- Open Access
An ectromelia virus profilin homolog interacts with cellular tropomyosin and viral A-type inclusion protein
© Butler-Cole et al; licensee BioMed Central Ltd. 2007
- Received: 14 May 2007
- Accepted: 24 July 2007
- Published: 24 July 2007
Profilins are critical to cytoskeletal dynamics in eukaryotes; however, little is known about their viral counterparts. In this study, a poxviral profilin homolog, ectromelia virus strain Moscow gene 141 (ECTV-PH), was investigated by a variety of experimental and bioinformatics techniques to characterize its interactions with cellular and viral proteins.
Profilin-like proteins are encoded by all orthopoxviruses sequenced to date, and share over 90% amino acid (aa) identity. Sequence comparisons show highest similarity to mammalian type 1 profilins; however, a conserved 3 aa deletion in mammalian type 3 and poxviral profilins suggests that these homologs may be more closely related. Structural analysis shows that ECTV-PH can be successfully modelled onto both the profilin 1 crystal structure and profilin 3 homology model, though few of the surface residues thought to be required for binding actin, poly(L-proline), and PIP2 are conserved. Immunoprecipitation and mass spectrometry identified two proteins that interact with ECTV-PH within infected cells: alpha-tropomyosin, a 38 kDa cellular actin-binding protein, and the 84 kDa product of vaccinia virus strain Western Reserve (VACV-WR) 148, which is the truncated VACV counterpart of the orthopoxvirus A-type inclusion (ATI) protein. Western and far-western blots demonstrated that the interaction with alpha-tropomyosin is direct, and immunofluorescence experiments suggest that ECTV-PH and alpha-tropomyosin may colocalize to structures that resemble actin tails and cellular protrusions. Sequence comparisons of the poxviral ATI proteins show that although full-length orthologs are only present in cowpox and ectromelia viruses, an ~ 700 aa truncated ATI protein is conserved in over 90% of sequenced orthopoxviruses. Immunofluorescence studies indicate that ECTV-PH localizes to cytoplasmic inclusion bodies formed by both truncated and full-length versions of the viral ATI protein. Furthermore, colocalization of ECTV-PH and truncated ATI protein to protrusions from the cell surface was observed.
These results suggest a role for ECTV-PH in intracellular transport of viral proteins or intercellular spread of the virus. Broader implications include better understanding of the virus-host relationship and mechanisms by which cells organize and control the actin cytoskeleton.
- Respiratory Syncytial Virus
- Inclusion Body
- Root Mean Square Deviation
- Gentle Agitation
- Conjugate Secondary Antibody
Profilins are critical to the cytoskeletal dynamics required for determination of cell shape and size, adhesion, cytokinesis, contractile force, morphogenesis and intracellular transport. Members of the profilin family of proteins are known to be key regulators of actin polymerization in eukaryotic organisms ranging from yeast to mammals, but little is known about profilin homologs found in the poxviridae and paramyxoviridae virus families [1, 2].
Poxviruses are complex viruses with large double-stranded DNA genomes that encode many proteins not required for virus replication in tissue culture . Some non-essential genes are involved in blocking host immune functions, while others function in pathogenesis-related pathways [4, 5]. Most poxvirus genes, in fact, are not universally conserved and, as might be expected, some are found only in phylogenetically related subgroups of the poxvirus family. The poxvirus gene that encodes a homolog of cellular profilin is such a gene and appears to have been acquired by an ancestral orthopoxvirus since it is present in all fully sequenced orthopoxvirus genomes (79 to date; [6, 7]), but absent from all other poxviruses. All of the poxvirus profilin homologs share 90% or greater protein sequence identity (data not shown).
Cellular profilins are believed to interact with three types of cellular molecules: actin monomers, phosphatidylinositol 4,5-bisphosphate (PIP2) and poly(L-proline) sequences . Profilins are thought to modulate actin filament dynamics (polymerization and depolymerization) via direct binding to actin through an actin-binding domain as well as by modulation of other actin-binding proteins . Over 50 proteins have been characterized as profilin ligands . Numerous proteins interact with profilin directly through the poly(L-proline) binding domain, while others may bind indirectly to profilin-regulated complexes or have their activities altered by these complexes . Profilins also assist in signalling between cell membrane receptors and the intracellular microfilament system by their interaction with phosphoinositides . Though many of the interactions with phosphoinositides and profilin-binding proteins remain poorly understood, profilin has been implicated in diverse processes involving actin, nuclear export receptors, endocytosis regulators, Rac and Rho effectors, and putative transcription factors .
In contrast to its cellular homolog, the vaccinia virus profilin-homolog (VACV-PH) binds actin only weakly, has no detectable affinity for poly(L-proline), and, although it has a similar affinity for PIP2 , does not show significant binding to phosphatidyl inositol (PI) or inositol triphosphate (IP3) . Little, therefore, is known about poxviral profilin function. However, RNA interference knockdown studies of the respiratory syncytial virus (RSV) profilin homolog showed that absence of this viral profilin had a small effect on reducing viral macromolecule synthesis and strongly inhibited maturation of progeny virions, cell fusion, and induction of stress fibers . The RSV profilin homolog has been found to interact with RSV phosphoprotein P and nucleocapsid protein N. These interactions are thought to help activate viral RNA-dependent RNA polymerase .
