Nuclear import of isoforms of the cytomegalovirus kinase pUL97 is mediated by differential activity of NLS1 and NLS2 both acting through classical importin-α binding

Webel, Rike; Solbak, Sara Marie Øie; Held, Christian; Milbradt, Jens; Gross, Andrea M.; Eichler, Jutta; Wittenberg, Thomas; Jardin, Christophe; Sticht, Heinrich; Fossen, Torgils; Marschall, Manfred

doi:10.1099/vir.0.040592-0

Cited by 22 publications

(33 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…7c). Finally, both proteins are fixed in this terminal localization at the nuclear rim and provide a scaffold for the association of further viral and cellular proteins to form an entire multi-component NEC (Milbradt et al, 2010(Milbradt et al, , 2012unpublished data).…”

Section: Comparison Of the Mechanisms Of Core Nec Formation In Hcmv Amentioning

confidence: 99%

“…Since it was demonstrated that interaction of pUL50 with pUL53 is required for export of HCMV capsids from the nucleus, the protein pair pUL50-pUL53 (or herpesviral homologues) was referred to as the viral core of the nuclear egress complex (NEC) (Lötzerich et al, 2006;Mettenleiter et al, 2009;Milbradt et al, 2007Milbradt et al, , 2012. Besides the potential direct involvement of pUL50 and pUL53 in destabilizing the nuclear lamina, recruitment of protein kinases with capacity to induce lamina-depleted areas is considered as one major activity of the HCMV-specific NEC (Lee & Chen, 2010;Marschall et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Schmeiser

Borst

Sticht

et al. 2013

Journal of General Virology

Self Cite

View full text Add to dashboard Cite

The nucleocytoplasmic export of cytomegaloviral capsids is regulated by formation of a multicomponent nuclear egress complex (NEC), essentially based on viral proteins pUL50 and pUL53. In this study, the generation of recombinant human cytomegaloviruses, expressing tagged versions of pUL50 and pUL53, enabled the investigation of NEC formation in infected primary fibroblasts. For these recombinant viruses, a wild-type-like mode of pUL50-pUL53 interaction and recruitment of both proteins to the nuclear envelope could be demonstrated. Importantly, pUL50 was translocated from an initial cytoplasmic distribution to the nuclear rim, whereas pUL53 accumulated in the nucleus before attaining overall rim colocalization with pUL50. Specified experimental settings illustrated that pUL50 and pUL53 were subject to different pathways of intracellular trafficking. Importantly, a novel nuclear localization signal (NLS) could be identified and functionally verified for pUL53 (amino acids 18-27), whereas no NLS was present in pUL50. Analysis of amino acid replacement mutants further illustrated the differential modes of nuclear import of the two essential viral egress proteins. Taken together, our findings suggest a combination of classical nuclear import (pUL53) and interaction-mediated recruitment (pUL50) as the driving forces for core NEC formation and viral nuclear egress.

show abstract

Section: Comparison Of the Mechanisms Of Core Nec Formation In Hcmv Amentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Schmeiser

Borst

Sticht

et al. 2013

Journal of General Virology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Both isoforms exhibited auto-phosphorylation activity and were incorporated in HCMV virions (4). Moreover, two nuclear localization sequences, NLS1 and NLS2, were identified in the N terminus of pUL97, indicating a differential, isoform-specific efficiency of nuclear import (5).…”

mentioning

confidence: 99%

“…It is expressed early in infection (2,3), localizes predominantly to the nucleus using amino-terminal nuclear localization signals (4,5), and is also observed in the cytoplasm later in the infection cycle (2,5,6). It is also present in the HCMV virion (7).…”

mentioning

confidence: 99%

Differential Properties of Cytomegalovirus pUL97 Kinase Isoforms Affect Viral Replication and Maribavir Susceptibility

