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Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6–specific IgA mAb
Ningguo Feng,1 Jeffrey A. Lawton,2 Joana Gilbert,1 Nelly Kuklin,1 Phuoc Vo,1 B.V. Venkataram Prasad,2 and Harry B. Greenberg1,3
1Department of Gastroenterology, Microbiology, and Immunology, Stanford University School of Medicine, Stanford, California, USA2Verna and Maars McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA3Veterans Administration Palo Alto Health Care System, Palo Alto, California, USA
Address correspondence to: Harry B. Greenberg, Department of Gastroenterology, Stanford University, 3801 Miranda Avenue, 154-C, Palo Alto, California 94304, USA. Phone: (650) 919-3711; Fax: (650) 852-3259; E-mail: hgreenberg@aviron.com.
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1Department of Gastroenterology, Microbiology, and Immunology, Stanford University School of Medicine, Stanford, California, USA2Verna and Maars McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA3Veterans Administration Palo Alto Health Care System, Palo Alto, California, USA
Address correspondence to: Harry B. Greenberg, Department of Gastroenterology, Stanford University, 3801 Miranda Avenue, 154-C, Palo Alto, California 94304, USA. Phone: (650) 919-3711; Fax: (650) 852-3259; E-mail: hgreenberg@aviron.com.
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1Department of Gastroenterology, Microbiology, and Immunology, Stanford University School of Medicine, Stanford, California, USA2Verna and Maars McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA3Veterans Administration Palo Alto Health Care System, Palo Alto, California, USA
Address correspondence to: Harry B. Greenberg, Department of Gastroenterology, Stanford University, 3801 Miranda Avenue, 154-C, Palo Alto, California 94304, USA. Phone: (650) 919-3711; Fax: (650) 852-3259; E-mail: hgreenberg@aviron.com.
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1Department of Gastroenterology, Microbiology, and Immunology, Stanford University School of Medicine, Stanford, California, USA2Verna and Maars McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA3Veterans Administration Palo Alto Health Care System, Palo Alto, California, USA
Address correspondence to: Harry B. Greenberg, Department of Gastroenterology, Stanford University, 3801 Miranda Avenue, 154-C, Palo Alto, California 94304, USA. Phone: (650) 919-3711; Fax: (650) 852-3259; E-mail: hgreenberg@aviron.com.
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1Department of Gastroenterology, Microbiology, and Immunology, Stanford University School of Medicine, Stanford, California, USA2Verna and Maars McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA3Veterans Administration Palo Alto Health Care System, Palo Alto, California, USA
Address correspondence to: Harry B. Greenberg, Department of Gastroenterology, Stanford University, 3801 Miranda Avenue, 154-C, Palo Alto, California 94304, USA. Phone: (650) 919-3711; Fax: (650) 852-3259; E-mail: hgreenberg@aviron.com.
Find articles by Vo, P. in: JCI | PubMed | Google Scholar
1Department of Gastroenterology, Microbiology, and Immunology, Stanford University School of Medicine, Stanford, California, USA2Verna and Maars McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA3Veterans Administration Palo Alto Health Care System, Palo Alto, California, USA
Address correspondence to: Harry B. Greenberg, Department of Gastroenterology, Stanford University, 3801 Miranda Avenue, 154-C, Palo Alto, California 94304, USA. Phone: (650) 919-3711; Fax: (650) 852-3259; E-mail: hgreenberg@aviron.com.
Find articles by Prasad, B. in: JCI | PubMed | Google Scholar
1Department of Gastroenterology, Microbiology, and Immunology, Stanford University School of Medicine, Stanford, California, USA2Verna and Maars McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA3Veterans Administration Palo Alto Health Care System, Palo Alto, California, USA
Address correspondence to: Harry B. Greenberg, Department of Gastroenterology, Stanford University, 3801 Miranda Avenue, 154-C, Palo Alto, California 94304, USA. Phone: (650) 919-3711; Fax: (650) 852-3259; E-mail: hgreenberg@aviron.com.
