Skip to main content
. Author manuscript; available in PMC: 2014 Aug 15.
Published in final edited form as: Cell Host Microbe. 2013 Aug 14;14(2):125–135. doi: 10.1016/j.chom.2013.06.008

Figure 1. MCV sT enhances LT-mediated MCV origin replication through an exon1A domain in a PP2A independent manor.

Figure 1

(A) An MCV sT exon1A-encoded domain enhances MCV LT-dependent origin replication. Diagram compares MCV and SV40 sT coding regions and sites of MCV point mutations used in replication assay. SV40 exon1A (83–174 a.a.) was replaced with MCV exon1A (80–186 a.a.) to make the chimeric construct, SV40/MCVsT. The obverse MCV exon1 and SV40 exon1A chimeric (MCV/SV40sT) could not be evaluated due to its protein instability. Dpn1-resistant MCV origin replication in 293 cells was assayed by Southern blotting (middle panel) in the absence of T antigens (lane 1), in the presence of LT alone (lane 2), or LT together with MCV or SV40 sT proteins (lanes 3–8). MCV sT point mutants that eliminate PP2A binding (lanes 4, 5) and Hsc70 binding (lane 6) have comparable activity to the wild-type sT protein (lane 3). SV40 sT did not induce enhanced LT-dependent MCV origin replication (lane 7) but the SV40/MCV sT chimeric protein possessing the C-terminal MCV peptide sequence (lane 8) restored origin replication activity. Corresponding immunoblots for LT (CM2B4) and sT proteins (mixture of anti-MCV sT (CM8E6) and anti-SV40 LT/sT (pAb419)) are shown (lower panels).

(B) Identification of an MCV sT LT-stabilization domain (LSD) responsible for enhanced LT-mediated origin replication. MCV origin replication was assayed in 293 cells by coexpression of LT together with sT mutants having sequential 5 amino acid deletions or 5 alanine substitutions in exon1A from 86 to 104 amino acids. Deletion or substitution at MCV sT amino acids 91 to 95 (LKDYM) ablated enhanced origin replication and increased steady-state MCV LT protein expression (lanes 5 and 6) compared to wild-type MCV sT (lane 2). Deletion (but not alanine substitutions) at residues 86–90 reduced sT protein stability as well as origin replication and LT expression. See also Figure S1.