isolate is similar to live attenuated vaccine av-S1133 and vaccine break isolates (SD09-1, LN09-1, and GX110116). For the p10- and p17-encoding genes, all isolates have identical fusion associated small transmembrane (FAST) protein and nuclear localization signal (SNL) motif to chicken-origin ARVs. Phylogenetic analysis of the amino acid sequences of the σC-encoding gene revealed that all isolates were belonged to genotypic cluster I. For the p10- and p17-encoding genes, the nucleotide sequences of all isolates indicated close relationship with commercial vaccine isolate S1133 and Chinese isolates. For the σNS-encoding gene, the nucleotide sequences of all isolates indicated close relationship with the Californian chicken-origin isolate K1600657 and belonged to chicken-origin ARV cluster. Our data indicates that wild birds ARVs were derived from the chicken farms. This finding suggests that wild birds serve as natural carriers of such viruses for domestic poultry.

Safe and Effective Delivery of Nucleic Acids Using Proteolipid Vehicles Formulated with Fusion-Associated Small Transmembrane Proteins

Safe and Effective Delivery of Nucleic Acids Using Proteolipid Vehicles Formulated with Fusion-Associated Small Transmembrane Proteins

Immunogenicity of a SARS-CoV-2 DNA Vaccine Formulated With the Fusion-Associated Small Transmembrane Protein Proteolipid Vehicle Delivery System

Immunogenicity of a SARS-CoV-2 DNA Vaccine Formulated With the Fusion-Associated Small Transmembrane Protein Proteolipid Vehicle Delivery System

Production of a replicating retroviral vector expressing Reovirus fast protein for cancer gene therapy

Reovirus fusion-associated small transmembrane (FAST) proteins induce syncytium formation. Recently, several studies have shown that the use of recombinant vectors engineered to express fusion proteins is becoming attractive for the development of enhanced oncolytic viruses. In this study, we investigated the cytotoxic effect of four different FAST proteins (p10 FAST of Avian reovirus [ARV], p10 FAST of Pulau virus [PuV], p13 FAST of Broome virus [BroV], and p14 FAST of reptilian reovirus [RRV]). Plasmids encoding FASTs were transfected into Vero cells. All FAST proteins induced syncytium formation at varying intensities. To achieve high levels of FAST expression, four different FAST genes were inserted into the murine leukemia virus (MLV)-based replication-competent retroviral (RCR) vector. Two days after transfection in 293 T cells, only the MoMLV-10A1-p10(PuV) RCR vector showed syncytia formation. Based on these results, p10(Puv) was selected from the four FASTs. Next, we investigated the cytotoxicity of p10(PuV) on HeLa cervical carcinoma cells, HT1080 human fibrosarcoma cells, and U87 human glioma cells. Although three human cancer cell lines induced syncytium formation, U87 cells were highly susceptible to syncytia formation by transfection with p10(PuV). In addition, the viral supernatants from MoMLV-10A-p10(PuV) RCR vector-transfected 293 T cells also induced syncytium formation in HT1080, TE671, and U87 cells. This RCR vector encoding p10(PuV) is a promising candidate for cancer gene therapy.

FAST Proteins: Development and Use of Reverse Genetics Systems for Reoviridae Viruses.

Reverse genetics systems for viruses, the technology used to generate gene-engineered recombinant viruses from artificial genes, enable the study of the roles of the individual nucleotides and amino acids of viral genes and proteins in infectivity, replication, and pathogenicity. The successful development of a reverse genetics system for poliovirus in 1981 accelerated the establishment of protocols for other RNA viruses important for human health. Despite multiple efforts, rotavirus (RV), which causes severe gastroenteritis in infants, was refractory to reverse genetics analysis, and the first complete reverse genetics system for RV was established in 2017. This novel technique involves use of the fusogenic protein FAST (fusion-associated small transmembrane) derived from the bat-borne Nelson Bay orthoreovirus, which induces massive syncytium formation. Co-transfection of a FAST-expressing plasmid with complementary DNAs encoding RV genes enables rescue of recombinant RV. This review focuses on methodological insights into the reverse genetics system for RV and discusses applications and potential improvements to this system.

