Vaccinia Virus A25 and A26 Proteins Are Fusion Suppressors for Mature Virions and Determine Strain-Specific Virus Entry Pathways into HeLa, CHO-K1, and L Cells (original) (raw)
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Membrane cell fusion activity of the vaccinia virus A17?A27 protein complex
Cellular Microbiology, 2007
Vaccinia virus enters cells by endocytosis and via a membrane fusion mechanism mediated by viral envelope protein complexes. While several proteins have been implicated in the entry/fusion event, there is no direct proof for fusogenic activity of any viral protein in heterologous systems. Transient coexpression of A17 and A27 in mammalian cells led to syncytia formation in a pH-dependent manner, as ascertained by confocal fluorescent immunomicroscopy. The pH-dependent fusion activity was identified to reside in A17 amino-terminal ectodomain after overexpression in insect cells using recombinant baculoviruses. Through the use of A17 ectodomain deletion mutants, it was found that the domain important for fusion spanned between residues 18 and 34. To further characterize A17–A27 fusion activity in mammalian cells, 293T cell lines stably expressing A17, A27 or coexpressing both proteins were generated using lentivectors. A27 was exposed on the cell surface only when A17 was coexpressed. In addition, pH-dependent fusion activity was functionally demonstrated in mammalian cells by cytoplasmic transfer of fluorescent proteins, only when A17 and A27 were coexpressed. Bioinformatic tools were used to compare the putative A17–A27 protein complex with well-characterized fusion proteins. Finally, all experimental evidence was integrated into a working model for A17–A27-induced pH-dependent cell-to-cell fusion.
Virology, 1990
The mechanism by which the large-size poxviruses enter animal cells is not known. In this investigation we show that acid pH treatment of wild-type vaccinia virus-infected cells triggers strong fusion of cells in culture, with an optimum at pH 4.8. We have identified the virus-induced fusion protein as a 14-kDa envelope protein, based on the ability of a 14-kDa specific monoclonal antibody (mAbC3) to block vaccinia virus-induced fusion-from-within and fusion-from-without. We provide genetic evidence for a role of the 14-kDa protein in cell fusion, since insertion of the 1CkDa encoding gene into the genome of nonfusogenic mutant viruses generates heterozygous viruses that now acquire acid pHdependent fusion activity. DNA sequence analyses of the 14-kDa encoding gene of the mutant viruses, 65-l 6 and 101-14, reveal N-terminal deletions of 46 and 10 amino acids, respectively. These deletions remove a small hydrophobic region at the N-terminus of the 14.kDa protein and prevent fusion. Our findings demonstrate that vaccinia virus can induce strong fusion of cells in culture at acid pH implying some entry of the virus by endocytosis, that the 14-kDa virus envelope protein is the fusogenic protein, and that the N-terminal proximal region is involved in fusion.
Journal of Virology, 2007
The proteins encoded by the A56R and K2L genes of vaccinia virus form a heterodimer (A56/K2) and have a fusion regulatory role as deletion or mutation of either causes infected cells to form large syncytia spontaneously. Here, we showed that syncytia formation is dependent on proteins of the recently described entry fusion complex (EFC), which are also required for virus-cell fusion and low-pH-triggered cell-cell fusion. This finding led us to consider that A56/K2 might prevent fusion by direct or indirect interaction with the EFC. To test this hypothesis, we made a panel of recombinant vaccinia viruses that have a tandem affinity purification tag attached to A56, K2, or the A28 EFC protein. Interaction between A56/K2 and the EFC was demonstrated by their copurification from detergent-treated lysates of infected cells and identification by mass spectrometry or Western blotting. In addition, a purified soluble transmembrane-deleted form of A56/K2 was shown to interact with the EFC. Tagged A56 did not interact with the EFC in the absence of K2, nor did tagged K2 interact with the EFC in the absence of A56. The finding that both A56 and K2 are required for efficient binding to the EFC fits well with prior experiments showing that mutation of either A56 or K2 results in spontaneous fusion of infected cells. Because A56 and K2 are located on the surface of infected cells, they are in position to interact with the EFC of released progeny virions and prevent back-fusion and syncytia formation. TAP and mass spectrometry. HeLa S3 cells (1.5 ϫ10 9 ) were infected at a multiplicity of 5 PFU for 24 h. Cells were collected, washed once with ice-cold buffer (150 mM NaCl and 50 mM Tris-HCl, pH 7.4), and lysed by incubating for 1 h at 4°C in streptavidin binding buffer (SBB) (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, with