REPLICATION-DEFICIENT ADENOVIRUS

Abstract

The present invention generally relates to the field of adenoviruses and adenoviral vectors that can be used as vaccines and gene therapy vectors. More specifically, the present invention relates to an adenovirus or an adenoviral vector that comprises a mutated DNA-binding protein that inhibits adenoviral DNA replication in a cell infected with a virus expressing said protein. The invention further relates to a nucleotide sequence encoding the mutated DNA-binding protein. In another aspect, the invention provides pharmaceutical compositions, vaccines and cells that comprise the mutated protein, a nucleotide sequence encoding same, or a modified adenovirus or adenoviral vector comprising any of those. The invention also relates to the use of the mutated protein, a nucleotide sequence encoding the same, or an adenovirus or recombinant adenoviral vector comprising any of those for the preparation of a vaccine.

Claims

1. Adenoviral DNA-binding protein (DBP) comprising a mutation in the sequence motif NH.sub.2-Ser-[Gly/Ser/Ala]-[Lys/Arg]-Ser-COOH which inhibits adenoviral DNA replication in a cell infected with a virus expressing said protein.

2. Adenoviral DNA-binding protein (DBP) of claim 1, wherein said mutation is an amino acid substitution or deletion of the Ser residue located at the COOH terminus of the sequence motif.

3. Adenoviral DNA-binding protein (DBP) of claim 2, wherein said mutation is an amino acid substitution from Ser to Ala.

4. Adenoviral DNA-binding protein (DBP) of claim 1, wherein said protein comprises the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 90% identity thereto.

5. Nucleotide sequence encoding the adenoviral DNA-binding protein (DBP) of claim 1.

6. Plasmid comprising the nucleotide sequence of claim 5.

7. Adenovirus or recombinant adenoviral vector comprising the nucleotide sequence of claim 5.

8. Adenovirus or recombinant adenoviral vector of claim 7, wherein said adenovirus or adenoviral vector belongs to a type selected from the group of HAdV types 1, 2, 3, 4, 5, 6, 7, 31, 40, and 41.

9. (canceled)

10. A method of treating an adenovirus infection, comprising administering to a subject in need thereof, an adenoviral DNA-binding protein (DBP) of claim 1 or a nucleotide sequence encoding the same.

11. A method of vaccinating a subject against a disease caused by an adenovirus, the method comprising administering to a subject in need thereof, an adenoviral DNA-binding protein (DBP) of claim 1 or a nucleotide sequence encoding the same.

12. The method of claim 11, wherein said disease is selected from the group consisting of keratoconjunctivitis epidemica, acute respiratory diseases, pharyngoconjunctival fever, follicular conjuncttivitis, gastroenteritis, gastroenteritis with mesenteric lymphadenopathy, pneumonia, and pharyngitis.

13. A gene therapy method, said method comprising administering to a subject in need thereof, an adenoviral DNA-binding protein (DBP) of claim 1 or a nucleotide sequence encoding the same.

14. Cell comprising an adenoviral DNA-binding protein (DBP) of claim 1 or a nucleotide sequence encoding the same.

15. Pharmaceutical composition or vaccine comprising an adenoviral DNA-binding protein (DBP) of claim 1.

16. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0044] FIG. 1 shows that DBP interacts with USP7 in transfected cells. H1299 cells (A) and HCT116 cells (B) were co-transfected with 10 μg of the plasmid constructs flag-DBP and myc-USP7. In order to transfect the cells with the same amount of plasmid-DNA between the different samples, the DNA amounts were adjusted with the expression vector pCMX3b-Flag. The cells were harvested 48 h p.t. (hours post transfection) and lysed. The flag-coupled DBP was immunoprecipitated using α-flag-antibody coupled agarose. The proteins were separated by SDS-PAGE and visualized by immunodetection. Precipitated and coprecipitated proteins were detected with the antibodies M2 (α-flag) and 3D8 (α-USP7).