Although the importance of actin filaments in poxvirus motion (and therefore cell-to-cell spread) is well understood, the specific interactions involved are not yet well-characterized [13–16]. Although viral profilin binds actin only weakly, its significant sequence similarity to cellular profilin suggested that it was a possible component in this pathway. Using the murine smallpox model, ectromelia virus, we initiated a search for proteins that interact with the ectromelia profilin homolog, ECTV-PH.
Herein we present evidence that ECTV-PH interacts with cellular α-tropomyosin and both full-length and truncated viral ATI proteins in infected cells and colocalizes to inclusion bodies and protrusions from the cells at putative actin-like tails. Many of the residues important for binding actin and other known mammalian substrates are not conserved in ECTV-PH; however, the ECTV-PH protein can be modelled onto the related structures of mammalian profilins 1 and 3.
Sequence analysis of profilin
Structural Analysis of the ECTV-PH
Root mean square deviation (RMSD) values for the superposition of human profilin 1 with ECTV-PH and human profilin 3. The right-most column gives the number of atoms over which the superposition was made
Number of atoms
Human profilin 1
Human profilin 3
Human profilin 1
Human profilin 3
Comparison of residues important in actin, poly(L-proline) and PIP2 binding in human profilin 1, human profilin 3 and ECTV-PH. Identical and functionally conserved residues are indicated with an asterisk
Residue in human profilin 1
Equivalent residue in human profilin 3
Equivalent residue in ECTV-PH
Function in human profilin 1
Actin and PIP2 binding
Actin and PIP2 binding
Actin and PIP2 binding
Actin and PIP2 binding*
Comparisons with human profilin regarding PIP2 binding are more difficult. A range of binding affinities has been reported for human profilin 1 (0.13 μM < Kd < 35 μM) depending on the experimental method used [2, 10, 23]. Most recently, a dissociation constant of 985 μM was obtained using a relatively more biologically relevant assay that employed sub-micellar concentrations of PIP2 . Because of this uncertainty in the literature, it is difficult to quantitatively compare the affinities of ECTV-PH and human profilin 1 for PIP2. Of the 6 amino acids important for PIP2 binding in human profilin 1 and 3, 5 residues are not conserved in ECTV-PH (Figure 4, Table 2) suggesting that it should have little or no binding affinity to PIP2. Given that Machesky observed a significant binding affinity of ECTV-PH for PIP2, (Kd = 1.3 μM) , it is probable that nearby residues contribute to PIP2 binding. The loop located between beta-strands 5 and 6 of human profilin 1 has been weakly implicated in PIP2 binding , and is substantially smaller in ECTV-PH (Figure 4). It has previously been suggested that a smaller, less obtrusive loop could contribute to a lower binding affinity to PIP2 , and the observed data would seem to fit this hypothesis.
Thus, despite low sequence similarity and lack of conserved binding residues for actin, poly(L-proline), and PIP2, a relatively high level of structural similarity between viral and mammalian profilin is maintained. Further studies may show this structural conservation reflects functional conservation or, alternatively, adaptation of a stable protein structure by the virus for new functionality.
Sequence analysis of viral A-type inclusion proteins
The next step was to investigate the poxviral ATI proteins that interact with ECTV-PH. The majority of orthopoxviruses encode an ATI protein that is expressed late in infection at approximately the same time as the profilin homolog . ATI proteins are present either as a full-length protein, found in cowpox virus (CPXV) and ECTV, or a truncated form of the protein found in most other orthopoxviruses. Full-length ATI proteins form large bodies in the cytoplasm that contain intracellular mature virions (IMV), and are thought to be important in survival and dissemination of the virions [26, 27]. Although the function of truncated ATI proteins is poorly understood, in VACV they do associate with mature virions , and the conservation of these truncated genes suggests the protein does confer an advantage to the virus during its life cycle.
Localization of the ECTV-PH and VACV-WR A-type inclusion proteins in infected cells
Localization of the ECTV-PH and ECTV-Moscow A-type inclusion proteins in infected cells
Taken together, the results of these two immunofluorescence experiments suggest that ECTV-PH localizes to inclusion bodies formed by both truncated and full-length versions of the viral ATI protein in the cytoplasm of the host cell. As the amino (N) terminus and first two tandem repeats are the only domains these proteins share, it is reasonable to conclude that this shared region contains the site of interaction with the profilin homolog. In addition, the colocalization of viral profilin and truncated ATI protein to protrusions from the cell surface suggests that these proteins may together be involved in intercellular transport of the virus.
Localization of the ECTV-PH and cellular tropomyosin proteins in infected cells
The role of tropomyosin is well understood in skeletal muscle, where it regulates the actin-myosin interaction, controlling muscle contraction. However, the role of tropomyosin in the cytoskeleton has remained elusive. Actin filaments vary in composition due to utilization of distinct isoforms of both actin and tropomyosin, which are temporally and spatially regulated . It has been demonstrated that tropomyosin isoforms differentially regulate actin filament function and stability . As ECTV-PH binds tropomyosin and may be involved in actin polymerization, we investigated the localization of ECTV-PH and cellular tropomyosin in poxvirus-infected cells using indirect immunofluorescence.
Intriguingly, some ECTV-PH and the endogenous cellular tropomyosin appear to colocalize in higher concentrations to structures resembling actin tails (Figure 11E, arrows labelled 1); these are known to support extracellular enveloped virus (EEV)-containing protrusions from the cell surface (Figure 11E, arrows labelled 2) that are important for the intercellular spread of poxviruses . ECTV-PH (but not tropomyosin) also localizes in high concentrations to structures resembling inclusion bodies (arrows labelled 3). These are presumably aggregates of the truncated ATI protein encoded by vTF7-3, the recombinant vaccinia virus used in the transient expression system. Though similar to the inclusion bodies formed when the truncated VACV-ATI is overexpressed (Figure 9E arrows 1 and 2), those seen here are more spherical, suggesting that overexpression of the protein may affect the morphology of the putative inclusion bodies.
