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.
Highlights
IntroductionThe E1B 55kDa-RNA interaction promotes viral DNA replication and viral late mRNA metabolism
To detect a possible interaction between E1B 55kDa (E1B 55K) and viral late mRNA in the context of infection, an RNA immunoprecipitation assay (RIP) was performed, in which total protein lysates of cells infected with the Ad5 WT or E1B− viruses or with the RNP mutants C288A, C288S, Δ284–289, and W289F were immunoprecipitated with the anti-E1B 55K MAb-2A6, and the pulled-down RNA were amplified by RT qPCR (Fig 1)
The RNA sequence amplified corresponds to the tripartite leader (TPL) RNA of 81nts (S1 Fig), which was selected because all five families of viral late mRNA from the adenovirus major late transcription unit contain this sequence in their 5’-noncoding region and it is necessary for their efficient export from the nucleus and translation [67, 68, 69]
Summary
The E1B 55kDa-RNA interaction promotes viral DNA replication and viral late mRNA metabolism. CONACyT CB-2011-01-168497 and Programa para el Desarrollo Profesional Docente-Secretarıa de Educacion Publica (PRODEP-SEP). Https://www.humboldtfoundation.de/web/home.html; NO - The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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