Detection of Nonstructural Protein 6 in Murine Coronavirus-Infected Cells and Analysis of the Transmembrane Topology by Using Bioinformatics and Molecular Approaches

Coronaviruses encode large replicase polyproteins which are proteolytically processed by viral proteases to generate mature nonstructural proteins (nsps) that form the viral replication complex. Mouse hepatitis virus (MHV) replicase products nsp3, nsp4, and nsp6 are predicted to act as membrane anchors during assembly of the viral replication complexes. We report the first antibody-mediated Western blot detection of nsp6 from MHV-infected cells. The nsp6-specific peptide antiserum detected the replicase intermediate p150 (nsp4 to nsp11) and two nsp6 products of approximately 23 and 25 kDa. Analysis of nsp6 transmembrane topology revealed six membrane-spanning segments and a conserved hydrophobic domain in the C-terminal cytosolic tail.Coronaviruses are enveloped, positive-stranded RNA viruses that sequester host cell membranes to assemble viral replication complexes in the cytoplasm of infected cells (2, 21). In the case of murine coronavirus mouse hepatitis virus (MHV), three viral proteases process the replicase polyproteins (3, 4, 5, 9, 12, 13, 14, 16, 18, 19, 24, 26) into intermediates and 16 mature nonstructural protein (nsp) products (Fig. ​(Fig.1A).1A). It is unclear whether the intermediate forms or the mature nsps are responsible for assembly of the viral replication complex. The replicase proteins nsp3, nsp4, and nsp6 contain transmembrane (TM)-spanning sequences that are proposed to be important for sequestering endoplasmic reticulum (ER) membranes to form the double-membrane vesicles which are the site of viral RNA synthesis (11, 17). However, the mechanism used by the nsps to generate double-membrane vesicles is not yet understood. Recent reports (8, 15, 22, 23, 28) and the study presented here have unraveled the membrane topology of these nsps. nsp4 is a glycoprotein with four TM domains (8, 22, 23, 28). nsp3 anchors its 213-kDa multidomain protein to ER membranes, likely using two TM domains (15, 22). Recently, nsp6 was shown to contain six TM domains (22); however, the authors were unable to resolve which of two C-terminal hydrophobic domains can act as the final membrane-spanning region.Open in a separate windowFIG. 1.Schematic diagram of MHV RNA genome, indicating the proteolytic processing scheme of the replicase polyprotein and Western blot detection of MHV nsp6. (A) MHV-A59 linear RNA genome with the canonical representation of replicase, structural, and accessory genes. The replicase polyprotein intermediates and mature nsps generated during processing are depicted. The mature nsp6 replicase protein (hatched box) and the antibodies used to detect nsp6 and nsp8 (solid black boxes) are indicated. aa''s, amino acids. (B) Western blot analysis of nsp6. Whole-cell lysates were prepared from mock-infected (M) and MHV-infected (I) HeLa-MHVR cells, and the lysates were separated by 12.5% SDS-PAGE. Products were detected by probing with nsp6- or nsp8-specific antibodies.In this report, we show the first antibody-mediated detection of MHV-A59 nsp6 in virally infected cells. We also report the TM topology of nsp6, as determined by glycosylation tagging and N-linked glycosylation sequence insertion mutagenesis approaches, providing evidence that nsp6 contains six membrane-spanning segments with a large C-terminal tail exposed to the cytosol. Multiple alignment of the nsp6 amino acid sequences from each coronavirus group revealed a high level of conservation at the C-terminal end, suggesting an evolutionarily conserved function.To detect nsp6 replicase protein in MHV-A59-infected cells, we used a polyclonal rabbit antiserum directed against a peptide (PLGVYNYKISVQEL) from the C-terminal region of nsp6. We detected the replicase intermediate p150 (nsp4 to nsp11) and two nsp6-specific products of 23 and 25 kDa (Fig. ​(Fig.1B,1B, lane 2) in MHV-infected HeLa-MHVR (25) cells by Western blot analysis. We found similar mature products of nsp6 in MHV-infected murine cell lines 17Cl-1 and DBT (data not shown). The same MHV-infected cell lysate was used to detect nsp8 replicase protein with a specific antibody that also recognizes p150 (Fig. ​(Fig.1B,1B, lane 4). The reason for the existence of multiple forms of nsp6 is currently unknown, although posttranslational modification or alternative processing of nsp6 cannot be ruled out at this point. Future experiments will be directed at purification and analysis of the two forms of nsp6 detected here.To develop a framework for understanding the membrane topology of nsp6, we first performed nsp6 bioinformatics analysis. Five out of the seven bioinformatics tools predicted that nsp6 would encode seven TM domains, whereas two programs predicted that it would encode eight TM domains (Fig. ​(Fig.2).2). However, because both the N and C termini of nsp6 must be processed in the cytosol by the viral 3C-like protease (3CLpro), we expected nsp6 to encode an even number of TM domains and established a consensus TM domain prediction for nsp6 (Fig. ​(Fig.2,2, bottom row). The consensus provided a working model for generating plasmid DNA constructs for evaluating the membrane topology of MHV nsp6. First, we employed enhanced green fluorescent protein (EGFP) glycosylation tagging (EGFPglyc) experiments as previously used for determining the membrane topology of other viral replicase TM proteins (20, 22). This approach allowed us to determine the localization of the tagged protein based on the differences in the mobility of the endoglycosidase H (endo H)-treated protein versus that of the untreated protein by the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Based on the consensus topology model (Fig. ​(Fig.3A)3A) suggesting a maximum of eight TM domains, we generated a series of plasmid DNA constructs starting with the N-terminal putative TM1 domain, and successively larger constructs were fused at their carboxyl terminus in frame with EGFPglycV5. The plasmid DNAs were individually transfected into BsrT7 cells (6), and the newly synthesized fusion proteins were radiolabeled with 100 μCi of [³⁵S]methionine-cysteine per ml from 20 to 22 h posttransfection. Chimeric proteins expressed from the cell lysates were immunoprecipitated with V5 antibody, either endo H treated or mock treated, separated using 12.5% SDS-PAGE, and analyzed by autoradiography as described previously (15).Open in a separate windowFIG. 2.Summary of TM predictions for MHV nsp6 obtained from membrane topology bioinformatics tools. The nsp6 amino acid sequence (amino acids 3637 to 3923 in the MHV A59 genome are numbered 1 to 287 for simplicity) was analyzed for TM-spanning domains by the use of various bioinformatics tools, and the residue numbers with predicted TM domains are displayed. The consensus TM topology of MHV nsp6 used as a basis for the topology experiments is depicted at the bottom row (shaded in gray).Open in a separate windowFIG. 