Summary of the Immunofluorescence Results
Our immunofluorescence results show that full-length ECTV-ATI and ECTV-PH colocalize to inclusion bodies, where IMVs are known to be sequestered . Truncated ATI proteins do not form stable inclusion bodies, and the structures formed are seen to be small and irregularly shaped in our study in agreement with previous work , yet we observed some colocalization of ECTV-PH and VACV-ATI proteins to putative inclusion bodies and protrusions on the cell surface. IMV particles have been shown to travel along microtubules and form intracellular enveloped virus (IEV) particles that then travel to the cell surface . Our results suggest that profilin may be involved with inclusion bodies and IMV transport. Though the immunofluorescence data for tropomyosin are less conclusive, it is possible that tropomyosin and ECTV-PH are also involved in release and/or intercellular transport of viral particles. Because ECTV-PH was overexpressed using a T7 promoter, it is possible that the protein was more widely distributed than when it is expressed at endogenous levels, in effect weakening the visualization of concentrated ECTV-PH and making interpretation of results more difficult.
In a previous study by Blasco et al., cells infected with a deletion mutant of VACV-WR lacking the profilin homolog showed normal plaque formation, infectivity, and IMV/EEV production, movement, and release . This study also showed by fluorescence microscopy that viral profilin does not associate with actin filaments within the infected cell. In agreement with this, we did not detect an interaction between actin and ECTV-PH. However, we did observe associations between ECTV-PH, tropomyosin and ATI proteins at cellular protrusions and putative actin tails. It is possible that VACV profilin used in the Blasco study has functional differences from ECTV-PH used in our study. It would be interesting to perform a similar deletion study with ECTV-PH and visualize viral particle movement using confocal microscopy or immuno-electron microscopy. Although the profilin protein sequences are 90% identical between VACV and ECTV, many genes in the large poxvirus genome are different, including the ATI protein that is full-length in ECTV and truncated in VACV. Another possibility is that VACV profilin is more connected to processes involving PIP2 than those involving actin (proposed by Machesky et al. and Blasco et al. [11, 32]). Our structural analysis (discussed earlier) does not dismiss this possibility, though most of the PIP2 binding residues in the active site are not conserved. Since both the Blasco study and our study were done in cell culture, it is possible that during the natural infection of the host, the associations we observed between ECTV-PH, tropomyosin and ATI protein may become important in actin-associated events. Although not necessarily required for viral particle production and movement, profilin may have a role to play when present in this context.
Taken as a whole, the immunofluorescence results support the idea that ECTV-PH may have some role in intracellular transport of viral proteins in the cytoplasm or intercellular spread of the virus. However, further studies are needed to demonstrate these functions, as well as confirm the colocalization of ECTV and tropomyosin within the cell. Subsequent immunofluorescence studies examining the association of ECTV-PH, tropomyosin, and ATI proteins with viral membrane proteins and actin, and examining the movement of these proteins in infected cells would provide valuable insight.
In this study, we characterize a profilin homolog, ECTV-PH, encoded by ectromelia virus. Poxviruses are known to utilize the cellular cytoskeleton for the transport of virions and viral components during viral infection, although the specific mechanisms are not well understood. The ability of cellular profilin to bind directly to actin and to modulate the activities of other actin-binding proteins makes the viral homolog a candidate for involvement in these processes.
Our investigation provides evidence that suggests viral profilin plays a role in protein or viral particle localization. We show that ECTV-PH associates with viral ATI protein in immunoprecitations of infected cells. Furthermore, immunofluorescence studies show strong colocalization of ECTV-PH with full-length and truncated ATI proteins in inclusion bodies. In the case of truncated ATI protein, colocalization with ECTV-PH also occurs at protrusions from the cell surface. The formation or utilization of these structures that are involved in the protection and spread of the virus may be facilitated by the action of the profilin homolog.
ECTV-PH also directly associates with cellular tropomyosin, an actin-binding protein and regulator of actin filament function and stability. The extent to which ECTV-PH and tropomyosin colocalize in immunoflourescence studies is less clear; however, our results raise the possibility that ECTV-PH and tropomyosin may associate at protrusions from the cell surface and putative actin tails. These data support a potential role for these proteins in intracellular transport of IMVs or viral proteins, or intercellular spread of the virus.
Three-dimensional modelling showed that although the viral profilin homolog shares only 31% and 23% amino acid identity with mammalian profilin 1 and 3 respectively, the overall structure of the proteins are very similar. The lack of conservation of residues known in human profilin to be involved in binding of actin, poly(L-proline), and PIP2 suggests that the function of the poxviral homolog may be quite different from cellular profilins and illustrates how genes that are hijacked by viruses may be rapidly modified and apparently new functions selected for (e.g. binding of viral ATI protein). It is interesting that although the primary sequences have diverged considerably, the structures have to a large degree been maintained. This suggests that there is an inherent value to a stable protein structure that can support a variety of functional interaction surfaces. Also, since cellular profilins are known to interact with multiple protein and phosphoinositide ligands, it is possible that the poxvirus profilin homologs have maintained some of these interactions that were not detected in our immunoprecipitation experiment. Low protein concentration or either weak or transient interactions could result in such interactions being undetected. The conservation of profilin-like genes in current orthopoxviruses (greater than 90% aa identity) indicates that these proteins perform an important function during viral infection. Further characterization of the mechanisms by which poxviruses manipulate the cytoskeleton will not only result in a deeper understanding of the virus-host relationship, but may also give a fresh insight into mechanisms by which uninfected cells organize and control the actin cytoskeleton.