The genome position of a therapeutic transgene strongly influences the level of expression in an armed oncolytic human adenovirus vector

Efficacy of oncolytic, conditionally-replicating adenovirus (CRAd) vectors can be enhanced by “arming” the vector with therapeutic transgenes. We examined whether inclusion of an intact early region 3 (E3) and the reptilian reovirus fusogenic p14 fusion-associated small transmembrane (FAST) protein enhanced vector efficacy. The p14 FAST transgene was cloned between the fiber gene and E4 region, with an upstream splice acceptor for replication-dependent expression from the major late promoter. In A549 cells, this vector expressed p14 FAST protein at very low levels, and showed a poor ability to mediate cell-cell fusion, relative to a similar vector encoding p14 FAST within the E3 deletion. Although expression of E3 proteins from the CRAd increased plaque size, poor expression of p14 FAST protein compromised the fusogenic capacity of the vector. Thus, location of a therapeutic transgene within a CRAd can significantly impact expression of the transgene and is an important consideration in vector design.

Inhibition of glioma using a novel non-neurotoxic vesicular stomatitis virus.

The aim of this study was to demonstrate the in vivo safety and antitumor effect of a novel recombinant vesicular stomatitis virus (VSV): G protein less (GLESS)-fusion-associated small transmembrane (FAST)-VSV. Viral infection efficiency and cell proliferation were detected using an inverted fluorescence microscope and alarmaBlue assay, respectively. To evaluate the safety of the virus, different doses of GLESS-FAST-VSV and a positive control virus (VSV∆M51) were injected into normal F344 rats and C57BL/6 mice, and each animal's weight, survival time, and pathological changes were examined on the following day. To evaluate the efficacy of the virus, RG2 and GL261 cells were used to construct rat and mouse glioma models, respectively, via a stereotactic method. After multiple intratumoral injections of the virus, tumor growth (size) and the survival time of the animals were observed. In vitro experiments showed that GLESS-FAST-VSV could infect and kill brain tumor cells and had less toxic effects on normal cells. After direct injection of GLESS-FAST-VSV into the animal brains, all animals tolerated the virus well, and no animal death, encephalitis, or ventriculitis was observed. In contrast, all animals that received brain injections of VSV∆M51 in the brain died. Moreover, multiple injections of GLESS-FAST-VSV in brain tumors significantly prolonged the survival of normal-immunity animals harboring brain tumors. GLESS-FAST-VSV exhibited little neurotoxicity and could be injected directly into the tumor to effectively inhibit tumor growth and prolong the survival of normal-immunity animals, laying a theoretical foundation for the early application of such viruses in clinical trials.

Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion

Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

Polycistronic Genome Segment Evolution and Gain and Loss of FAST Protein Function during Fusogenic Orthoreovirus Speciation.

The Reoviridae family is the only non-enveloped virus family with members that use syncytium formation to promote cell–cell virus transmission. Syncytiogenesis is mediated by a fusion-associated small transmembrane (FAST) protein, a novel family of viral membrane fusion proteins. Previous evidence suggested the fusogenic reoviruses arose from an ancestral non-fusogenic virus, with the preponderance of fusogenic species suggesting positive evolutionary pressure to acquire and maintain the fusion phenotype. New phylogenetic analyses that included the atypical waterfowl subgroup of avian reoviruses and recently identified new orthoreovirus species indicate a more complex relationship between reovirus speciation and fusogenic capacity, with numerous predicted internal indels and 5’-terminal extensions driving the evolution of the orthoreovirus’ polycistronic genome segments and their encoded FAST and fiber proteins. These inferred recombination events generated bi- and tricistronic genome segments with diverse gene constellations, they occurred pre- and post-orthoreovirus speciation, and they directly contributed to the evolution of the four extant orthoreovirus FAST proteins by driving both the gain and loss of fusion capability. We further show that two distinct post-speciation genetic events led to the loss of fusion in the waterfowl isolates of avian reovirus, a recombination event that replaced the p10 FAST protein with a heterologous, non-fusogenic protein and point substitutions in a conserved motif that destroyed the p10 assembly into multimeric fusion platforms.