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Published May 1, 2002 - More info
J Clin Invest. 2002;109(9):1203–1213. https://doi.org/10.1172/JCI14397.
© 2002 The American Society for Clinical Investigation
Received: October 11, 2001; Accepted: March 25, 2002
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Methods
Cells and viruses. Tissue culture–adapted murine rotavirus EC,VP7 serotype 3 and VP4 genotype 16 (3,[16]), semian rotavirus RRV (3,[3]), SA11-4F (3,[1]), human rotavirus Wa (1,[8]), and bovine rotavirus NCDV (5,[1]) were propagated in the MA104 green monkey kidney cell line as previous described (11). MA104 cells were maintained in media M199 (BioWhittaker Inc., Walkersville, Maryland, USA) with penicillin/streptomycin (M199, GIBCO BRL; Life Technologies Inc., Grand Island, New York, USA) and supplemented with 7% FCS. Virus culture yields were freeze-thawed twice, and rotaviruses were either concentrated by centrifugation at 140,000 g for 2 hours through a 40% sucrose cushion for neutralization assays (see below) or purified on a cesium chloride density gradient for RNA transcription assays (24). To make rotavirus DLPs, purified or concentrated virus was treated with 5 mM EDTA and incubated at 37°C for 20 minutes and then dialyzed against PBS without Ca++ and Mg++. The resulting DLP preparation was not infectious (25) when directly inoculated on MA104 cells at a concentration greater than or equal to tenfold more than that used in the lipofection-mediated neutralization assay (see below). Propagation and ID50 assays of a wild-type murine rotavirus ECw were described previously (26).
mAb’s. The mAb’s 7D9 and 8D3 (VP6-specific, IgA), 1E11 and SGI-255/60 (VP6-specific, IgG), B4-2 (NSP4-specific, IgG), and 159 (VP7-specific, IgG) have been described previously (4, 17, 27, 28). All mAb’s used in neutralization assays and RNA transcription assays were purified from ascites using protein L (CLONTECH Laboratories Inc., Palo Alto, California, USA) or protein A (Pierce Chemical Co., Rockford, Illinois, USA), dialyzed against PBS and stored in PBS. Antibody concentrations of purified mAb’s or ascites were determined by ELISA. Polyethylene microtiter plates were coated with goat anti-mouse IgG, A, and M (Kirkegaard & Perry Laboratories Inc., Gaithersburg, Maryland, USA) at 4°C overnight and then blocked with 5% dry skim milk (Carnation; Nestle USA Inc., Solon, Ohio, USA) in PBS. After washing with PBS–0.05% Tween-20 (PBS-T), serial dilutions of purified mAb or ascites were added to the plates. IgG or IgA standards (purified mouse IgG [Sigma Chemical Co., St. Louis, Missouri, USA] or mouse IgA myeloma protein [Sigma Chemical Co.]) were also added to each plate. Plates were incubated at 37°C for 2 hours. After incubation, plates were washed with PBS-T and peroxidase-conjugated goat anti-mouse IgG (γ chain–specific; Kirkegaard & Perry Laboratories Inc.) or IgA (α chain–specific; Kirkegaard & Perry Laboratories Inc.) antibody was added. After a 1-hour incubation at 37°C, plates were washed and the color reaction was developed with 2,2′-azino-di-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) substrate (Kirkegaard & Perry Laboratories Inc.). OD was measured on a microtiter plate reader (Bio-Tek Instruments Inc., Winooski, Vermont, USA) at wavelength of 405 nm. Concentrations of the mAb preparations were calculated based on the standard curves generated from purified mouse IgG or IgA standards and adjusted to 1 mg/ml for purified mAb’s and 3 mg/ml for ascites.
