The human adenovirus type 5 E1B 55kDa protein interacts with RNA promoting timely DNA replication and viral late mRNA metabolism

Figures

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  Fig 1. E1B 55K interacts with RNA in Ad5 WT-infected cells and RNP substitutions affect RNA binding. HFF cells infected with the indicated viruses were harvested at 36hpi. E1B 55K was immunoprecipitated with the 2A6 MAb, RNA was isolated and RT qPCR were performed to detect a sequence corresponding to intron 2 in the TPL. (A) Western blot of immunoprecipitated samples with the anti-E1B 55K 2A6 MAb. (B) RT qPCR of immunoprecipitated viral RNA. Immunoprecipitation data was normalized as described in Materials and Methods and it is represented as the percentage of the input RNA. Standard deviations from three independent experiments performed in triplicate are shown. *** P<0.001, **** P<0.0001.

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  Fig 2. E1B 55K-RNP peptides interact with TPL RNA 196nts in vitro. (A) Expanded region of an overlay of TOCSY spectra of free WT peptide (black) and WT peptide bound to the TPL RNA 196nts probe (blue). (B) Heat exchanged from each injection of WT peptide into a solution containing the TPL RNA 196nts probe. (C) Expanded region of an overlay of TOCSY spectra of free C287S/C288S peptide (black) and C287S/C288S peptide bound to the TPL RNA 196nts (blue). (D) Heat exchanged from each injection of C287S/C288S peptide into a solution containing the TPL RNA 196nts. The thermograms were best fit to a one binding site model.

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  Fig 3. Effect of substitutions in the E1B 55K RNP motif on viral progeny production. HFF cells were infected at a MOI 30 FFU/cell and harvested at 36 and 48 hpi. Viral titers were determined in 293 cells by fluorescent foci using a mouse monoclonal anti-E2 72K (DBP) antibody. The standard deviations from two independent titration experiments are shown. * P <0.05, **P<P0.01, ***P<0.001.

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  Fig 4. E1B 55k RNP substitutions do not impair degradation of Mre11 and p53. HFF cells were infected and harvested at 16, 24 and 36 hpi. Total protein extracts were obtained and Western blot assays were performed employing anti-Mre11 (Novus Biologicals) and anti-p53 DO1 (Santa Cruz Biotechnology) antibodies. β actin (Santa Cruz Biotechnology) was used as the loading control.

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  Fig 5. Effect of substitutions in the E1B 55K RNP on kinetics of accumulation of viral proteins, DBP and fiber. HFF cells infected with Ad5 WT or E1B 55K mutants were harvested at 16, 24 and 36 hpi. Total protein extracts were obtained and western blot assays were performed using the anti-DBP (B6) and anti-fiber (Abcam) antibodies. β actin (Santa Cruz Biotechnology) was used as the loading control.

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  Fig 6. Localization of DBP and E1B 55K to viral RC in Ad5 WT- or RNP mutants-infected HFF cells. HFF cells infected with Ad5 WT or E1B 55K mutant viruses were fixed and processed for immunofluorescence as described in materials and methods. Blue (DAPI), green (DBP), red (E1B 55K). (A) 24hpi, (B) 36hpi. Results shown are representative of at least two independent experiments.

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  Fig 7. Kinetics of viral DNA accumulation are altered by mutations of the E1B 55K RNP motif. HFF cells infected with Ad5 WT or E1B 55K mutants were harvested at 16, 24 and 36 hpi and total DNA was isolated. Viral DNA was amplified through a quantitative PCR and a viral DNA absolute quantification was performed. Data are shown as viral DNA copy number per cell of duplicate samples from two independent experiments. * p<0.05, ** p<0.01, ** p<0.001, **** p<0.0001, t-test.

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  Fig 8. Substitutions in E1B 55K RNP affect viral late mRNA biogenesis. HFF cells infected with Ad5 WT or E1B 55K mutants were harvested at 36 hpi and total RNA was isolated. Viral late pre-mRNA levels were determined for L5 RNA by RT qPCR against an (A) intron-exon (L5NP) or (B) Exon-exon (L5P) junction, for the unspliced and spliced L5 mRNA species, respectively. (C) To compare the splicing efficiency the L5P:L5NP ratios were calculated. β actin mRNA was used as endogenous control. Data from two independent experiments performed in triplicate are shown. ** P<0.01, ****P<0.0001.

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  Fig 9. Intrinsic disorder in E1B 55K. PONDR VLXT, PONDR XL1_XT, PONDR VL3-BA, PONDR VSL2, IUPred and DISOPRED predictors were used for disorder analysis. All predictors indicate a high level of intrinsic disorder in the N- and C-terminus.

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  Fig 10. E1B 55K-RNA interaction model. (A) Superposition of the model for the wild-type central domain that contains the putative dsRNA binding motif (solid cyan ribbons) and the parent LH3 structure (transparent gray ribbons). (B) Superposition of the models for the wild-type (cyan and red ribbons) and deletion Δ284–289 (cyan and yellow ribbons) versions of the putative dsRNA binding motif of E1B 55K, in the same orientation as above facing the surface designated as PB3. N- and C-termini, as well as loops belonging to T2 and T3 surfaces are indicated. PB2 lies at the right of the figure and PB1 at the back, following the nomenclature of LH3. (C) Interaction of dsRNA with the putative RNA-binding motif in E1B 55K. Left: best ranked complex for the wild-type protein. Right: best ranked complex for the mutant protein. dsRNA is shown in sticks, the protein domain in a translucent ribbon (N-terminus to the right) and the RNP motif in spacefilling representation, carbon in cyan, nitrogen in blue, oxygen in red, sulfur in yellow. (D). Sample of the classes of RNA-protein conformations or poses found for the wild-type domain, showing different angles of interaction between dsRNA and the long…

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  Fig 11. Representation of amino acids with highest probability of coevolution on the E1B 55K model. Three-dimensional model of the E1B 55K central region with VDW representation of amino acid with highest probability of coevolution. A) F264-F285-F307. B) R281-K303-R323. C) C283-C305-N325.