Rotavirus Species B Encodes a Functional Fusion-Associated Small Transmembrane Protein.

Rotavirus is an important cause of diarrheal disease in young mammals. Rotavirus species A (RVA) causes most human rotavirus diarrheal disease and primarily affects infants and young children. Rotavirus species B (RVB) has been associated with sporadic outbreaks of human adult diarrheal disease. RVA and RVB are predicted to encode mostly homologous proteins but differ significantly in the proteins encoded by the NSP1 gene. In the case of RVB, the NSP1 gene encodes two putative protein products of unknown function, NSP1-1 and NSP1-2. We demonstrate that human RVB NSP1-1 mediates syncytium formation in cultured human cells. Based on sequence alignment, NSP1-1 proteins from species B, G, and I contain features consistent with fusion-associated small transmembrane (FAST) proteins, which have previously been identified in other genera of the Reoviridae family. Like some other FAST proteins, RVB NSP1-1 is predicted to have an N-terminal myristoyl modification. Addition of an N-terminal FLAG peptide disrupts NSP1-1-mediated fusion. NSP1-1 from a human RVB mediates fusion of human cells but not hamster cells and, thus, may serve as a species tropism determinant. NSP1-1 also can enhance RVA replication in human cells, both in single-cycle infection studies and during a multicycle time course in the presence of fetal bovine serum, which inhibits rotavirus spread. These findings suggest potential yet untested roles for NSP1-1 in RVB species tropism, immune evasion, and pathogenesis.IMPORTANCE While species A rotavirus is commonly associated with diarrheal disease in young children, species B rotavirus has caused sporadic outbreaks of adult diarrheal disease. A major genetic difference between species A and B rotaviruses is the NSP1 gene, which encodes two proteins for species B rotavirus. We demonstrate that the smaller of these proteins, NSP1-1, can mediate fusion of cultured human cells. Comparison with viral proteins of similar function provides insight into NSP1-1 domain organization and fusion mechanism. These comparisons suggest that there is a fatty acid modification at the amino terminus of the protein, and our results show that an intact amino terminus is required for NSP1-1-mediated fusion. NSP1-1 from a human virus mediates fusion of human cells, but not hamster cells, and enhances species A rotavirus replication in culture. These findings suggest potential, but currently untested, roles for NSP1-1 in RVB host species tropism, immune evasion, and pathogenesis.

Fusogenic Reoviruses and Their Fusion-Associated Small Transmembrane (FAST) Proteins.

With no limiting membrane surrounding virions, nonenveloped viruses have no need for membrane fusion to gain access to intracellular replication compartments. Consequently, nonenveloped viruses do not encode membrane fusion proteins. The only exception to this dogma is the fusogenic reoviruses that encode fusion-associated small transmembrane (FAST) proteins that induce syncytium formation. FAST proteins are the smallest viral membrane fusion proteins and, unlike their enveloped virus counterparts, are nonstructural proteins that evolved specifically to induce cell-to-cell, not virus-cell, membrane fusion. This distinct evolutionary imperative is reflected in structural and functional features that distinguish this singular family of viral fusogens from all other protein fusogens. These rudimentary fusogens comprise specific combinations of different membrane effector motifs assembled into small, modular membrane fusogens. FAST proteins offer a minimalist model to better understand the ubiquitous process of protein-mediated membrane fusion and to reveal novel mechanisms of nonenveloped virus dissemination that contribute to virulence.