complete protease inhibitor; Roche, Indianapolis, IN). The lysate was centrifuged for 15 min at 3,000 ϫ g, and the clarified supernatant was collected. The latter, except for 0.3 ml reserved for later analysis, was added to 0.5 ml to 1 ml of streptavidin-Sepharose (GE Healthcare, Piscataway, NJ) that had been washed with SBB, and the mixture was rotated overnight at 4°C. The beads were washed three times with 10 ml of ice cold SBB, and the bound proteins were eluted by three washes with 1 ml of SBB containing 1 mg/ml of D-biotin (USB Corp., Cleveland, OH). The 3 ml of streptavidin eluate was supplemented with Mg acetate, imidazole, and CaCl 2 to final concentrations of 1 mM, 1 mM, and 2 mM, respectively. Calmodulin-Sepharose (0.5 ml to 1 ml of packed resin; GE Healthcare) was washed with calmodulin binding buffer ([CBB], which consists of SBB without protease inhibitors and is supplemented with Mg acetate, imidazole, and CaCl 2 at final concentrations of 1 mM, 1 mM, and 2 mM, respectively). Calmodulin-Sepharose was added to the supplemented streptavidin eluate along with an additional 2 ml of CBB, and the mixture was rotated overnight at 4°C. The beads were washed three times with 10 ml of CBB and three times with 0.75 ml of CBB containing 25 mM EGTA to elute proteins. The proteins in the calmodulin eluate were concentrated by trichloroacetic acid precipitation, resuspended in lithium-dodecyl sulfate sample buffer (Invitrogen, Carlsbad, CA) containing NuPage sample reducing agent (Invitrogen), and separated on a 4 to 12% NuPage gel (Invitrogen) with 2(N-morpholino)ethanesulfonic acid buffer. Gels were stained with Coomassie blue (GelCode blue stain reagent; Pierce, Rockford, IL), and bands of interest were excised from the polyacrylamide gel and subsequently digested with trypsin. Tandem mass spectrometry and database searching were performed at the National Institute of Allergy and Infectious Diseases (NIAID) core facility.
Journal of Virology, 2008
The recently described vaccinia virus entry/fusion complex (EFC) comprises at least eight polypeptides that are conserved in all poxviruses. Neither the structure of the complex nor the roles of individual subunits are known. Here we provide evidence for an interaction between the H2 and A28 subunits in the context of a virus infection as well as in uninfected cells transfected with plasmids expressing the corresponding genes. We focused on a highly conserved 21-amino acid-segment in H2 that is flanked by cysteine residues. The effect of amino acid substitutions within the 21-amino-acid segment was determined by an infectivity complementation assay using a conditional H2-null mutant of vaccinia virus. Mutations that had no, moderate, or large negative effects on complementation were found. The latter group included glutamic acid substitutions of leucine and individual glycines and alanine substitution of both glycines within a LGYSG sequence. Mutations with the most pronounced effect on infectivity disrupted the interaction of H2 with A28 to the greatest extent in both infected and uninfected cells. These data indicate that the LGYSG sequence is important for the interaction of H2 with A28 and suggest that this sequence is buried within the EFC complex.
Expression of the A56 and K2 Proteins Is Sufficient To Inhibit Vaccinia Virus Entry and Cell Fusion
Journal of Virology, 2009
associated with mutations affecting the A56 and K2 proteins, which form a multimer (A56/K2) on the surface of infected cells. Recent evidence that A56/K2 interacts with the entry/fusion complex (EFC) and that the EFC is necessary for syncytium formation furnishes a strong connection between virus entry and cell fusion. Among the important remaining questions are whether A56/K2 can prevent virus entry as well as cell-cell fusion and whether these two viral proteins are sufficient as well as necessary for this. To answer these questions, we transiently and stably expressed A56 and K2 in uninfected cells. Uninfected cells expressing A56 and K2 exhibited resistance to fusing with A56 mutant virus-infected cells, whereas expression of A56 or K2 alone induced little or no resistance, which fits with the need for both proteins to bind the EFC. Furthermore, transient or stable expression of A56/K2 interfered with virus entry and replication as determined by inhibition of early expression of a luciferase reporter gene, virus production, and plaque formation. The specificity of this effect was demonstrated by restoring entry after enzymatically removing a chimeric glycophosphatidylinositolanchored A56/K2 or by binding a monoclonal antibody to A56. Importantly, the antibody disrupted the interaction between A56/K2 and the EFC without disrupting the A56-K2 interaction itself. Thus, we have shown that A56/K2 is sufficient to prevent virus entry and fusion as well as formation of syncytia through interaction with the EFC.
