The wtAd5 VA-RNAs are known to interact with several parts of the RNAi pathway. After export into the cytoplasm, VA-RNAs were processed by Dicer into 19-29 nucleotide long small virus associated RNAs (sva-RNAs), which are able to enter the RNA induced silencing complex RISC. As VA-RNAs share structural features with cellular micro RNAs (miRNAs), we speculated that they could also be bound by P19 and may contribute to the observed effect on virus replication. Therefore, we isolated His-tagged P19 from infected B6 cells or as control infected HEK293 cells, using magnetic beads. After purifcation, small RNAs were purified from the protein fraction using different conditions and inserted into a polyA adding reaction followed by cDNA synthesis. In order to solely amplify sva-RNAs, specific primers for the VAI-RNA (3´VAn-r, Table 5.1) and the VAII-RNA (5´VAII, Table 5.1) were used and the generated fragments were subcloned and sequenced (Figure 5.7A and 5.7B, upper panel). Using this method we could show that sva-RNAs from VAI-RNA as well as from VAII- RNA origin are bound to P19 and may be inhibited during infection.
Figure 5.6: Quantification of viral RNA expression of the Bwtp19ΔE3 in comparison to AdΔfiberIL. HEK293 cells were infected with Bwtp19ΔE3 or AdΔfiberIL at a MOI of 3 and RNA was isolated at given time points post infection. RNAs were subjected to reverse transcription and viral mRNA amounts were quantified from cDNA using different adenovirus primers displayed in Table 5.1. Quantitative Real-Time PCR results were normalized to 10 000 molecules of hB2m as internal control. * indicates a p-value < 0.05.
It is estimated that miRNAs incorporated into the P19 dimer were translocated to the cellular processing bodies, where downstream processing of the RNA takes place (Beckham and Parker 2008; Eulalio et al., 2008). Thus, we aimed at analyzing the fate of the sva-RNAs after interaction with the P19 dimer. HEK293 cells and the p19 expressing cell line B6 were infected with wtAd5 at an MOI of 0.05 and small RNAs were isolated at different time points post infection (Figure 5.7, lower panel). After performing Northern Blot analysis using an sva-RNA specific probe either for VAI-RNA (3´Van probe) or VAII-RNA (3´VAII probe) (see
Figure 5.7: Binding of sva-RNAs to P19 and the fate of the svaRNAs during infection. Upper panel: structure of the VAI-RNA (A) and VAII- RNA (B). Highlighted by a grey circle are the predicted sva-RNAs generated from the mature VA-RNA by Dicer processing. Middle panel: PCR fragments of sva-RNAs. B6 cells were infected with wtAd5 at an MOI of 10 and P19 protein was purified 24 hours post infection. Small RNA molecules were isolated from the protein fraction and inserted into a polyA polymerase reaction followed by cDNA synthesis. PCR was then performed with an sva-RNA specific primer for 3´stem of VAI-RNA (3´Van-r) (A) or 5´stem of VAII-RNA (5´VAII) and a primer of the polydT adapter. M: Marker; 293: HEK293 cells; B6f/t: B6 cells were lysed by 4 consecutive freeze/thaw cycles; B6s: B6 cells were sonificated 5 times on ice; PK: Proteinkinase buffer was used to isolate miRNAs from P19. Other miRNAs were isolated using Qiazol reagent and following the manufacturer instructions. Lower panel: Northern Blot analysis to track the fate of sva-RNAs during adenovirus infection in HEK293 cells and B6 cells. HEK293 cells and the RNAi knockdown cell line B6 were infected at an MOI of 0.05 and small RNAs were isolated at time points displayed in the figure. Equal RNA amounts were loaded on a Northern gel, blotted to a NylonBond+ membrane and detected with a-P32 labbeled probe specific for either the stem of the VAI-RNA (3´Van) (A) or the VAII-RNA (5´VAII) (B). Degradation products observed in the B6 cell line were highlighted in boxes.
also Table 5.1) we observed VA-RNAs, sva-RNAs and also products of smaller size compared to sva-RNAs, which may refere to degraded sva-RNAs (black boxes in Figure 5.7, lower panel). Furthermore, svaRNAs from VAI-RNA origin (Figure 5.7A, lower panel) are degraded much earlier than the ones from VAII-RNA (3´VAII probe (Figure 5.7B, lower panel) as the small fragment appears at 15 hours post infection whereas the ones from VAII- RNA can be observed not until 42 hours post infection (Figure 5.7B. lower panel). The binding of the sva-RNAs to P19 suggests a contribution of the small VA-RNA derived fragments to the enhanced replication, however a direct proof is missing.
Therefore, we investigated a VAII-deleted adenovirus obtained from Göran Akusjärvi (Uppsala University, Uppsala, Sweden). The deletion was shown by Northern Blot analysis (data not shown). Replication was determined in HEK293 cells and the RNAi knockdown cell line B6 infected at an MOI of 0.5 (Figure 5.8). The quantification of viral genome copy numbers revealed a strong increase up to 100-fold in viral genome copies 10 hours post infection. However, replication adopted to slightly lower levels of viral genome copy numbers compared to virus derived from HEK293 cells 24 and 48 hours post infection (Figure 5.8). Thus, we speculated that VAII-RNA are rather important at later time points post infection as the lack of VAII-RNA leads to abrogation of the virus replication at later time points. This is in concordance with our Northern analysis, which showed that svaRNAs were processed only at late time points during infection.
Figure 5.8: Replication of a VAII-deleted virus in HEK293 cells and the RNAi knockdown cell line B6. The VAII-deleted Virus dl705 was infected at an MOI of 0.1 into HEK293 cells and B6 cells. Whole cellular DNA was isolated at standard time points and viral genome copy numbers were quantified using gexon specific primers. Results were normalized to B2m as internal control and viral genome copies were calculated on the basis of 104 cells. * indicates a p-value < 0.05.
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5.2.6 Application of p19 expression to improve production of high-capacity