[0045] FIG. 2 shows that DBP-S76A does not bind to USP7. H1299 cells were transfected with 10 μg of each of the plasmid constructs pcDNA3 or myc-USP7. 24 h after transfection the cells were infected with the viruses H5pg4100 (WT), H5pm4250 (UBM2) or H5pm4251 (UBM5) (m.o.i. 20), harvested 24 h.p.i/48 h p.t. and lysed. The DBP was immunoprecipitated using α-DBP-Ab coupled protein A-Sepharose, the proteins were separated by SDS-PAGE and visualized by immunodetection. Equilibrium amounts of specific proteins were detected with the antibodies B6-8 (α-DBP), 3D8 (α-USP7) and α-β-actin. Precipitated and coprecipitated protein (IP) was detected with the antibodies B6-8 (α-DBP) and 3D8 (α-USP7). The molecular weight in kDa is indicated on the left and the detected proteins on the right side of the figure.

[0046] FIG. 3 shows the localization of USP7 and DBP in H5pg4100 (WT), H5pm4250 (UBM2) or H5pm4251 (UBM5) infected HCT116 cells. HCT116 cells were infected with H5pg4100 (WT), H5pm4250 (UBM2) or H5pm4251 (UBM5) (m.o.i. 10) and fixed 48 h p.i. with 4% PFA. The immunodetection was carried out with the primary antibodies α-DBP (B6-8) and α-USP7 (3D8) as well as the secondary antibodies α-mouse-Texas red coupled and α-rat-Alexa488 coupled. The position of the cell nucleus was detected using the dye DAPI. Representative DBP and USP7 localization patterns of an average of 70 analyzed cells are shown (magnification ×1000).

[0047] FIG. 4 shows that the viral mutant UBM5 has a defect in the viral DNA synthesis. H1299 cells were infected with H5pg4100 (WT), H5pm4250 (UBM2) or H5pm4251 (UBM5) (m.o.i. 10) or transfected with 5 pg of the plasmid construct Flag-DBP WT followed by an UBM5 infection 24 h p.t. (m.o.i. 10). The cells were harvested 8-72 h p.i.. The viral DNA from the prepared lysates served as a template for a PCR with gene-specific oligonucleotides so that a PCR product of 389 bp from the HAdV-C5 E1B gene could be amplified and visualized on an agarose gel.

[0048] FIG. 5 shows altered equilibrium quantities of viral and cellular proteins after UBM5 infection compared to the wild-type. H1299 cells (A) or HCT116 cells (B) were infected with H5pg4100 (WT), H5pm4250 (UBM2) or H5pm4251 (UBM5) at an m.o.i. of 20, harvested 8-72 h p.i. and lysed. The total protein cell extracts were separated by SDS-PAGE, and the proteins were visualized by immunodetection. Equilibrium quantities of specific proteins were detected with the antibodies M73 (α-E1A), 2A6 (α-E1B-55K), B6-8 (α-DBP), a—E4orf4, RSA3 (α-E4orf6), 6B10 (α-L4-100K), L133 (α-capsid proteins), α-β actin, 3D8 (α-USP7), α-Daxx and α-PML. The molecular weight in kDa is indicated on the left and the detected proteins on the right side of the figure.

[0049] FIG. 6 shows restoration of expression of late viral proteins in the viral mutant UBM5. H1299 cells were transfected with 5 pg of the plasmid construct flag-DBP WT or 10 μg of the plasmid construct flag-DBP S354A. In order to transfect the cells with the same amount of plasmid DNA in the different samples, the expression vector pCMX3b-flag was co-transfected if necessary. the transfected cells were infected with H5pg4100 (WT) or H5pm4251 (UBM5) (m.o.i. 10) 24 h p.t., harvested 8-72 h p.i. and lysed. The total protein cell extracts were separated by SDS-PAGE and the proteins were visualized by immunodetection. Equilibrium amounts of specific proteins were detected with the antibodies M73 (α-E1A), 2A6 (α-E1B-55K), B6-8 (α-DBP), α-E4orf4, RSA3 (α-E4orf6), 6B10 (α-L4-100K), L133 (α-capsid proteins), α-β actin, 3D8 (α-USP7), α-Daxx and α-PML.