Lipofection-mediated neutralization assay. Classical focus reduction neutralization assay for rotavirus was carried out as described (29). Infection of MA104 cells with rotavirus DLPs using lipofectin-mediated transfection was carried out as described (30). The lipofection-mediated intracellular neutralization assay was based on the rotavirus DLP transfection method. Rotavirus DLPs diluted in M199 (to give approximately 50–100 foci per field in an “unneutralized” control using an inverted microscope) were mixed with lipofectin (GIBCO BRL; Life Technologies Inc.) (15% vol/vol) and incubated at room temperature for 40 minutes. Selected mAb’s (starting concentration 1 mg/ml) were diluted fourfold from 1:40 to 1:163,840 in M199 and added at 30 μl per well to 96-well plates. An equal volume of lipofectin-treated DLPs was added to the 96-well plates and incubated at 37°C for 1 hour. The mixtures were then transferred to MA104 cell monolayers in 96-well plates (40 μl per well) and incubated at 37°C and 5% CO2 for 4 hours. An additional 40 μl of M199 with 20% FCS was then added to each well, and the incubation was continued for 6 hours. The plates were then washed once and fixed with cold methanol. Fixed plates were washed with HBSS. Rabbit anti-rotavirus hyperimmune serum was added to the plates and incubated for 1 hour at 37°C. Plates were then washed, and peroxidase-conjugated goat anti-rabbit IgG antibody (Kirkegaard & Perry Laboratories Inc.) was added for 1 hour at 37°C. After washing, the color reaction was developed with 3-amino-9-ethylcarbazole substrate, and viral antigen–positive foci were counted in each field as above. The neutralization titer of an antibody was defined as the highest dilution at which there was a >50% reduction of viral antigen–positive foci as compared with the no-antibody control.
Quantitation of intracellular antibody. Four hours after transfection of the virus-antibody mixture by lipofectin (see above), MA104 cells were fixed with cold methanol and permeabilized with 1% Triton X in PBS. Peroxidase-conjugated goat anti-mouse IgG (γ chain–specific) or IgA (α chain–specific) was added for 1 hour at 37°C. The plates were then washed five times, and ABTS substrate was added. After 10–15 minutes of color development, the reaction mixture was transferred to a polyethylene ELISA plate and the absorbance measured with a microtiter plate reader. As a control, DLPs and antibodies with identical dilution were added to separate wells in the same plate without lipofectin and were processed and stained in the same fashion. Intracellular antibody titers in transfected wells were expressed as the highest dilution that had a specific OD greater than 0.1. The specific OD was defined as the OD from antibody-transfected well minus the OD from control wells. The OD readings from nontransfected control wells were not significantly different from those from wells with MA104 cell alone without antibody added.
Administration of mAb’s and wild-type rotavirus challenge in adult mice. Six- to eight-week-old female BALB/c mice were injected intraperitoneally twice per day with 500 μl of ascites (total daily dose of 3 mg of antibody) from 2 days before challenge until 7 days after challenge. Two days after the initial inoculation (total 4 doses), mice were orally inoculated with 100 μl of 1.33% sodium bicarbonate followed by 102 ID50 of ECw. Fecal samples were collected from 1 day before challenge until 7 days after challenge. Fecal rotavirus antigen shedding was measured by ELISA as previously described (26). The area under the antigen shedding curve was calculated as total viral antigen shedding (TVS) (26). The TVS was used as the measure of shedding for statistical analysis. Percentage reduction of fecal virus shedding as compared with control group for each antibody-treated mouse was calculated as (mean TVS of control group – TVS of treated mouse) ÷ mean TVS of control group.
Inhibition of transcription assay. For the studies of the ability of different mAb’s to inhibit rotavirus transcription (Figure 1) we used an in vitro transcription assay (31). Purified EC or RRV DLPs were activated with 5 mM EDTA at 37°C for 30 minutes. Equal volumes of EDTA-activated viruses and purified antibodies were mixed and incubated at 37°C for 60 minutes. Rotavirus in vitro transcription was initially performed using a Riboprobe system (Promega Corp., Madison, Wisconsin, USA) (including optimized ×5 transcription buffer, 100 mM DTT, recombinant RNasin ribonuclease inhibitor, and rNTP) according to the manufacturer’s instructions. Transcription products were labeled with 32P rCTP (50 μCi), and the reaction was carried at 40°C for 1 hour. Labeled viral RNA was precipitated with cold trichloroacetic acid and absorbed on filter paper disks as described in the protocol for the Riboprobe system. The filter paper disks were washed extensively with cold trichloroacetic acid and air-dried. The incorporated 32P was measured by a Wallac MicroBeta TriLux liquid scintillation counter (Wallac Inc., Turku, Finland). Data were expressed as cpm.