All poxviral protein sequences were obtained from the Viral Orthologous Clusters (VOCs) database [6, 7]. Other sequences were retrieved from GenBank: human profilin 1 (NP_005013), profilin 2 isoform a (NP_444252), profilin 2 isoform b (NP_002619), and profilin 3 (NP_001025057); Bos taurus profilin 1 (NP_001015592), profilin 2 (Q09430), and profilin 3 (NP_001071413); Mus musculus profilin 1 (NP_035202), profilin 2 (NP_062283), and profilin 3 (NP_083579); Rattus norvegicus profilin 1 (NP_071956), profilin 2 (NP_110500), and profilin 3 (XP_001065833). Multiple sequence alignments and percent identity tables were created with Base-By-Base  using the T-Coffee alignment algorithm  with minor manual adjustments to the profilin alignment based on structural analysis.
Phylogenetic trees were constructed from 14 aligned profilin amino acid sequences using the PHYLIP package . The maximum likelihood tree was calculated with the "proml" program, using the Jones-Taylor-Thornton model of amino acid substitution , with a constant rate of change across sites, and allowing global rearrangements. The input sequence order was randomized 5 times, and from the resulting 5 output trees, the highest-scoring tree was selected. The maximum parsimony tree (data not shown) was calculated with the "protpars" program, using ordinary parsimony, with all sites equally weighted. Input order was again randomized 5 times, and the best tree chosen from the 5 trials. Bootstrap values were created using "seqboot" to generate 100 bootstrap samples of the input sequences. For both maximum parsimony and maximum likelihood, trees were computed for all bootstrap data sets using the same parameters as the original data; the consensus trees and bootstrap values were calculated using "consense". Trees were drawn with the "drawtree" program, and edited with the Xfig Drawing Program for the X Window System .
The ECTV-PH primary protein sequence (NP_671660.1) was submitted to the SWISS-MODEL  server using the "First Approach" mode with default settings. The server identified 4 profilin proteins as having a high degree of sequence identity with ECTV-PH based on BLASTp results . These 4 profilins were then used for the ECTV-PH structural model: human platelet profilin 1 (high salt; 1FIL; , human platelet profilin 1 (low salt; 1FIK; ), human profilin NMR structure (1PFL; ), and bovine profilin complexed with beta-actin (1HLU; ). The homology model of ECTV-PH was superimposed and subsequently compared to the crystal structure of human profilin 1 (1FIL) using the MatchMaker feature of the Chimera visualization software . The structural model of ECTV-PH was confirmed by modelling the primary sequence of ECTV-PH using the Robetta protein structure prediction server [20–22] using the human platelet profilin crystal structure as a template (1CJF chain A; ).
The primary protein sequence of the human profilin 3 protein (NP_001025057.1) was also modelled with SWISS-MODEL  using default settings. The SWISS-MODEL server modelled the structure of human profilin 3 based on 4 profilin proteins; human platelet profilin 1 (low salt; 1FIK; ), human platelet profilin 1 (high salt; 1FIL; ), human profilin 1 NMR structure (1PFL; ), and human platelet profilin 1 complexed with a proline-rich ligand (1CF0; ). A structural model for human profilin 2a (data not shown) was created using SWISS-MODEL in the same manner as both ECTV-PH, and human profilin 3, using the following protein crystal structures as templates: human profilin 2b (1D1J; ) and bovine profilin complexed with beta-actin (1HLU; ).
Expression and purification of recombinant proteins
The polymerase chain reaction (PCR) was utilized to amplify the target genes from ECTV-Moscow DNA and incorporate a primer sequence encoding a 6-histidine tag onto the 5' end of each gene. PCR products were cloned into the pENTR/SD/D-Topo entry vector, and then subcloned into the pDEST14 destination vector to generate expression clones as per the manufacturer's instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). A recombinant VACV strain WR vTF7-3 (ATCC VR-2153; ) expressing a T7 polymerase was used to transiently overexpress His-tagged ECTV-PH in BS-C-1 cells. Tissue culture reagents were obtained from Gibco BRL Inc. (Gaithersburg, MD, USA). The African green monkey kidney cell line BS-C-1 (ATCC CCL 26), was grown in complete Dulbecco's modified Eagle medium supplemented with 10% newborn bovine serum, 50 U/ml penicillin, 50 μg/ml streptomycin and GlutaMAX-II (Gibco). BS-C-1 cells were lysed using a French Pressure Cell (American Instrument Company, Silver Spring, Maryland, USA) and His-tagged ECTV-PH was purified using a Ni-NTA column (Invitrogen Life Technologies) with a Bio-Rad Biologic low-pressure chromatography system and a 0–300 mM imidazole elution gradient (Bio-Rad, Richmond, CA, USA). EDTA and glycerol were added to the fractions (final concentrations of 1 mM and 10%, respectively) to prevent degradation and aggregation of the proteins, which were then stored at -20°C. Protein concentrations were determined using Bradford Reagent (Sigma-Aldrich, Oakville, ON, Canada) following manufacturer's instructions.
