Nuclear transport of adenovirus DNA polymerase is facilitated by interaction with preterminal protein

The mRNAs for the 80 kd adenovirus preterminal protein (pTP) and the 140 kd DNA polymerase (AdPol) contain several exons spliced to the main open reading frames (m-ORFs) located in the early transcription unit E2B. These proteins were transiently expressed in monkey kidney cells (CV1) utilizing the first ATG (pTP1 and AdPol1) or the ATG of a linker inserted at the beginning of the m-ORFs (pTP2 and AdPol2). Only pTP2 and AdPol2 were functionally active in an in vitro replication initiation assay. Both pTP1 and pTP2 were transported to the nucleus. The sequence RLPV(R)6VP, which is present in both pTPs, is identified as their nuclear localization signal. In contrast, AdPol1 was cytoplasmically localized, whereas AdPol2 was distributed in both compartments, suggesting that the nuclear localization signal for AdPol is within the first 139 amino acids. Interestingly, when AdPol1 and pTP1 or AdPol2 and pTP2 were coexpressed in the transfected cells, the nuclear distribution of AdPol1 or AdPol2 was significantly increased. We demonstrate that the nuclear transport of AdPol is facilitated, irrespective of the presence of its nuclear localization signal, by interaction with pTP.

[1]  F. Tamanoi,et al.  The origin of adenovirus DNA replication. , 1984, Current topics in microbiology and immunology.

[2]  Roger D. Kornberg,et al.  Synthetic peptides as nuclear localization signals , 1986, Nature.

[3]  B. Stillman,et al.  Identification of the gene and mRNA for the adenovirus terminal protein precursor , 1981, Cell.

[4]  W. Richardson,et al.  Nuclear protein migration involves two steps: Rapid binding at the nuclear envelope followed by slower translocation through nuclear pores , 1988, Cell.

[5]  F. Tamanoi,et al.  Purification of an adenovirus-coded DNA polymerase that is required for initiation of DNA replication , 1982, Cell.

[6]  J. M. Velazquez,et al.  hsp70: Nuclear concentration during environmental stress and cytoplasmic storage during recovery , 1984, Cell.

[7]  H. Ginsberg,et al.  Codon insertion mutants of the adenovirus terminal protein. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[8]  J. Hurwitz,et al.  Complementation of the temperature-sensitive defect in H5ts125 adenovirus DNA replication in vitro. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[9]  F. Tamanoi,et al.  Function of adenovirus terminal protein in the initiation of DNA replication. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[10]  E. Nigg,et al.  Rapid and reversible translocation of the catalytic subunit of cAMP‐dependent protein kinase type II from the Golgi complex to the nucleus. , 1985, The EMBO journal.

[11]  J. Hurwitz,et al.  Adenovirus DNA replication in vitro: identification of a host factor that stimulates synthesis of the preterminal protein-dCMP complex. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[12]  B. Howard,et al.  High efficiency DNA-mediated transformation of primate cells. , 1983, Science.

[13]  C. Feldherr,et al.  Movement of a karyophilic protein through the nuclear pores of oocytes , 1984, The Journal of cell biology.

[14]  I. Pastan,et al.  The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[15]  M. Challberg,et al.  Adenovirus DNA replication in vitro. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[16]  N. Jones,et al.  Mapping of functional domains in adenovirus E1A proteins. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[17]  William D. Richardson,et al.  A short amino acid sequence able to specify nuclear location , 1984, Cell.

[18]  L. Cox,et al.  Nucleoplasmin cDNA sequence reveals polyglutamic acid tracts and a cluster of sequences homologous to putative nuclear localization signals. , 1987, The EMBO journal.

[19]  J. Hurwitz,et al.  Separation of the adenovirus terminal protein precursor from its associated DNA polymerase: role of both proteins in the initiation of adenovirus DNA replication. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[20]  I. Herskowitz,et al.  Targeting of E. coli β-galactosidase to the nucleus in yeast , 1984, Cell.

[21]  J. Hurwitz,et al.  The in vitro replication of adenovirus DNA. , 1984, Current topics in microbiology and immunology.

[22]  Y. Sasaguri,et al.  Immunological analysis of 140-kDa adenovirus-encoded DNA polymerase in adenovirus type 2-infected HeLa cells using antibodies raised against the protein expressed in Escherichia coli. , 1987, Virology.

[23]  R A Laskey,et al.  Protein import into the cell nucleus. , 1986, Annual review of cell biology.

[24]  J. Hurwitz,et al.  Formation of a covalent complex between the 80,000-dalton adenovirus terminal protein and 5'-dCMP in vitro. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[25]  R. Kennedy,et al.  Induction of nuclear transport with a synthetic peptide homologous to the SV40 T antigen transport signal , 1986, Cell.

[26]  G. Pruijn,et al.  Nuclear factor III, a novel sequence-specific DNA-binding protein from HeLa cells stimulating adenovirus DNA replication , 1986, Nature.

[27]  M. Kozak.  Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes , 1986, Cell.

[28]  B. Stillman,et al.  Adenovirus terminal protein precursor. Partial amino acid sequence and the site of covalent linkage to virus DNA. , 1982, The Journal of biological chemistry.

