Assembly Protein Precursor (pUL80.5 Homolog) of Simian Cytomegalovirus Is Phosphorylated at a Glycogen Synthase Kinase 3 Site and Its Downstream “Priming” Site: Phosphorylation Affects Interactions of Protein with Itself and with Major Capsid Protein

Casaday, Rebecca J.; Bailey, Justin R.; Kalb, Suzanne R.; Brignole, Edward J.; Loveland, Amy N.; Cotter, Robert J.; Gibson, Wade

doi:10.1128/jvi.78.24.13501-13511.2004

Cited by 19 publications

(12 citation statements)

References 59 publications

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“…1, lanes p62 and p26). This is probably due to posttranslational modifications, such as phosphorylation, which has been reported for other herpesvirus scaffold proteins (5,15).…”

Section: Resultsmentioning

confidence: 99%

Small Capsid Protein pORF65 Is Essential for Assembly of Kaposi's Sarcoma-Associated Herpesvirus Capsids

Perkins

Anacker

Davis

et al. 2008

J Virol

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Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent for KS tumors, multicentric Castleman's disease, and primary effusion lymphomas. Like other herpesvirus capsids, the KSHV capsid is an icosahedral structure composed of six proteins. The capsid shell is made up of the major capsid protein, two triplex proteins, and the small capsid protein. The scaffold protein and the protease occupy the internal space. The assembly of KSHV capsids is thought to occur in a manner similar to that determined for herpes simplex virus type 1 (HSV-1). Our goal was to assemble KSHV capsids in insect cells using the baculovirus expression vector system. Six KSHV capsid open reading frames were cloned and the proteins expressed in Sf9 cells: pORF25 (major capsid protein), pORF62 (triplex 1), pORF26 (triplex 2), pORF17 (protease), pORF17.5 (scaffold protein), and also pORF65 (small capsid protein). When insect cells were coinfected with these baculoviruses, angular capsids that contained internal core structures were readily observed by conventional electron microscopy of the infected cells. Capsids were also readily isolated from infected cells by using rate velocity sedimentation. With immuno-electron microscopy methods, these capsids were seen to be reactive to antisera to pORF65 as well as to KSHV-positive human sera, indicating the correct conformation of pORF65 in these capsids. When either virus expressing the triplex proteins was omitted from the coinfection, capsids did not assemble; similar to observations made in HSV-1-infected cells. If the virus expressing the scaffold protein was excluded, large open shells that did not attain icosahedral structure were seen in the nuclei of infected cells. The presence of pORF65 was required for capsid assembly, in that capsids did not form if this protein was absent as judged by both by ultrastructural analysis of infected cells and rate velocity sedimentation experiments. Thus, a novel outcome of this study is the finding that the small capsid protein of KSHV, like the major capsid and triplex proteins, is essential for capsid shell assembly.Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of KS tumors, an angiogenic skin malignancy, as well primary effusion lymphomas and multicentric Castleman's disease (7,9,40). Malignancies due to KSHV are a major clinical problem for AIDS patients and consequently represent the predominant cancer found in southern Africa. KSHV (or human herpesvirus 8), like the other herpesviruses, assembles icosahedral capsids that display a Tϭ16 shell. For all herpesviruses studied to date the capsids are comprised of six polypeptides (reviewed in references 17, 34, and 42). The capsid shell subunits or capsomeres are composed of the major capsid protein (51). There are 150 hexons of the major capsid protein and 11 pentons, which are located at the vertices of the icosahedral structure. The portal protein occupies the 12th vertex (4, 8, 11) and forms a dodecameric ring structure (48), and it is the portal for DNA entry into ...

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“…1, lanes p62 and p26). This is probably due to posttranslational modifications, such as phosphorylation, which has been reported for other herpesvirus scaffold proteins (5,15).…”

Section: Resultsmentioning

confidence: 99%

Small Capsid Protein pORF65 Is Essential for Assembly of Kaposi's Sarcoma-Associated Herpesvirus Capsids

Perkins

Anacker

Davis

et al. 2008

J Virol

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“…Furthermore, we observed that the C-tail of FGF14 contains a consensus GSK–3 phosphorylation motif ( S/T )XXX(S/T), the first residue in this sequence corresponding to S226. (The phosphorylation motif for GSK–3 contains two serine/threonines: the first, at the P position, corresponds to the site phosphorylated by GSK–3, and the second, at the P + 4 position, corresponds to a priming phosphorylation site that increases the efficiency of GSK–3-dependent phosphorylation (Casaday et al, 2004; St-Denis et al, 2015; ter Haar et al, 2001)). Based on these observations, we conducted an in vitro phosphorylation assay using recombinant GSK–3β with a FGF14 peptide with the sequence KPGVTP S KSTSASAIMNGGK.…”

