An Antibody to the Putative Aphid Recognition Site on Cucumber Mosaic Virus Recognizes Pentons but Not Hexons

Abstract

Cucumber mosaic virus (CMV), the type member of the genus Cucumovirus (family Bromoviridae), is transmitted by aphids in a nonpersistent manner. Mutagenesis experiments identified the ␤H-␤I loop of the capsid subunit as a potential key motif responsible for interactions with the insect vector. To further examine the functional characteristics of this motif, we generated monoclonal antibodies that bound to native virions but not to ␤H-␤I mutants. Fab fragments from these antibodies were complexed with wild-type CMV and the virus-Fab structure was determined to 12-Å resolution by using electron cryomicroscopy and image reconstruction techniques. The electron density attributed to the bound antibody has a turret-like appearance and protrudes from each of the 12 fivefold axes of the icosahedral virus. Thus, the antibody binds only to the pentameric clusters (pentons) of A subunits of the T‫3؍‬ quasisymmetric virus and does not appear to bind to any of the B and C subunits that occur as hexameric clusters (hexons) at the threefold (quasi-sixfold) axes. Modeling and electron density comparisons were used to analyze the paratope-epitope interface and demonstrated that the antibody binds to three ␤H-␤I loops in three adjacent A subunits in each penton. This antibody can discriminate between A and B/C subunits even though the ␤H-␤I loop adopts the same structure in all 180 capsid subunits and is therefore recognizing differences in subunit arrangements. Antibodies with such character have potential use as probes of viral assembly. Our results may provide an additional rationale for designing synthetic vaccines by using symmetrical viral particles.Cucumber mosaic virus (CMV), the type member of the genus Cucumovirus (family Bromoviridae), infects more than 800 plant species and causes economically important diseases of many crops worldwide (26). CMV is transmitted by aphids in a nonpersistent manner. The virus does not circulate or replicate in the aphid and is quickly acquired from and transmitted to a host during feeding. Virus interacts with the anterior portion of the alimentary tract (food canal to foregut), from which it can be subsequently inoculated by egestion. Unlike some other plant viruses that are transmitted in a nonpersistent manner, CMV does not require helper proteins for transmission. Hence, the aphid recognition motifs must reside on the capsid itself. The ϳ24-kDa coat protein also appears to play a role in normal cell-to-cell and systemic movement independent of particle formation (17, 29).The structures of CMV and cowpea chlorotic mottle virus (CCMV), another member of the family Bromoviridae, were both determined by using cryoelectron microscopy (37, 39) and X-ray crystallography (32, 37). Though the CMV and CCMV capsid proteins share only 19% amino acid sequence identity, they have highly homologous central ␤-barrel structures. Despite this, a number of significant differences distinguish these Tϭ3, icosahedral viruses. First, CMV is transmitted by aphids, whereas bromoviruses are transmitted by bee...

Reovirus polymerase λ3 localized by cryo-electron microscopy of virions at a resolution of 7.6 Å

“…At the end of refinement, 7,939 of 8,996 particle images were included in the reconstruction reported here. During all cycles of refinement, the effects of the microscope CTF on each image were compensated in part by means of a pair of filtering operations (δ 1 = 0.05, δ 2 = 0.1) as described 53 . The distribution of particle orientations was sufficiently random as measured by the eigenvalue spectrum (all inverse eigenvalues <0.01) to allow computation of the final reconstruction to a resolution of 7.6 Å (ref.…”

“…These parameters were refined by means of a modified version of the original PFT protocol: the Fourier transform of each image was adjusted to correct for effects of the microscope contrast transfer function (CTF) by means of FILTER2 (δ 2 = 0.1) as described 53 , and the real and imaginary components of each image transform were correlated against unmodified components from corresponding data derived from a 7 × 7 search grid of model projections.…”

“…The origin and orientation parameters (x, y, θ, ϕ and ω) for each image were initially determined using polar Fourier transform (PFT) procedures 52 , with a reconstruction of the reovirion at a resolution of 18 Å used as the initial model. These parameters were refined by means of a modified version of the original PFT protocol: the Fourier transform of each image was adjusted to correct for effects of the microscope contrast transfer function (CTF) by means of FILTER2 (δ 2 = 0.1) as described 53 , and the real and imaginary components of each image transform were correlated against unmodified components from corresponding data derived from a 7 × 7 search grid of model projections.…”

“…At the end of each cycle of refinement, two independent reconstructions were computed using the EM3DR Fourier-Bessel program (http://bilbo.bio.purdue.edu/~baker/programs/ programs.html) or a parallelized version of it (http://bilbo.bio.purdue.edu/~viruswww/ Rossmann_home/river_programs/para-pft-em3dr.shtml), and the new resolution limit was assessed 40,53 . The subsequent cycle of refinement included all Fourier data to this resolution limit.…”

“…Third, amplitude thresholds were imposed at each spatial frequency in both image and projection transforms such that data with an amplitude <0.6 of the average at a given frequency were omitted (for projection data, average amplitudes were computed from the entire set of 49 projections in the search grid for each image). Finally, transform data were multiplied by an inverse temperature factor to enhance the weight of the high spatial frequency components used in refinement, and its magnitude was gradually increased from 1/300 Å 2 to 1/900 Å 2 during the course of refinement.At the end of each cycle of refinement, two independent reconstructions were computed using the EM3DR Fourier-Bessel program (http://bilbo.bio.purdue.edu/~baker/programs/ programs.html) or a parallelized version of it (http://bilbo.bio.purdue.edu/~viruswww/ Rossmann_home/river_programs/para-pft-em3dr.shtml), and the new resolution limit was assessed 40,53 . The subsequent cycle of refinement included all Fourier data to this resolution limit.…”