3.Determining the topology of nsp6 by the use of EGFPglyc and insertion of glycosylation consensus sites. (A) Schematics of a working topology model of MHV nsp6 (obtained from our consensus experiments) and nsp6-EGFPglycV5 fusion constructs generated for endo H assay. (B) Metabolic labeling and endo H treatment of nsp6-EGFPglycV5 fusion proteins. The nsp6-EGFPglycV5 fusion proteins expressed in transfected BsrT7 cells were radiolabeled from 20 to 22 h posttransfection, and then cell lysates were subjected to immunoprecipitation with V5 antibody, treated with endo H or left untreated, separated by 12.5% SDS-PAGE, and analyzed by autoradiography. (C) Map of plasmid DNA construct showing the sites of inserted glycosylation acceptor consensus sequences (NXS). The locations of glycosylation insertion in the nsp6-V5 construct are represented, with the amino acid number at the site of insertion. (D) Metabolic labeling and endo H analysis of glycosylation sequence insertion expression constructs of nsp6-V5. The plasmid DNAs (iNsp6-V5 constructs) were transfected and analyzed as described for panel B. (E) MHV-A59 nsp6 topology model, summarizing the results of EGFPglycV5 and glycosylation sequence insertion experiments. Amino acid positions indicated by the symbol “Y” were glycosylated and were positive by endo H assay, whereas those positions tested but found not glycosylated and negative by endo H assay are depicted by solid black horizontal lines. The inserted glycosylation acceptor sequence positions precede the letter i. Selected charged residues are shown in white characters on a black background. K, lysine residues; R, arginine residues; E, glutamic acid residues.We found that fusion protein products expressed from the reporter constructs (nsp6-35glycV5, nsp6-86glycV5, and nsp-165glycV5) were glycosylated, as shown by sensitivity to endo H treatment, indicating that the C-terminal end of these chimeric proteins must extend into the ER lumen (Fig. ​(Fig.3B,3B, lanes 4, 8, and 10). In contrast, the remaining reporter constructs were not sensitive to endo H treatment; therefore, the C-terminal end of the chimeric constructs must extend into the cytoplasm (Fig. ​(Fig.3B,3B, lanes 6, 12, 14, 16, and 18). Thus, these results indicate the presence of three luminal loops in nsp6. Identical results were obtained when we used PNGaseF (data not shown), which indicates that the lack of endo H sensitivity was not attributable to the protein transiting through the Golgi body, thereby rendering the protein insensitive to endo H treatment.To further investigate nsp6 topology in detail, we exploited a glycosylation sequence insertion mutagenesis approach (7) to create acceptor sequences in the region between amino acids 86 and 200 of nsp6 by the use of site-directed mutagenesis as described in reference 32 in order to independently investigate the topology, since bioinformatics predictions of the TM domains within this region differ (Fig. ​(Fig.2).2). Consensus glycosylation acceptor sites (NXS) were generated at four sites in the nsp6-V5 plasmid backbone by introducing single-codon insertions as depicted in Fig. ​Fig.3C.3C. All the glycosylation insertion constructs were expressed and analyzed by use of the endo H assay as described above. As expected, the parental nsp6-V5 protein is not glycosylated and did not show a mobility shift after endo H treatment (Fig. ​(Fig.3D,3D, lanes 1 and 2). In contrast, expression of 99iNsp6-V5 revealed evidence of endo H sensitivity (Fig. ​(Fig.3D,3D, lanes 3 and 4), indicating the ER luminal localization of the N99 introduced into MHV nsp6. This result is in agreement with those obtained with the nsp6-86glycV5 construct that is also endo H sensitive (Fig. ​(Fig.3B,3B, lanes 7 and 8). The insertion of glycosylation acceptor sequences at other sites yielded endo H-negative results (lanes 6, 8, and 10), indicating the possibility that the introduced NX(S/T) motifs (i) are localized in the cytosol, (ii) are localized within the membrane, or (iii) are not used, as the glycosylation site is not at least 12 amino acids away from the end of the preceding TM and 14 amino acids away from the beginning of the following TM (12 + 14 rule), thus rendering it inaccessible for glycosylation (1, 7, 30). Our results confirm and extend the results of a recent study (22) in which authors were unable to resolve whether TM6 or TM7 acted as the final TM domain. Our results indicate that TM6 is the final TM domain for MHV nsp6. We propose a topology model of MHV-A59 nsp6 in Fig. ​Fig.3E3E which is in accordance with the distribution of positively charged residues (positive inside rule; reviewed in reference 31), depicting the higher number of lysine and arginine residues facing the cytosolic side of the membrane and the majority of charged residues excluded from the TM domain. Taken together, the results presented above are consistent with a six-TM domain model of MHV nsp6. This report provides new information on the membrane topology of nsp6 and provides potential clues with respect to the assembly of the coronavirus replication complex.To determine whether the experimentally determined six-TM-spanning domain topology of MHV-A59 is conserved among coronaviruses, we performed MUSCLE (10) and ClustalW (29) multiple sequence alignment of nsp6 amino acid sequences representing group 1, group 2, and group 3 coronaviruses obtained from PATRIC (http://patric.vbi.vt.edu/) (27). The most striking observations were the amino acid sequence conservation in the C terminus of all nsp6 proteins and the conservation in the hydrophobicities within the putative TM domains (Fig. ​(Fig.4).4). This analysis revealed several conserved sites that may be important for the function of nsp6. We designated the conserved region between TM2 and TM3 the “KH loop” because of the invariant lysine and histidine residues that are present in the cytosolic loop (Fig. ​(Fig.3E),3E), although the function of these amino acids is not yet known. We also designated the hydrophobic region in the C-terminal tail the “conserved hydrophobic domain” (Fig. ​(Fig.4).4). We speculate that cysteine residue(s) within the region we designated the “conserved G(X)C(X)G motif” may be modified by palmitoylation, indicating that this region of nsp6 may have important functions in establishing protein-protein or protein-membrane interactions during the assembly of the viral replication complex. Additionally, for the MHV-A59 nsp6 protein, the NetPhosK 1.0 server (http://www.cbs.dtu.dk/services/NetPhosK/) predicted serine and tyrosine residues (serine 244 and tyrosine 250; see Fig. ​Fig.4)4) at the C-terminal region as sites of possible phosphorylation by epidermal growth factor receptor kinase and protein kinase C, respectively. Both predicted sites are highly conserved in all coronavirus nsp6 proteins (Fig. ​(Fig.4).4). Overall, our analysis revealed conserved features in the nsp6 C-terminal region whose importance in viral replication can be investigated using a coronavirus reverse genetics system.Open in a separate windowFIG. 4.Multiple sequence alignment (MSA) and percent sequence identity of coronavirus nsp6. The nsp6 amino acid sequences of 18 different coronaviruses were obtained from PATRIC (http://patric.vbi.vt.edu/) and aligned using MUSCLE and ClustalW software. The experimentally determined TM domains of MHV-A59 nsp6 were used as a reference for alignment. Unshaded boxes indicate the conserved TM domains that aligned with other coronavirus nsp6 sequences; the conserved hydrophobic domain (CHD) predicted by all the topology programs is indicated by gray shading. The residues of the peptide against which the nsp6 antibody was raised are boxed, with residue designations shown in boldface. Putative sites for palmitoylation (cysteine residue[s]) within the GXCXG motif) and phosphorylation (serine 244 and tyrosine 250 in MHV-A59 nsp6) are indicated. Percent identity (% ID) values are indicated. In MSA, the following notations were used: asterisk indicate invariant amino acids, colons indicate highly similar amino acids, and dots indicate similar amino acids. HCoV, human coronavirus; PHEV, porcine hemagglutinating encephalomyelitis virus; BCoV, bovine coronavirus; BatSARS, bat severe acute respiratory syndrome coronavirus; BatCoV, bat coronavirus; SARSCoV, severe acute respiratory syndrome coronavirus; FIPV, feline infectious peritonitis virus; PRCoV, porcine respiratory coronavirus; TGEV, transmissible gastroenteritis virus; PEDV, porcine epidemic diarrhea virus; IBV, infectious bronchitis virus.     