Vaccinia Virus Entry into Cells via a Low-pH-Dependent Endosomal Pathway
Journal of Virology, 2006
Previous studies established that vaccinia virus could enter cells by fusion with the plasma membrane at neutral pH. However, low pH triggers fusion of vaccinia virus-infected cells, a hallmark of viruses that enter by the endosomal route. Here, we demonstrate that entry of mature vaccinia virions is accelerated by brief low-pH treatment and severely reduced by inhibitors of endosomal acidification, providing evidence for a predominant low-pH-dependent endosomal pathway. Entry of vaccinia virus cores into the cytoplasm, measured by expression of firefly luciferase, was increased more than 10-fold by exposure to a pH of 4.0 to 5.5. Furthermore, the inhibitors of endosomal acidification bafilomycin A1, concanamycin A, and monensin each lowered virus entry by more than 70%. This reduction was largely overcome by low-pH-induced entry through the plasma membrane, confirming the specificities of the drugs. Entry of vaccinia virus cores with or without brief low-pH treatment was visualized by electron microscopy of thin sections of immunogold-stained cells. Although some virus particles fused with the plasma membrane at neutral pH, 30 times more fusions and a greater number of cytoplasmic cores were seen within minutes after low-pH treatment. Without low-pH exposure, the number of released cores lagged behind the number of virions in vesicles until 30 min posttreatment, when they became approximately equal, perhaps reflecting the time of endosome acidification and virus fusion. The choice of two distinct pathways may contribute to the ability of vaccinia virus to enter a wide range of cells.
Journal of Virology, 2008
to form large multinucleated syncytia. A56 and K2 polypeptides bind to one another (A56/K2) and together are required for interaction with the VACV entry fusion complex (EFC); this association has been proposed to prevent the fusion of infected cells. At least eight viral polypeptides comprise the EFC, but no information has been available regarding their interactions either with each other or with A56/K2. Utilizing a panel of recombinant VACVs designed to repress expression of individual EFC subunits, we demonstrated that A56/K2 interacted with two polypeptides: A16 and G9. Both A16 and G9 were required for the efficient binding of each to A56/K2, suggesting that the two polypeptides interact with each other within the EFC.
Journal of virology, 1998
The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the f...
Vaccinia virus strain differences in cell attachment and entry
Virology, 2009
Vaccinia virus (VACV) strain WR can enter cells by a low pH endosomal pathway or direct fusion with the plasma membrane at neutral pH. Here, we compared attachment and entry of five VACV strains in six cell lines and discovered two major patterns. Only WR exhibited pH 5-enhanced rate of entry following neutral pH adsorption to cells, which correlated with sensitivity to bafilomycin A1, an inhibitor of endosomal acidification. Entry of IHD-J, Copenhagen and Elstree strains were neither accelerated by pH 5 treatment nor prevented by bafilomycin A1. Entry of the Wyeth strain, although not augmented by pH 5, was inhibited by bafilomycin A1. WR and Wyeth were both relatively resistant to the negative effects of heparin on entry, whereas the other strains were extremely sensitive due to inhibition of cell binding. The relative sensitivities of individual vaccinia virus strains to heparin correlated inversely with their abilities to bind to and enter glycosaminoglycan-deficient sog9 cells but not other cell lines tested. These results suggested that that IHD-J, Copenhagen and Elstree have a more limited ability than WR and Wyeth to use the low pH endosomal pathway and are more dependent on binding to glycosaminoglycans for cell attachment.