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To evaluate the amounts of 10.4K and 14.5K in cells infected with the Ad2 recombinant viruses encoding Ad4 10.4K and/or 14.5K proteins A549 cells were either infected at a MOI of 25 pfu/cell with Ad2, Ad4, and Ad2-recombinants or were mock-infected. Cells were lysed in Triton X-100 buffer at 17 hours post infection. Cell lysates were subjected to immunoprecipitation and western blot detection of E3/19K, 10.4K and 14.5K. Indicative of equal infection efficiency and E3 gene expression, all five Ad2/Ad4 chimeric viruses exhibited a similar level of E3/19K expression as wt Ad2 (Fig. 42A, 19K). Detection of the Ad4 E3/19K homologue required the use of Ad4 E3/19K specific serum and thus the intensity of the band could not be directly compared to the Ad2 E3/19K signals.

Ad4 14.5K was detected with polyclonal serum Bur4 directed against the cytoplasmic tail peptide (CEISYFNLTGGDD) of Ad2 14.5K (Fig. 42A, 14.5K), which is nearly identical to the

Fig. 42 10.4 and 14.5K levels in A549 cells infected with Ad2 recombinant viruses

Parallel immunoprecipitation and western blot analysis of E3/19K, 10.4K and 14.5K in Triton X-100 lysates of mock-infected A549 cells (lane 1), or A549 cells infected (MOI 25 pfu/cell) with Ad4 (lane 2), Ad2 (lane 3), Ad2/(Ad4-14.5) #7-1 (lane 4), Ad2/(Ad4-14.5) #3-8 (lane 5), Ad2/(Ad4-10.4) #12-1 (lane 6), Ad2/(Ad4- 10.4-14.5) #7-4 (lane 7 ), Ad2/(Ad4-10.4-14.5) #16-1 (lane 8). Immunoprecipitates were separated on a 15% minigel by SDS-PAGE, prior to transfer to a nitrocellulose membrane for western blot detection.

(A) Immunoprecipitation and western blot detection of E3/19K employing rabbit serum against Ad4 19K (lanes 1,2) or Ad2 19K (lanes 3-8). Rabbit serum Bur4 was used in IP/WB of E3/14.5K and E3/10.4K was analyzed using rabbit serum R59 in IP and Bur3 for western blot detection.

(B) Different exposures of the left and right portion of the western blot anti-14.5K depicted in (A) to visualize bands of different intensities.

corresponding Ad4 sequence (see Fig. 8). In the immunoblot three major protein species were obtained from lysates of Ad4-infected cells, which migrated with the expected MW of about 14 to 18 kD. The Ad4 14.5K bands differed from those of the Ad2 14.5K protein (Fig. 42 B, left panel). Instead of one more intense faster migrating species and two of higher MW, as seen for Ad2 14.5K, the Ad4 14.5K protein was represented by three bands of equal intensity. Thus, in addition to the expected variation in size, Ad4 14.5K being 16 amino acids longer than the Ad2 homologue, the processing of Ad4 14.5K might differ from that of Ad2 14.5K. In any case, the Ad4 14.5K protein is in fact synthesized in Ad4-infected cells, and lack of 14.5K expression is not the reason for the impaired down-regulation of Fas, EGFR and DR5 by this virus.

10.4-like protein species were also detected in Ad4-infected cells, using the Ad2 10.4K specific reagents R59 and Bur3. The two protein species (faint bands) migrated somewhat faster than their Ad2 counterparts (compare lanes 2 and 3). As the Ad2 10.4K homologue, the Ad4 10.4K protein is predicted to consist of 91 amino acids. The cytoplasmic tail sequence of the Ad4 10.4K protein differs in 9 residues from the Ad2 peptide sequence, which was used to generate

polyclonal serum R59. Bur3 serum recognizes a stretch of 13 amino acids at the C-terminus of 10.4K, but the corresponding Ad4 sequence differs in 6 positions. Therefore, the Ad2-specific sera are likely to bind the Ad4 10.4K protein with lower affinity, which is consistent with the faint appearance. However, the apparent MW of the detected bands is lower than expected, although the Ad5 version of the 10.4K protein, which also consists of 91 amino acids, has been reported to migrate with apparent MW of 7kD (a doublet) and 15kD (Tollefson et al., 1990b). Thus, by analogy and as the doublet bands were not present in lysates of uninfected A549 cells, they may correspond to the Ad4 10.4K protein.