Ganglioside GT1b Is a Putative Host Cell Receptor for the Merkel Cell Polyomavirus

The Merkel cell polyomavirus (MCPyV) was identified recently in human Merkel cell carcinomas, an aggressive neuroendocrine skin cancer. Here, we identify a putative host cell receptor for MCPyV. We found that recombinant MCPyV VP1 pentameric capsomeres both hemagglutinated sheep red blood cells and interacted with ganglioside GT1b in a sucrose gradient flotation assay. Structural differences between the analyzed gangliosides suggest that MCPyV VP1 likely interacts with sialic acids on both branches of the GT1b carbohydrate chain. Identification of a potential host cell receptor for MCPyV will aid in the elucidation of its entry mechanism and pathophysiology.Members of the polyomavirus (PyV) family, including simian virus 40 (SV40), murine PyV (mPyV), and BK virus (BKV), bind cell surface gangliosides to initiate infection (2, 8, 11, 15). PyV capsids are assembled from 72 pentamers (capsomeres) of the major coat protein VP1, with the internal proteins VP2 and VP3 buried within the capsids (7, 12). The VP1 pentamer makes direct contact with the carbohydrate portion of the ganglioside (10, 12, 13) and dictates the specificity of virus interaction with the cell. Gangliosides are glycolipids that contain a ceramide domain inserted into the plasma membrane and a carbohydrate domain that directly binds the virus. Specifically, SV40 binds to ganglioside GM1 (2, 10, 15), mPyV binds to gangliosides GD1a and GT1b (11, 15), and BKV binds to gangliosides GD1b and GT1b (8).Recently, a new human PyV designated Merkel cell PyV (MCPyV) was identified in Merkel cell carcinomas, a rare but aggressive skin cancer of neuroendocrine origin (3). It is as yet unclear whether MCPyV is the causative agent of Merkel cell carcinomas (17). A key to understanding the infectious and transforming properties of MCPyV is the elucidation of its cellular entry pathway. In this study, we identify a putative host cell receptor for MCPyV.Because an intact infectious MCPyV has not yet been isolated, we generated recombinant MCPyV VP1 pentamers in order to characterize cellular factors that bind to MCPyV. VP1 capsomeres have been previously shown to be equivalent to virus with respect to hemagglutination properties (4, 16), and the atomic structure of VP1 bound to sialyllactose has demonstrated that the capsomere is sufficient for this interaction (12, 13). The MCPyV VP1 protein (strain w162) was expressed and purified as described previously (1, 6). Briefly, a glutathione S-transferase-MCPyV VP1 fusion protein was expressed in Escherichia coli and purified using glutathione-Sepharose affinity chromatography. The fusion protein was eluted using glutathione and cleaved in solution with thrombin. The thrombin-cleaved sample was then rechromatographed on a second glutathione-Sepharose column to remove glutathione transferase and any uncleaved protein. The unbound VP1 was then chromatographed on a P-11 phosphocellulose column, and peak fractions eluting between 400 and 450 mM NaCl were collected. The purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie blue staining (Fig. ​(Fig.1A,1A, left) and immunoblotting using an antibody (I58) that generally recognizes PyV VP1 proteins (Fig. ​(Fig.1A,1A, right) (9). Transmission electron microscopy (Philips CM10) analysis confirmed that the purified recombinant MCPyV VP1 formed pentamers (capsomeres), which did not assemble further into virus-like particles (Fig. ​(Fig.1B).1B). In an initial screening of its cell binding properties, we tested whether the MCPyV VP1 pentamers hemagglutinated red blood cells (RBCs). The MCPyV VP1 pentamers were incubated with sheep RBCs and assayed as previously described (5). SV40 and mPyV recombinant VP1 pentamers served as negative and positive controls, respectively. We found that MCPyV VP1 hemagglutinated the RBCs with the same efficiency as mPyV VP1 (protein concentration/hemagglutination unit) (Fig. ​(Fig.1C,1C, compare rows B and C from wells 1 to 11), suggesting that MCPyV VP1 engages a plasma membrane receptor on the RBCs. The recombinant murine VP1 protein used for comparison was from the RA strain, a small plaque virus (4). Thus, MCPyV VP1 has the hemagglutination characteristics of a small plaque mPyV (12, 13).Open in a separate windowFIG. 1.Characterization of MCPyV VP1. Recombinant MCPyV VP1 forms pentamers and hemagglutinates sheep RBCs. (A) Coomassie blue-stained SDS-PAGE and an immunoblot of the purified recombinant MCPyV VP1 protein are shown. Molecular mass markers are indicated. (B) Electron micrograph of the purified MCPyV VP1. MCPyV VP1 (shown in panel A) was diluted to 100 μg/ml and absorbed onto Formvar/carbon-coated copper grids. Samples were washed with phosphate-buffered saline, stained with 1% uranyl acetate, and visualized by transmission electron microscopy at 80 kV. Bar = 20 nm. (C) Sheep RBCs (0.5%) were incubated with decreasing concentrations of purified recombinant SV40 VP1 (row A), mPyV VP1 (row B), and MCPyV VP1 (row C). Wells 1 to 11 contain twofold serial dilutions of protein, starting at 2 μg/ml (well 1). Well 12 contains buffer only and serves as a negative control. Well 7 (rows B and C) corresponds to 128 hemagglutination units per 2 μg/ml VP1 protein.To characterize the chemical nature of the putative receptor for MCPyV, total membranes from RBCs were purified as described previously (15). The plasma membranes (30 μg) were incubated with MCPyV VP1 (0.5 μg) and floated on a discontinuous sucrose gradient (15). After fractionation, the samples were analyzed by SDS-PAGE, followed by immunoblotting with I58. VP1 was found in the bottom of the gradient in the absence of the plasma membranes (Fig. ​(Fig.2A,2A, first panel). In the presence of plasma membranes, a fraction of the VP1 floated to the middle of the gradient (Fig. ​(Fig.2A,2A, second panel), supporting the hemagglutination results that suggested that MCPyV VP1 binds to a receptor on the plasma membrane.Open in a separate windowFIG. 2.MCPyV VP1 binds to a protease-resistant, sialic acid-containing receptor on the plasma membrane. (A) Purified recombinant MCPyV VP1 was incubated with or without the indicated plasma membranes. The samples were floated in a discontinuous sucrose gradient, and the fractions were collected from the top of the gradient, subjected to SDS-PAGE, and immunoblotted with the anti-VP1 antibody I58. (B) Control and proteinase K-treated plasma membranes were subjected to SDS-PAGE, followed by Coomassie blue staining. (C) HeLa cells treated with proteinase K (4 μg/ml) were incubated with MCPyV at 4°C, and the resulting cell lysate was probed for the presence of MCPyV VP1. (D) As described in the legend to panel C, except 293T cells were used. (E) Purified MCPyV VP1 was incubated with plasma membranes pretreated with or without α2-3,6,8 neuraminidase and analyzed as described in the legend to panel A.To determine whether the receptor is a protein or a lipid, plasma membrane preparations (30 μg) were incubated with proteinase K (Sigma), followed by analysis with SDS-PAGE and Coomassie blue staining. Under these conditions, the majority of the proteins in the plasma membranes were degraded by the protease (Fig. ​(Fig.2B,2B, compare lanes 1 and 2). Despite the lack of proteins, the proteinase K-treated plasma membranes bound MCPyV VP1 as efficiently as control plasma membranes (Fig. ​(Fig.2A,2A, compare the second and third panels), demonstrating that MCPyV VP1 interacts with a protease-resistant receptor. The absence of VP1 in the bottom fraction in Fig. ​Fig.2A2A (third panel) is consistent with the fact that the buoyant density of the membranes is lowered by proteolysis. Of note, a similar result was seen with binding of the mPyV to the plasma membrane (15). Binding of MCPyV to the cell surface of two human tissue culture cells (i.e., HeLa and 293T) was also largely unaffected by pretreatment of the cells with proteinase K (Fig. 2C and D, compare lanes 1 and 2), further indicating that a nonproteinaceous molecule on the plasma membrane engages the virus.We next determined whether the protease-resistant receptor contains a sialic acid modification. Plasma membranes (10 μg) were incubated with a neuraminidase (α2-3,6,8 neuraminidase; Calbiochem) to remove the sialic acid groups. In contrast to the control plasma membranes, the neuraminidase-treated membranes did not bind MCPyV VP1 (Fig. ​(Fig.2E,2E, compare first and second panels), indicating that the MCPyV receptor includes a sialic acid modification.Gangliosides are lipids that contain sialic acid modifications. We asked if MCPyV VP1 binds to gangliosides similar to other PyV family members. The structures of the gangliosides used in this analysis (gangliosides GM1, GD1a, GD1b, and