Initial, reference-free, two-dimensional (2D) class averages were calculated using particles binned by two via the Xmipp Clustering 2D Alignment[27]and sorted into classes. We propose a model in which D8 oligomerization on the IMV would allow VACV to adhere to heterogeneous population of CS, including CS-C and potentially CS-A, while overall increasing binding efficiency to CS-E. == Author Summary == Vaccinia virus (VACV) is an orthopox virus and considered the gold standard of vaccines as it was used to eradicate smallpox from the human population. Inoculation with VACV leads to K-Ras(G12C) inhibitor 12 a strong B cell immune response and the production of potent antibodies that simultaneously target several envelope proteins of the virus. Among those viral proteins, D8 is an adhesion molecule that binds chondroitin sulfate, a glycosaminoglycan, on the host cell surface. Here, we identified chondroitin sulfate E (CS-E), as the preferred ligand for D8 and assessed the role of a panel of anti-D8 antibodies in preventing D8 binding to CS-E. We further mapped the binding site of each antibody on the D8 surface to reveal the targeted epitopes. Finally, using several truncated D8 constructs, we identified that the C-terminal domain of D8 that is not involved in CS-E binding is in fact involved in oligomerization of K-Ras(G12C) inhibitor 12 native D8in vitroand likely, also on the virion, as a means of increasing binding affinity to increase viral adhesion to CS on the host cell. == Introduction == Vaccinia virus (VACV) is a low virulence orthopoxvirus that was used to eradicate smallpox[1]. Immunization with VACV leads to the production of potent protective antibodies that target the VACV envelope proteins A27, A33, B5, D8, H3 and L1, among others[1]. VACV has two forms of infectious virions. The intracellular mature virion (IMV) is the most abundant K-Ras(G12C) inhibitor 12 form of VACV and mainly responsible for viral spread between hosts. A27, D8, H3 and L1 are expressed on the outer membrane of IMV. A33 and B5 are embedded in the more fragile extracellular enveloped virion (EEV), K-Ras(G12C) inhibitor 12 which has an additional host cell derived envelope. EEV is thought to be involved in cell-to-cell spread within the host ACVRLK4 and is critical for virulence. Antibody responses against VACV potently target both infectious forms of the virus, likely contributing to the efficacy of the smallpox vaccine[2]. Among the VACV envelope proteins, A27, H3 and D8 are viral adhesion molecules that bind to glycosaminoglycans (GAG) for attachment to host cells. GAGs are linear polysaccharides with repeating disaccharide units predominantly found on cell surfaces and as constituents of the extracellular matrix[3]. While A27 and H3 interact with heparan sulfate (HS) or heparin (HP)[4],[5], D8 binds to chondroitin sulfate (CS)[6]. Viral adhesion to GAGs represents a major route of entry for a range of pathogens[7]. As a result, GAG adhesion is an early and important step that initiates viral infection. We have recently determined the crystal structure of the CS adhesion protein D8[8]. The N-terminal ectodomain contains a carbonic anhydrase fold (CAH, residues 1234), followed by a smaller domain of unknown function (residues 235273). A single transmembrane domain (TM, 274294) and a small intra-virion tail (295304) constitute the rest of the protein. The CAH domain was suggested to be responsible for CS binding, as it has a central positively charged crevice that complements the negative charge of CS[8]. We have recently identified four different antibody specificity groups among an ensemble of twelve murine monoclonal antibodies specific to D8[9]. D8 MAbs were first sorted by competitive ELISA according to their targeted epitopes on the basis of which other MAbs they cross-block. Four main specificity groups resulted from this experiment (group I: JE11; II: AB12 and CC7.1; III: BG9.1, BH7.2, EB2.1, EE11, JA11.2, JE10, and JF11; IV: FH4.1 and LA5)[9]. Partial epitope definitions for each of the antibody groups were previously determined. For group I MAbs, the epitope definition was mapped by Deuterium Exchange Mass Spectrometry (DXMS) to residues 1014 and 8090[9]. Peptide ELISA, using 20-mer peptides that overlap by 10 amino acids and cover the entire D8 protein sequence, suggested that group II antibodies target a linear epitope called peptide 58 (residues 91110), while groups I, III and IV target a conformational epitope[9]. Alanine scanning of peptide 58, and D8 point mutation K-Ras(G12C) inhibitor 12 analysis (PMA) refined the group II epitope to 10 residues (H95, W96, N97, K99, Y101, S102, S103, E106, H110 and D112). While the group III MAb epitope is conformational, group III MAbs can also bind peptide 58, albeit much weaker than group II antibodies. Interestingly, group II and III.