EXAMPLES

[0050] The present invention is further illustrated by the following examples, which in no way should be construed as limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co pending patent applications) cited throughout this application are hereby expressly incorporated by reference. The following materials and methods were used for performing the experiments described herein.

[0051] Cell Lines and Culture Conditions

[0052] H1299 (Mitsudomi et al., 1992), HCT116 (Brattain et al., 1981), HEK-293 (Graham et al., 1977) and 2E2 cells (Catalucci, 2005) were grown in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 5 to 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 pg/ml streptomycin in a 5% CO.sub.2 atmosphere at 37° C. For 2E2 cells, the medium was additionally supplemented with 90 pg/ml of hygromycin B and 250 pg/ml of geneticin (G418). In 2E2 cells, expression of the E2-gene region was induced by 1 pg/ml doxycycline.

[0053] Plasmids and Transient Transfections

[0054] Both, pcDNA3 (Invitrogen) and pCMX3b encoding the cytomegalovirus (CMV) immediate-early promoter were used in the present study. HAdV-DBPs expressed from pCMX3b-based plasmids are flag tagged (Terzic, 2014). Human USP7 (tagged with myc; Zapata et al., 2001) was expressed from pcDNA3 based plasmid. Flag-DBP mutants were derived through nucleotide exchanges by site-directed mutagenesis using the following oligonucleotides:

TABLE-US-00001 E2A-UBM2 fwd primer 5′-CCAGCCCGCGGCCATCGACCGCGGCGGCGGATTTGGCC-3′, E2A-UBM2 rev primer 5′-GGCCAAATCCGCCGCCGCGGTCGATGGCCGCGGGCTGG-3′; E2A-UBM5 fwd primer 5′-CCAATCAGTTTTCCGGCAAGGCTTGCGGCATGTTCTTCTC-3′, E2A-UBM5 rev primer 5′-GAGAAGAACATGCCGCAAGCCTTGCCGGAAAACTGATTGG-3′.

[0055] Transient transfection of subconfluent cells was performed with linear polyethylenimine (PEI; 25 kDa; Polysciences). The transfection solution was prepared by incubating a mixture of DNA, PEI and DMEM in a ratio of 1:6:60 (DNA:PEI:DMEM) for 10 min at room temperature (RT). Prior to transfection, the culture medium was replaced by DMEM without FBS and antibiotics. Transfection solution was added to the cells and incubated for 4 h in a 5% CO.sub.2 atmosphere at 37° C. before replacement of the medium with DMEM supplemented with 10% FCS, 100 U of penicillin and 100 pg of streptomycin per ml.

[0056] Viruses and Infections

[0057] The following viruses were used in the present study: H5pg4100 (wt), H5pm4250 (UBM2) and H5pm4251 (UBM5). H5pg4100 is an HAdV-C5-derived virus with deletions in the E3-coding region (Kindsmüller et al., 2007) and served as wild type virus. A nucleotide exchange in the DBP open reading frame of H5pm4250 or H5pm4251 resulted in a serine to alanine substitution at position 76 or 354, respectively. Mutagenesis of the DBP gene via site-directed mutagenesis PCR (Groitl & Dobner, 2007) was carried out with the following two primers for UBM2:

TABLE-US-00002 3151 - 5′-CCA GCC CGC GGC CAT CGA CCG CGG CGG CGG ATT TGG CC-3′ 3152 - 5′-GGC CAA ATC CGC CGC CGC GGT CGA TGG CCG CGG GCT GG-3′

[0058] and the following two primers for UBM5:

TABLE-US-00003 3157 - 5′-CCA ATC AGT TTT CCG GCA AGG CTT GCG GCA TGT TCT TCT C-3′ 3158 - 5′-GAG AAG AAC ATG CCG CAA GCC TTG CCG GAA AAC TGA TTG G-3′.