GT1b) are depicted in Fig. ​Fig.3A.3A. To assess a possible ganglioside-VP1 interaction, we employed a liposome flotation assay established previously (15). When liposomes (consisting of phosphatidyl-choline [19 μl of 10 mg/ml], -ethanolamine [5 μl of 10 mg/ml], -serine [1 μl of 10 mg/ml], and -inositol [3 μl of 10 mg/ml]) were incubated with MCPyV VP1 and subjected to the sucrose flotation assay, the VP1 remained in the bottom fraction (Fig. ​(Fig.3B,3B, first panel), indicating that VP1 does not interact with these phospholipids. However, when liposomes containing GT1b (1 μl of 1 mM), but not GM1 (1 μl of 1 mM) or GD1a (1 μl of 1 mM), were incubated with MCPyV VP1, the vesicles bound this VP1 (Fig. ​(Fig.3B).3B). A low level of virus floated partially when incubated with liposomes containing GD1b (Fig. ​(Fig.3B),3B), perhaps reflecting a weak affinity between MCPyV and GD1b. Importantly, MCPyV binds less efficiently to neuraminidase-treated GT1b-containing liposomes than to GT1b-containing liposomes (Fig. ​(Fig.3B,3B, sixth panel), suggesting that the GT1b sialic acids are involved in virus binding. This finding is consistent with the ability of neuraminidase to block MCPyV binding to the plasma membrane (Fig. ​(Fig.2E).2E). The level of virus flotation observed in the neuraminidase-treated GT1b-containing liposomes is likely due to the inefficiency of the neuraminidase reaction with a high concentration of GT1b used to prepare the vesicles.Open in a separate windowFIG. 3.MCPyV VP1 binds to ganglioside GT1b. (A) Structures of gangliosides GM1, GD1a, GD1b, and GT1b. The nature of the glycosidic linkages is indicated. (B) Purified MCPyV VP1 protein was incubated with liposomes only or with liposomes containing the indicated gangliosides. The samples were analyzed as described in the legend to Fig. ​Fig.2A.2A. Where indicated, GT1b-containing liposomes were pretreated with α2-3,6,8 neuraminidase and analyzed subsequently for virus binding. (C to E) The indicated viruses were incubated with liposomes and analyzed as described in the legend to panel B.As controls, GM1-containing liposomes bound SV40 (Fig. ​(Fig.3C),3C), GD1a-containing liposomes bound mPyV (Fig. ​(Fig.3D),3D), and GD1b-containing liposomes bound BKV (Fig. ​(Fig.3E),3E), demonstrating that the liposomes were functionally intact. We note that, while all of the MCPyV VP1 floated when incubated with liposomes containing GT1b (Fig. ​(Fig.3B,3B, sixth panel), a significant fraction of SV40, mPyV, and BKV VP1 remained in the bottom fraction despite being incubated with liposomes containing their respective ganglioside receptors (Fig. 3C to E, second panels). This result is likely due to the fact that in contrast to MCPyV, which are assembled as pentamers (Fig. ​(Fig.1B),1B), the SV40, mPyV, and BKV used in these experiments are fully assembled particles: their larger and denser nature prevents efficient flotation. Nonetheless, we conclude that MCPyV VP1 binds to ganglioside GT1b efficiently.The observation that GD1a does not bind to MCPyV VP1 suggests that the monosialic acid modification on the right branch of GT1b (Fig. ​(Fig.3A)3A) is insufficient for binding. Similarly, the failure of GD1b to bind MCPyV VP1 suggests that the sialic acid on the left arm of GT1b is necessary for binding. Together, these observations suggest that MCPyV VP1 interacts with sialic acids on both branches of GT1b (Fig. ​(Fig.4).4). A recent structure of SV40 VP1 in complex with the sugar portion of GM1 (10) demonstrated that although SV40 VP1 binds both branches of GM1 (Fig. ​(Fig.4),4), only a single sialic acid in GM1 is involved in this interaction. In the case of mPyV, structures of mPyV VP1 in complex with different carbohydrates (12, 13) revealed that the sialic acid-galactose moiety on the left branch of GD1a (and GT1b) is sufficient for mPyV VP1 binding (Fig. ​(Fig.4).4). Although no structure of BKV in complex with the sugar portion of GD1b (or GT1b) is available, in vitro binding studies (8) have suggested that the disialic acid modification on the right branch of GD1b (and GT1b) is responsible for binding BKV VP1 (Fig. ​(Fig.4).4). Thus, it appears that the unique feature of the MCPyV VP1-GT1b interaction is that the sialic acids on both branches of this ganglioside are likely involved in capsid binding.Open in a separate windowFIG. 4.A potential model of the different VP1-ganglioside interactions (see the text for discussion).The identification of a potential cellular receptor for MCPyV will facilitate the study of its entry mechanism. An important issue for further study is to determine whether MCPyV targets Merkel cells preferentially, and if so, whether GT1b is found in higher levels in these cells to increase susceptibility.     

Acetolactate Synthase from Bacillus subtilis Serves as a 2-Ketoisovalerate Decarboxylase for Isobutanol Biosynthesis in Escherichia coli

A pathway toward isobutanol production previously constructed in Escherichia coli involves 2-ketoacid decarboxylase (Kdc) from Lactococcus lactis that decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde. Here, we showed that a strain lacking Kdc is still capable of producing isobutanol. We found that acetolactate synthase from Bacillus subtilis (AlsS), which originally catalyzes the condensation of two molecules of pyruvate to form 2-acetolactate, is able to catalyze the decarboxylation of KIV like Kdc both in vivo and in vitro. Mutational studies revealed that the replacement of Q487 with amino acids with small side chains (Ala, Ser, and Gly) diminished only the decarboxylase activity but maintained the synthase activity.We have previously shown that 2-keto acids generated from amino acid biosynthesis can serve as precursors for the Ehrlich degradation pathway (15) to higher alcohols (3). In order to produce isobutanol, the valine biosynthesis pathway was used to generate 2-ketoisovalerate (KIV), the precursor to valine, which was then converted to isobutanol via a decarboxylation and reduction step (Fig. ​(Fig.1A).1A). The entire pathway to isobutanol from glucose is shown in Fig. ​Fig.1A.1A. To produce isobutanol, we overexpressed five genes, alsS (Bacillus subtilis), ilvC (Escherichia coli), ilvD (E. coli), kdc (Lactococcus lactis), and ADH2 (Saccharomyces cerevisiae) (Fig. ​(Fig.1A).1A). This E. coli strain produced 6.8 g/liter isobutanol in 24 h (Fig. ​(Fig.1B)1B) and more than 20 g/liter in 112 h (3). More recently, we have found that an alcohol dehydrogenase (Adh) encoded by yqhD on the E. coli genome can convert isobutyraldehyde to isobutanol efficiently (5) (Fig. ​(Fig.1B1B).Open in a separate windowFIG. 1.Schematic representation of the pathway for isobutanol production. (A) The Kdc-dependent synthetic pathway for isobutanol production. (B) Isobutanol production with the Kdc-dependent and -independent synthetic pathways. IlvC, acetohydroxy acid isomeroreductase; IlvD, dihydroxy acid dehydratase. (C) Enzymatic reaction of Als, Ahbs, and Kdc activities.One key reaction in the production of isobutanol is the conversion of KIV to isobutyraldehyde catalyzed by 2-ketoacid decarboxylase (Kdc) (Fig. ​(Fig.1C).1C). Since E. coli does not have Kdc, kdc from L. lactis was overexpressed. Kdc is a nonoxidative thiamine PP_i (TPP)-dependent enzyme and is relatively rare in bacteria, being more frequently found in plants, yeasts, and fungi (8, 19). Several enzymes with Kdc activity have been found, including pyruvate decarboxylase, phenylpyruvate decarboxylase (18), branched-chain Kdc (8, 19), 2-ketoglutarate decarboxylase (10, 17, 20), and indole-3-pyruvate decarboxylase (13).In this work, unexpectedly, we find that Kdc is nonessential for E. coli to produce isobutanol (Fig. ​(Fig.1).1). An E. coli strain overexpressing only alsS (from B. subtilis), ilvC, and ilvD (both from E. coli) is still able to produce isobutanol. Since E. coli is not a natural producer of isobutanol, it cannot be detected from the culture media in any unmodified strain. We identify that AlsS from B. subtilis, which was introduced in E. coli for acetolactate synthesis (Als), catalyzes the decarboxylation of 2-ketoisovalerate like Kdc both in vivo and in vitro. AlsS is part of the acetoin synthesis pathway and catalyzes the aldo condensation of two molecules of pyruvate to 2-acetolactate (Als activity) (Fig. ​(Fig.1C)1C) (11). The overall reaction catalyzed by AlsS is irreversible because of CO₂ evolution. The first step in catalysis is the ionized thiazolium ring of TPP reacting with the first pyruvate, followed by decarboxylation. This intermediate then reacts with the second pyruvate. Deprotonation followed by C-C bond breakage produces 2-acetolactate. In this work, mutational approaches were used to assess the importance of Q487 in the Kdc activity of AlsS.     

