P14 Fusion-associated Small Transmembrane Research Articles

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.

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.

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.

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.

Expression of the fusogenic p14 FAST protein from a replication-defective adenovirus vector does not provide a therapeutic benefit in an immunocompetent mouse model of cancer.

When injected directly into a tumor mass, adenovirus (Ad) vectors only transduce cells immediately along the injection tract. Expression of fusogenic proteins from the Ad vector can lead to syncytium formation, which efficiently spreads the therapeutic effect. Fusogenic proteins can also cause cancer cell death directly, and enhance the release of exosome-like particles containing tumor-associated antigens, which boosts the anti-tumor immune response. In this study, we have examined whether delivery of an early region 1 (E1)-deleted, replication-defective Ad vector encoding the reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein can provide therapeutic efficacy in an immunocompetent mouse tumor model. A high multiplicity of infection of AdFAST is required to induce cell fusion in mouse mammary carcinoma 4T1 cells in vitro, and FAST protein expression caused a modest reduction in cell membrane integrity and metabolic activity compared with cells infected with a control vector. Cells expressing FAST protein released significantly higher quantities of exosomes. In immunocompetent Balb/C mice harboring subcutaneous 4T1 tumors, AdFAST did not induce detectable cancer cell fusion, promote tumor regression or prolong mouse survival compared with untreated mice. This study suggests that in the context of the 4T1 model, Ad-mediated FAST protein expression did not elicit a therapeutic effect.

Adenovirus-Mediated Expression of the p14 Fusion-Associated Small Transmembrane Protein Promotes Cancer Cell Fusion and Apoptosis In Vitro but Does Not Provide Therapeutic Efficacy in a Xenograft Mouse Model of Cancer.

Adenoviruses (Ads) are used in numerous preclinical and clinical studies for delivery of anti-cancer therapeutic genes. Unfortunately, Ad has a poor ability to distribute throughout a tumor mass after intratumoral injection, and infects cells primarily within the immediate area of the injection tract. Thus, Ad-encoded transgene expression is typically limited to only a small percentage of cells within the tumor. One method to increase the proportion of the tumor impacted by Ad is through expression of fusogenic proteins. Infection of a single cell with an Ad vector encoding a fusogenic protein should lead to syncytium formation with adjacent cells, effectively spreading the effect of Ad and Ad-encoded therapeutic transgenes to a greater percentage of the tumor mass. Moreover, syncytium formation can be cytotoxic, suggesting that such proteins may be effective sole therapeutics. We show that an early region 1 (E1)-deleted Ad expressing reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein caused extensive cell fusion in the replication-permissive 293 cell line and at high multiplicity of infection in non-permissive human lung adenocarcinoma A549 cells in vitro. FAST protein expression in the A549 cancer cell line led to a loss of cellular metabolic activity and membrane integrity, which correlated with induction of apoptosis. However, in an A549 xenograft CD-1 nude mouse cancer model, Ad-mediated FAST gene delivery did not induce detectable cell fusion, reduce tumor burden nor enhance mouse survival compared to controls. Taken together, our results show that, although AdFAST can enhance cancer cell killing in vitro, it is not effective as a sole therapeutic in the A549 tumor model in vivo.

A novel tribasic Golgi export signal directs cargo protein interaction with activated Rab11 and AP-1-dependent Golgi-plasma membrane trafficking.

The reovirus fusion-associated small transmembrane (FAST) proteins comprise a unique family of viral membrane fusion proteins dedicated to inducing cell-cell fusion. We recently reported that a polybasic motif (PBM) in the cytosolic tail of reptilian reovirus p14 FAST protein functions as a novel tribasic Golgi export signal. Using coimmunoprecipitation and fluorescence resonance energy transfer (FRET) assays, we now show the PBM directs interaction of p14 with GTP-Rab11. Overexpression of dominant-negative Rab11 and RNA interference knockdown of endogenous Rab11 inhibited p14 plasma membrane trafficking and resulted in p14 accumulation in the Golgi complex. This is the first example of Golgi export to the plasma membrane that is dependent on the interaction of membrane protein cargo with activated Rab11. RNA interference and immunofluorescence microscopy further revealed that p14 Golgi export is dependent on AP-1 (but not AP-3 or AP-4) and that Rab11 and AP-1 both colocalize with p14 at the TGN. Together these results imply the PBM mediates interactions of p14 with activated Rab11 at the TGN, resulting in p14 sorting into AP1-coated vesicles for anterograde TGN-plasma membrane transport.

Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor.

Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation.

Lysophosphatidylcholine Reversibly Arrests Pore Expansion during Syncytium Formation Mediated by Diverse Viral Fusogens

Using lysophosphatidylcholine, a curvature-inducing lysolipid, we have isolated a reversible, "stalled pore" phenotype during syncytium formation induced by the p14 fusion-associated small transmembrane (FAST) protein and influenza virus hemagglutinin (HA) fusogens. This is the first evidence that lateral propagation of stable fusion pores leading to syncytiogenesis mediated by diverse viral fusogens is inhibited by promotion of positive membrane curvature in the outer leaflets of the lipid bilayer surrounding intercellular fusion pores.

Abstract 3224: Targeted non-invasive therapy of prostate cancer using fusogenic liposomes

Abstract Prostate cancer is the most common cancer in Canadian men. Most chemotherapeutic drugs do not preferentially target the tumour and therefore subject the patient's healthy cells to significant toxicity. There is therefore an urgent need for a novel strategy to deliver chemotherapeutic drugs specifically to the tumour. P14 is a fusion associated small transmembrane (FAST) protein which mediates efficient cell-cell interaction. When we incorporate this p14 FAST protein into liposomes we increase the fusion of the liposome to cell membranes. I hypothesize that targeted ablation of prostate cancer may be achieved by delivering chemotherapeutics encapsulated inside fusogenic liposomes targeted to the gastrin releasing peptide receptors. The objectives of the study are to i) validate the fusogenic properties of p14-containg liposomes ii) express and purify p14-bomesin fusion protein, iii) assemble targeted fusogenic lipsoomes iv) and assess the targeted ablation of human prostate cancer in a xenograft model. To validate the fusogenic properties of p14-containing liposomes in our cell lines, we encapsulated fluorescent dextran inside liposomes containing either no p14, mutant p14 (fusogenic incompetent) or an active p14. We incubated the liposomes on human fibrosarcoma cells (HT1080) and measured the uptake of fluorescent dextran using fluorescent microscopy. To construct a fusion protein, we used PCR amplification to fuse bombesin to the C-terminus of the p14 protein. Our analysis demonstrates that the incorporation of the p14 protein significantly increased the fluorescent dextran uptake in the cytoplasm of the HT1080 cells compared to the liposomes that contained no p14 and the mutant p14. Protein purification of the p14-bombesin fusion protein is still in progress. I propose to use these targeted fusogenic liposomes for future studies to evaluate the ability of bombesin-coated fusogenic liposomes to target prostate cells in vitro and deliver a toxic therapeutic payload to prostate tumours in vivo and assess tumour reduction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3224. doi:10.1158/1538-7445.AM2011-3224

Helix-Destabilizing, β-Branched, and Polar Residues in the Baboon Reovirus p15 Transmembrane Domain Influence the Modularity of FAST Proteins

The fusogenic reoviruses induce syncytium formation using the fusion-associated small transmembrane (FAST) proteins. A recent study indicated the p14 FAST protein transmembrane domain (TMD) can be functionally replaced by the TMDs of the other FAST proteins but not by heterologous TMDs, suggesting that the FAST protein TMDs are modular fusion units. We now show that the p15 FAST protein is also a modular fusogen, as indicated by the functional replacement of the p15 ectodomain with the corresponding domain from the p14 FAST protein. Paradoxically, the p15 TMD is not interchangeable with the TMDs of the other FAST proteins, implying that unique attributes of the p15 TMD are required when this fusion module is functioning in the context of the p15 ecto- and/or endodomain. A series of point substitutions, truncations, and reextensions were created in the p15 TMD to define features that are specific to the functioning of the p15 TMD. Removal of only one or two residues from the N terminus or four residues from the C terminus of the p15 TMD eliminated membrane fusion activity, and there was a direct correlation between the fusion-promoting function of the p15 TMD and the presence of N-terminal, hydrophobic β-branched residues. Substitution of the glycine residues and triserine motif present in the p15 TMD also impaired or eliminated the fusion-promoting activity of the p15 TMD. The ability of the p15 TMD to function in an ecto- and endodomain-specific context is therefore influenced by stringent sequence requirements that reflect the importance of TMD polar residues and helix-destabilizing residues.