Figure 1Protective efficacy following intraperitoneal injection of selected rotavirus-specific mAb’s. BALB/c female mice (three mice per group) were intraperitoneally injected with mAb’s (3 mg/d) twice a day starting 2 days prior to 102 ID50 ECw challenge (see Methods). Treatment continued until day 7 after challenge. Fecal virus shedding for each mouse was detected by ELISA, and the area under the 7-day antigen shedding curve was calculated. Figure shows the mean percentage reduction of virus shedding for each group + SEM. *Percentage reduction in these 7D9-inoculated groups was significantly different from that in non–7D9-inoculated groups (P < 0.05). Results represent two separate experiments.
Electron cryomicroscopy. Complexes of DLP with 7D9 IgA were formed by mixing DLPs and IgA in a stoichiometric ratio equivalent to five antigen-binding regions per VP6 molecule in order to ensure complete saturation of all available epitopes. Such a molar excess of IgA should inhibit viral aggregation. Mixtures were incubated for 2 hours at room temperature and then concentrated to about 1012 particles per milliliter by ultrafiltration using a MicroCon-100 microconcentrator (Millipore Corp., Bedford, Massachusetts, USA). Specimens for microscopy were embedded in vitreous ice on holey carbon films according to standard procedures (32).
Specimens were observed on a JEOL 1200EX transmission electron microscope (JEOL USA Inc., Peabody, Massachusetts, USA) operated at ×30,000 magnification using 100 kilo-electron volts electrons and a beam dose of approximately 5 electrons/Å2. Images were collected using Kodak SO-163 electron film (Eastman Kodak Co., Rochester, New York, USA) with a 1-second exposure time. Micrographs were developed for 12 minutes using Kodak D-19 developer at 21°C and fixed for 10 minutes in Kodak Fixer (Eastman Kodak Co.). For each imaging field, two micrographs were collected, one at about 1 μm underfocus and the other at about 2 μm underfocus.
Three-dimensional structural analysis. Micrographs suitable for three-dimensional structural analysis were digitized using a Perkin-Elmer 1010M microdensitometer (Perkin-Elmer Inc., Shelton, Connecticut, USA) operated at a raster scanning interval of 5.33 Å per pixel in the object. Three-dimensional reconstructions were produced using software in the ICOS Toolkit suite (33) according to standard procedures (34–37). Particle centers and orientations were initially estimated from further-from-focus image data, and the parameters were then applied to the closer-to-focus image set for refinement and structural determination. The three-dimensional reconstruction was computed to a resolution of about 21 Å using 137 particle images. The effects of the contrast transfer function were corrected as previously described (38), assuming 10% amplitude contrast and a noise-to-signal ratio of 0.3. The resolution achieved in the final structure was confirmed by Fourier ring correlation analysis (39). The distribution of image data throughout the asymmetric unit was sufficient for the resolution achieved, as 97% of the mean inverse eigenvalue spectrum was less than 0.01.
The three-dimensional structure was viewed using IRIS Explorer 3.5 (Numerical Algorithms Group Ltd., Oxford, United Kingdom) on a Silicon Graphics Indigo2 (Silicon Graphics Inc., Mountain View, California, USA) workstation. Surface representations were displayed using mass density threshold chosen to account for the expected number of VP6 molecules within the VP6 capsid region. To compare internal regions of mass between native and antibody-bound structures, mass density values were converted to variance and the maps were scaled to have the same minima and maxima. As with the surface representations, the surface contour level was chosen to account for the expected number of VP6 molecules within the VP6 capsid region, and contour intervals were chosen to represent density differences of about 0.37 ς.