A549 cells infected with Ad2 recombinants Ad2/(Ad4-14.5) #3-8 and #7-1 encoding the Ad4 14.5K protein, produced qualitatively and quantitatively the same 10.4 species as seen in Ad2 (Fig. 42A, 10.4K, compare lane 3 with lanes 4, 5). However, the corresponding Ad4 14.5K signal was different from that obtained by infection with wt Ad4 (Fig. 42, 14.5K, lane 4,5). Instead of 3 bands of equal intensity two major species were detected, which migrated at an apparent MW different from any of the bands observed in Ad4-infected cells. Moreover, the amounts of Ad4 14.5K isolated from lysates of cells infected with Ad2/Ad4 recombinants were lower than those obtained with Ad4-infected cells. Similar amounts of 14.5K were detected for both chimeric constructs, which differed in the spacing of 10.4-14.5K ORFs, but contained the natural context of translation initiation of Ad4 14.5K (TAAGC).

Thus, the Ad4 14.5K protein encoded within the Ad2/E3 region is expressed in infected cells, but is less abundant and might be differently processed compared to that produced during Ad4 infection. None of the Ad2 recombinants in which the Ad2 10.4K sequence was replaced by that of Ad4 10.4K did reveal the doublet band observed after IP/WB of lysates from Ad4-infected cells (Fig. 42A, 10.4K, compare lane 2 with lanes 6-8). Moreover, signal intensities for both Ad2 and Ad4 14.5K proteins were very faint (Fig. 42A, 14.5K, lanes 6-8). Qualitatively, the 14.5K pattern seemed to resemble that observed after infection with wt Ad2 (compare Fig. 42B lanes 3 and 6) or with chimeric constructs Ad2/(Ad414.5) #7-1, Ad2/(Ad414.5) #3-8 (compare Fig. 42B, lanes 7, 8 to 4, 5), respectively. The drastic reduction in signal intensity cannot be explained by reduced protein stability due to the absence of the Ad2 10.4K protein, as in 293 (E3-10.4*)- transfectants, lacking Ad2 10.4K 14.5K was stable and synthesized at a similar rate as in wt E3 transfectants (Elsing and Burgert, 1998). Thus, replacing the Ad2 10.4K ORF and intercistronic sequence upstream of Ad2 14.5K with the corresponding Ad4 sequence seemed to interfere with Ad2 E3/14.5K expression.

Independent of whether the intervening sequence was derived from Ad2 or Ad4, insertion of the Ad4(10.4-14.5) encoding sequence as a whole into the E3 region of Ad2 (Fig. 42, lane 7, 8)

did not allow expression of the Ad4 proteins in a manner similar to that found in Ad4-infected cells. Ad4 14.5K levels were drastically reduced and migrated as differently processed forms.

In conclusion, the Ad4 10.4-14.5K proteins were not sufficiently expressed when placed within the Ad2/E3 region. For all chimeric virus constructs, the Ad2/E3 19K protein was constantly expressed, suggesting that the splicing of the E3 19K encoding mRNA is normal. Normal 10.4K expression was detected as long as the Ad2-10.4 sequence remains intact (Fig. 42, compare lane 3 with lanes 4,5), and was not significantly altered by insertion of Ad4 sequences downstream of the 10.4K ORF. By contrast, replacement of the Ad2 10.4K coding sequence and intercistronic sequence by the corresponding Ad4 nucleotide sequence strongly reduced the amounts of the Ad2 14.5K protein isolated from infected cells. Similarly, Ad4 14.5K levels were also drastically reduced when the Ad4 10.4K CDS was preceeding the 14.5K ORF. This reduction of 14.5K levels was not influenced by the type of intercistronic sequences (Ad2- or Ad4-like), or spacing of 10.4-14.5K coding sequences. The coordinated lack of Ad4 10.4 and Ad4 14.5 expression suggested, that the mRNA(s) for Ad4 10.4-14.5K is not synthesized and inappropriate expression of the Ad4 10.4K protein may contribute to differential processing of Ad4 14.5K encoded by Ad2. In subgroup C viruses Ad2 10.4-14.5K have been reported to be translated from the same bicistronic mRNA, but so far it is unknown, how initiation of translation at the downstream 14.5K ORF occurs. The Ad2/Ad4 chimeric viruses reveal that expression of 14.5K in the Ad2 E3 region is influenced by sequences preceding the 14.5K ORF. Taken together, the data suggest, that expression of Ad4 10.4-14.5K is differently regulated in the Ad4 E3 region, which differs in size and composition from E3 region of subgenus C viruses (Fig. 4).

Discussion

7.1. Importance of strictly conserved amino acids for the function of Ad2 10.4-14.5K