Construction and expression of a recombinant DNA gene encoding a polyomavirus middle-size tumor antigen with the carboxyl terminus of the vesicular stomatitis virus glycoprotein G.

Abstract

We constructed a molecular clone encoding the N-terminal 379 amino acids of the polyomavirus middlesize tumor antigen, followed by the C-terminal 60 amino acids of the vesicular stomatitis virus glycoprotein G. This hybrid gene contained the coding region for the C-terminal hydrophobic membrane-spanning domain of the G protein in place of the C-terminal hydrophobic domain of the middle-size tumor antigen. The hybrid gene was expressed in COS-1 cells under the control of the simian virus 40 late promoter. The hybrid protein was located in cell membranes and was associated with a tyrosine-specific protein kinase activity, as was the middle-size tumor antigen. Plasmids encoding the hybrid protein failed to transform mouse NIH 3T3 or rat F2408 cells.The polyomavirus genome encodes three early proteins, the small, middle-size (MT), and large T antigens. The coding regions for these three proteins overlap, so that all three share an N-terminal region of 79 amino acids. The small and MT antigens share an additional 112 amino acids not found in the large T antigen. The three T antigens have unique C-terminal regions translated from different reading frames in the viral DNA (29).The three proteins are at different locations in the cell: the large T antigen is found in the nucleus, the small T antigen is found in the cytoplasm, and the MT antigen is found in a cell membrane fraction (14,27). The mechanisms by which the three proteins are directed to different intracellular locations are not understood.The MT antigen is of interest because it is primarily responsible for cell transformation by polyomavirus. Recombinant DNA plasmids encoding only the MT antigen can transform established cell lines (32). Many mutations affecting the MT antigen reduce or abolish cell transformation (1,5,11,15,(17)(18)(19)31). Although the MT antigen alone can transform established cell lines, a portion of the large T antigen is required in addition for transforming primary cells (20).The MT antigen is associated with a protein kinase activity (6,24,28). The protein kinase phosphorylates a tyrosine residue of the MT antigen in immunoprecipitates (6). With few exceptions (18,24), mutations in the MT antigen that affect cell transformation also affect the protein kinase activity, suggesting that the activity may be involved in transformation (1,6,24,28,31). However, polyomavirustransformed cells do not have elevated levels of phosphotyrosine in total cellular protein, as do cells transformed by retroviruses encoding tyrosine-specific protein kinases (26).The MT antigen has an uninterrupted stretch of 22 hydrophobic and uncharged amino acids bounded by positively charged amino acids near its C-terminus (29). The size and location of this C-terminal hydrophobic region are similar to the size and location of regions involved with membrane association in other proteins. In particular, the vesicular * Corresponding author. 282 stomatitis virus (VSV) glycoprotein G has an uninterrupted stretch of 20 hydrophobic and uncharged amino acids which are also...

At the onset of transformation polyomavirus middle-T recruits shc and src to a perinuclear compartment coincident with condensation of endosomes

“…Although extensively studied, the site of mT membrane localization remains unresolved with morphologic studies favouring intracellular membranes (Dilworth et al, 1986;Templeton et al, 1984;Zhu et al, 1984) while biochemical analyses continue to be interpreted as suggesting that kinase active mT containing complexes are located at the plasma membrane (Ballmer-Hofer and Benjamin, 1985;Ito et al, 1977;Scha hausen et al, 1982;Segawa and Ito, 1982). Recently, we have demonstrated a role for cytoskeletal proteins, particularly actin, in the localization of mT (Andrews et al, 1993).…”

Evidence that the middle T antigen of polyomavirus interacts with the membrane skeleton.

“…Subcellular fractionation, immunofluorescence, and immunoelectron microscopy of mT-expressing cells suggest that mT is found in association with subcellular membranes (4,9,18,41,44,47,55). It has been assumed that mT is an integral membrane protein because of a contiguous 22-amino-acid hydrophobic segment near the carboxyl end of the molecule.…”

“…Moreover, it appears that accumulation of the phosphorylated products of the PI-3-kinase is required for transformation (32). Nevertheless, only a fraction of mT molecules in cells are complexed to either the src family kinases or PI-3-kinase; therefore, it remains possible that additional interactions are involved in cellular transformation (23).Subcellular fractionation, immunofluorescence, and immunoelectron microscopy of mT-expressing cells suggest that mT is found in association with subcellular membranes (4,9,18,41,44,47,55). It has been assumed that mT is an integral membrane protein because of a contiguous 22-amino-acid hydrophobic segment near the carboxyl end of the molecule.…”

Cell transformation by the middle T-antigen of polyoma virus

“…Without these hydrophobic amino acids, MT no longer binds to membranes, fails to associate with most of the cellular proteins that it normally binds, and does not transform. However, this hydrophobic region does more than simply mediate membrane interaction, as replacement with the membrane targeting domains of vesicular stomatitis virus glycoprotein G (Templeton et al, 1984), cytochrome b5 (Zhu et al, 1998), or the myristylation signal of pp60 c-src (Elliott et al, 1998) all locate MT to a membrane site, but fail to restore transformation. In addition, mutations within the hydrophobic domain can also abolish transforming activity without disrupting membrane binding (reviewed in Markland and Smith, 1987), and a signi®cant homology exists between the regions in murine and hamster MT.…”