[0059] The mutated DBP open reading frames were used to generate the virus mutants as described previously (Groitl & Dobner, 2007; Zeller, 2005; Koyuncu & Dobner, 2009).

[0060] Sequence analyses of the whole adenoviral genome ensured that only the desired mutation was inserted into the genome of these virus mutants during the whole process. H5pg4100 (wt) was propagated in H1299, HEK-293, whereas H5pm4251 (UBM5) was first generated in 2E2 cells. All viruses were titrated in H1299 cells. To measure viral progeny of infected cells, the cells were harvested, released viral particles and viral titer were determined as described before (Kindsmuller et al., 2009). Infection of cells was carried out with virus dilutions in DMEM without additive at indicated multiplicities of infection (m.o.i.). Two hours post infection DMEM containing FCS, penicillin and streptomycin was added to the virus-containing medium (1:1). Cells were harvested at the indicated time points.

[0061] Antibodies

[0062] Primary antibodies specific for adenoviral proteins used in the present study included anti-DBP mouse mAb B6-8, α-E1A mouse mAb M73, α-E1B-55K mouse mAb 2A6, α-E4orf4 rabbit pAb, α-E4orf6 mouse mAb RSA3, α-L4-100K rat mAb 6610 and α-capsid proteins rabbit pAb L133 (Kindsmuller, 2007). Primary antibodies for the detection of cellular and ectopically expressed proteins included α-β-actin mouse mAb (Sigma-Aldrich), α-flag mouse mAb M2 (Sigma-Aldrich), α-Daxx rabbit pAb, α-PML rabbit pAb, and α-USP7 rat mAb 3D8. Secondary antibodies conjugated to horseradish peroxidase (HRP) for detection of proteins by immunoblotting were anti-mouse IgG, α-rabbit IgG and α-rat IgG (Jackson, ImmunoResearch).

[0063] Protein Analysis and Immunoprecipitation

[0064] Cell pellets of transfected or infected cells were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl (pH 7,6), 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0,5% sodium deoxycholate and 0,1% sodium dodecyl sulfate (SDS)) with protease inhibitors (1% (vol/vol) PMSF (phenylmethylsulfonyl fluoride), 0,1% (vol/vol) aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin) added upon usage on ice for 30 min. Cell lysates were sonicated for 30 sec at 4° C. (output 0.6; 0.8 impulses/s; Branson Sonifier 450) and centrifuged subsequently to pellet the cell debris (11000 rpm, 3 min, 4° C.). Protein concentration was determined photometrically with Bradford Reagent (BioRad). Protein samples were separated by SDS-PAGE after boiling for 3 min at 95° C. in Laemmli buffer (5×). Proteins were transferred to nitrocellulose blotting membranes (0.45 μm pore size) and visualized by immunoblotting (Western blot) as described previously (Freudenberger et al., 2017). Protein lysates were additionally used for immunoprecipitation analyses. To investigate protein-protein interaction, proteins were immunoprecipitated as described previously (Berscheminski et al., 2012) with the exception that the Ab-coupled protein A-sepharose or flag-M2-coupled agarose (Sigma-Aldrich) was rotated overnight at 4° C. with the precleared protein lysates.

[0065] Analysis of Viral DNA Synthesis by PCR

[0066] Adenoviral DNA replication was determined by PCR. At the indicated time points, infected cells were harvested and lysed in RIPA buffer as described above. Then, 10 pg of total cell lysates were treated with Tween-20 (final concentration: 0.5%; Applichem) and proteinase K (final concentration: 100 pg/ml; Roche) together with nucleic acid-free water (Promega) to a final volume of 100 μl. The samples were incubated for 1 h at 55° C. prior to enzyme inactivation for 10 min at 100° C. 24.5 μl of each sample were used as DNA matrices for a PCR with specific oligonucleotides to amplify a fragment of 389 bp (E1B-55K-gene of HAdV-C5) as described before (Ching et al., 2013). The PCR products were analyzed on 1% agarose gels containing ethidium bromide.

