Marek's disease virus latent protein MEQ: delineation of an epitope in the BR1 domain involved in nuclear localization.
Marek's disease virus latent protein MEQ (MDV Eco Q) is abundantly expressed and consistently detected in MDV-induced tumors and cell lines. Deletion mutants were constructed to study the domain structure of MEQ. Four deletion mutants were obtained in the basic regions of MEQ, namely basic region 1 (DeltaBR1), basic region 2 (DeltaBR2), basic regions 1 and 2 (DeltaBR1 and 2), and the C-terminal (bZIP) domain. The BR1 and BR2 are nuclear localization signals and either is sufficient to cause transport of MEQ into the nucleus. In addition, the BR2 is also responsible for MEQ's nucleolar localization. A monoclonal antibody (Mab 23B46) was produced using recombinant fowlpox virus (rFPV) expressing MEQ (rFPV/MEQ) as a source of protein. The isotype of Mab 23B46 is IgG1 and immunoprecipitated a band in rFPV/MEQ infected cells with molecular weight of 60 kDa specific to MEQ protein. We detected abundant expression of MEQ in (rFPV/MEQ), recombinant baculovirus (rBac) (rBac/MEQ), and lymphoid tumors induced by MDV. In order to delineate the epitope of MEQ reactive with Mab 23B46, we used four deletion mutants from the basic and bZIP domains. We found the deletions in the N-terminal region including BR1 (DeltaBR1), and (DeltaBR1 and 2) completely abolished the specific binding with Mab 23B46 as shown by Western blot analysis and immunofluoresence test. Deletion of BR2 (DeltaBR2) and the C-terminal (bZIP) domain had no effect on antibody binding. These data provide direct evidence that monoclonal antibody reactive epitope is localized in the BR1 domain of the molecule. Since both BR1 and BR2 domains contain sequences important for nuclear entry, we now have reagent to further study and elucidate the mechanism of MEQ's involvement in nuclear and nucleolar localization.
Lee
Lucy F
LF
Avain Disease and Oncology Laboratory, U.S. Department of Agriculture, Agricultural Research Service, East Lansing, MI 48823, USA.
Liu
J-L
JL
Cui
X-P
XP
Kung
H-J
HJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D005814:Genes, Viral; D008381:Herpesvirus 2, Gallid / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity; D008380:Marek Disease / Q000821:virology; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D017434:Protein Structure, Tertiary; D051381:Rats; D017384:Sequence Deletion
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; C100369:Eco-Q protein, Gallid herpesvirus 2; D000939:Epitopes; D019913:Nuclear Localization Signals; D009856:Oncogene Proteins, Viral