Analysis of transcription products. A second transcription assay was used to examine the effect of 7D9 on elongation (see Figure 6). Complexes of DLP with 7D9 IgA were formed by mixing purified DLPs and IgAs in a stoichiometric ratio equivalent to three antigen-binding regions per VP6 molecule. The mixture was incubated at room temperature for 2 hours and then sonicated for 1 minute. Virus:IgA complexes derived from 5 μg DLP were suspended in a standard transcription reaction buffer (30 μl) containing 100 mM Tris·HCl (pH 8), 10 mM MgOAc, 0.5 mM S-adenosylmethionine, 1 mM ATP, 100 μM each of GTP, CTP, and UTP, and 40 μCi [α-32P]GTP (3000 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, New Jersey, USA) and incubated at 37°C for 20 minutes. The reaction mixture was passed through a Micro Bio-Spin P-30 column (Bio-Rad Laboratories Inc., Hercules, California, USA) equilibrated in 10 mM Tris buffer (pH 7) to remove salt and unincorporated radionucleotides. The eluate was lyophilized and resuspended in 1× Tris-Borate-EDTA sample buffer containing 7 M urea and 0.1% SDS and boiled for 5 minutes to disrupt the particles and denature the nucleic acid. The transcription reaction products were resolved on a high-resolution denaturing 20% polyacrylamide gel and autoradiographed. For comparison purposes, equivalent quantities of native DLPs and mature TLPs were analyzed using the same procedures.
Figure 6Denaturing PAGE analysis of transcription products in the native DLP (lane 1), 7D9 DLP:IgA complex (lane 2), mature TLP (lane 3), and control reaction containing no particles (lane 4). The positions of full-length mRNA and unincorporated nucleotides are noted with arrows. The position of short oligonucleotide transcription products in lanes 1–3 is marked with a bracket. The band corresponding to unincorporated nucleotides at the bottom of the autoradiograph is not of the same intensity in each reaction because of the differing efficiency with which gel filtration columns removed the nucleotide precursors after transcription.
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Results
Non-neutralizing anti-VP6 IgA mAb 7D9 effectively protects mice from rotavirus infection. Previously, in a “backpack” model in which mice were transplanted with hybridoma cells secreting selected mAb’s, mAb 7D9 was shown to protect mice from virulent rotavirus challenge, while mice treated with other hybridoma cells failed to be protected (17). To rule out the possibility that the lack of protection was caused by variation in viability or Ig production in vivo by the transplanted hybridoma cells, we treated mice daily with 3 mg of selected mAb injected intraperitoneally. The treated animals were challenged with wild-type murine rotavirus after 2 days of pretreatment as described. The protective efficacy of the antibodies expressed as the percent reduction of total fecal antigen shedding as compared with noninjected controls is presented in Figure 1. The non-neutralizing anti-VP6 IgA 7D9 completely protected mice from challenge, confirming our prior studies using the “backpack” model and a cell transfer approach (Figure 1). Even after a 1:50 dilution of ascites (0.06 mg daily dose), mice treated with 7D9 shed significantly less viral antigen than did controls (62% reduction of shedding). Treatment with other non-neutralizing antibodies to VP6 or NSP4, including mAb 8D3, 1E11, SGI-255/60, and B4-2, did not significantly reduce fecal viral antigen shedding after challenge (Figure 1). Of note, VP7 serotype G3–specific neutralizing mAb 159 also failed to protect mice, even though the in vitro neutralizing titer against tissue culture–adapted EC virus was greater than 1:500,000 (Table 1). The protective efficacy of 7D9 was, however, affected by the dose size of challenge virus. When 7D9-treated mice received 105 ID50 of EC virus rather than 102 ID50, no reduction of shedding was observed (data not shown). These findings confirm and extend our previous observations and indicate that the protective effect of 7D9 is not dependent on the transfer of living hybridoma cells to recipient mice but is dependent on the titer of the challenge dose administrated.