An Oncolytic Adenovirus Vector Expressing p14 FAST Protein Induces Widespread Syncytium Formation and Reduces Tumor Growth Rate In Vivo

Intratumoral injection of oncolytic viruses provides a direct means of tumor cell destruction for inoperable tumors. Unfortunately, oncolytic vectors based on human adenovirus (HAdV) typically do not spread efficiently throughout the tumor mass, reducing the efficacy of treatment. In this study, we explore the efficacy of a conditionally replicating HAdV vector expressing the p14 Fusion-Associated Small Transmembrane (FAST) protein (CRAdFAST) in both immunocompetent and immunodeficient mouse models of cancer. The p14 FAST protein mediates cell-cell fusion, which may enhance spread of the virus-mediated, tumor cell-killing effect. In the murine 4T1 model of cancer, treatment with CRAdFAST resulted in enhanced cell death compared to vector lacking the p14 FAST gene, but it did not reduce the tumor growth rate in vivo. In the human A549 lung adenocarcinoma model of cancer, CRAdFAST showed significantly improved oncolytic efficacy in vitro and in vivo. In an A549 xenograft tumor model in vivo, CRAdFAST induced tumor cell fusion, which led to the formation of large acellular regions within the tumor and significantly reduced the tumor growth rate compared to control vector. Our results indicate that expression of p14 FAST from an oncolytic HAdV can improve vector efficacy for the treatment of cancer.

Cell-cell fusion induced by reovirus FAST proteins enhances replication and pathogenicity of non-enveloped dsRNA viruses.

Fusogenic reoviruses encode fusion-associated small transmembrane (FAST) protein, which induces cell–cell fusion. FAST protein is the only known fusogenic protein in non-enveloped viruses, and its role in virus replication is not yet known. We generated replication-competent, FAST protein-deficient pteropine orthoreovirus and demonstrated that FAST protein was not essential for viral replication, but enhanced viral replication in the early phase of infection. Addition of recombinant FAST protein enhanced replication of FAST-deficient virus and other non-fusogenic viruses in a fusion-dependent and FAST-species-independent manner. In a mouse model, replication and pathogenicity of FAST-deficient virus were severely impaired relative to wild-type virus, indicating that FAST protein is a major determinant of the high pathogenicity of fusogenic reovirus. FAST-deficient virus also conferred effective protection against challenge with lethal homologous virus strains in mice. Our results demonstrate a novel role of a viral fusogenic protein and the existence of a cell–cell fusion-dependent replication system in non-enveloped viruses.

Reovirus FAST Protein Enhances Vesicular Stomatitis Virus Oncolytic Virotherapy in Primary and Metastatic Tumor Models

The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral fusogens (∼100–150 amino acids) and efficiently induce cell-cell fusion and syncytium formation in multiple cell types. Syncytium formation enhances cell-cell virus transmission and may also induce immunogenic cell death, a form of apoptosis that stimulates immune recognition of tumor cells. These properties suggest that FAST proteins might serve to enhance oncolytic virotherapy. The oncolytic activity of recombinant VSVΔM51 (an interferon-sensitive vesicular stomatitis virus [VSV] mutant) encoding the p14 FAST protein (VSV-p14) was compared with a similar construct encoding GFP (VSV-GFP) in cell culture and syngeneic BALB/c tumor models. Compared with VSV-GFP, VSV-p14 exhibited increased oncolytic activity against MCF-7 and 4T1 breast cancer spheroids in culture and reduced primary 4T1 breast tumor growth in vivo. VSV-p14 prolonged survival in both primary and metastatic 4T1 breast cancer models, and in a CT26 metastatic colon cancer model. As with VSV-GFP, VSV-p14 preferentially replicated in vivo in tumors and was cleared rapidly from other sites. Furthermore, VSV-p14 increased the numbers of activated splenic CD4, CD8, natural killer (NK), and natural killer T (NKT) cells, and increased the number of activated CD4 and CD8 cells in tumors. FAST proteins may therefore provide a multi-pronged approach to improving oncolytic virotherapy via syncytium formation and enhanced immune stimulation.