Departamento de Caldas.

To analyze whether the different effects of Ad4 versus Ad2 on target receptor modulation is an intrinsic property of the corresponding subunits of the 10.4-14.5K complex the homologous Ad4 CDS were introduced into the adenovirus 2 genome. This approach may offer several advantages: (i) Both 10.4K and 14.5K are expressed from a single vector backbone, allowing to maintain a constant ratio of 10.4K and 14.5K expression. (ii) The splicing of the Ad2 E3 region is well-characterized (Imperiale et al., 1995). (iii) E3 gene expression could be standardized by monitoring expression of the Ad2 E3/19K protein. iv) In infected cells a high number of Ad

Fig. 38 Sequence composition of the mTn-containing pAd2 intermediates (A) and pAd2/Ad4 chimeric constructs (B).

Nucleotide sequences are given for the coding strand (5’ to 3’), start and stop codons of the ORFs are highlighted in boldface. (A) mTn encoding sequences are indicated by checked boxes and adjacent nucleotide sequences are given to specify their position. (B) 11.6K, 10.4K and 14.5K represent Ad2 ORFs, whereas Ad4 CDS are designated Ad410.4K and Ad414.5K (shaded boxes). Refer to text for details.

genome copies is present, thus a high E3 protein expression level can be attained. Infection can be carried out at equal infection efficiency (equal MOI). (v) Since the target specificity of Ad2 is well- known, the effect of the gene replacement on down-regulation of plasma membrane receptors can be analysed. The adenovirus 2 genome had been cloned into a BAC vector facilitating manipulation of the viral genomic DNA (Ruzsics et al., in preparation). In order to exchange the coding regions, the Ad2 10.4 and 14.5 ORFs were removed from the pAd2-BAC vector in a first step by ET recombination with a kanamycin cassette encoding a mini-transposonsequence (mTn), generating mTn-containing intermediate BAC vectors (Fig. 38A).

The mTn-containing replacement sequences were amplified by PCR using pGPS1.1 as a template. The linear recombination fragments had 40-43 base pair homology arms to the sequence flanking the target site within the pAd2-BAC, which served to direct ET recombination (described

in Materials and Methods). For ET recombination the mTn containing PCR products were

transformed into DH10B containing pBADαβγ and the target BAC pAd2. mTn-containing intermediate pAd2BAC vectors could be selected by growth of the bacteria in Cm/Km-containing media. The transposon sequence was excised from the pAd2-BAC vector in an in vitro reaction using TnsABC* (see chapter 5.2.5 Tn removal) and the gap in the pAd2 vector was ligated with an insert encoding the Ad4 replacement sequence. Transposase excises the transposon with trinucleotide 5’ overhangs on both sides of the transposon. Thereby, distinct trinucleotide single stranded overhangs are generated in the open vector, which preclude recircularization of the pAd2- BAC vector and allow directed insertion of the Ad4 sequence with its correct 5’→3’ orientation. Ad4 inserts were amplified by PCR on plasmid pA4E3, containing part of the Ad4 E3 region (Genbank AF361223). Primers for synthesis of Ad4-CDS inserts (see also Materials and Methods) encompassed a priming region with 20 bp homology to the Ad4 sequence and SapI recognition sites at the 5’ ends. A trinucleotide sequence complementary to the corresponding 3 nt overhang in the pAd2-BAC vector was incorporated in the primer 3’ to the SapI recognition site. By this approach, referred to as exposon cloning (Ruszics et al., manuscript in preparation), different pAd2/Ad4 chimeric constructs were generated with 10.4, 14.5 or both CDS replaced by the corresponding Ad4 sequences. Interestingly, the Ad4-14.5K coding sequence was found to be 48 nucleotides longer than the Ad2-14.5K ORF and in addition the spacing of the Ad4-10.4 and Ad4- 14.5 ORFs is different from the one in Ad2. In Ad4 the intercistronic sequence is 5 nt (nucleotide sequence TAAGC) in length instead of 2 nt (TT) in Ad2. In order to be able to generate Ad2/Ad4 constructs with different intercistronic sequences, the position of the mTn sequence was defined in a way (Fig. 38A) that allowed to modify the 10.4-14.5K intercistronic sequence by incorporating sequence changes in the corresponding Ad4 insert. The desired sequence was included in the inserted fragment between the SapI cleavage site and the Ad4-specific sequence by modification of insert amplification primers (see also Materials and Methods).The pAd2/(10.4Tn) construct was used for generation of pAd2/(Ad4-10.4) by Tns excision and ligation with an Ad4-10.4 encoding insert (Fig. 38B, see Materials and Methods). pAd2/(Ad4-14.5) constructs #3-8 and #7-1, were obtained by Tn removal from pAd2/(14.5Tn) and ligation of the open vector with two different types of Ad4- 14.5 inserts. These were designed to yield pAd2/(Ad4-14.5) constructs #3-8 and #7-1, with 10.4- 14.5K intercistronic sequences of 5 or 2 nucleotides in length, respectively (refer to Fig. 38B and