Lipofection-mediated intracellular neutralization. Because 7D9 is directed against the rotavirus intermediate layer protein VP6, which is only exposed on DLPs and is not the target of classical neutralization activity (Table 1 and ref. 17), we reasoned that the antibody may neutralize rotavirus only after the virus has entered the cell and has shed the outer capsid protein VP4 and VP7. Previous studies have shown that “noninfectious” DLPs were rendered infectious if introduced into the cytoplasm of permissive cells (30). Based on these results, we developed a lipofection-mediated intracellular neutralization assay to determine whether antibodies to VP6 had antiviral activity if introduced into the cytoplasm (see Methods). The intracellular neutralization titers of selected antibodies against a panel of rotaviruses including the challenge strain EC are provided in Table 2. Only mAb 7D9 effectively neutralized viruses at high titer in this lipofection-based assay. The neutralization titers of 7D9 were similar against the different virus strains tested, which represented a variety of serotype and subgroup specificities (Table 2). The nonprotective VP6 IgA antibody 8D3 had low intracellular neutralization titers, and this low-level neutralization was not consistently detectable. Antibodies 1E11 and B4-2 did not neutralize virus intracellularly, while mAb SGI-255/60 showed low neutralization activity against RRV only (a subgroup I virus). mAb 159, which effectively neutralizes infectious serotype G3 viruses in classical neutralization assay (Table 1), did not inhibit DLP viruses (Table 2) or TLP viruses (data not shown) in the lipofection assay. The reciprocal titers of the mAb’s detected inside MA104 cells after transfection (data not shown) were between 2560 and 10,240, suggesting that transfection efficiencies for different antibodies were similar and that the differences in intracellular neutralization activity were not due to differences in the amount of antibody transfected.
Kinetic of intracellular neutralization. To investigate at which stage of infection rotaviruses were being inhibited by IgA 7D9, we infected cells with RRV TLPs and, at selected times after infection, transfected purified 7D9 (0.025 mg/ml) into the infected monolayers (Figure 2). When 7D9 was transfected between 0 and 2 hours after infection, it significantly inhibited virus infection. However, by 3 hours after infection, reduction was less than 50% (below neutralizing titer cutoff) (Figure 2). The intracellular neutralizing activity became undetectable when 7D9 was transfected 4 hours after infection. Since the antibody was effective even when added to cells after infection, its effects are not likely to be due to extracellular aggregation.
Figure 2Efficiency of lipofection-mediated intracellular neutralization by 7D9 at different time points after infection. MA104 cells were infected with TLPs of RRV. At indicated time points after infection, 7D9 mAb was transfected into cells (0.025 mg/ml) using lipofectin. Viral antigen in the cells was detected 10 hours after initial RRV infection by immunostaining. Data are expressed as percentage of focus reduction as compared with control. *Focus reduction was below 50% and significantly different from that at time 0 (P < 0.05). Results were calculated from triplicate measurements and represent two separate experiments.
In vitro inhibition of viral RNA transcription. The kinetic studies indicated that IgA 7D9 neutralizes rotavirus at an early stage of viral replication following viral infection. We hypothesized that IgA 7D9 may function, at least in part, by inhibiting viral mRNA transcription after the virus uncoats in the cytoplasm. To initially evaluate the inhibitory effects of selected rotavirus-specific antibodies on viral mRNA transcription, we incubated various antibodies with transcriptionally competent DLPs of RRV and EC and measured the incorporation of 32P into viral mRNA transcripts (Figure 3, a and b). The level of transcription of RRV and EC in the presence of antibodies B4-2 and 159 was not significantly different from that of the control with no antibody added. The mRNA transcription of DLPs treated with antibodies 8D3 and 1E11 was significantly greater than that of the control. SGI slightly suppressed mRNA transcription of RRV only (62%, P < 0.05). IgA 7D9, however, completely suppressed the in vitro rotavirus mRNA transcription of both RRV and EC (Figure 3, a and b; P > 0.01). This finding is consistent with the hypothesis that IgA 7D9 mediates its antiviral effect by blocking early-phase transcription of the rotavirus replication cycle.
