Hy. Li et al., Effect on polyomavirus T-antigen function of mutations in a conserved leucine-rich segment of the DnaJ domain, J VIROLOGY, 75(5), 2001, pp. 2253-2261
The N-terminal part of the mouse polyomavirus T antigens contains a highly
conserved segment (-LLELLKL-), including amino acid residues 13 to 19, The
sequence motif is predicted to form alpha helix I in the DnaJ domain of the
T antigens, Four mutants with conservative substitutions of amino acid res
idues 13 and 14 were constructed. Of the four substitutions, L13M, L13I, L1
3V, and L14V, only L13V resulted in a phenotypic change. In transfected mou
se cells, L13V large T antigen showed a more than 100-fold-reduced viral DN
A synthesis. The viral replication could not be rescued by cotransfection o
f the cells with DNA expressing small t antigen or a large T antigen trunca
ted at the C terminus that would compensate for a defect in host cell stimu
lation. In contrast to the effect on DNA replication, the L13V substitution
in large T antigen did not prevent complex formation with Hsc70 and the Rb
protein. Also, the activity of the protein in transactivation of transcrip
tion from the adenovirus E2 promoter was unimpaired, showing that the trans
cription factor E2F was released from pRb, The L13V substitution also cause
d a defect in small t antigen. However, this phenotypic change was due to p
rotein instability. In contrast, middle T antigen with the L13V substitutio
n remained stable and functional in cellular transformation. Together, the
data show that the effect of the L13V substitution did not abrogate the Hsc
70 interaction of the DnaJ domain. However, it is possible that the substit
ution of amino acid residue 13 affected specific DnaJ functions of large T
antigen.