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Journal of Virology, May 2002, p. 4507-4519, Vol. 76, No. 9
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.9.4507-4519.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Effects of Mutations in the Adenoviral E1B 55-Kilodalton Protein Coding Sequence on Viral Late mRNA Metabolism

Ramon A. Gonzalez,{dagger} and S. J. Flint*

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544

Received 3 August 2001/ Accepted 30 January 2002

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.

* Corresponding author. Mailing address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544. Phone: (609) 258-6113. Fax: (609) 258-4575. E-mail: sjflint{at}molbio.princeton.edu.

{dagger} Present address: Facultad de Ciencias, Universidad Autonoma del Estado de Morelos, Au. Universidad, Colonia Chamilpa, Cuernavaca, Morelos 62010, Mexico.

Journal of Virology, May 2002, p. 4507-4519, Vol. 76, No. 9
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.9.4507-4519.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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