Webel

Hakki

Prichard

et al. 2014

J Virol

Self Cite

View full text Add to dashboard Cite

The human cytomegalovirus (HCMV)-encoded kinase pUL97 is required for efficient viral replication. Previous studies described two isoforms of pUL97, the full-length isoform (M1) and a smaller isoform likely resulting from translation initiation at codon 74 (M74). Here, we report the detection of a third pUL97 isoform during viral infection resulting from translation initiation at codon 157 (isoform M157). The consistent expression of isoform M157 as a minor component of pUL97 during infection with clinical and laboratory-adapted HCMV strains was suppressed when codon 157 was mutagenized. Viral mutants expressing specific isoforms were generated to compare their growth and drug susceptibility phenotypes, as well as pUL97 intracellular localization patterns and kinase activities. The exclusive expression of isoform M157 resulted in substantially reduced viral growth and resistance to the pUL97 inhibitor maribavir while retaining susceptibility to ganciclovir. Confocal imaging demonstrated reduced nuclear import of amino-terminal deletion isoforms compared to isoform M1. Isoform M157 showed reduced efficiency of various substrate protein interactions and autophosphorylation, whereas Rb phosphorylation was preserved. These results reveal differential properties of pUL97 isoforms that affect viral replication, with implications for the antiviral efficacy of maribavir. IMPORTANCEThe HCMV UL97 kinase performs important functions in viral replication that are targeted by the antiviral drug maribavir. Here, we describe a naturally occurring short isoform of the kinase that when expressed by itself in a recombinant virus results in altered intracellular localization, impaired growth, and high-level resistance to maribavir compared to those of the predominant full-length counterpart. This is another factor to consider in explaining why maribavir appears to have variable antiviral activity in cell culture and in vivo.T he product of the human cytomegalovirus (HCMV) UL97 gene (pUL97) is a serine/threonine protein kinase that phosphorylates itself and multiple viral and host proteins (1). It is expressed early in infection (2, 3), localizes predominantly to the nucleus using amino-terminal nuclear localization signals (4, 5), and is also observed in the cytoplasm later in the infection cycle (2, 5, 6). It is also present in the HCMV virion (7). Genetic deletion of UL97 results in a severe (10-to 1,000-fold) replication defect in vitro (8-10), as does mutation of the K355 lysine residue, which is critical for kinase activity (6). UL97 is therefore an attractive target for antiviral drug development (1,(11)(12)(13)(14) as an alternative to the UL54 DNA polymerase target of all currently licensed HCMV antivirals, including ganciclovir (GCV).Maribavir (MBV) is a benzimidazole riboside pUL97 inhibitor that inhibits HCMV replication potently (15) but variably depending on cell culture conditions (16). While MBV showed promise in early clinical trials, two recently published phase III trials demonstrated that MBV was no more...

show abstract

“…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).…”

mentioning

confidence: 99%

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

Bigley

Reitsma

Mirza

et al. 2013

J Virol

View full text Add to dashboard Cite

Human cytomegalovirus (HCMV) is a common agent of congenital infection and causes severe disease in immunocompromised patients. Current approved therapies focus on inhibiting viral DNA replication. The HCMV kinase pUL97 contributes to multiple stages of viral infection including DNA replication, controlling the cell cycle, and virion maturation. Our studies demonstrate that pUL97 also functions by influencing immediate early (IE) gene expression during the initial stages of infection. Inhibition of kinase activity using the antiviral compound maribavir or deletion of the UL97 gene resulted in decreased expression of viral immediate early genes during infection. Expression of pUL97 was sufficient to transactivate IE1 gene expression from the viral genome, which was dependent on viral kinase activity. We observed that pUL97 associates with histone deacetylase 1 (HDAC1). HDAC1 is a transcriptional corepressor that acts to silence expression of viral genes. We observed that inhibition or deletion of pUL97 kinase resulted in increased HDAC1 and decreased histone H3 lysine 9 acetylation associating with the viral major immediate early (MIE) promoter. IE expression during pUL97 inhibition or deletion was rescued following inhibition of deacetylase activity. HDAC1 associates with chromatin by protein-protein interactions. Expression of active but not inactive pUL97 kinase decreased HDAC1 interaction with the transcriptional repressor protein DAXX. Finally, using mass spectrometry, we found that HDAC1 is uniquely phosphorylated upon expression of pUL97. Our results support the conclusion that HCMV pUL97 kinase regulates viral immediate early gene expression by phosphorylation-mediated disruption of HDAC1 binding to the MIE promoter.

show abstract

Nuclear import of isoforms of the cytomegalovirus kinase pUL97 is mediated by differential activity of NLS1 and NLS2 both acting through classical importin-α binding

Cited by 22 publications

References 38 publications

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

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

Differential Properties of Cytomegalovirus pUL97 Kinase Isoforms Affect Viral Replication and Maribavir Susceptibility

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

Contact Info

Product

Resources

About