Purified protein or cellular lysates were separated by SDS-PAGE on pre-cast NuPAGE Novex 4 – 12% Bis-Tris 12 well gels (Cat # NP0322BOX, Invitrogen Life Technologies) using an Xcell SureLock Mini-cell apparatus as per the manufacturer's instructions (Invitrogen Life Technologies). The proteins were transferred to a Trans-Blot nitrocellulose membrane (Cat #12011, Bio-Rad) using a Bio-Rad mini trans-blot cell apparatus as per the manufacturer's instructions (Bio-Rad). The blot was detected with a 1:1500 dilution of primary antibody and a 1:2500 dilution of secondary antibody as follows. His-tagged proteins were detected and visualized using mouse IgG1 anti-penta His primary antibody (Cat # 34660, QIAGEN, Chatsworth, CA, USA) and rabbit anti-mouse IgG (H&L) IRDye 800 conjugate secondary antibody (Cat # 610432020, Rockland Immunochemicals Inc., Gilbertsville, PA, USA). Myc-tagged proteins were detected and visualized using rabbit polyclonal anti-Myc primary antibody (Cat # 2272, Cell Signalling Technology, Beverly, MA, USA) and goat anti-rabbit IgG (H&L) IRDye 800 conjugate secondary antibody (Cat # 611132122, Rockland Immunochemicals Inc.). HA-tagged proteins were detected and visualized using mouse IgG1 anti-HA Alexa Fluor 488 conjugate antibody (Cat # A21287, Molecular Probes Inc., Eugene, OR, USA) and rabbit anti-mouse IgG (H&L) IRDye 800 conjugate secondary antibody (Cat # 610432020, Rockland Immunochemicals Inc.). Actin was detected and visualized using rabbit IgG anti-actin primary antibody (Cat # A5060, Sigma-Aldrich) and goat anti-rabbit IgG (H&L) IRDye 800 conjugate secondary antibody (Cat # 611132122, Rockland Immunochemicals Inc.). Blots were visualized and digitally photographed using the Odyssey Infrared Imaging System (model 9120, Li-COR Biosciences, Lincoln, NB, USA).
BS-C-1 cells were seeded in 9 × 100 mm tissue culture dishes and grown to 90% confluency (approximately 6.3 × 107 cells/dish). Cells were infected with a recombinant VACV strain WR vTF7-3 (ATCC VR-2153) expressing a T7 polymerase, at a multiplicity of infection (MOI) of 10, and then transfected with 25 μg ECTV-Moscow 141 (His-tagged) pDEST14 Expression Clone plasmid DNA per 100 mm dish. After 16 h incubation, cells were washed with PBS and lysed with non-denaturing lysis buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1% Triton ×-100, 10 mM imidazole) containing protease inhibitor cocktail (Cat # 1836153, Roche Applied Science, Indianapolis, IN, USA). After centrifugation at 20,000 ×g for 10 min at 4°C, the lysate supernatant fluid was added to 15 μl Protein G-Plus Agarose (Cat # sc2002, Santa Cruz Biotechnology, Santa Cruz, CA, USA) to pre-clear the extract. Following rotation of the tube at 4°C for 1 h, the Protein G-Plus Agarose was pelleted by centrifuging at 1000 × g for 1 min and Penta-His Antibody (Cat # 34660, mouse Penta-His Antibody IgG1, QIAGEN) was added to the supernatant fluid to a final concentration of 5 μg/ml. The tube was rotated for 3 h at 4°C before 60 μl Protein G-Plus Agarose was added and the incubation continued overnight. After ~ 16 h, the Protein G-Plus Agarose was pelleted by centrifuging at 1000 × g for 1 min and the supernatant fluid was removed. The agarose was washed 4 times in 3 ml of wash buffer (0.1% Triton ×-100, 50 mM Tris-HCl, pH 7.5, 300 mM NaCl) and once with PBS. The pellet was resuspended in 80 μl of 1 × NuPAGE LDS sample buffer (Cat # NP0007, Invitrogen Life Technologies) containing 10 mM DTT (dithiothreitol), then heated to 70°C for 10 min and subjected to SDS-PAGE and western blot. The control used herring sperm DNA instead of pDEST14 expression clone DNA.
Coomassie blue-stained bands were excised from an SDS-PAGE gel using a new scalpel for each band and were prepared and analyzed by the Genome BC Proteomics Centre (Victoria, BC, Canada). The gel slices were subjected to an automated in-gel trypsin digestion, and the proteins obtained were analyzed by MALDI-TOF using a Voyager-DE STR mass spectrometer (Applied Biosystems, Foster City, CA, USA). The Mascot search engine  was used to identify the primary protein sequences of the samples from the mass spectrometry data by searching primary sequence databases.
