Cytomegaloviral protein kinase pUL97 interacts with the nuclear mRNA export factor pUL69 to modulate its intranuclear localization and activity

Thomas, Marco; Rechter, Sabine; Milbradt, Jens; Auerochs, Sabrina; Müller, Regina; Stamminger, Thomas; Marschall, Manfred

doi:10.1099/vir.0.005827-0

Cited by 48 publications

(75 citation statements)

References 35 publications

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“…pcDNA-UL97-FLAG, pcDNA-UL97(K355M)-FLAG, pcDNA-UL97-(1-595)-FLAG, and pcDNA-UL97-(181-707)-FLAG)), hemagglutinin-tagged pUL50 (pcDNA-UL50-HA), and PKC␣ fused to enhanced green fluorescent protein (EGFP; pEGFP-N1-PKC␣) were described previously (19,21,22,27). Plasmid pHM990, coding for a fusion protein of IE2p86 and GFP, was kindly provided by Dr. N. Tavalai (Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg) (28).…”

Section: Methodsmentioning

confidence: 99%

Novel Mode of Phosphorylation-triggered Reorganization of the Nuclear Lamina during Nuclear Egress of Human Cytomegalovirus

Milbradt

Webel

Auerochs

et al. 2010

Journal of Biological Chemistry

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148

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The nucleocytoplasmic egress of viral capsids is a rate-limiting step in the replication of the human cytomegalovirus (HCMV). As reported recently, an HCMV-specific nuclear egress complex is composed of viral and cellular proteins, in particular protein kinases with the capacity to induce destabilization of the nuclear lamina. Viral protein kinase pUL97 and cellular protein kinase C (PKC) play important roles by phosphorylating several types of nuclear lamins. Using pUL97 mutants, we show that the lamin-phosphorylating activity of pUL97 is associated with a reorganization of nuclear lamin A/C. Either pUL97 or PKC has the potential to induce distinct punctate lamina-depleted areas at the periphery of the nuclear envelope, which were detectable in transiently transfected and HCMV-infected cells. Using recombinant HCMV, which produces green fluorescent protein-labeled viral capsids, the direct transition of viral capsids through these areas could be visualized. This process was sensitive to an inhibitor of pUL97/PKC activity. The pUL97-mediated phosphorylation of lamin A/C at Ser 22 generated a novel binding motif for the peptidyl-prolyl cis/trans-isomerase Pin1. In HCMV-infected fibroblasts, the physiological localization of Pin1 was altered, leading to recruitment of Pin1 to viral replication centers and to the nuclear lamina. The local increase in Pin1 peptidyl-prolyl cis/trans-isomerase activity may promote conformational modulation of lamins. Thus, we postulate a novel phosphorylation-triggered mechanism for the reorganization of the nuclear lamina in HCMVinfected cells. Human cytomegalovirus (HCMV)2 belongs to the ␤-herpesvirus subfamily, exhibiting worldwide distribution. When infecting immunocompetent individuals, HCMV possesses low pathogenicity, causing mainly asymptomatic infections. In immunocompromised or immunosuppressed hosts, HCMV infection can cause severe and even life-threatening diseases, including pneumonitis, retinitis, hepatitis, encephalitis, and gastroenteritis (1-3).HCMV replication is based on a nuclear phase, a characteristic of most DNA viruses. Transition from the nuclear to the cytoplasmic phase is determined by the nuclear exit of DNAfilled capsids budding through the inner nuclear membrane (INM) (4 -6). The site-specific budding of viral capsids through distinct locally occurring invaginations in the INM of HCMVinfected cells was clearly illustrated by Buser et al. (7) using electron microscopic analysis. The proteinaceous network of the nuclear lamina, underlying the INM, constitutes a major obstacle for the nuclear egress of capsids. Lamins, belonging to type V intermediate filament proteins, are the main constituents of the nuclear lamina and are grouped into A and B types. A-type lamins (A, C, A⌬10, and C2; collectively lamin A/C) result from alternative splicing of the LMNA gene. B-type lamins are encoded by the LMNB1 (B1) or LMNB2 (B2, B3) gene (8, 9). A major function of the nuclear lamina is to maintain the structure of the nuclear envelope. During mitosis, the nuclear lamin...

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Section: Methodsmentioning

confidence: 99%

Novel Mode of Phosphorylation-triggered Reorganization of the Nuclear Lamina during Nuclear Egress of Human Cytomegalovirus

Milbradt

Webel

Auerochs

et al. 2010

Journal of Biological Chemistry

Self Cite

148

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“…The mechanism by which pUL69 blocks cell cycle progression is not known. It may result from interaction with cellular cyclin-dependent kinases, such as CDK1, which phosphorylates pUL69 (6), or with the HCMVcoded pUL97 kinase (9). pUL97, which has a structural resemblance to cellular cyclin-dependent kinases, hyperphosphorylates and inactivates the cellular retinoblastoma protein, which favors cell cycle progression (10,11).…”

Section: H Uman Cytomegalovirus (Hcmv) Is a Ubiquitous β-Herpesvirusmentioning

confidence: 99%

“…pUL69 shuttles between nucleus and cytoplasm (16), binds RNA (17), and interacts with the DExD/Hbox RNA helicase UAP56 or the related URH49 protein (12), cellular proteins involved in RNA transport. The pUL69 transport function is stimulated by both cellular cyclin-dependent kinases (6) and pUL97 (9).…”

Section: H Uman Cytomegalovirus (Hcmv) Is a Ubiquitous β-Herpesvirusmentioning

confidence: 99%

Human cytomegalovirus UL69 protein facilitates translation by associating with the mRNA cap-binding complex and excluding 4EBP1

Aoyagi

Gaspar

Shenk

2010

Proc. Natl. Acad. Sci. U.S.A.

