Another study (7) showed that Ad2 pIX trimers are buried within the GON

Another study (7) showed that Ad2 pIX trimers are buried within the GON. imaging (33). pIX is a Rabbit Polyclonal to EPHB1/2/3 14.3-kDa minor component of the groups of nine hexons (GONs) that form the central Kv3 modulator 2 region of each facet of the icosahedral capsid (6, 7). pIX functions as a cement stabilizing hexon-hexon interactions (19) and is essential for viral DNA packaging (20). A mutant of Ad serotype 5 (Ad5) containing a deletion in the gene for pIX produced virions that are more heat labile than those of the wild type (11). The mutant can grow only in 293 cells, which produce pIX coded for by their resident Ad5 sequences; however, the amount is too small to be incorporated into virions (21). Recently it was shown that pIX of Ad5, in addition to its structural contribution, exhibits transcriptional properties (27). We have also found that Ad2 pIX and Ad3 pIX are O-glycosylated and phosphorylated (1). Antisera directed to virions or purified capsid proteins were used to elucidate the topographical organization of the structural proteins in studies based on the currently accepted model of the architecture of adenoviruses (6, 7, 18, 19). The pIX has not been characterized in as much detail as the Kv3 modulator 2 hexon, fiber, or pentonbase with regard to its antigenicity (38). Polyclonal serum was produced against purified Ad2 pIX to study the immunological properties of pIX (6). On the basis of the results of immunodiffusion tests with Ad2 pIX-specific antibodies it was assumed that both type- and group-specific determinants exist on pIX. However, no information is available on whether specific regions of the protein are important for serotype or subgenus specificity or what may constitute a type-specific, as opposed to a cross-reactive, epitope. Furthermore, pIX is known to be buried within the capsid, although there is still the question of whether it is located on the inner or outer surface of the GON (6, 8). We used bacterially expressed recombinant pIX of Ad2 and Ad3 as a subgenus-specific antigen in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis (2). In this report we present data on the immunogenic regions and the orientation of pIX in virions, based on immunological analysis of the C- and N-terminal parts of pIX by ELISA, immunoblotting, immunogold electron microscopy, and neutralization assay. Construction of plasmids, expression of pIX, and production of polyclonal rabbit sera.The full-length gene encoding pIX was amplified by PCR with either an Ad2 genome or an Ad3 genome as a template, using primers as reported previously (2). A C-terminal fragment was prepared by PCR amplification by using the sense primers Ad2pIXC, 5[3810]-TT aga tct ACC GCC CGC GGG ATT GTG A-3 (with a em Bgl /em II restriction site [underlined]), and Ad3pIXC, 5[3691]-TT aga tct AAC ACC ATC CTT GGA ATG G-3 (with a em Bgl /em II restriction site [underlined]). The Kv3 modulator 2 sequence encoding the N-terminal part of pIX was amplified by PCR by using the antisense primers Ad2pIXN, 5[3672]-TTT aag ctt GGC GGC AGC AGT AGC-3 (with a em Hin /em dIII restriction site [underlined]), and Ad3pIXN, 5[3771]-TTT aag ctt TTA GGC TGC AGC GGC TGA-3 (with a em Hin /em dIII restriction site [underlined]). The antisense primers for the C-terminal fragments and the sense primers for the N-terminal fragments were the same as those for the full-length pIX gene (2). PCR-amplified em Bam /em HI- em Hin /em dIII- and em Eco /em RI- em Hin /em dIII-restricted fragments of the pIX genes of Ad2 and Ad3, respectively, were cloned into appropriately digested pQE30 expression vector (QIAGEN, Hilden, Germany), generating Kv3 modulator 2 Ad2pIX/ pQE30 and Ad3pIX/pQE30. In these constructs, pIX was fused with six histidine residues, enabling purification Kv3 modulator 2 by metal chelate affinity chromatography. PCR fragments encoding only the N- and C-terminal halves of Ad2 pIX and Ad3 pIX were cleaved with the appropriate corresponding enzymes and cloned into pQE40 expression vector, generating Ad2pIXN/pQE40 or Ad2pIXC/pQE40 and Ad3pIXN/pQE40 or Ad3pIXC/pQE40. In these constructs, residues 2 to 70 and 71 to 140 of Ad2 pIX and residues 2 to 70 and 71 to 138 of Ad3 pIX were fused in frame to the C terminus of dihydrofolate reductase (DHFR), which has six histidine residues (His6 tag), at its N terminus. Polyclonal antisera.