Figure 3Effects of selected mAb’s on in vitro rotavirus transcription. RRV (a) and EC (b) were treated with EDTA and mixed with selected mAb’s. Transcription assay was conducted using a Riboprobe system at 40°C for 1 hour as described. Transcripts were labeled with 32P rCTP and measured by liquid scintillation counter. Data are expressed as cpm. As control, reactions with no virus or with virus but no antibody were also included. *Mean cpm in this group was significantly different from that of nonantibody control (P < 0.05). Results were calculated from four replicates and represent four separate experiments.
Three-dimensional structural analysis of 7D9 DLP:IgA complexes. To directly visualize the interactions between 7D9 IgA and the VP6 capsid layer, we imaged DLP:IgA complexes in vitreous ice by electron cryomicroscopy (data not shown), computed the three-dimensional structure to a resolution of about 21 Å from 137 independent particle images, and compared it with the structure of the native DLP (Figure 4). The VP6 capsid is made up of 260 trimers arranged on a T=13 (levo) icosahedral lattice. This arrangement of capsomers defines 132 channels that penetrate the VP6 layer. In the outer regions of the VP6 capsid layer, the trimers are well separated from one another, while in the lower regions, near the VP2 layer, the trimers form a network of interactions with one another.
Figure 4(a and b) Surface representations of the three-dimensional structure of native DLP (a) and 7D9 DLP:IgA complex (b), viewed along the icosahedral threefold axis. The VP2 capsid layer is shown in green, VP6 in blue, and IgA in pink. Scale bar, 200 Å. (c and d) Surface representations of the mRNA release channel computationally isolated from the native DLP (c) and the 7D9 DLP:IgA complex (d). Scale bar, 50 Å.
In the surface representation of the 7D9 DLP:IgA complex (Figure 4b), a total of 240 individual Fab domains (pink) were clearly visible on the viral surface. The Fab domains were attached at the tops of the VP6 trimers, with the epitope positioned along the edge. The Fab domains were oriented vertically on the viral surface and did not adversely affect the width of any of the channels penetrating the VP6 capsid layer.
The Fab domains had the expected bi-lobed morphology seen in the atomic model of IgA (40), and the molecular envelope was also similar to that observed for IgGs at a comparable resolution (22). Though bivalent IgA molecules were bound to the viral surface, only the Fab domains were visible in the three-dimensional structure. The most reasonable explanation for this is that both the Fc domains and the extended hinge regions, being highly flexible, were disordered and hence were averaged away during the reconstruction process.
Surrounding each icosahedral threefold axis was a cluster of three small spheres of mass density. The volume of each cluster was equivalent to that of an individual Fab domain, suggesting that one Fab domain is also bound to the VP6 trimer located at each of the 20 icosahedral threefold axes, though the morphology cannot be visualized in the three-dimensional structure. A Fab domain interacting with a trimer at the icosahedral threefold axis may adopt any one of three different orientations, depending upon which underlying VP6 monomer is specifically bound. The disrupted morphology of these Fab domains in the three-dimensional structure is likely a consequence of the fact that only one of three symmetrically equivalent positions is occupied around each icosahedral threefold axis. Taken together, these results indicate that a total of 260 IgA antigen-binding domains, or one per VP6 trimer, are bound to the VP6 surface at saturation.
Comparison of the VP6 capsid region in the 7D9 DLP:IgA complex and the native DLP. The attachment of various ligands, such as the VP7 capsid protein or anti-VP6 Fab’s, to the VP6 surface has been reported to introduce architectural changes in the body of the VP6 capsid layer (22). In these studies, mass translocations were observed within the network of trimer-trimer interactions present near the VP6-VP2 interface region in the vicinity of the mRNA release channels at the icosahedral fivefold axes. To investigate whether the attachment of 7D9 IgA causes similar structural changes in VP6, we compared the distribution of mass in the native DLP and the 7D9 DLP:IgA complex at various radii within the VP6 capsid region.
