Replication of Bwtp19 Δ E3 is significantly enhanced in comparison to wtAd and

To further improve Ad5 replication, we attempted to increase P19 levels by introducing the

p19 cDNA into the adenovirus genome and couple its transcription to the adenovirus fiber protein. Therefore, an expression cassette containing parts of the flanking regions in the adenovirus genome (58 nt overlapping sequence to the 3´end of the fiber and 58 nt overlapping nucleotides to the sequence downstream of the fiber gene), the internal ribosomal entry site (IRES) of the encephalo myocarditis virus (EMCV), a spacer fragment, the p19 cDNA and a polyA signal was introduced into an E3-deleted Ad5 bacterial artifical chromosome (BAC) by homologous recombination (Figure 5.3A). We used an E3-deleted adenovirus BAC to ensure proper packaging of the adenoviral genome, because we introduced a DNA fragment of 1400 bp length, which may exceed the Ad packaging capacity. After reconstitution of single plaque forming viruses, respective lyates were used to re-infect HEK293 cells. After isolation of whole cellular DNA the specific 1700 bp fragment (including parts of the fiber) introduced into the Ad5 BAC could be confirmed by PCR (data not shown). Furthermore p19 mRNA expression of two independent single plaques after reverse transcription (RT) and PCR with p19 specifiic primers confirmed functional viruses (Figure 5.3B). Hence, viral lysate of clone 2 was amplified, purified and titrated resulting in the virus Bwtp19ΔE3 (Figure 5.3A, upper panel). Viral titers obtained from Bwtp19ΔE3 were comparable to wtAd5 (data not shown).

Figure 5.2: Quantification of adenoviral genome copy numbers derived from B6 cells in comparison to HEK293 and analysis of the p19 RNA status during infection. All results are statistically relevant with a p-value < 0.05. (A) Quantification of adenoviral genome copy numbers in B6 cells and HEK293 cells after infection. HEK293 and B6 cells were infected at an MOI of 0.05 and genomic DNA was isolated 2 hrs, 10 hrs, 24 hrs and 48 hrs post infection. Viral genome copy numbers were quantified using hexon specific primers for qRT-PCR and were normalized to expression levels of human B2m. (B) Analysis of p19 RNA molecules during infection of adenovirus at an MOI of 0.05. B6 cells were infected at an MOI of 0.05 and RNA was isolated at indicated time points. RNA was then subjected to reverse transcription and cDNA was normalized to endogenous human B2m expression levels of 10 000 molecules.

To prove that enhanced virus replication in cells infected with the P19 expressing virus Bwtp19ΔE3 is due to enhanced P19 expression levels compared to the stably expressing cell line B6, p19 mRNA levels were determined by qRT-PCR during virus infection. We infected HEK293 cells with the virus Bwtp19ΔE3 at an MOI of 3 and isolated RNA at the ndicated time points. After RT reaction, p19 RNA molecules were quantified and the results confirmed a strong increase of p19 mRNA levels compared to B6 cells, reflecting the expression pattern of the fiber protein (Figure 5.3C). During infection with the virus

Figure 5.3: Design and characterization of a p19 expressing adenovirus deleted in E3 and analyzing its replication properties in comparison to wtAd5. (A) Constructs used to analyze replication efficiency of a p19 expressing adenovirus. Upper panel: Bwtp19ΔE3: adenovirus containing a p19 expression cassette expressed under the control of the fiber promoter. The spacer, the internal ribosomal entry site (IRES), P19 and a polyadenylation signal (polyA) containing fragment was inserted into the E3-deleted bacterial artificial chromosome (BAC) by homologous recombination and virus was reconstituted. Lower panel: wildtype adenovirus genome (wtAd5). (B) P19 mRNA expression from the BAC-derived virus Bwtp19ΔE3 in comparison to stably p19 expressing B6 cells. HEK293 cells were infected with the virus Bwtp19ΔE3 at a MOI of 1 and 24 hours post-infection RNA was isolated and reverse transcribed. The generated cDNA was then used as template for a PCR with p19 specific primers. As control, reverse transcribed RNA from B6 cells and genomic DNA of Bwtp19ΔE3 infected cells are shown. (C) Quantification of p19 mRNA molecules during infection with Bwtp19ΔE3. HEK293 cells were infected with Bwtp19ΔE3 at an MOI of 3 and RNA was isolated and reverse transcribed. The cDNA was subjected to qRT-PCR reaction and normalized to 10,000 molecules of the internal control (human B2m). Quantification of viral genome copy numbers after infection of HEK293 cells at an MOI of 0.05 is displayed in (D) and at an MOI 3 in (E). After infection whole cellular DNA was isolated after given time points and DNA was quantified with hexon specific primers and normalized to human B2m. The amount of viral genomes was adopted to 104 cells. * indicates a statistically significant result; * p-value< 0.05.

Bwtp19ΔE3, p19 RNA expression levels were enhanced up to 100-fold 24 hrs and 48 hrs post infection when compared to the B6 cell line.

As the generated Bwtp19ΔE3 showed a high P19 expression level we hypothesized that there could also be a significant effect in virus replication. Subsequently, we investigated whether the newly designed virus Bwtp19ΔE3 displays an advanced replication profile in comparison to a control virus. As already mentioned before the amount of P19 protein is a major hurdle within the experimental set up and therefore, we first tested pre-conditions concerning the adenovirus transducing units required in our assay. We infected HEK293 cells with Bwtp19ΔE3or wtAd5 (Figure 5.3A, lower panel) either at an MOI of 0.05 (Figure 5.3D) or 3 (Figure 5.3E), isolated whole genomic DNA and quantified viral genome copy numbers at the standard time points. QRT-PCR results confirmed our hypothesis that at an MOI of 3, and therefore higher P19 expression levels, produce sufficient amounts of P19 to enhance viral replication up to 10-fold 24 hours post infection, whereas an MOI of 0.05 was not sufficient (Figure 5.3D and E). In fact, we observed a slight increase in wtAd5 replication levels. This clearly indicated that the P19 effect on virus replication is controlled by the amount of P19 protein. Furthermore, we speculated that the slight decrease of Bwtp19ΔE3 replication compared to wtAd5 using an MOI of 0.05 could be due to an attenuation of virus replication because of the deletion of the E3 gene or introduction of a DNA sequence into the adenoviral genome, which may destabilize the viral DNA. Therefore, we explored another control virus, the virus AdΔfiberIL, harboring the same DNA fragment connected to the fiber but with a luciferase cDNA instead of the p19 (Figure 5.4A, upper panel) (Rivera et al., 2004). In addition, AdΔfiberIL contains a 24 bp deletion in the E1A gene, however, this has no effect on replication in the HEK293 cell line (data not shown). After infection of these viruses into HEK293 cells at an MOI of 3, quantification of viral genome copy numbers revealed significantly enhanced virus replication of Bwtp19ΔE3 compared to AdΔfiberIL, with up to 100-fold more viral genomes 24 hours post infection (Figure 5.4B). Furthermore, after titration of the viral lysates we obtained an up to 10-fold higher amount of viral particles from Bwtp19ΔE3 infection at 48 hours post infection, indicating a faster replication cycle in the presence of proper levels of P19 (Figure 5.4C).