Example 1: DBP-USP7 Binding Studies

[0067] In order to investigate the hypothesis that USP7 relocalization is mediated by DBP, a putative protein-protein interaction of DBP with transfected and endogenous USP7 was investigated in human H1299 and HCT116 cells. These two cell lines represent different infection targets of adenoviruses (Brattain et al., 1981; Mitsudomi et al., 1992). First, immunoprecipitation of DBP with wt-virus (H5pg4100) infected H1299 and HCT116 cells and subsequent staining of USP7 showed an interaction between both (data not shown). To test whether this interaction is independent of other viral proteins, H1299 and HCT116 cells were transfected with a plasmid encoding flag-DBP and myc-USP7, respectively. In H1299 cells both endogenous and overexpressed USP7 could be coprecipitated with DBP (FIG. 1A) whereas in HCT cells only overexpressed USP7 was coprecipitated with DBP (FIG. 1B). The infection analyses confirmed USP7-DBP interaction during the infection cycle. Additionally, the transfection analysis with the lack of further viral proteins shows that the aforementioned interaction is independent from other viral proteins.

[0068] To characterize the interaction in more detail, the DBP binding domain of USP7 was investigated. USP7 consists of the N-terminal TRAF (tumor necrosis factor receptor (TNFR)-associated factor)-like domain, central catalytic domain and five C-terminal UBL (ubiquitin-like)-domains (Holowaty et al., 2003a; Zapata et al., 2001; Faesen et al., 2011). GST fusion proteins corresponding to these USP7-domains were used and their interaction with DBP was evaluated by GST pull-down experiments (data not shown). The first 215 residues of USP7, which correspond to the TRAF-like domain of USP7, precipitated strongly and specifically DBP from transfected and wt-virus (H5pg4100) infected cells (data not shown). Whereas binding with the other GST-fused USP7-fragments or GST alone is not detectable (data not shown). This result is in accordance with already published USP7-interaction partners (e.g. p53, MDM2), which had also been shown to interact with the TRAF-like domain of USP7 (Sheng et al., 2006; Holowaty et al., 2003a).

Example 2: Generation of DBP Variants

[0069] Several USP7-interaction partners like p53, MDM2 and EBNA1 encode the consensus motifs “X-Gly-X-Ser” or “Pro/Ala-X-X-Ser” which provide for USP7 binding (Sheng et al., 2006). Therefore, the amino acid sequence of DBP was analyzed for USP7-binding motifs (UBM) and five potential USP7-binding sites were identified (31-Pro-Ser-Pro-Ser-36, 72-Pro-Ser-Thr-Ser-77, 118-Val-Gly-Phe-Ser-123, 175-Pro-Iso-Val-Ser-180, 350-Ser-Gly-Lys-Ser-355). In order to analyze the putative USP7-binding sites in DBP, the last serine of each motif was substituted (from Ser to Ala) and the interaction of these DBP variants with USP7 was investigated in immunoprecipitation experiments. The expression levels of the DBP variants were comparable except for the mutant DBP-S354A whose protein level was reduced (data not shown). USP7 interaction could be proven with almost all DBP variants although binding to the mutant DBP-S354A was decreased. Strikingly, the interaction of USP7 with mutant DBP-S76A is abolished.

[0070] To further verify the abolished interaction between USP7 and the mutant DBP-S76A, GST pull-down assays were performed. For this purpose, the GST-fused TRAF-like domain of USP7 was used and lysates of H1299 cells were transfected with the DBP-variants. Consistent with the immunoprecipitation analyses shown before, DBP-variants were pulled-down with the GST-tagged TRAF-like domain of USP7 (data not shown) except of the mutant DBP-S76A. Although the protein levels of DBP-S354A were decreased in comparison to the other DBP-variants, the amount of pulled-down DBP-S354A was comparable to DBP-wt (data not shown). These results indicate that the TRAF-like domain of USP7 interacts with the “Pro-Ser-Thr-Ser” motif of DBP.