Homomultimerization of the reovirus p14 fusion-associated small transmembrane protein during transit through the ER-Golgi complex secretory pathway

The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral membrane-fusion proteins. How these diminutive fusogens mediate cell-cell fusion and syncytium formation is unclear. Ongoing efforts are aimed at defining the roles of the FAST protein ecto-, endo- and transmembrane domains in the membrane-fusion reaction. We now provide direct evidence for homomultimer formation by the FAST proteins by using an anti-haemagglutinin (HA) mAb to co-precipitate the untagged p14 FAST protein from cells co-transfected with HA-tagged p14. Disrupting the intracellular endoplasmic reticulum-Golgi complex vesicle transport pathway prevented p14 homomultimer formation, while lower pH disrupted p14 multimers. The p14 endodomain or transmembrane domains are not required for multimer formation, which, along with the pH sensitivity and the distribution of histidine residues, suggests the 36 aa p14 ectodomain is a multimerization motif.

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.

The p14 FAST Protein of Reptilian Reovirus Increases Vesicular Stomatitis Virus Neuropathogenesis

The fusogenic orthoreoviruses express nonstructural fusion-associated small transmembrane (FAST) proteins that induce cell-cell fusion and syncytium formation. It has been speculated that the FAST proteins may serve as virulence factors by promoting virus dissemination and increased or altered cytopathology. To directly test this hypothesis, the gene encoding the p14 FAST protein of reptilian reovirus was inserted into the genome of a heterologous virus that does not naturally form syncytia, vesicular stomatitis virus (VSV). Expression of the p14 FAST protein by the VSV/FAST recombinant gave the virus a highly fusogenic phenotype in cell culture. The growth of this recombinant fusogenic VSV strain was unaltered in vitro but was significantly enhanced in vivo. The VSV/FAST recombinant consistently generated higher titers of virus in the brains of BALB/c mice after intranasal or intravenous infection compared to the parental VSV/green fluorescent protein (GFP) strain that expresses GFP in place of p14. The VSV/FAST recombinant also resulted in an increased incidence of hind-limb paralysis, it infected a larger volume of brain tissue, and it induced more extensive neuropathology, thus leading to a lower maximum tolerable dose than that for the VSV/GFP parental virus. In contrast, an interferon-inducing mutant of VSV expressing p14 was still attenuated, indicating that this interferon-inducing phenotype is dominant to the fusogenic properties conveyed by the FAST protein. Based on this evidence, we conclude that the reovirus p14 FAST protein can function as a bona fide virulence factor.

The p14 Fusion-associated Small Transmembrane (FAST) Protein Effects Membrane Fusion from a Subset of Membrane Microdomains

The p14 Fusion-associated Small Transmembrane (FAST) Protein Effects Membrane Fusion from a Subset of Membrane Microdomains

The p14 Fusion-associated Small Transmembrane (FAST) Protein Effects Membrane Fusion from a Subset of Membrane Microdomains

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipid-anchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.

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.

Myristoylation, a Protruding Loop, and Structural Plasticity Are Essential Features of a Nonenveloped Virus Fusion Peptide Motif

Members of the fusion-associated small transmembrane (FAST) protein family are a distinct class of membrane fusion proteins encoded by nonenveloped fusogenic reoviruses. The 125-residue p14 FAST protein of reptilian reovirus has an approximately 38-residue myristoylated N-terminal ectodomain containing a moderately apolar N-proximal region, termed the hydrophobic patch. Mutagenic analysis indicated sequence-specific elements in the N-proximal portion of the p14 hydrophobic patch affected cell-cell fusion activity, independent of overall effects on the relative hydrophobicity of the motif. Circular dichroism (CD) of a myristoylated peptide representing the majority of the p14 ectodomain suggested this region is mostly disordered in solution but assumes increased structure in an apolar environment. From NMR spectroscopic data and simulated annealing, the soluble nonmyristoylated p14 ectodomain peptide consists of an N-proximal extended loop flanked by two proline hinges. The remaining two-thirds of the ectodomain peptide structure is disordered, consistent with predictions based on CD spectra of the myristoylated peptide. The myristoylated p14 ectodomain peptide, but not a nonmyristoylated version of the same peptide nor a myristoylated scrambled peptide, mediated extensive lipid mixing in a liposome fusion assay. Based on the lipid mixing activity, structural plasticity, environmentally induced conformational changes, and kinked structures predicted for the p14 ectodomain and hydrophobic patch (all features associated with fusion peptides), we propose that the majority of the p14 ectodomain is composed of a fusion peptide motif, the first such motif dependent on myristoylation for membrane fusion activity.