Far western analysis
2 μg each of porcine muscle tropomyosin (Cat # T2400, Sigma-Aldrich), ECTV-PH (His-tagged, metal chelation chromatography purified), rabbit muscle actin (Cat # A2522, Sigma-Aldrich), RelA (His-tagged bacterial protein, a gift from Dr. Edward Ishiguro, Dept. Biochemistry and Microbiology, University of Victoria), and Bovine Serum Albumin (BSA; Cat # A9647, Sigma-Aldrich) were separated by SDS-PAGE. After transfer to a nitrocellulose membrane, proteins were refolded by a denaturation and renaturation cycle in guanidine hydrochloride as described by Rea et al. . The membrane was washed twice in 50 ml of denaturation buffer (6 M guanidine hydrochloride, 20 mM HEPES pH 7.5, 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.1% Nonidet P-40) for 10 min at 4°C with gentle agitation. The denaturation buffer was diluted 1:1 with basic buffer (20 mM HEPES pH 7.5, 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.1% Nonidet P-40) and the membrane was washed as before. This dilution and wash cycle was repeated four more times until the final wash contained 175 mM guanidinium hydrochloride. Porcine muscle tropomyosin was used as the probe protein, and was diluted to a final concentration of 20 μg/ml in interaction buffer (1% nonfat dry milk in basic buffer, 5% glycerol, 1 mM PMSF) and was incubated with the membrane for 5 h at 4°C with gentle agitation. The tropomyosin solution was removed and the membrane was washed 4 × 10 min in buffer #1 (0.2% Triton ×-100 in PBS) at 4°C with gentle agitation, followed by 2 × 10 min in buffer #2 (0.2% Triton ×-100, 100 mM KCl in PBS) at 4°C with gentle agitation. The membrane was exposed to mouse monoclonal anti-tropomyosin IgG1 primary antibody (Cat # T2780, Sigma-Aldrich) diluted 1:1000 in 1:1 Odyssey Blocking Buffer and PBS + 0.2% TWEEN-20, overnight at 4°C with gentle agitation. The next day the primary antibody was removed and the membrane was washed 4 × 5 min with PBS + 0.1% TWEEN-20. The membrane was then exposed to rabbit anti-mouse IgG (H&L) IRDye 800 conjugate secondary antibody (Cat # 610432020, Rockland Immunochemicals Inc.) at a 1:2500 dilution for 1 h at 4°C with gentle agitation. The secondary antibody was removed and the membrane was washed 4 × 5 min with PBS + 0.1% TWEEN-20 and once with PBS alone. The blot was visualized and digitally photographed using the Odyssey Infrared Imaging System (Li-COR Biosciences).
BS-C-1 cells were grown on cover slips to 80% confluency, infected with recombinant VACV-WR strain vTF7-3 (ATCC VR-2153; MOI = 10), transfected with 200 ng pDEST14 expression clone plasmid DNA per chamber and incubated for approximately 16 h. After removing growth medium, cells were washed once with RT Tris-buffered saline (TBS; 150 mM Tris-HCl pH 7.4, 150 mM NaCl), fixed for 10 min in 4% paraformaldehyde (in PBS), washed 5 min with TBS, permeabilized in 0.2% Triton ×-100 (in PBS) for 5 min at RT and then washed 3 × 5 min each with TBS. Cells were quenched in fresh 0.1% sodium borohydride (in PBS) for 5 min, washed 3 × 5 min with TBS, blocked (in 10% fetal bovine serum, 1% BSA, 0.02% NaN3, in PBS) for 1 h at RT with gentle agitation and finally washed once for 5 min with TBS.
Cells were incubated in primary antibody diluted in 1% BSA (in TBS) overnight at 4°C with gentle agitation, washed 3 × 5 min with TBS and then incubated with secondary antibody diluted in 1% BSA (in TBS) at RT in the dark for 45 min. Proteins containing a Myc tag were visualized using rabbit polyclonal anti-Myc primary antibody (Cat # 2272, Cell Signalling Technology) 1:100 dilution, and Alexa Fluor 568 conjugate goat anti-rabbit IgG (H+L) secondary antibody (Cat # A11011, Molecular Probes) 1:200 dilution. Proteins containing a HA tag were visualized using Alexa Fluor 488 conjugate mouse monoclonal IgG1 anti-HA antibody (Cat # A21287, Molecular Probes) 1:200 dilution. Endogenous cellular tropomyosin was visualized using mouse monoclonal anti-tropomyosin IgG1 primary antibody (Cat # T2780, Sigma-Aldrich) 1:200 dilution, and goat anti-mouse IgG (whole molecule) FITC conjugate secondary antibody (Cat # F2012, Sigma-Aldrich) 1:40 dilution. After incubation with secondary antibody, cells were washed 3 × 5 min each with TBS in low lighting, and DNA was visualized by incubation of cells with DAPI (Cat # D5964, Sigma-Aldrich) at 1 ng/ml in TBS for 5 min in the dark. Controls used herring sperm DNA in place of the expression plasmids. After staining with DAPI, cells were washed 3 × 5 min each with TBS in low lighting and coverslips were mounted on slides using the Prolong Antifade Kit (Cat # P7481, Molecular Probes). Pictures of cells were taken at 1000× magnification using the Leica DM6000 B microscope (Leica Microsystems, Richmond Hill, ON, Canada) using the autoexposure option.
The authors are grateful to Angelika Ehlers for bioinformatics support and to Cristalle Watson for editorial assistance. The authors also thank Shan Sundararaj (current address: Department of Computing Science and Biological Sciences, University of Alberta, Edmonton, Alberta, Canada) for preliminary protein structure analysis. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). The project was supported by funding from the Protein Engineering Network Centre of Excellence and NIH/NAID Contract HHSN266200400036C.
