
Full text loading...
The pUL97 protein kinase encoded by human cytomegalovirus is a multifunctional determinant of the efficiency of viral replication and phosphorylates viral as well as cellular substrate proteins. Here, we report that pUL97 is expressed in two isoforms with molecular masses of approximately 90 and 100 kDa. ORF UL97 comprises an unusual coding strategy in that five in-frame ATG start codons are contained within the N-terminal 157 aa. Site-directed mutagenesis, transient expression of point and deletion mutants and proteomic analyses accumulated evidence that the formation of the large and small isoforms result from alternative initiation of translation, with the start points being at amino acids 1 and 74, respectively. In vitro kinase assays demonstrated that catalytic activity, in terms of autophosphorylation and histone substrate phosphorylation, was indistinguishable for the two isoforms. An analysis of the intracellular distribution of pUL97 by confocal laser-scanning microscopy demonstrated that both isoforms have a pronounced nuclear localization. Surprisingly, mapping experiments performed to identify the nuclear localization signal (NLS) of pUL97 strongly suggest that the mechanism of nuclear transport is distinct for the two isoforms. While the extreme N terminus (large isoform) comprises a highly efficient, bipartite NLS (amino acids 6–35), a second sequence apparently conferring a less efficient mode of nuclear translocation was identified downstream of amino acid 74 (small and large isoforms). Taken together, the findings argue for a complex mechanism of nuclear translocation for pUL97 which might be linked with fine-regulatory differences between the two isoforms.
Article metrics loading...
Full text loading...
References
Data & Media loading...
Supplements
Journal of General Virology vol. 92 , part 3, pp. 638–649
Supplementary Fig. S1. Multiple sequence alignment. pUL97 sequences of human and simian cytomegaloviruses were analysed by using Tcoffee multiple sequence alignment tools.
Supplementary Fig. S2. Mass spectrometry analysis of the small isoform of pUL97.
Supplementary Fig. S3. Wild-type pUL97 showing nuclear localization as indistinguishable from the two individually expressed isoforms.
Supplementary Fig. S4. Confirmation of a functional NLS in amino acid region 6–35 of pUL97.
Supplementary Table S1. Oligonucleotide primers used in this study
[ Single PDF file] (210 KB)