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. 2011 Aug 15;417(1):9-17.
doi: 10.1016/j.virol.2011.04.014. Epub 2011 May 24.

Role of the RNA recognition motif of the E1B 55 kDa protein in the adenovirus type 5 infectious cycle

Sayuri E M Kato  1 , Wenying Huang, S J Flint
Affiliations

Role of the RNA recognition motif of the E1B 55 kDa protein in the adenovirus type 5 infectious cycle

Sayuri E M Kato et al. Virology. .

Abstract

Although the adenovirus type 5 (Ad5) E1B 55 kDa protein can bind to RNA in vitro, no UV-light-induced crosslinking of this E1B protein to RNA could be detected in infected cells, under conditions in which RNA binding by a known viral RNA-binding protein (the L4 100 kDa protein) was observed readily. Substitution mutations, including substitutions reported to inhibit RNA binding in vitro, did not impair synthesis of viral early or late proteins or alter significantly the efficiency of viral replication in transformed or normal human cells. However, substitutions of conserved residues in the C-terminal segment of an RNA recognition motif specifically inhibited degradation of Mre11. We conclude that, if the E1B 55 kDa protein binds to RNA in infected cells in the same manner as in in vitro assays, this activity is not required for such well established functions as induction of selective export of viral late mRNAs.

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Figures

Figure 1
Figure 1. The E1B 55 kDa protein cannot be cross-linked to RNA in Ad5-infected cells
HeLa cells were infected with 20 pfu/cell Ad5 for 18 hrs (I) or mock-infected (M) and either exposed (+) or not exposed (−) to 254nm light for 4 minutes. The E1B 55kDa or He L4 100kDa protein was immunoprecipitated (IP) from nuclear lysates, and any covalently attached RNA fragments indirectly labeled with [32P], as described in Materials and Methods. Following electrophoresis in an 8% SDS-polyacrylamide gel, proteins were transferred to nitrocellulose and the membrane subjected sequentially to autoradiography (A) and immunoblotting to detect the E1B 55 kDa protein (B). The positions of molecular mass markers are listed at the left of panel A. The black and gray arrows at the right of panel A indicate the positions of the L4 100 kDa protein and a protein of some 50 kDa discussed in the text, respectively.
Figure 2
Figure 2. The RNA recognition motif of the E1B 55 kDa protein
A. The Ad5 E1B 55 kDa protein is represented by the box at the top, with the positions of the nuclear export signal (NES) (Dobbelstein et al., 1997; Kratzer et al., 2000), the RRM (Horridge and Leppard, 1998), and sites of sumoylation (Sumo-1) (Teodoro and Branton, 1997) and phosphorylation (P) (Teodoro and Branton, 1997) indicated. The sequence of the RRM is shown below, with the RNPI and RNPII motifs boxed, and amino acids conserved among RRMs indicated in bold, italic face. The underlined amino acids are aromatic residues important for RNA binding (Maris, Dominguez, and Allain, 2005). The conserved secondary structure of the RRM (Maris, Dominguez, and Allain, 2005) is shown below. B. The sequences of the E1B 55 kDa proteins of human adenoviruses of species C (Ad5 and Ad2), species A (Ad9 and Ad37), species B (Ad3 and Ad7), species D (Ad9 and Ad37), species E (Ad4 and Ad25) and species F (Ad41) corresponding to residues 248 to 308 of the Ad5 protein were aligned using Clustal W (1.83) (Larkin et al., 2007; Thompson, Higgins, and Gibson, 1994). The sequences of the RRM are shown, with the RNPI and RNPII motifs in bold face and the alpha2 helix in italic face. Below are indicated amino acids that are identical in all sequences (*) or represent conservative (:) or semi-conservative (.) substitutions. The amino acid substitutions introduced by the E1BSub12 and E1BSub13 mutations are listed below.
Figure 3
Figure 3. Steady State concentrations of viral proteins in wild type- and mutant virus- infected cells
A. HeLa cells were infected with 10 pfu/cell AdEasyE1 (WT), AdEasyE1Sub12 or AdEasyE1Sub13 for the periods indicated at the top, or mock infected (M). Total protein extracts were prepared and the E1B 55 kDa protein and β-actin examined by immunoblotting, as described in Materials and Methods. B. As panel A, except that HFFs were infected with 30 pfu/cell of the viruses listed at the top of panel A. C. The viral E2 DNA binding protein (DBP) and late protein V were examined by immunoblotting of the samples used in the experiment shown in panel B. In all panels, the positions of molecular mass markers are indicated at the left.
Figure 4
Figure 4. The RRM mutations do not impair viral replication
A. HeLa cells were infected with 10 pfu/cell AdEasyE1 (WT), AdEasyE1Sub12 or AdEasyE1Sub13, and samples harvested after increasing periods. The yield of intracellular infectious particles was determined as described in Materials and Methods. In both panels, the values represent the means of at least three technical replicates, with the standard deviations shown by the error bars. B. Yields of intracellular infectious particles were determined 44 hrs after infection of HeLa cells with 0.1 or 1.0 pfu/cell, and of HFFs with 3 or 30 pfu/cell wild type or mutant viruses. The asterisks indicate difference between wild-type and mutant virus replication determined to be significant (P=0.005 and 8 × 10−6 for 3 and 30 Pfu/cell, respectively) by application of the Students t test.
Figure 5
Figure 5. Effects of the RRM mutations on degradation of Mre11 and p53
A. HFFs were infected with AdEasyE1 (WT) or AdEasyE1Δ2347 (Δ2347) for the periods indicated, or mock infected (M). Total cell extracts were prepared and the proteins listed at the right examined by immunoblotting. B. HFFs were infected with the same wild-type and E1B 55 kDa-null mutant or AdEasyE1Sub13 (Sub13) for the periods indicated, or mock infected (M22, M44). The proteins listed at the right were examined by immunoblotting of total cell extracts.
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