Example 3: Generation of Virus Mutants

[0071] To clarify the role of the DBP-variants in virus infection, virus mutants were prepared. DBP-variants with a different phenotype in transfection compared to wild-type DBP (DBP-wt) were selected. Hence, the DBP amino acid exchange mutant S76A (UBM2), which is deficient in USP7 binding, and the mutant S354A (UBM5), which exerts reduced protein levels, were selected. USP7-binding of the newly generated DBP-mutants in infection was investigated via immunoprecipitation analyses. Consistent with the transfection experiments, DBP-S76A of UBM2 did not bind to USP7 in infection (FIG. 2). Despite of decreased protein levels of DBP-S354A, the levels of coprecipitated USP7 with the UBM5-DBP are comparable to the DBP-wt (FIG. 2). These data demonstrate that the “PSTS” motif (aa 73-76) in the N-terminal domain of DBP is crucial for USP7-binding. Furthermore, these data showed that the amino acid exchange of aa 354 in the C-terminal domain of DBP influences the expression or stability of this protein.

Example 4: Functional Studies

[0072] In order to investigate the implication of DBP binding to the intranuclear relocalization of USP7 into viral replication centers, H1299 cells were infected with wt-, UBM2- or UBM5-virus and analyzed via immunofluorescence analysis. Non-infected cells showed a diffuse nuclear distribution of USP7, while it was relocalized to viral replication centers in wt-infected cells independent of the investigated time point. During the infection progress, different morphologies of the replication centers could be observed, nevertheless USP7 and DBP always colocalized in wt-infected cells.

[0073] In UBM2-infected cells DBP-S76A localized comparable to DBP-wt, but the relocalization of USP7 into viral replication centers was completely abolished at 8, 16 and 24 h p.i. in 100% of the infected cells (data not shown). A partial relocalization of USP7 into ring-like or rosette-shaped replication centers was observed 48 h p.i. in 55% of the UBM2-infected cells (FIG. 3). However, USP7 was still diffusely distributed within the nucleus in 45% of the UBM2-infected cells. Therefore, it was concluded that the binding of USP7 by DBP is necessary for relocalization of USP7 during infection, as DBP-S76A from the UBM2-virus is unable to bind USP7.

[0074] In UBM5-infected cells both DBP and USP7 localized diffusely in the nucleus at all investigated time points and in all evaluated cells. (data not shown). Thus, the UBM5-virus is completely defective in the establishment of replication centers. To finally show that the amino acid exchange S354A is responsible for the affected replication centers formation, a plasmid encoding DBP-wt was transfected before UBM5 infection (data not shown). This led to detectable replication centers and a relocalization of USP7 in UBM5-infected cells.

[0075] In summary, it was demonstrated that the amino acid exchange S76A in DBP prevents the relocalization of USP7, especially at early time points of infection, whereas the amino acid exchange S354A in DBP abolishes the establishment of viral replication centers.

Example 5: Analysis of DNA Replication and Virus Progeny Production

[0076] Since immunofluorescence analyses showed impaired USP7 relocalization in UBM2-virus infected cells and failure to establish replication centers for the UBM5-virus, the consequences of these phenotypes on viral DNA replication were further analyzed. To investigate this, H1299 cells were infected with wt-, UBM2- or UBM5-virus, and the isolated viral DNA was analyzed by standard PCR with E1B-gene-specific oligonucleotides. PCR products could be detected starting 16 h p.i. from the wt-virus and the UBM2-virus infected cell extracts (FIG. 4). As expected, PCR products were absent in the UBM5-virus infected cell extracts (FIG. 4). To narrow down this effect on the mutation of DBP, H1299 cells were transfected with a plasmid encoding DBP-wt before infecting with UBM5-virus. As a consequence, viral DNA could be detected in the UBM5-virus infected cells starting 24 h p.i.