[29]  T. Kelly.  Adenovirus DNA Replication , 1984 .

[30]  J. Hurwitz,et al.  Adenoviral protein-primed initiation of DNA chains in vitro. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[31]  J. Davey,et al.  Identification of the sequence responsible for the nuclear accumulation of the influenza virus nucleoprotein in Xenopus oocytes , 1985, Cell.

[32]  J. Sussenbach,et al.  The mechanism of adenovirus DNA replication and the characterization of replication proteins. , 1984, Current topics in microbiology and immunology.

[33]  R. Wides,et al.  Adenovirus origin of DNA replication: sequence requirements for replication in vitro , 1987, Molecular and cellular biology.

[34]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[35]  K. Yamamoto,et al.  Two signals mediate hormone‐dependent nuclear localization of the glucocorticoid receptor. , 1987, The EMBO journal.

[36]  E. A. O'neill,et al.  Purification and characterization of nuclear factor III (origin recognition protein C), a sequence-specific DNA binding protein required for efficient initiation of adenovirus DNA replication. , 1988, The Journal of biological chemistry.

[37]  E. A. O'neill,et al.  Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication , 1987, Molecular and cellular biology.

[38]  W. Richardson,et al.  Sequence requirements for nuclear location of simian virus 40 large-T antigen , 1984, Nature.

[39]  C. Wychowski,et al.  The intranuclear location of simian virus 40 polypeptides VP2 and VP3 depends on a specific amino acid sequence , 1987, Journal of virology.

[40]  T. Kelly,et al.  In vitro complementation as an assay for purification of adenovirus DNA replication proteins. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[41]  K. Scheidtmann,et al.  Kinetics of nuclear transport and oligomerization of simian virus 40 large T antigen. , 1986, Virology.

[42]  M. Green,et al.  Immunological and Chemical Identification of Intracellular Forms of Adenovirus Type 2 Terminal Protein , 1981, Journal of virology.

[43]  T. Gingeras,et al.  Nucleotide sequences from the adenovirus-2 genome. , 1982, The Journal of biological chemistry.

[44]  W. Richardson,et al.  Nuclear location signals in polyoma virus large-T , 1986, Cell.

[45]  C. Wychowski,et al.  A domain of SV40 capsid polypeptide VP1 that specifies migration into the cell nucleus. , 1986, The EMBO journal.

[46]  C. Dingwall,et al.  Accumulation of the isolated carboxy‐terminal domain of histone H1 in the Xenopus oocyte nucleus. , 1984, The EMBO journal.

[47]  J. Hurwitz,et al.  Replication of adenovirus DNA-protein complex with purified proteins. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[48]  D. Klessig,et al.  Identification of two nuclear subclasses of the adenovirus type 5-encoded DNA-binding protein , 1986, Journal of virology.

[49]  J. Hurwitz,et al.  Adenovirus DNA replication in vitro: synthesis of full-length DNA with purified proteins. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[50]  J. Engler,et al.  Nucleotide sequence of the genes encoded in early region 2b of human adenovirus type 12. , 1986, Gene.

[51]  J. Hurwitz,et al.  Adenovirus DNA replication in vitro: purification of the terminal protein in a functional form. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[52]  D. Newmeyer,et al.  Nuclear import can be separated into distinct steps in vitro: Nuclear pore binding and translocation , 1988, Cell.

[53]  E. G. Westaway,et al.  Replication strategy of Kunjin virus: evidence for recycling role of replicative form RNA as template in semiconservative and asymmetric replication. , 1985, Virology.

[54]  H. Nam,et al.  Identification of a nuclear localization signal of a yeast ribosomal protein. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[55]  W. Richardson,et al.  The effect of protein context on nuclear location signal function , 1987, Cell.

[56]  D. Newmeyer,et al.  Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation. , 1986, The EMBO journal.

[57]  S. Munro,et al.  Use of peptide tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila hsp 70. , 1984, The EMBO journal.

[58]  J. Butel,et al.  Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen , 1984, Cell.

[59]  E. D. De Robertis,et al.  The nuclear migration signal of Xenopus laevis nucleoplasmin. , 1987, The EMBO journal.

[60]  J. Engler,et al.  Expression of enzymatically active adenovirus DNA polymerase from cloned DNA requires sequences upstream of the main open reading frame. , 1987, Virology.

[61]  E. Robertis,et al.  Nuclear segregation of U2 snRNA requires binding of specific snRNP proteins , 1985, Cell.

[62]  S. Pettit,et al.  Adenovirus preterminal protein synthesized in COS cells from cloned DNA is active in DNA replication in vitro , 1988, Journal of virology.

[63]  J. Hurwitz,et al.  Evidence for an altered adenovirus DNA polymerase in cells infected with the mutant H5ts149. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[64]  J. Hurwitz,et al.  Specific binding of a cellular DNA replication protein to the origin of replication of adenovirus DNA. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[65]  R. Wides,et al.  Structure and function of the adenovirus origin of replication , 1984, Cell.

[66]  R. Laskey,et al.  A polypeptide domain that specifies migration of nucleoplasmin into the nucleus , 1982, Cell.