A Conserved Gene Cluster Rules Anaerobic Oxidative Degradation of l-Ornithine

For the ornithine fermentation pathway, described more than 70 years ago, genetic and biochemical information are still incomplete. We present here the experimental identification of the last four missing genes of this metabolic pathway. They encode l-ornithine racemase, (2R,4S)-2,4-diaminopentanoate dehydrogenase, and the two subunits of 2-amino-4-ketopentanoate thiolase. While described only for the Clostridiaceae to date, this pathway is shown to be more widespread.The catabolism of ornithine by anaerobic bacteria can be accomplished through the Stickland reaction, the main chemical reaction by which Clostridium sporogenes obtains its energy (16, 17). The Stickland reaction usually involves one amino acid which acts as an electron donor while another acts as an electron acceptor, as described for Clostridium sporogenes (16, 20), Clostridium botulinum (4, 5), and Clostridium sticklandii (6, 7). However, l-ornithine, as a single substrate, is converted into both an electron donor and acceptor and metabolized in a way similar to the Stickland reaction: it is oxidized to acetate, alanine, and ammonia (oxidative pathway) and reduced to 5-aminovalerate through the formation of proline (reductive pathway) (Fig. ​(Fig.1).1). This study focuses on the oxidative degradation pathway, starting with the conversion of l-ornithine to the d isomer by ornithine racemase (OR) (EC 5.1.1.12) (Fig. ​(Fig.1,1, step 1) (2). d-Ornithine is next converted to (2R,4S)-2,4-diaminopentanoate (DAP) through the action of d-ornithine aminomutase (OA) (EC 5.4.3.5) (Fig. ​(Fig.1,1, step 2), an adenosylcobalamine and pyridoxal phosphate (PLP)-dependent enzyme (3, 14). DAP then undergoes a NAD⁺- or NADP⁺-dependent oxidative deamination by DAP dehydrogenase (DAPDH) (EC 1.4.1.12) (Fig. ​(Fig.1,1, step 3), leading to 2-amino-4-ketopentanoate (AKP) (13, 18). This compound is metabolized by AKP thiolase (AKPT), a PLP-dependent enzyme, through a thiolytic cleavage with coenzyme A (CoA) to form acetyl-CoA and alanine (Fig. ​(Fig.1,1, step 4) (9).Open in a separate windowFIG. 1.The ornithine fermentation pathway. Enzymes involved are OR (encoded by or-5) (EC 5.1.1.12) in step 1, OA (encoded by oraS and oraE) (EC 5.4.3.5) in step 2, DAPDH (encoded by or-1) (EC 1.4.1.12) in step 3, AKPT (encoded by or-2 and or-3) in step 4, ornithine transaminase (EC 2.6.1.13) in step 5, spontaneous in step 6, pyrroline-5-carboxylate reductase (EC 1.5.1.2) in step 7, proline racemase (EC 5.1.1.4) in step 8, d-proline reductase (EC 1.21.4.1) in step 9, and ornithine cyclodeaminase (EC 4.3.1.12) in step 10.Although the proteins of this oxidative pathway were characterized biochemically 30 years ago for C. sticklandii, only the genes corresponding to the two subunits of OA (oraS and oraE) have been identified to date (3). In this article, we present the analysis of genes which are colocalized with oraS and oraE and which are hypothesized to be involved in the conversion of l-ornithine to d-ornithine, the oxidative deamination of DAP, and the thiolytic cleavage of AKP. The proteins encoded by these genes were purified and their enzymatic activity characterized, which made it possible to reconstitute the whole oxidative branch of the l-ornithine fermentation pathway in vitro. The occurrence of this oxidative metabolic pathway in bacterial genomes which have been sequenced to date is discussed.     

Molecular Chaperone BiP Interacts with Borna Disease Virus Glycoprotein at the Cell Surface

Borna disease virus (BDV) is characterized by highly neurotropic infection. BDV enters its target cells using virus surface glycoprotein (G), but the cellular molecules mediating this process remain to be elucidated. We demonstrate here that the N-terminal product of G, GP1, interacts with the 78-kDa chaperone protein BiP. BiP was found at the surface of BDV-permissive cells, and anti-BiP antibody reduced BDV infection as well as GP1 binding to the cell surface. We also reveal that BiP localizes at the synapse of neurons. These results indicate that BiP may participate in the cell surface association of BDV.Borna disease virus (BDV) belongs to the Bornaviridae family of nonsegmented, negative-strand RNA viruses and is characterized by highly neurotropic and noncytopathic infection (18, 33). BDV infects a wide variety of host species and causes central nervous system (CNS) diseases in animals, which are frequently associated with behavioral disorders (14, 19, 29, 31). BDV cell entry is mediated by endocytosis, following the attachment of viral envelope glycoprotein (G) to the cellular receptor (2, 7, 8). BDV G is translated as a precursor protein, GP, which is posttranslationally cleaved by the cellular protease furin to generate two functional subunits of the N (GP1) and C (GP2) termini (28). Recent studies revealed that GP1 is involved in virus interaction with as-yet-unidentified cell surface receptor(s) and that GP2 mediates a pH-dependent fusion event between viral and cell membranes (2, 7, 27). In addition, a previous work using a hippocampal culture system suggested that BDV G is required for viral dissemination in neurons (2); however, cellular factors involved in BDV cell entry, especially cell surface association, remain to be elucidated.To extend our understanding of the role of BDV G in the interaction with the cell plasma membrane, we transfected GP1 fused with hemagglutinin-tobacco etch virus protease cleavage site-FLAG tags (GP1-TAP) into human oligodendroglioma OL cells. GP1-TAP was purified using anti-FLAG M2 affinity gel (Sigma). To verify that GP1-TAP binds to OL cells, the cells were incubated with 4 μg/ml GP1-TAP, and binding was detected by anti-FLAG M2 antibody (Sigma). A flow cytometric analysis indicated that GP1-TAP binds to OL cells (Fig. ​(Fig.1A).1A). To further validate the binding of GP1-TAP, we tested whether GP1-TAP inhibits BDV infection. OL cells were pretreated with 4 μg/ml GP1-TAP for 30 min. Proteins purified from mock-transfected cells using an anti-FLAG M2 affinity gel served as a control. The cells were then mixed with cell-free BDV. After 1 h of absorption, the supernatants were removed and fresh medium was added. At 3 days postinfection, the viral antigens were stained with anti-nucleoprotein (N) monoclonal and anti-matrix (M) polyclonal antibodies. As shown in Fig. ​Fig.1B,1B, GP1-TAP reduced BDV infection by 40% compared to levels for mock-treated cells. This result was consistent with earlier reports showing that recombinant GP1 protein binds to the cell surface and inhibits BDV infection (6, 20).Open in a separate windowFIG. 1.BDV GP1 binds to the cell surface. (A) Binding of BDV GP1 to OL cells. OL cells were incubated with GP1-TAP (solid line), and its binding was detected using anti-FLAG M2 antibody and flow cytometry. As a control, cells incubated with proteins purified from mock-transfected cells were detected by an anti-FLAG M2 antibody (dotted line). (B) Inhibition of BDV infection by GP1. OL cells pretreated with GP1-TAP were inoculated with the BDV huP2br strain. Values are the means + standard deviations (SD) from three independent experiments. **, P < 0.01.To investigate the host factor(s) that mediates the interaction of GP1 with the cell surface, a combination of tandem affinity purification (TAP) and liquid chromatography tandem mass spectrometry analyses was designed (13). We transfected GP1-TAP into OL cells and then purified GP1 from cell homogenates using a TAP strategy. We compared the purified proteins from the whole-cell and cytosol fractions (Fig. ​(Fig.2A),2A), and the bands detected only in the whole-cell fraction were determined as GP1-binding proteins in the membrane and/or nuclear fractions. In addition to GP1 protein (Fig. ​(Fig.2A,2A, arrow), we identified a specific band around 80 kDa in the whole-cell homogenate, but not in the cytosol fraction (Fig. ​(Fig.2A,2A, arrowhead), and determined that the band corresponded to the BiP (immunoglobulin heavy chain-binding protein) molecular chaperone, also called glucose-regulated protein 78 (GRP78), by mass spectrometry analysis. We confirmed the specific interaction between endogenous BiP and BDV G in infected cells by immunoprecipitation analysis (Fig. ​(Fig.2B).2B). To map the binding domain on BiP to GP1, we constructed a series of deletion mutants of the green fluorescent protein (GFP)-tagged BiP plasmid (Fig. ​(Fig.2C).2C). We transfected the mutant plasmids into BDV-infected OL cells and then performed an immunoprecipitation assay using anti-GFP antibody (Invitrogen). As shown in Fig. ​Fig.2D,2D, BDV G was coimmunoprecipitated with truncated BiP mutants, except for BiPΔN-GFP, which lacks the ATP-binding domain of BiP (lane 3), suggesting that BiP interacts with GP1 via its N-terminal region.Open in a separate windowFIG. 