- Bitko V, Oldenburg A, Garmon NE, Barik S: Profilin is required for viral morphogenesis, syncytium formation, and cell-specific stress fiber induction by respiratory syncytial virus. BMC Microbiol. 2003, 3: 9-10.1186/1471-2180-3-9.PubMedPubMed CentralView ArticleGoogle Scholar
- Polet D, Lambrechts A, Vandepoele K, Vandekerckhove J, Ampe C: On the origin and evolution of vertebrate and viral profilins. FEBS Lett. 2007, 581: 211-217. 10.1016/j.febslet.2006.12.013.PubMedView ArticleGoogle Scholar
- Bugert JJ, Darai G: Poxvirus homologues of cellular genes. Virus Genes. 2000, 21: 111-133. 10.1023/A:1008140615106.PubMedView ArticleGoogle Scholar
- Johnston JB, McFadden G: Technical knockout: understanding poxvirus pathogenesis by selectively deleting viral immunomodulatory genes. Cell Microbiol. 2004, 6: 695-705. 10.1111/j.1462-5822.2004.00423.x.PubMedView ArticleGoogle Scholar
- Johnston JB, McFadden G: Poxvirus immunomodulatory strategies: current perspectives. J Virol. 2003, 77: 6093-6100. 10.1128/JVI.77.11.6093-6100.2003.PubMedPubMed CentralView ArticleGoogle Scholar
- Viral Orthologous Clusters database. [http://www.virology.ca]
- Upton C, Slack S, Hunter AL, Ehlers A, Roper RL: Poxvirus orthologous clusters: toward defining the minimum essential poxvirus genome. J Virol. 2003, 77: 7590-7600. 10.1128/JVI.77.13.7590-7600.2003.PubMedPubMed CentralView ArticleGoogle Scholar
- Witke W: The role of profilin complexes in cell motility and other cellular processes. Trends Cell Biol. 2004, 14: 461-469. 10.1016/j.tcb.2004.07.003.PubMedView ArticleGoogle Scholar
- Yarmola EG, Bubb MR: Profilin: emerging concepts and lingering misconceptions. Trends Biochem Sci. 2006, 31: 197-205. 10.1016/j.tibs.2006.02.006.PubMedView ArticleGoogle Scholar
- Moens PDJ, Bagatolli LA: Profilin binding to sub-micellar concentrations of phosphatidylinositol (4,5) bisphosphate and phosphatidylinositol (3,4,5) trisphosphate. Biochim Biophys Acta. 2007, 1768: 439-449. 10.1016/j.bbamem.2006.12.012.PubMedView ArticleGoogle Scholar
- Machesky LM, Cole NB, Moss B, Pollard TD: Vaccinia virus expresses a novel profilin with a higher affinity for polyphosphoinositides than actin. Biochemistry. 1994, 33: 10815-10824. 10.1021/bi00201a032.PubMedView ArticleGoogle Scholar
- Machesky LM, Goldschmidt-Clermont PJ, Pollard TD: Theaffinities of human platelet and Acanthamoeba profilin isoforms for polyphosphoinositides account for their relative abilities to inhibit phospholipase C. Cell Regul. 1990, 1: 937-950.PubMedPubMed CentralGoogle Scholar
- Döhner K, Nagel CH, Sodeik B: Viral stop-and-go along microtubules: taking a ride with dynein and kinesins. Trends Microbiol. 2005, 13: 320-327. 10.1016/j.tim.2005.05.010.PubMedView ArticleGoogle Scholar
- Radtke K, Döhner K, Sodeik B: Viral interactions with the cytoskeleton: a hitchhiker's guide to the cell. Cell Microbiol. 2006, 8: 387-400. 10.1111/j.1462-5822.2005.00679.x.PubMedView ArticleGoogle Scholar
- Smith GL, Law M: The exit of vaccinia virus from infected cells. Virus Res. 2004, 106: 189-197. 10.1016/j.virusres.2004.08.015.PubMedView ArticleGoogle Scholar
- Ward BM, Moss B: Visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-B5R membrane protein chimera. J Virol. 2001, 75: 4802-4813. 10.1128/JVI.75.10.4802-4813.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Schwede T, Kopp J, Guex N, Peitsch MC: SWISS-MODEL: Anautomated protein homology-modeling server. Nucleic Acids Res. 2003, 31: 3381-3385. 10.1093/nar/gkg520.PubMedPubMed CentralView ArticleGoogle Scholar
- Fedorov AA, Pollard TD, Almo SC: Purification, characterization and crystallization of human platelet profilin expressed in Escherichia coli. J Mol Biol. 1994, 241: 480-482. 10.1006/jmbi.1994.1522.PubMedView ArticleGoogle Scholar
- Nodelman IM, Bowman GD, Lindberg U, Schutt CE: X-ray structure determination of human profilin II: A comparative structural analysis of human profilins. J Mol Biol. 1999, 294: 1271-1285. 10.1006/jmbi.1999.3318.PubMedView ArticleGoogle Scholar
- Chivian D, Kim DE, Malmström L, Bradley P, Robertson T, Murphy P, Strauss CEM, Bonneau R, Rohl CA, Baker D: Automated prediction of CASP-5 structures using the Robetta server. Proteins. 2003, 53 (Suppl 6): 524-533. 10.1002/prot.10529.PubMedView ArticleGoogle Scholar
- Chivian D, Kim DE, Malmström L, Schonbrun J, Rohl CA, Baker D: Prediction of CASP6 structures using automated Robetta protocols. Proteins. 2005, 61 (Suppl 7): 157-166. 10.1002/prot.20733.PubMedView ArticleGoogle Scholar
- Kim DE, Chivian D, Baker D: Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res. 2004, 32: W526-W531. 10.1093/nar/gkh468.PubMedPubMed CentralView ArticleGoogle Scholar
