To ascertain that cellCcell fusion was the consequence of peptideCpseudoreceptor interaction, we introduced, into the tagged MeV-H, protein mutations that ablate natural tropism (MeV-Haals; Y481A, R533A, S548L, F549S) [39] and compared them with that obtained by the untagged MeV-H on binding to CD46

To ascertain that cellCcell fusion was the consequence of peptideCpseudoreceptor interaction, we introduced, into the tagged MeV-H, protein mutations that ablate natural tropism (MeV-Haals; Y481A, R533A, S548L, F549S) [39] and compared them with that obtained by the untagged MeV-H on binding to CD46. shed further light on the factors constraining the evolution of new morbillivirus serotypes. family, are highly infectious respiratory viruses and cause devastating disease in humans and animals (recently reviewed by Pfeffermann et al. [1]). Measles virus (MeV) is the prototype morbillivirus and infects humans exclusively. In contrast, canine distemper virus (CDV) is a rather promiscuous carnivore morbillivirus, able to infect even primates and BAY-876 cause a disease outbreak [2,3]. This characteristic poses a more than theoretical risk that CDV could extend into the human population after MeV eradication [4,5,6]. Even though high titers of MeV-neutralizing antibodies have been shown to protect against CDV infection [7], those levels are not routinely achieved through vaccination [4]. The morbillivirus envelope comprises two envelope glycoproteins that encompass the so-called virus fusion-membrane apparatus: the attachment (H) and the fusion (F). The attachment glycoprotein is required for the initial binding step to the host cell receptor, whereas the F glycoprotein mediates the fusion event between the viral and host cell membranes. F glycoprotein also can fuse adjacent cell membranes, forming syncytia when cells expressing H and F interact with cells expressing the receptors [8,9]. The signaling lymphocytic activation molecule family member 1 (SLAMF1) on immune cells and the nectin cell adhesion molecule 4 (NECTIN-4) in epithelial cells are the known receptors in morbilliviruses [10,11]. Vaccine strains of MeV and CDV have gained the ability to infect other cell types in a CD46-dependent and independent manner, respectively [12,13,14,15,16]. Cross-talk between H and F seems virus-species specific, and albeit heterologous combinations typically compromise fusion activity, successful examples exist [17,18,19,20,21]. Targeting virus surface proteins and neutralization antibodies are crucial for vaccine-bestowed protection [22]. On the basis of epidemiologic studies, a microneutralization titer of 210 mIU/mL or greater has been assigned as an absolute correlate of protection against measles [23,24,25]. The knowledge on the antigenic determinants recognized after natural infection or vaccination has depended heavily on the availability of monoclonal antibodies and DNA-based vaccines BAY-876 [18,26,27,28,29,30,31,32]. Since the morbillivirus coat consists of hetero-oligomers of the H and F glycoproteins that are organized into tetramers and trimers, respectively, single expression of antigens could alter the overall immunogenicity of the protein [33,34]. Moreover, the hybridoma technology is known to be subjected to bias, and therefore their representativeness with the in vivo immune responses is questioned [35,36]. Thus, the exact antigenic determinants involved in morbillivirus protection are poorly characterized. To address this gap in the knowledge, we used an improved reverse genetic system to generate recombinant MeV-encoding chimeric envelope glycoprotein from the close, but not cross-reactive, CDV. With the BAY-876 use PPP3CC of virus-specific antisera induced in different species, our study provides the neutralizing-antibody response against the virus envelope glycoproteins. 2. Materials and Methods 2.1. Cell Lines Baby hamster kidney cells (BHK; american type culture collection [ATCC], Cat. #CCL-10, Manassas, VA, USA) and Vero African green monkey kidney cells (Vero; ATCC, Cat. #CCL-81) and their derivatives (either expressing human nectin-4 BAY-876 [37], canine SLAM [38], or a membrane-anchored single-chain antibody specific for a hexahistidine peptide [39]) were kept in Dulbeccos modified Eagles medium (DMEM) (GE Healthcare Life Sciences, Cat. #SH30022.01, Pittsburg, PA, United States). The human melanoma Mel-JuSo cell line was a kind gift from Rik de Swart, PhD (Erasmus MC, Rotterdam, The Netherlands), and was grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Corning Inc, Cat. #10-040-CV, Corning, NY, United States). Cells were supplemented with 1 penicillin/streptomycin (Corning Inc, Cat. #30-002-CI) and 5 mM HEPES (Life Technologies, Cat. #15630-080, Carlsbad, CA, United States). Additionally, Vero, Vero/anti-His, and Vero/NECTIN-4 were maintained in the presence of 5% fetal bovine serum (FBS) (Thermo Fisher Scientific, Cat. #10437-028, Waltham, MA, United States). BHK, Mel-JuSo, and Vero/dogSLAM cells were grown in 10% FBS plus 1 mg/mL of Zeocin (InvivoGen, San Diego, CA, United States) BAY-876 (Vero/dogSLAM). Vero/NECTIN-4 cells were propagated in the presence of 0.5 mg/mL G418 (Mediatech Inc, Cat. #MT-61-234-RG, Manassas, VA, United States). Cells were kept at 37 C and 5% CO2 with saturating humidity. 2.2. Construction of Plasmids The chimeric MeV/CDV-H (MeV-H stalk combined with CDV-H head domain) and CDV/MeV-H (CDV-H stalk combined with MeV-H head domain) genes were generated through overextension polymerase chain reaction (PCR). For the MeV/CDV-H chimera, the amino acid (aa) 1C159 portion of vaccine-lineage MeV-H Nse strain [40,41] was amplified.