2.BDV GP1 interacts with BiP molecular chaperone. (A) TAP analysis of BDV GP1. Proteins coimmunoprecipitated with GP1-TAP in OL cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by silver staining. Cyt, cytosol fraction; Wc, whole-cell homogenate. Arrow, GP1-TAP; arrowhead, BiP. (B) Coimmunoprecipitation (IP) of BDV G and endogenous BiP. BDV G was immunoprecipitated from BDV-infected OL cells by anti-BDV G polyclonal antibody. Endogenous BiP was then detected by anti-BiP monoclonal antibody (Becton Dickinson). IgG, immunoglobulin G. (C) Schematic representation of deletion mutants of recombinant BiP-GFP. The known functional regions are indicated. (D) Immunoprecipitation analysis of BiP-GFP mutants in BDV-infected OL cells. The deletion plasmids were transfected and immunoprecipitated by anti-GFP antibody. Specific binding was detected using anti-BDV G antibody. Lane 1, GFP; lane 2, BiP-GFP; lane 3, BiPΔN-GFP; lane 4, BiPΔPB-GFP; lane 5, BiPΔC-GFP.BiP is known to be resident primarily in the endoplasmic reticulum and functions as a molecular chaperone involved in the folding process of nascent proteins, mostly through interaction with its peptide-binding domain (12, 17, 21). On the other hand, BiP has been reported to serve as a coreceptor of certain viruses at the plasma membrane (15, 34). Recent studies also revealed that cell surface BiP mediates the internalization of its ligands into cells (1, 10). We first investigated whether BiP is expressed on the cell surface of BDV-permissive OL and 293T cells using an anti-BiP polyclonal antibody (H-129; Santa Cruz Biotechnology, Inc.). As shown in Fig. ​Fig.3A,3A, BiP expression is detected on the surface of both cell lines. This result is in agreement with recent observations that BiP is expressed on the surface of various types of cells (9, 10, 15, 23, 24, 34). We also investigated whether BiP is expressed on the cell surface of BDV-nonpermissive cell lines, such as HeLa and CHO cells. As shown in Fig. ​Fig.3A,3A, we detected BiP expression on the surface of HeLa, but not CHO, cells. These observations were confirmed by immunofluorescence analysis (Fig. ​(Fig.3B).3B). Note that BiP is clearly detected at the endoplasmic reticulum in the permeabilized CHO cells by the antibody (see Fig. S1 in the supplemental material), suggesting that BiP is expressed at a very low level, if at all, on the surface of CHO cells. We next examined whether cell surface BiP serves as a binding molecule of BDV GP1. To test this, we performed an inhibition assay using an anti-BiP polyclonal antibody (N-20; Santa Cruz Biotechnology, Inc.) which recognizes the N terminus of BiP. As shown in Fig. ​Fig.3C,3C, the antibody inhibited GP1 binding to the cell surface by 40%. Furthermore, BDV infection was found to decrease by 70% when cells were treated with the antibody (Fig. ​(Fig.3D3D).Open in a separate windowFIG. 3.Cell surface BiP mediates cell association of BDV. (A) Flow cytometric analysis was performed with anti-BiP antibody (H-129) in BDV-permissive (OL and 293T) and -nonpermissive (HeLa and CHO) cells (solid lines). Cells stained with normal rabbit immunoglobulin G were used as a control (dotted lines). (B) Immunofluorescence analysis was performed by using anti-BiP antibody (H-129) with BDV-permissive and -nonpermissive cells. Arrows indicate BiP staining at the membrane. Scale bars, 10 μm. (C) Inhibition of GP1 binding by anti-BiP antibody (N-20). OL cells were pretreated with anti-BiP antibody, followed by labeling with GP1. GP1 binding on the cell surface was detected using flow cytometry. Values are the means + SD from three independent experiments. *, P < 0.05. (D) Inhibition of BDV infection by anti-BiP antibody. OL cells were incubated with 10 μg/ml anti-BiP antibody or normal goat immunoglobulin G and then the cells were mixed with cell-free BDV. After 1 h absorption, the supernatants were replaced with fresh medium. Virus infection was measured by immunofluorescence analysis using anti-N and -M antibodies at 3 days postinfection. Values are the means + SD from three independent experiments. *, P < 0.05. IgG, immunoglobulin G.To investigate the role of cell surface BiP in the infection of BDV, the BiP expression was inhibited by short interfering RNA (siRNA) in OL cells (see Fig. S2A in the supplemental material). We selected an siRNA (Hs_HSPA5_4; Qiagen, Inc.) which could partially downregulate the cell surface expression of BiP (see Fig. S2B in the supplemental material). However, siRNA treatment of BiP did not influence the infectivity of BDV in OL cells (see Fig. S2C in the supplemental material). This may be due to an incomplete reduction of BiP expression on the cell surface. Alternatively, while BiP mediates at least in part the cell surface association of BDV particles, this result may exhibit the presence of another, as-yet-unidentified BDV G-binding protein that is involved in the binding and subsequent cell entry of BDV.Previous studies demonstrated that BDV can be traced centripetally and transsynaptically after olfactory, ophthalmic, or intraperitoneal inoculation (3, 25). Migration of BDV to the CNS after footpad infection can be prevented by sciatic nerve transection (3). These observations suggest that BDV may disseminate primarily via neural networks. Recently, it has been demonstrated that BDV G was expressed at the termini of neurites or at contact sites of neurites (2), suggesting that local assembly of BDV may take place at the presynaptic terminals of synapses, similar to assembly of other neurotropic viruses (22, 26, 32). If BiP localizes at synapse sites, BiP may efficiently participate in the transmission of BDV particles at the synapses. To evaluate this hypothesis, we examined BiP localization in primary culture of mouse hippocampal neurons. After in vitro culture for 17 days, BiP localization was determined by an immunofluorescence assay without permeabilization. As shown in Fig. ​Fig.4A,4A, BiP signals were clearly detected at neurites, including the contact sites between dendrites and axons, as punctate staining (arrows), suggesting that BiP is expressed at the neuronal surface, most likely at the synapses. We next examined the localization of BiP with postsynaptic density 95 (PSD-95), a marker of postsynaptic density (5). Although BiP signals were detected mainly in the perinuclear area of the hippocampal neurons, punctate staining was also found at neurites colocalized with PSD-95 (Fig. ​(Fig.4B,4B, arrows). Taken together, these observations suggested that BiP is distributed at the synaptic surface, including the postsynaptic membrane, of neurons, a possible site for BDV budding and entry (2).Open in a separate windowFIG. 4.BiP localizes at the synaptic surface of hippocampus neurons. (A) Localization of BiP at synaptic surface. Hippocampal neurons were immunostained with anti-BiP antibody (N-20) without permeabilization. A differential interference contrast (DIC) image is shown. Dotted lines in the Merge panel indicate the dendrite outline. Arrows indicate BiP staining at the contact sites between axons and dendrites. (B) Colocalization between BiP and a postsynaptic protein. Hippocampal neurons were immunostained with anti-BiP (N-20) and anti-PSD-95 (Millipore) antibodies. Arrows indicate colocalized signals of BiP and PSD-95 at neurites. Scale bars, 10 μm.In summary, this study demonstrates that BiP is a GP1-binding protein at the synaptic surface. This is the first report showing the BDV G-binding factor on the cell surface. The first step of BDV entry might be mediated by the interaction of GP1 with as-yet-unidentified cell surface receptors, which may form a complex with other molecules, such as BiP. We showed that treatment with anti-BiP antibody affects BDV infection as well as GP1 binding to the cell surface (Fig. ​(Fig.3).3). Furthermore, synaptic distribution of BiP was found in hippocampal primary neurons (Fig. ​(Fig.4).4). These findings strongly suggest that BiP plays critical roles in BDV association with the neuronal surface via interaction with GP1. On the other hand, a BDV-nonpermissive cell line, HeLa, appeared to express BiP on the cell surface, suggesting that the cell surface BiP may not be necessarily involved in the infectivity of BDV. A recent study by Clemente et al. (6) revealed that following initial attachment to the cell surface, BDV is recruited to the plasma membrane lipid raft (LR) prior to internalization of the particles. The study suggested that BDV may use the LR as a platform to interact with additional host cell factor(s) required for efficient BDV internalization. Because BiP does not contain transmembrane regions, BiP needs another host protein(s) with transmembrane regions on the cell surface. It has been reported that cell surface BiP interacts with diverse proteins, such as major histocompatibility complex class I molecules (34), the voltage-dependent anion channel (9), and the DnaJ-like protein MTJ-1 (4), all of which associate with LR in the plasma membrane (16, 24, 35). Once BDV has attached to the cell surface, it might utilize such BiP-associated LR proteins for efficient cell surface attachment or internalization. Previously, it has been proposed that kainate 1 (KA-1) receptor might represent the BDV receptor within the CNS (11). Because some glutamate receptors are shown to bind to BiP (30), KA-1 receptors might interact with BiP and serve as a receptor complex for BDV. Further studies are required for a full understanding of the cell association processes, especially receptor binding, of BDV.      