- Lu PJ, Shieh WR, Rhee SG, Yin HL, Chen CS: Lipid products of phosphoinositide 3-kinase bind human profilin with high affinity. Biochemistry. 1996, 35: 14027-14034. 10.1021/bi961878z.PubMedView ArticleGoogle Scholar
- Sohn RH, Chen J, Koblan KS, Bray PF, Goldschmidt-Clermont PJ: Localization of a binding site for phosphatidylinositol 4,5-bisphosphate on human profilin. J Biol Chem. 1995, 270: 21114-21120. 10.1074/jbc.270.36.21114.PubMedView ArticleGoogle Scholar
- Meyer H, Rziha HJ: Characterization of the gene encoding the A-type inclusion protein of camelpox virus and sequence comparison with other orthopoxviruses. J Gen Virol. 1993, 74 (Pt 8): 1679-1684.PubMedView ArticleGoogle Scholar
- Martin KH, Franke CA, Hruby DE: Novel acylation of poxvirus A-type inclusion proteins. Virus Res. 1999, 60: 147-157. 10.1016/S0168-1702(99)00013-1.PubMedView ArticleGoogle Scholar
- Ulaeto D, Grosenbach D, Hruby DE: The vaccinia virus 4c and A-type inclusion proteins are specific markers for the intracellular mature virus particle. J Virol. 1996, 70: 3372-3377.PubMedPubMed CentralGoogle Scholar
- Patel DD, Pickup DJ, Joklik WK: Isolation of cowpox virus A-type inclusions and characterization of their major protein component. Virology. 1986, 149: 174-189. 10.1016/0042-6822(86)90119-4.PubMedView ArticleGoogle Scholar
- Osterrieder N, Meyer H, Pfeffer M: Characterization of the gene encoding the A-type inclusion body protein of mousepox virus. Virus Genes. 1994, 8: 125-135. 10.1007/BF01703611.PubMedView ArticleGoogle Scholar
- Gunning PW, Schevzov G, Kee AJ, Hardeman EC: Tropomyosin isoforms: divining rods for actin cytoskeleton function. Trends Cell Biol. 2005, 15: 333-341. 10.1016/j.tcb.2005.04.007.PubMedView ArticleGoogle Scholar
- Gouin E, Welch MD, Cossart P: Actin-based motility of intracellular pathogens. Curr Opin Microbiol. 2005, 8: 35-45. 10.1016/j.mib.2004.12.013.PubMedView ArticleGoogle Scholar
- Blasco R, Cole NB, Moss B: Sequence analysis, expression, and deletion of a vaccinia virus gene encoding a homolog of profilin, a eukaryotic actin-binding protein. J Virol. 1991, 65: 4598-4608.PubMedPubMed CentralGoogle Scholar
- Brodie R, Smith AJ, Roper RL, Tcherepanov V, Upton C: Base-By-Base: single nucleotide-level analysis of whole viral genome alignments. BMC Bioinformatics. 2004, 5: 96-10.1186/1471-2105-5-96.PubMedPubMed CentralView ArticleGoogle Scholar
- Notredame C, Higgins DG, Heringa J: T-Coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol. 2000, 302: 205-217. 10.1006/jmbi.2000.4042.PubMedView ArticleGoogle Scholar
- Felsenstein : PHYLIP – Phylogeny Inference Package (Version3.2). Cladistics. 1989, 5: 164-166.Google Scholar
- Jones DT, Taylor WR, Thornton JM: The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci. 1992, 8: 275-282.PubMedGoogle Scholar
- The Xfig Drawing Program for the X Window System. [http://www.xfig.org]
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215: 403-410.PubMedView ArticleGoogle Scholar
- Metzler WJ, Farmer BT, Constantine KL, Friedrichs MS, Lavoie T, Mueller L: Refined solution structure of human profilin I. Protein Sci. 1995, 4: 450-459.PubMedPubMed CentralView ArticleGoogle Scholar
- Chik JK, Lindberg U, Schutt CE: The structure of an open state of beta-actin at 2.65 A resolution. J Mol Biol. 1996, 263: 607-623. 10.1006/jmbi.1996.0602.PubMedView ArticleGoogle Scholar
- Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE: UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem. 2004, 25: 1605-1612. 10.1002/jcc.20084.PubMedView ArticleGoogle Scholar
- Mahoney NM, Rozwarski DA, Fedorov E, Fedorov AA, Almo SC: Profilin binds proline-rich ligands in two distinct amide backbone orientations. Nat Struct Biol. 1999, 6: 666-671. 10.1038/10722.PubMedView ArticleGoogle Scholar
- Fuerst TR, Niles EG, Studier FW, Moss B: Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci USA. 1986, 83: 8122-8126. 10.1073/pnas.83.21.8122.PubMedPubMed CentralView ArticleGoogle Scholar
- Perkins DN, Pappin DJ, Creasy DM, Cottrell JS: Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis. 1999, 20: 3551-3567. 10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2.PubMedView ArticleGoogle Scholar
- Rea G, Iacovacci P, Ferrante P, Zelli M, Brunetto B, Lamba D, Boffi A, Pini C, Federico R: Refolding of the Cupressus arizonica major pollen allergen Cup a1.02 overexpressed in Escherichia coli. Protein Expr Purif. 2004, 37: 419-425. 10.1016/j.pep.2004.06.034.PubMedView ArticleGoogle Scholar
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