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4EBP1 is phosphorylated by the mTORC1 kinase. When mTORC1 activity is inhibited, hypophosphorylated 4EBP1 binds and sequesters eIF4E, a component of the mRNA cap-binding complex, and blocks translation. As a consequence, mTORC1 activity is needed to maintain active translation. The human cytomegalovirus pUL38 protein preserves mTORC1 activity, keeping most of the E4BP1 in the infected cell in a hyperphosphorylated, inactive state. Here we report that a second viral protein, pUL69, also antagonizes the activity of 4EBP1, but by a separate mechanism. pUL69 interacts directly with eIF4A1, an element of the cap-binding complex, and the poly(A)-binding protein, which binds to the complex. When pUL69 accumulates during infection with wild-type virus, 4EBP1 is excluded from the complex. However, 4EBP1 is present in the cap-binding complex after infection with a pUL69-deficient virus, coincident with reduced accumulation of several late virus-coded proteins. We propose that pUL69 supports translation in human cytomegalovirus-infected cells by excluding hypophosphorylated 4EBP1 from the cap-binding complex.translational control | two-hybrid screen | eukaryotic initiation factor 4A

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“…Both tegument proteins are known to associate with pUL97, and pUL69 is phosphorylated by the viral kinase (Chevillotte et al, 2009;Thomas et al, 2009). To investigate whether the three proteins formed a complex and whether the two tegument proteins were phosphorylated by the viral kinase in that complex, coimmunoprecipitation (CoIP) experiments in conjunction with in vitro kinase assays (IVKA) were performed on lysates of infected cells.…”

Section: Rv-vm1 Is Resistant To Inhibition Of Pul97 By Gö 6976mentioning

confidence: 99%

Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells

Becke

Fabre-Mersseman

Aue

et al. 2010

Journal of General Virology

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The tegument protein pp65 of human cytomegalovirus (HCMV) is abundant in lytically infected human foreskin fibroblasts (HFF), as well as in virions and subviral dense bodies (DB). Despite this, we showed previously that pp65 is dispensable for growth in HFF. In the process of refining a DB-based vaccine candidate, different HCMV mutants were generated, expressing a dominant HLA-A2-presented peptide of the IE1 protein fused to pp65. One of the mutant viruses (RV-VM1) surprisingly showed marked impairment in virus release from HFF. We hypothesized that analysis of the phenotypic alterations of RV-VM1 would provide insight into the functions of pp65, poorly defined thus far. RV-VM1 infection resulted in nuclear retention of the fusion protein and reorganization of nuclear inclusion bodies. Coimmunoprecipitation experiments suggested that wild-type (wt) pp65 and pp65-VM1 were substrates of the viral pUL97 kinase in vitro and formed a complex with the viral RNA-export protein pUL69 and with pUL97 in lysates of infected cells. No evidence for an impairment of pUL97 within this complex was found. However, RV-VM1 replication in infected cells was resistant to a pUL97 inhibitor, and pUL97 inhibitors mimicked the mutant in terms of pp65 being retained in the nucleus. The results suggest that the life cycle of RV-VM1 was impeded at the stages of early-late transcription, RNA export or capsid maturation. wt-pp65 may play a role at these stages of infection, and complex formation with pUL69 and pUL97 may be important for that function. INTRODUCTIONHuman cytomegalovirus (HCMV) is a ubiquitous pathogen that severely affects individuals with impaired or immature immune-defence functions (Griffiths et al., 2009;Mocarski et al., 2007). The components of the HCMV tegument have recently attracted considerable attention, as these proteins carry functions important for various stages of the virus replication cycle (reviewed by Kalejta, 2008). The phosphoprotein pp65 (ppUL83) has long been known as an abundant tegument constituent (Roby & Gibson, 1986), yet it is dispensable for HCMV growth in human foreskin fibroblast (HFF) cell cultures (Schmolke et al., 1995b).pp65 is an important target antigen of cellular and humoral immune responses (Beninga et al., 1995; McLaughlinTaylor et al., 1994; Plachter et al., 1990;Wills et al., 1996). It would thus be reasonable to assume that modification or deletion of the protein would be favourable for the virus. However, the sequence of pp65 is highly conserved in laboratory strains and clinical isolates (Dolan et al., 2004;Pande et al., 1991) and lack of pp65 expression in such strains has never been reported. Indeed, detection of pp65 has been used as a reliable diagnostic marker for acute HCMV infection in transplant recipients for over 15 years (Grefte et al., 1992a, b Initial studies described an association of pp65 with serine/ threonine kinase activity (Britt & Auger, 1986;Somogyi et al., 1990). Subsequent analyses identified the cellular Polo-like kinase 1 (Gallina et al., 1999) and the vi...

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Cytomegaloviral protein kinase pUL97 interacts with the nuclear mRNA export factor pUL69 to modulate its intranuclear localization and activity

Cited by 48 publications

References 35 publications

Novel Mode of Phosphorylation-triggered Reorganization of the Nuclear Lamina during Nuclear Egress of Human Cytomegalovirus

Novel Mode of Phosphorylation-triggered Reorganization of the Nuclear Lamina during Nuclear Egress of Human Cytomegalovirus

Human cytomegalovirus UL69 protein facilitates translation by associating with the mRNA cap-binding complex and excluding 4EBP1

Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells

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