Antibody Recognition Force Microscopy Shows that Outer Membrane Cytochromes OmcA and MtrC Are Expressed on the Exterior Surface of Shewanella oneidensis MR-1

Antibody recognition force microscopy showed that OmcA and MtrC are expressed on the exterior surface of living Shewanella oneidensis MR-1 cells when Fe(III), including solid-phase hematite (Fe₂O₃), was the terminal electron acceptor. OmcA was localized to the interface between the cell and mineral. MtrC displayed a more uniform distribution across the cell surface. Both cytochromes were associated with an extracellular polymeric substance.Shewanella oneidensis MR-1 is a dissimilatory metal-reducing bacterium that is well known for its ability to use a variety of anaerobic terminal electron acceptors (TEAs), including solid-phase iron oxide minerals, such as goethite and hematite (8, 10). Previous studies suggest that S. oneidensis MR-1 uses outer membrane cytochromes OmcA and MtrC to catalyze the terminal reduction of Fe(III) through direct contact with the extracellular iron oxide mineral (2, 8, 10, 15, 16, 20, 21, 23). However, it has yet to be shown whether OmcA or MtrC is actually targeted to the external surface of live S. oneidensis MR-1 cells when Fe(III) serves as the TEA.In the present study, we used atomic force microscopy (AFM) to probe the surface of live S. oneidensis MR-1 cells, using AFM tips that were functionalized with cytochrome-specific polyclonal antibodies (i.e., anti-OmcA or anti-MtrC). This technique, termed antibody recognition force microscopy (Ig-RFM), detects binding events that occur between antibodies (e.g., anti-OmcA) on an AFM tip and antigens (e.g., OmcA) that are exposed on a cell surface. While this is a relatively new technique, Ig-RFM has been used to map the nanoscale spatial location of single molecules in complex biological structures under physiological conditions (5, 9, 11, 13).Anti-MtrC or anti-OmcA molecules were covalently coupled to silicon nitride (Si₃N₄) cantilevers (Veeco or Olympus) via a flexible, heterofunctional polyethylene glycol (PEG) linker molecule. The PEG linker consists of an NHS (N-hydroxysuccinimide) group at one end and an aldehyde group at the other end (i.e., NHS-PEG-aldehyde). AFM tips were functionalized with amine groups, using ethanolamine (6, 7). The active NHS ester of the NHS-PEG-aldehyde linker molecule was then used to form a covalent linkage between PEG-aldehyde and the amine groups on the AFM tips (6, 7). Next, anti-MtrC or anti-OmcA molecules were covalently tethered to these tips via the linker molecule''s aldehyde group. This was accomplished by incubating the tips with antibody (0.2 mg/ml) and NaCNBH₃ as described previously (7). The cantilevers were purchased from Veeco and had spring constant values between 0.06 and 0.07 N/m, as determined by the thermal method of Hutter and Bechhoefer (12).Prior to conducting the Ig-RFM experiments, the specificity of each polyclonal antibody (i.e., anti-OmcA and anti-MtrC) for OmcA or MtrC was verified by Western blot analysis as described previously (24, 28). Proteins were resolved by both denaturing and nondenaturing polyacrylamide gel electrophoresis (PAGE). Briefly, 2.5 μg of purified OmcA or MtrC (23) was resolved by sodium dodecyl sulfate-PAGE or native PAGE, transferred to a polyvinylidene difluoride membrane, incubated with either anti-OmcA or anti-MtrC, and then visualized using the Amersham ECL Plus Western blotting detection kit. Anti-OmcA bound exclusively to OmcA, anti-MtrC bound exclusively to MtrC, and neither antibody showed cross-reactivity with the other cytochrome. Antibody specificities of anti-OmcA and anti-MtrC were also validated by immunoblot analysis of S. oneidensis whole-cell lysate (28).To determine if MtrC or OmcA was expressed on the external surface of live bacteria when Fe(III) served as the TEA, Ig-RFM was conducted on wild-type versus ΔomcA ΔmtrC double mutant cells. For these experiments, bacteria were cultivated anaerobically with Fe(III), in the form of Fe(III) chelated to nitrilotriacetic acid (NTA), serving as the TEA (19, 23). Growth conditions have been described elsewhere (3, 15) and were based on previous studies (3, 15, 16, 18) that suggest that S. oneidensis MR-1 targets OmcA and MtrC to the cell surface when Fe(III) serves as the TEA.An Asylum Research MFP-3D-BIO AFM or a Digital Instruments Bioscope AFM (16, 17) was used for these experiments. The z-piezoelectric scanners were calibrated as described previously (17). Cells were deposited on a hydrophobic glass coverslip and immersed in imaging buffer (i.e., phosphate-buffered saline [pH 7.4]). The hydrophobic glass coverslips were made as described previously (17) using a self-assembling silane compound called octadecyltrichlorosilane (OTS; Sigma-Aldrich). S. oneidensis MR-1 cells readily adsorbed onto OTS glass coverslips and remained attached to the coverslips during the entire experiment. No lateral cell movement was observed during the experiment, consistent with previous studies that used OTS glass to immobilize bacteria (15, 17, 18, 27).The AFM tip was brought into contact with the surface of a bacterium, and the antibody-functionalized tip was repeatedly brought into and out of contact with the sample, “fishing” for a binding reaction with cytochrome molecules that were exposed on the external cell surface. Binding events were observed upon separating anti-OmcA- or anti-MtrC-functionalized tips from wild-type S. oneidensis MR-1 cells (Fig. ​(Fig.1).1). For the wild-type cells, we observed both nonspecific and specific interactions (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Retraction force curves for anti-MtrC-functionalized tips (A) and anti-OmcA-functionalized tips (B) that are being pulled away from the surface of living ΔomcA ΔmtrC double mutant (gray dotted line) or wild-type (solid black line) S. oneidensis MR-1. These bacteria were adsorbed onto OTS glass coverslips. (C) Retraction curves exhibiting nonspecific binding, specific binding, or no binding between the AFM tip and the cell surface.The distinction between “specific” and “nonspecific” adhesion is made by observing the change in slope of the force curve during the retraction process (26). During specific binding (Fig. ​(Fig.1C),1C), the cantilever is initially relaxed as it is pulled away from the sample. Upon further retraction, the ligand-receptor complex becomes stretched and unravels, resulting in a nonlinear force profile as noted in references 26 and 16. On the other hand, nonspecific adhesion (Fig. ​(Fig.1C)1C) maintains the same slope during the retraction process because only the cantilever flexes (26).Figure ​Figure22 summarizes the frequency or probability of observing a binding event for both anti-OmcA and anti-MtrC tips. Each bar in Fig. ​Fig.22 represents one experiment in which 500 to 1,000 force curves were collected between one AFM tip and two to four live bacterial cells. This figure does not make a distinction between specific and nonspecific binding. It simply shows the frequency of observing an attractive interaction as the antibody-functionalized tip was pulled away from the surface of S. oneidensis MR-1. Binding events occurred with roughly the same frequency when wild-type S. oneidensis MR-1 cells were probed with anti-MtrC-functionalized tips as when they were probed with anti-OmcA-functionalized tips (Fig. ​(Fig.22).Open in a separate windowFIG. 