[0077] Therefore, it can be concluded that the DNA replication of the UBM2-virus is intact, despite the affected recruitment of USP7 into replication centers. However, the UBM5-virus has a severe defect in viral DNA replication consistent with the defective replication centers formation in UBM5-infected cells. To verify this data with a quantitative and more sensitive method, the viral DNA replication was investigated with real time qPCRs. It was found that the amount of incoming viral DNA at 1 h p.i. was comparable for wt, UBM2 and UBM5 infected cells. During the course of infection however, the UBM2-virus showed a slightly increased DNA synthesis compared to wt-virus. Especially 48 h p.i. about 20% more viral DNA was detected in the UBM2-infected cells. In contrast, the amount of viral DNA did not increase in the UBM5-infected cells during the infection cycle. The amount of detected DNA corresponded to the amount of incoming viral DNA at 1 h p.i. In line with the results shown before, the UBM5-virus is affected severely in replicating viral DNA, whereas the UBM2-virus seems to replicate slightly more efficient than the wt-virus.

[0078] Subsequently, H1299 cells were infected with wt-, UBM2- or UBM5-virus to determine the progeny production by titration analyses/fluorescence focus identification assay. Consistent with the replication defect, virus progeny production is completely abolished in UBM5-virus infected cells. As for the UBM2-virus, slightly increased progeny production of 28% 24 h p.i. and 17% 48 h p.i. was observed compared to wt-virus. This is in agreement with the observed increase in DNA synthesis for the UBM2-virus compared to wt-virus. In light of this, it can be concluded that the amino acid exchange S76A in DBP has a mild effect on viral progeny production, while the amino acid exchange S354A in DBP prevents virus progeny production as a result of defective DNA replication.

Example 6: Analysis of Late Viral Protein Expression

[0079] It is well known that the onset of viral DNA replication is crucial for the expression of late viral genes and thereby induces the transition from early to late phase of infection (Seth, 1999). As the virus mutants showed altered or defective viral DNA synthesis, we investigated their expression of early and late viral proteins. H1299 cells were infected with wt-, UBM2- or UBM5-virus, and cell lysates were investigated with Western Blot-analyses. The early and late protein levels of UBM2-virus compared to wt-virus infected cells were similar (FIG. 5A, compare lane 2-6 with lane 7-11). However, UBM5-virus infected cells showed a few differences in early protein expression and huge differences in late protein expression in comparison to wt-virus infected cells (FIG. 5A, compare lane 2-6 with lane 12-16). The first protein expressed during infection is the early protein E1A, which was expressed during the entire infection cycle in UBM5-infected cells in contrast to wt-virus infected cells in which the E1A levels peaked at 16 h p.i. and decreased over time. As previously observed, the UBM5-virus showed decreased DBP protein levels during the infection cycle compared to the wt-virus. Strikingly, UBM5-virus infection is entirely defective in late protein expression, which is represented by the absence of late protein L4-100K and capsid proteins expression (FIG. 5A, lane 12-16). Thus, as a consequence of defective DNA replication of the UBM5-virus is incapable to induce late viral protein expression. Almost identical results were obtained when HCT116 cells were infected the wild-type virus (FIG. 5B, lanes 2-6) and the UBM2 and UBM5 mutant viruses (FIG. 5B, lanes 7-11 and 12-16, respectively).

[0080] To verify that the observed defect in late protein expression is due to the amino acid exchange in DBP, the experiment was repeated with UBM5-virus infected cells additionally transfected with a plasmid encoding wild-type DBP. The results showed that late viral protein expression could be rescued in the UBM5-virus infected cells after transfection with DBP-wt proving that the UBM5 mutation induces the defect (FIG. 6). Furthermore, this experiment was performed to exclude that a threshold of DBP is needed for the formation of replication centers and therefore the transition from early to late phase of infection. Hence, UBM5-virus infected H1299 cells were transfected with a plasmid encoding the mutant DBP-S354A to adjust the expression level of DBP to wt-infection. Although, the UBM5-DBP level was similar to DBP-wt, late protein expression was still defective after UBM5-virus infection excluding the possibility that the reduced UBM5-DBP level causes the defect in late protein expression. In summary, the amino acid exchange S354A in DBP abolishes the transition into the late phase of infection and therefore affects late protein expression during UBM5-virus infection.

LITERATURE

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