Date Published: April 3, 2019
Publisher: Public Library of Science
Author(s): Berto Tejera, Raúl E. López, Paloma Hidalgo, Reinier Cárdenas, Grisel Ballesteros, Lina Rivillas, Leidys French, Carlos Amero, Nina Pastor, Ángel Santiago, Peter Groitl, Thomas Dobner, Ramón A. Gonzalez, Michael Nevels.
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 early 1B protein, E1B 55kDa (E1B 55K), from species C human adenoviruses participates in key steps of the virus replication cycle, as it is implicated in the regulation of cellular processes that produce a favorable environment that results in efficient virus gene expression and progeny production [1–3]. E1B 55K has been reported to interact with over 30 proteins, including the viral E4 Orf3, E4 Orf6, DBP, L4 100K, pVI, pVII and a variety of cellular proteins . The role played by the interaction of E1B 55K with each viral or cellular protein in viral replication has not been fully characterized; however, most can be grouped into a few major biological processes that range from regulation of gene expression to polyubiquitin-dependent proteasomal degradation of target proteins. The late phase of infection is characterized by the establishment of a selective expression program in which E1B 55K is required for viral late mRNA intranuclear trafficking, cytoplasmic accumulation  and translation . However, E1B 55K is also implicated in the regulation of the anti-viral response of the infected cell. In the absence of this protein, the expression of interferon-sensitive genes like GBP1-5, IFIH1 (MDA5), IFIT2, MX2 and TAP1 increases , and its role as inhibitor of the interferon-pathway has been described . A repression domain for interferon target genes has been mapped to the E1B 55K C-terminus , while its N-terminus can interact with the tumor supressor p53, tethering a repressor domain that inhibits p53-dependent transcription [10,11]. The interaction with the viral early E4 Orf6 protein and the cellular proteins Cullin 5, Elongins B and C, and Rbx1 is required for assembly of an E3 Ubiquitin ligase complex  that promotes polyubiquitylation and degradation of a growing number of cellular substrates that include components of the DNA Damage Response (DDR), such as Mre11, Rad 50 [13,14], the Bloom helicase  and DNA ligase IV [16,17], as well as, ATRX , p53 [12,19], Tip 60 , SPOC 1 , Tab182 , and α3 integrin . Interestingly, E1B 55K is also responsible for degradation of the PML nuclear bodies component Daxx without the assembly of the E4 Orf6-dependent Cullin5 E3 Ubiquitin ligase complex .
HFF cells grown on glass coverslips to approximately 90% confluence were mock- infected or infected with Ad5 WT or the E1B 55K mutant viruses. The infected cells were processed at the indicated times postinfection, as described previously . After the application of specific primary antibodies, cells were incubated with secondary antibodies coupled to fluorophores as indicated. The coverslips were mounted on glass slides in PBS–10% glycerol, and samples were examined using a Zeiss Axiovert 200M inverted microscope with a 63x/1.4-numerical-aperture oil-immersion objective lens with an Axiocam MRM and Axiovision 3.1 software (Carl Zeiss, Inc.).
The E1B 55K protein makes several contributions to the viral replication cycle; however, the molecular mechanisms that are altered or regulated by this protein in the infected cell are incompletely understood. Here we have shown that E1B 55K interacts with viral RNA in the infected cell (Fig 1) and that the RNP motif can participate in direct protein-RNA contacts (Fig 2). Substitution of amino acid residues at positions in the RNP motif that were previously shown to either reduce or increase RNA interaction in vitro  displayed similar patterns of altered binding, confirming that the RNP and the positions that were substituted are relevant for the interaction in infected cells. Nevertheless, deletion of the RNP motif increased RNA binding, indicating that its exclusion results in changes that promote the interaction. Interestingly, molecular modeling of E1B 55K with dsRNA suggests the protein can associate with two such molecules and that the deletion of the RNP may increase the conformational arrangements that the E1B 55K-dsRNA complexes can adopt (Fig 10).