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. 2002 May;76(9):4507-19.
doi: 10.1128/jvi.76.9.4507-4519.2002.

Effects of mutations in the adenoviral E1B 55-kilodalton protein coding sequence on viral late mRNA metabolism

Ramon A Gonzalez  1 , S J Flint
Affiliations

Effects of mutations in the adenoviral E1B 55-kilodalton protein coding sequence on viral late mRNA metabolism

Ramon A Gonzalez et al. J Virol. 2002 May.

Abstract

The human subgroup C adenoviral E1B 55-kDa protein cooperates with the viral E4 Orf6 protein to induce selective export of viral, late mRNAs from the nucleus to the cytoplasm. Previous studies have suggested that such preferential transport of viral mRNA and the concomitant inhibition of export of cellular mRNAs are the result of viral colonization of specialized microenvironments within the nucleus. However, neither the molecular basis of this phenomenon nor the mechanism by which the E1B 55-kDa protein acts has been elucidated. We therefore examined viral late mRNA metabolism in HeLa cells infected with a series of mutant viruses that carry insertions at various positions in the E1B protein coding sequence (P. R. Yew, C. C. Kao, and A. J. Berk, Virology 179:795-805, 1990). All the mutations examined impaired cytoplasmic accumulation of viral L2 mRNAs and reduced L2 mRNA export efficiency. However, in most cases these defects could be ascribed to reduced E1B 55-kDa protein concentration or the unexpected failure of the altered E1B proteins to enter the nucleus efficiently. The latter property, the pleiotropic defects associated with all the mutations that impaired nuclear entry of the E1B protein, and consideration of its primary sequence suggest that these insertions result in misfolding of the protein. Insertion of four amino acids at residue 143 also inhibited viral mRNA export but resulted in increased rather than decreased accumulation of the E1B 55-kDa protein in the nucleus. This mutation specifically impaired the previously described association of the E1B protein with intranuclear structures that correspond to sites of adenoviral DNA replication and transcription (D. Ornelles and T. Shenk, J. Virol. 65:424-439, 1991) and the colocalization of the E1B and E4 Orf6 proteins. As this insertion has been shown to inhibit the interaction of the E1B with the E4 Orf6 protein in infected cell extracts (S. Rubenwolf, H. Schütt, M. Nevels, H. Wolf, and T. Dobner, J. Virol. 71:1115-1123, 1997), these phenotypes provide direct support for the hypothesis that selective viral mRNA export is determined by the functional organization of the infected cell nucleus.

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Figures

FIG. 1.
FIG. 1.
Accumulation of altered E1B 55-kDa proteins in mutant virus-infected HeLa cells. HeLa cells were mock infected or infected with Ad5 or the mutants listed for the periods indicated (A) or for 18 h (B). Total cell lysates were prepared and analyzed by immunoblotting with the anti-E1B 55-kDa protein monoclonal antibody 2A6 as described in Materials and Methods.
FIG. 2.
FIG. 2.
Localization of altered E1B 55-kDa proteins. HeLa cells infected with Ad5 or the mutant viruses indicated were labeled with [35S]methionine from 13 to 17 h after infection and separated into nuclear and cytoplasmic fractions as described in Materials and Methods. The E1B 55-kDa protein was immunoprecipitated from each sample with monoclonal antibody 2A6.
FIG. 3.
FIG. 3.
Intracellular localization of E2 72-kDa DBP and E1B 55-kDa proteins in HeLa cells infected with Ad5 or with the A143 or A262 mutants or mock-infected HeLa cells was examined 14 h after infection by immunofluorescence, using monoclonal antibodies B6-8 and 2A-6, respectively, as described in Materials and Methods.
FIG. 4.
FIG. 4.
Intracellular localization of the E4 Orf6 and E1B 55-kDa proteins in HeLa cells infected with the viruses indicated or mock-infected HeLa cells was examined 14 h after infection by immunofluorescence using monoclonal antibodies M45 and 2A6, respectively.
FIG. 5.
FIG. 5.
Cytoplasmic accumulation and transcription of viral L2 RNA. (A) Cytoplasmic L2 mRNAs were examined 18 h after infection by Northern blotting. Cells were infected with Ad5 or the E1B 55-kDa mutants listed, and the four L2 mRNAs and the human ribosomal protein 3 mRNAs were detected and quantified as described in Materials and Methods. Shown are penton and pre-Mu mRNA signals, corrected using the RpS3 mRNA internal control and expressed relative to the wild-type value, which was set at 1.0. Similar changes were observed in concentrations of the L2 V and pVII mRNAs. (B) Rates of L2 transcription were determined 18 h after infection of HeLa cells with Ad5 or the mutant viruses listed by run-on transcription in isolated nuclei and hybridization of the labeled RNA to membrane-immobilized L2 DNA. Signals were quantified and corrected as described in Materials and Methods and expressed relative to the wild-type value, which was set at 1.0. The values shown are the means of two independent experiments. Error bars show standard deviations.
FIG. 6.
FIG. 6.
Alterations in the ratio of cytoplasmic to nuclear L2 penton mRNA induced by E1B 55-kDa protein coding sequence mutations. Steady-state concentrations of mature L2 penton mRNA in the cytoplasm and nucleus were determined 14 h after infection of HeLa cells with Ad5 or the mutant viruses indicated by primer extension. Signals were corrected using the stable, cellular β-actin mRNA as an internal control and used to calculate the ratios of cytoplasmic to nuclear penton mRNA shown.
FIG. 7.
FIG. 7.
Phenotypes exhibited by E1B 55-kDa protein sequence insertion mutants. The 496-residue protein is represented to scale at the top by the open box, within which are shown the positions of a leucine-rich sequence (NES) necessary and sufficient for export of E1B-GFP and glutathione S-transferase-E1B peptide-GFP fusion proteins, respectively (37); a sequence that matches the ribonucleoprotein (RNP) motif of many RNA-binding proteins in both the conserved RNP1 and RNP2 sequences and in which specific substitutions impair nonspecific RNA-binding activity of the E1B protein in vitro (32); and a potential sequence matching the consensus for C2H2 zinc fingers identified by inspection and a protein motif search in SeqWeb, version 2. The dashed, double-headed arrows drawn above indicate the region containing the epitope recognized by the monoclonal antibody 2A6 (34) that has been used in the majority of studies of the protein and a sequence required for nuclear localization (NLS) of an E1B-GFP fusion protein transiently synthesized in human cells (37). The short, vertical lines drawn immediately below the protein list the sites of insertions introduced into an Ad2/5 E1B 55-kDa protein coding sequence in the viral genome. With the exception of the 18-amino-acid addition at residue 215, these insertions comprise four amino acids (81). The effects of these alterations on the concentration of the protein and other properties listed at the left are summarized below (−− and −, severe and moderate defects, respectively; +, twofold increase in defects; ND, effect not determined; ?, effect uncertain). The sources of these data, which were collected under a variety of experimental conditions using HeLa or other lines of human tumor cells, are listed in parentheses at the left.
FIG. 8.
FIG. 8.
Predicted properties of the E1B 55-kDa protein. The properties listed were predicted from the amino acid sequence of the 496-residue Ad5 E1B 55-kDa protein with the algorithms indicated, using the program MacVector 7.0.
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