Fig. 39 Restriction cut analysis of mTn-containing intermediate BAC vectors

EcoRV, HindIII (A) and XhoI (B) were used for analytical restriction cut of BAC DNA and digested samples were separated on a 0.8% agarose gel. Lane numbers indicate the type of construct: 1, pAd2/(10.4Tn) clone #1B; 2, pAd2/((10.4-14.5)Tn)#2A; 3, pAd2/(14.5Tn)#1A; 4, pAd2/F14.5. In (A) the gel was photographed twice to visualize bands of the top and bottom part. Arrows indicate the 3 kb band of M as point of reference. M = 1kb DNA ladder. (C) Schematic drawing of restriction sites and fragment sizes (written in boldface) expected for the mTn-containing pAd2-BACs. The mTn sequence is represented by a black bar. For a complete list of restriction sites refer to annexe.

Materials and Methods). Following transposon removal from the pAd2/((10.4-14.5)Tn)-BAC vector,

the Ad4-(10.4-14.5) coding sequence was inserted as a whole generating pAd2/(Ad4- 10.4)TAAGC(Ad4-14.5)#7-4 (Fig. 38B).

Fig. 40 Circular Map of pGPS1.1, illustrating the position of EcoRV, HindIII and XhoI sites which also cleave within the transposon (Tn) sequence.

The Tn sequence is contained in pGPS1.1 between map position 3068-4767. L and R denote the left and right transposon ends, respectively.

In order to generate pAd2/(Ad4-10.4)TT(Ad4-14.5) #16-1 with a modified intercistronic sequence between the Ad4 ORFs, an additional mTn--containing intermediate, pAd2/(10.4Tn)Ad414.5, was created (Fig. 38A). pAd2/(10.4Tn)(Ad4-14.5) was obtained from pAd2/10.4GC(Ad4-14.5)#7-1 by ET recombination. The mTn-containing linear recombination fragment consisted of a 40 bp homology region to the Ad2 sequence preceding the 10.4 ORF upstream of the mTn sequence and at the 3’ mTn end a homology to the first 40 nucleotides of the Ad4-14.5 CDS. Following transposon removal from pAd2/(10.4Tn)(Ad4-14.5) the ends of the BAC vector were ligated with a (Ad4-10.4)-containing insert, which had a dinucleotide (TT) insertion preceding the 3’ SapI site (Fig. 38B, pAd2/(Ad4-10.4)TT(Ad4-14.5) #16-1).

The Tn-containing pAd2-BAC vectors were analysed by restriction cut with EcoRV, HindIII and XhoI to prove that they contained the full-length genome with the desired transposon insertions (Fig. 39). The inserted mTn sequence contains two EcoRV, one HindIII and one XhoI site (Fig. 40). EcoRV and HindIII do not cut within the Ad2-(10.4-14.5), whereas one XhoI site is present in the Ad2 10.4K sequence. For a list of the expected fragment sizes refer to annexe.

Accurate incorporation of the Ad4 inserts was ascertained by sequencing of the corresponding region in the chimeric pAd2-BAC vectors containing Ad4 coding sequences. In addition the BAC vectors were cleaved by EcoRV and XhoI yielding the correct restriction pattern (Fig. 41). Neither Ad2 nor Ad4 10.4-14-5K coding sequences contain EcoRV sites. Therefore the

EcoRV restriction pattern was similar among all constructs (Fig. 41A), and comparable to the that of the wt pAd2-BAC, although depending on the type of Ad4 insert, the fragment which

pGPS1-1 4814 bp TetR R L oriR6K Tn XhoI (4427) EcoRV (1518) EcoRV (3344) EcoRV(4583) HindIII (404) HindIII (758) HindIII (1360) HindIII (3907)

Fig. 41 Analytical restriction cut of pAd2/Ad4 BAC DNA

BAC DNA was digested with EcoRV (A) or XhoI (B) and separated on a 0.8% agarose gel. Numbers on top of the lanes indicate the type of construct analyzed. 1, pAd2/(Ad410.4) #12-1; 2, pAd2/(Ad414.5) #3-8; 3, pAd2/(Ad414.5) #7-1; 4, pAd2/(Ad410.4-14.5) #7-4; 5, pAd2/(Ad410.4-14.5) #16-1; 6, pAd2-BAC. M = 1kb DNA ladder.

encomprises the 10.4-14.5K CDS slightly varied in length (4755-4803 bp). All the constructs containing the Ad4-10.4K sequence lack one XhoI site, which is present in Ad2-10.4K. Therefore, upon XhoI cleavage of these constructs one ~13.8 kb band was obtained instead of two smaller fragments (Fig. 41B). For a list of the exact fragment sizes refer to annexe.

For reconstitution of infectious viral particles the modified Ad2 genomes were released from the BAC vector by SnaB1 digest and the linear double-stranded genome was transfected into 293 cells.

6.3. Expression of Ad4 10.4K and Ad4 14.5K proteins in cells infected with Ad2/Ad4