In the vicinity of the mRNA release channels (Figure 5), structural differences were evident at nearly all radii examined, and the types of domain movements were very similar to those observed in previous structural studies of DLP-ligand complexes (22). As in previous studies, the most striking mass translocations were observed within the network of trimer-trimer interactions extending between the radii ∼275 Å to ∼310 Å. In this region, individual molecules of VP6 appeared to be shifted with respect to each other, with some displacements exceeding 10 Å (Figure 5a, section III). As a result of these movements, some of the intermolecular interactions within trimers appeared to be weakened, and some of the intermolecular interactions between trimers appeared to grow stronger. Furthermore, the movement of the VP6 molecules closest to the icosahedral fivefold axes caused the diameter of the mRNA release channel to narrow by about 10 Å in the middle of this region. Mass translocations were not observed at the present resolution in the VP6-VP2 interface (∼260 Å; Figure 5a, section V) or the VP2 capsid layer (data not shown).
Figure 5(a) Contoured cross sections of mass density from the three-dimensional structures of native DLP (blue) and 7D9 DLP:IgA complex (red), viewed perpendicular to the icosahedral fivefold axis. Each contour represents a mass density difference of about 0.37 ς. The cross sections, labeled I–V, were extracted from radii between 261 Å and 325 Å (b). The upper panel of cross sections depicts the distribution of mass surrounding the mRNA release channel, with the icosahedral fivefold axis passing through the center. The lower panel of cross sections provides a close-up view of the mass distribution in one of the five trimers (indicated in the corresponding upper panel by a dashed box). The arrowheads in cross section III, lower panel, denote representative intermolecular interactions that are different in the two structures. Measurements of three specific mass translocations are also given. Scale bars, 50 Å. (b) Graphical representation of the radii from which cross sections were extracted in a. The cross sections are shown as semitransparent gray planes. The five VP6 trimers surrounding the mRNA release channel in the native DLP are colored in blue, and the underlying VP2 capsid layer is shown in green. (c) A portion of the surface representation of the native DLP, viewed along the icosahedral fivefold axis. The position of the fivefold axis along which the sections shown in a were taken is marked. The pair of VP6 trimers surrounding the twofold axis is also indicated.
Interestingly, the sorts of mass translocations observed within the VP6 trimers surrounding the mRNA release channels did not occur in other regions of the capsid (data not shown). The distribution of mass within the VP6 trimers surrounding the icosahedral twofold axes was virtually identical in both the native DLP and the 7D9 DLP:IgA complex structures. Likewise, few structural changes were observed within the VP6 trimers located at other positions examined. The preponderance of structural changes in the regions of VP6 immediately adjacent to the mRNA release channels suggests that the trimers near the icosahedral vertices are more flexible than trimers at other locations and are more susceptible to alteration upon ligand attachment.
Analysis of transcription products in 7D9 DLP:IgA complex. In previous structural and biochemical studies of DLP-ligand complexes, the appearance of architectural rearrangements in the VP6 capsid layer directly correlated with an inhibition of genome transcription at the stage of transcript elongation (21, 22). Our initial studies indicated that 7D9 efficiently inhibited transcription of RRV and EC while other anti-VP6 mAb’s did not. To determine whether the attachment of 7D9 IgA to the DLP surface likewise blocks elongation but not initiation of transcription, we next compared the length distribution of transcription products in native DLPs and 7D9 DLP:IgA complexes following a period of incubation in a standard in vitro transcription reaction buffer (Figure 6). While native DLPs, as expected, were able to produce full-length mRNA transcripts (lane 1), 7D9 DLP:IgA complexes synthesized predominantly short oligonucleotide transcripts having an apparent length of about six nucleotides and only a small quantity of full-length mRNA (lane 2). The transcriptional behavior of the 7D9 DLP:IgA complex is similar to that of the mature TLP, which is not able to elongate mRNA transcripts beyond about 6 bases in length (lane 3). Given the similarities between the mature TLP and the 7D9 DLP:IgA complex in regard to the architecture of the VP6 capsid region, these results suggest that 7D9 IgA causes an inhibition of genome transcription by the same mechanism as does VP7 in the mature particle.
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