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

Abstract

The E1B 55kDa produced by human adenovirus type 5 is a multifunctional protein that participates in the regulation of several steps during the viral replication cycle. Previous studies suggest this protein plays an important role in postranscriptional regulation of viral and cellular gene expression, as it is required for the selective accumulation of maximal levels of viral late mRNA in the cytoplasm of the infected cell; however the molecular mechanisms that are altered or regulated by this protein have not been elucidated. A ribonucleoprotein motif that could implicate the direct interaction of the protein with RNA was initially predicted and tested in vitro , but the interaction with RNA could not be detected in infected cells, suggesting the interaction may be weak or transient. Here it was determined that the E1B 55kDa interacts with RNA in the context of the viral infection in non-transformed human cells, and its contribution to the adenovirus replication cycle was evaluated. Using recombinant adenoviruses with amino acid substitutions or a deletion in the ribonucleoprotein motif the interaction of E1B 55kDa with RNA was found to correlate with timely and efficient viral DNA replication and viral late mRNA accumulation and splicing.

The biology of the adenovirus E1B 55K protein

“…E1B 93R shares 79 amino acids with the N‐terminal region of full‐length E1B, suggesting that the N terminus of E1B is also disordered . Algorithms for prediction of intrinsic disorder support these findings and suggest that the N terminus of E1B (1–150 aa) is disordered (Fig B); furthermore, the C terminus also displays some degree of predicted disorder, especially in the region encompassing residues 390–496 (Fig B). Experimental data and in silico analysis of the sequence of E1B from a large variety of adenoviruses have helped identify potential motifs that might be functionally relevant.…”

“…However, it has not been established if the putative zinc finger binds zinc or if it participates in interaction with proteins or nucleic acids. The central region of the polypeptide, from amino acids 215 to 345, has been predicted to constitute a hydrophobic core that contains a ribonucleoprotein (RNP) motif that is directly implicated in the nonsequence‐specific interaction with RNA in vitro and with viral RNA in vivo (Fig A). This central region, which is particularly susceptible to amino acid substitutions , has been modeled as a β solenoid fold using the structure of the hexon‐interlacing protein LH3 (LH3) from a snake adenovirus (Fig C).…”

“…The central region of the polypeptide, from amino acids 215 to 345, has been predicted to constitute a hydrophobic core that contains a ribonucleoprotein (RNP) motif that is directly implicated in the nonsequence‐specific interaction with RNA in vitro and with viral RNA in vivo (Fig A). This central region, which is particularly susceptible to amino acid substitutions , has been modeled as a β solenoid fold using the structure of the hexon‐interlacing protein LH3 (LH3) from a snake adenovirus (Fig C). The rationale for this comparative modeling was that the fold displayed by LH3 was very similar to that predicted by the iterative threading ASSEmbly refinement (I‐TASSER) server for the central region of E1B , and that the LH3 and E1B genes share equivalent positions in the genome of atadenovirus and mastadenovirus, which suggests a common ancestor .…”

“…This central region, which is particularly susceptible to amino acid substitutions , has been modeled as a β solenoid fold using the structure of the hexon‐interlacing protein LH3 (LH3) from a snake adenovirus (Fig C). The rationale for this comparative modeling was that the fold displayed by LH3 was very similar to that predicted by the iterative threading ASSEmbly refinement (I‐TASSER) server for the central region of E1B , and that the LH3 and E1B genes share equivalent positions in the genome of atadenovirus and mastadenovirus, which suggests a common ancestor . Other conserved sequences include a nuclear export signal (NES) and a nuclear localization signal (NLS), as well as amino acid residues that are modified by PTMs (Fig A).…”

Formation of adenovirus DNA replication compartments

“…Although the composition of AdRCs at different times postinfection has not been reported, AdRCs contain nucleic acids and proteins, some of which are known to contain IDRs (e.g. DBP and E1B 55K ). Moreover, similar to the liquid characteristics of NBs, the dynamic nature of AdRCs suggests that their formation is mediated by weak, multivalent interactions between its constituents.…”