A tumor host range selection procedure identifies p150(sal2) as a target of polyoma virus large T antigen

Abstract

Cancer cells may undergo loss or alterations in functions that certain viruses normally target to promote virus replication. Virus mutants that have lost the targeting function(s) should be able to grow in such cancer cells but not in normal cells. A "tumor host range" (t-hr) selection procedure has been devised and applied to polyoma virus based on this rationale. Studies of one t-hr mutant have led to the identification of the mSal2 gene product (p150(sal2)) as a binding partner of the large T antigen. mSal2 encodes a multizinc finger protein and putative transcription factor homologous to the Drosophila homeotic gene Spalt. The t-hr mutant encodes an altered large T protein that fails to interact with p150(sal2) and is defective in replication and tumor induction in newborn mice.

Figure 1

Outline of the tumor host range selection procedure.

Figure 2

Growth of a t-hr mutant is restricted to transformed or tumor-derived cells. TCMK-1, SV40 transformed baby mouse kidney cells; A6241, derived from a spontaneous mammary tumor. Cultures were either uninfected (Mock) or infected by wild-type or mutant polyoma virus. Photos taken 12 days postinfection show extensive cytopathic effects indicative of virus growth in all infected cultures except for BMK infected by the mutant.

Figure 3

The t-hr mutant has an altered large T C terminus that prevents interaction with host factor mSal2. (A) Sequencing of the original mutant shows a 20-bp duplication of coding sequences at the large T C terminus causing a reading frame shift shown in red. (B) The coding region of mSal2 cDNA is shown schematically in blue with the zinc fingers in orange. Overlapping clones from the C-terminal region of mSal2 interact with the wild-type large T C-terminal fragment (amino acids 333–781) but not with that of the mutant in yeast two-hybrid assays. +, growth on His⁻ plates; His+ colonies were also lac Z positive (not shown). (C) Deletion analysis of the wild-type large T C terminus indicating that a minimal deletion of amino acids 774–776 abolishes the binding of mSal2 in yeast.

Figure 4

Interaction of wild-type but not mutant large T with mSal2. (A) In vitro binding. GST alone and GST-fusion protein containing the large T binding domain of mSal2 (amino acids 839–969) are used to pull down large T from cell extracts. The filter is blotted with F4 anti-T monoclonal antibody. *, crossreactive cellular band. Lane a, input extract from wild-type infected BMK cells; lane b, wild-type extract from lane a pulled down with GST alone; lane c, wild-type extract from lane a pulled down with GST-mSal2 fusion protein; lane d, input extract from 3T3 transfected with wild-type large T cDNA; lane e, input extract from 3T3 cells transfected with TMD-25 large T cDNA; lane f, wild-type extract from lane d pulled down with GST alone; lane g, wild-type extract from lane d pulled down with GST-mSal2 fusion protein; lane h, mutant extract from lane e pulled down with GST-mSal2 fusion protein. (B) In vivo binding. (Left) 3T3 cells were cotransfected with full-length GST-mSal2 fusion construct and either wild-type or TMD 25 large T. Following GST pull-down, blots were developed with anti-T antibody (Upper) and with monoclonal anti-mSal2 (Lower) to show similar amount of GST-mSal2 was present in both pull-down lanes. Input lanes contain 3% of the extract used for pull-down. (Right) A 24-h wild-type virus-infected BMK cell extract was immunoprecipitated with normal rabbit IgG or purified rabbit polyclonal antibody against the N terminus of mSal2. The blot was probed with anti-T monoclonal antibody and reprobed with anti-mSal2 monoclonal antibody.

Figure 5

p150^sal2 imposes a block to viral DNA replication. (A) TMD-25 fails to replicate in the mouse. Newborn mice were inoculated intraperitoneally with TMD25 or PTA (1 × 10⁶ pfu/animal) and killed 10 days later. Whole mouse sections were hybridized with ³⁵S-labeled viral DNA with overnight exposure (32). Wild-type PTA shows extensive replication especially in kidney, skin, and bones. TMD25 mutant shows no signs of viral DNA replication. (B Left) TMD25 fails to replicate in BMK cells 36 h postinfection. BMK cells were infected with TMD25 and wild-type virus (Wt Py). Low molecular weight DNA was isolated at 0, 18, and 36 h postinfection (p.i.) for Southern blot with viral DNA probe. (Right) p150^sal2 represses viral origin replication. Polyoma origin (Ori) and large T-expressing plasmid (Wt LT cDNA) were cotransfected with increasing amounts of plasmid expressing mSal2. Newly replicated DNA was detected with origin specific probe (Upper). Total amount of plasmids is adjusted to the same level in each transfection using empty vector. The filter was stripped and reprobed with LT and origin-specific probe to show that similar amounts of origin and LT DNA were present in each transfection. (C) Inhibition of replication by mSal2 requires the large T interaction domain and can be overcome by wild-type but not mutant large T. Origin replication assay was carried out similarly to B, with increasing amounts of plasmids expressing either wild-type mSal2 or mSal2 with deleted C-terminal zinc finger (Del Sal2) as shown in the left six lanes. With the amount of mSal2 held constant, increasing the amount of wild-type LT (Wt LT) but not TMD LT abolishes mSal2 inhibition as shown in the right six lanes.

Figure 6

Expression of p150^sal2 in mouse tissues. Tissues were taken from 2-week-old mice except in the first lane, which was from a newborn mouse. Protein (100 μg) was loaded on each lane. The blot was probed with the monoclonal antibody against the N terminus of mSal2.