Two isoforms of the protein kinase pUL97 of human cytomegalovirus are differentially regulated in their nuclear translocation

Abstract

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.

Epstein-Barr Virus Protein Kinase BGLF4 Targets the Nucleus through Interaction with Nucleoporins

“…For example, a putative NLS was mapped in a green fluorescent protein (GFP)-UL97 fusion protein at aa 48 to 110 by immunofluorescence analysis (46). However, it was recently reported that human cytomegalovirus UL97 can be translated from two alternative ATGs, which results in isoforms of 90 kDa and 100 kDa (56). In a GFP-␤-galactosidase (␤-Gal) fusion cassette, the N-terminal region from aa 6 to 35 is a functional bipartite NLS, while aa 164 to 198 and 190 to 213 are required to coordinate for optimal nuclear translocation.…”

The cytomegalovirus egress proteins pUL50 and pUL53 are translocated to the nuclear envelope through two distinct modes of nuclear import

“…5c). It has to be stressed, however, that the nuclear translocation efficiency of the GFP-bGal 53/2 construct occurred at a moderate level compared to previously identified NLS sequences of other HCMV proteins (Lischka et al, 2003;Webel et al, 2011Webel et al, , 2012. In order to optimize NLS activity (by removing putatively down-modulating flanking sequences), additional test constructs were generated to express amino acids 13-27, 18-27 or 22-27.…”

“…To measure the intracellular activity of these sequences, an established NLS mapping system was applied (Sorg & Stamminger, 1999;Webel et al, 2011). In this mapping system, putative NLS sequences were cloned in fusion to flanking GFP and bgalactosidase (bGal) sequences resulting in a reporter protein which could be easily detected and located using fluorescence microscopy ( Fig.…”

Human Cytomegalovirus pUL97 Regulates the Viral Major Immediate Early Promoter by Phosphorylation-Mediated Disruption of Histone Deacetylase 1 Binding

“…pUL97 is a tegument protein delivered to infected cells, and newly expressed pUL97 protein begins increasing around 5 hours postinfection (hpi) (5,6). The kinase exists in multiple isoforms, which have distinct expression patterns within the cell (7,8). Deletion or inactivation of the kinase results in an ϳ6-fold decrease in viral DNA accumulation and up to 100-fold decrease in viral yield (9,10).…”