2.Histograms showing the frequency of observing a binding event for anti-MtrC-functionalized (blue) or anti-OmcA-functionalized (red) AFM tips on live wild-type S. oneidensis MR-1 (solid bars) or ΔomcA ΔmtrC double mutant (diagonally hatched bars) cells. The downward arrows designate injection of free antibody into the imaging buffer. The solid gray bars correspond to results obtained with unbaited AFM tips.A number of control experiments were performed to verify the detection of OmcA and MtrC on the surface of wild-type S. oneidensis MR-1. First, 0.1 μM of free anti-OmcA (or anti-MtrC) was added to the imaging fluid to block binding between the antibody-functionalized AFM tip and surface-exposed cytochromes (11, 16). This decreased the adhesion that was observed between the antibody-functionalized tip and the cell surface (Fig. ​(Fig.22).Second, we performed force measurements on ΔomcA ΔmtrC double mutant S. oneidensis MR-1 cells. This mutant is deficient in both OmcA and MtrC (19, 23, 24) but produces other proteins native to the outer surface of S. oneidensis MR-1. The resulting force spectra showed a noticeable reduction in binding events for the ΔomcA ΔmtrC double mutant cells (Fig. ​(Fig.2).2). The binding events that were observed for the double mutant were only nonspecific in nature (Fig. ​(Fig.1).1). This indicates that the antibodies on the tip do not participate in specific interactions with other proteins on the surface of S. oneidensis MR-1 cells.As a final control experiment, force measurements were conducted on wild-type S. oneidensis MR-1 cells, using Si₃N₄ tips conjugated with the PEG linker but not functionalized with polyclonal antibody (unbaited tips). Like the results with the double mutant, the unbaited tips were largely unreactive with the surface of the bacteria (Fig. ​(Fig.2).2). Those binding events that were observed were nonspecific in nature. Taken together, these results demonstrate that the antibody-coated tips have a specific reactivity with OmcA and MtrC molecules. Furthermore, these force measurements show that MtrC and OmcA are present on the external cell surface when Fe(III) serves as the TEA.To map the distribution of cytochromes on living cells, Ig-RFM was conducted on living S. oneidensis MR-1 cells that were growing on a hematite (α-Fe₂O₃) thin film. The conditions for these experiments were as follows. A hematite film was grown on a 10-mm by 10-mm by 1-mm oxide substrate via oxygen plasma-assisted molecular beam epitaxy (14, 16). The cells were grown anaerobically to mid-log phase with Fe(III)-NTA serving as the TEA. Cells were deposited onto the hematite thin film along with anaerobic growth medium that lacked Fe(III)-NTA. The cells were allowed to attach to the hematite surface (without drying) overnight in an anaerobic chamber. The following day, the liquid was carefully removed and immediately replaced with fresh anaerobic solution (pH 7.4). Ig-RFM was performed on the cells by raster scanning an antibody-functionalized AFM tip across the sample surface, thereby creating an affinity map (1). Force curves were collected for a 32-by-32 array. The raw pixilated force-volume data were deconvoluted using a regularized filter algorithm. The total time to acquire a complete image was approximately 20 min.As noted above, attractive interactions between an antibody tip and cell resulted in relatively short-range, nonspecific and longer-range, specific adhesive forces (Fig. ​(Fig.1C).1C). To distinguish between these two interactions, we integrated each force curve beginning at >20 nm and ending at the full retraction of the piezoelectric motor (∼1,800 nm). This integration procedure quantifies the work of binding, measured in joules, between the antibody tip and a particular position on the sample. While this integration procedure does not totally exclude nonspecific binding, it does select for those events associated primarily with specific antibody-antigen binding. Figure ​Figure33 is the antibody-cytochrome recognition images for MtrC and OmcA. The corresponding height (or topography) images of the bacterial cells are also shown in Fig. ​Fig.33.Open in a separate windowFIG. 3.Ig-RFM of live S. oneidensis MR-1 cells deposited on a hematite (α-Fe₂O₃) thin film. Height image (A) and corresponding Ig-RFM image (B) for a bare unfunctionalized Si₃N₄ tip. Height and corresponding Ig-RFM image for a tip functionalized with anti-MtrC (C and D) or anti-OmcA (E and F). Each panel contains a thin white oval showing the approximate location of the bacterium on the hematite surface. A color-coded scale bar is shown on the right (height in micrometers [μm], and the work required to separate the tip from the surface in attojoules [aJ]).OmcA molecules were concentrated at the boundary between the bacterial cell and hematite surface (Fig. 3E and F). MtrC molecules were also detected at the edge of a cell (Fig. 3C and D). Some MtrC, unlike OmcA, was observed on the cell surface distal from the point of contact with the mineral (Fig. 3C and D). Both OmcA and MtrC were also present in an extracellular polymeric substance (EPS) on the hematite surface (Fig. 3D and F), which is consistent with previous results showing MtrC and OmcA in an EPS produced by cells under anaerobic conditions (19, 24). This discovery is interesting in light of the research by Rosso et al. (22) and Bose et al. (4), who found that Shewanella can implement a nonlocal electron transfer strategy to reduce the surface of hematite at locations distant from the point of cell attachment. Rosso et al. (22) proposed that the bacteria utilize unknown extracellular factors to access the most energetically favorable regions of the Fe(III) oxide surface. The Ig-AFM results (Fig. ​(Fig.3)3) suggest the possibility that MtrC and/or OmcA are the “unknown extracellular factors” that are synthesized by Shewanella to reduce crystalline Fe(III) oxides at points distal from the cell. Additional experiments showing reductive dissolution features coinciding with the extracellular location of MtrC and/or OmcA would need to be performed to test this hypothesis.It is important to note that these affinity maps were collected on only a few cells because it so challenging to produce large numbers of quality images. Future work should be conducted on a population of cells. Until this time, these affinity maps can be used to provide a crude, lowest-order estimate of the number of cytochromes on the outer surface of living S. oneidensis MR-1. For example, there were 236 force curves collected on the bacterium shown in Fig. ​Fig.3D.3D. Thirty-eight of these curves exhibited a distinct, sawtooth-shaped, antibody-antigen binding event. In other words, MtrC molecules were detected in one out of every six force curves (16%) that were collected on the cell surface.This probability can be compared to other independent studies that estimated the density and size of MtrC and OmcA molecules from S. oneidensis MR-1. Lower et al. (16) estimated that S. oneidensis has 4 × 10¹⁵ to 7 × 10¹⁵ cytochromes per square meter by comparing AFM measurements for whole cells to force curves on purified MtrC and OmcA molecules. Wigginton et al. (25) used scanning tunneling microscopy to determine that the diameter of an individual cytochrome is 5 to 8 nm. These values can be used to create a simple, geometric, close-packing arrangement of MtrC or OmcA molecules on a surface. Using this approach, cytochromes could occupy 8 to 34% of the cell surface.This estimate is consistent with the observed number of putative MtrC molecules shown in Fig. ​Fig.3D.3D. Therefore, it appears that these affinity maps can be used as a lowest-order estimate for the number of cytochromes on S. oneidensis MR-1 even though we do not know a priori the exact configuration of the antibody tip (e.g., the concentration of antibody on the tip, the exact shape of the tip, the binding epitopes within the antibody).In summary, the data presented here show that S. oneidensis MR-1 localizes OmcA and MtrC molecules to the exterior cell surface, including an EPS, when Fe(III) is the TEA. Here, the cytochromes presumably serve as terminal reductases that catalyze the reduction of Fe(III) through direct contact with the extracellular iron-oxide mineral.