Section: Resultsmentioning

confidence: 99%

PPARgamma agonists rescue increased phosphorylation of FGF14 at S226 in the Tg2576 mouse model of Alzheimer's disease

Hsu

Wildburger

Haidacher

et al. 2017

Experimental Neurology

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Background Cognitive impairment in humans with Alzheimer's disease (AD) and in animal models of Aβ-pathology can be ameliorated by treatments with the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ) agonists, such as rosiglitazone (RSG). Previously, we demonstrated that in the Tg2576 animal model of AD, RSG treatment rescued cognitive deficits and reduced aberrant activity of granule neurons in the dentate gyrus (DG), an area critical for memory formation. Methods We used a combination of mass spectrometry, confocal imaging, electrophysiology and split-luciferase assay and in vitro phosphorylation and Ingenuity Pathway Analysis. Results Using an unbiased, quantitative nano-LC-MS/MS screening, we searched for potential molecular targets of the RSG-dependent rescue of DG granule neurons. We found that S226 phosphorylation of fibroblast growth factor 14 (FGF14), an accessory protein of the voltage-gated Na+ (Nav) channels required for neuronal firing, was reduced in Tg2576 mice upon treatment with RSG. Using confocal microscopy, we confirmed that the Tg2576 condition decreased PanNav channels at the AIS of the DG, and that RSG treatment of Tg2576 mice reversed the reduction in PanNav channels. Analysis from previously published data sets identified correlative changes in action potential kinetics in RSG-treated T2576 compared to untreated and wildtype controls. In vitro phosphorylation and mass spectrometry confirmed that the multifunctional kinase GSK–3β, a downstream target of insulin signaling highly implicated in AD, phosphorylated FGF14 at S226. Assembly of the FGF14:Nav1.6 channel complex and functional regulation of Nav1.6-mediated currents by FGF14 was impaired by a phosphosilent S226A mutation. Bioinformatics pathway analysis of mass spectrometry and biochemistry data revealed a highly interconnected network encompassing PPARγ, FGF14, SCN8A (Nav 1.6), and the kinases GSK–3 β, casein kinase 2β, and ERK1/2. Conclusions These results identify FGF14 as a potential PPARγ-sensitive target controlling Aβ-induced dysfunctions of neuronal activity in the DG underlying memory loss in early AD.

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“…The predicted size of the scaffold protein (BdRF1) is 36 kDa, but based on its migration in the gel, it appeared to have a higher molecular mass. One explanation for this could be the observed posttranslational modifications of herpesvirus scaffold proteins (5,16). Accumulation of the protease (BVRF2) was not discernible in these experiments.…”

Section: Resultsmentioning

confidence: 99%

Self-Assembly of Epstein-Barr Virus Capsids

Henson

Perkins

Cothran

et al. 2009

J Virol

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Epstein-Barr virus (EBV), a member of the Gammaherpesvirus family, primarily infects B lymphocytes and is responsible for a number of lymphoproliferative diseases. The molecular genetics of the assembly pathway and high-resolution structural analysis of the capsid have not been determined for this lymphocryptovirus. As a first step in studying EBV capsid assembly, the baculovirus expression vector (BEV) system was used to express the capsid shell proteins BcLF1 (major capsid protein), BORF1 (triplex protein), BDLF1 (triplex protein), and BFRF3 (small capsid protein); the internal scaffold protein, BdRF1; and the maturational protease (BVRF2). Coinfection of insect cells with the six viruses expressing these proteins resulted in the production of closed capsid structures as judged by electron microscopy and sedimentation methods. Therefore, as shown for other herpesviruses, only six proteins are required for EBV capsid assembly. Furthermore, the small capsid protein of EBV (BFRF3), like that of Kaposi's sarcoma-associated herpesvirus, was found to be required for assembly of a stable structure. Localization of the small capsid protein to nuclear assembly sites required both the major capsid (BcLF1) and scaffold proteins (BdRF1) but not the triplex proteins. Mutational analysis of BFRF3 showed that the N-terminal half (amino acids 1 to 88) of this polypeptide is required and sufficient for capsid assembly. A region spanning amino acids 65 to 88 is required for the concentration of BFRF3 at a subnuclear site and the N-terminal 65 amino acids contain the sequences required for interaction with major capsid protein. These studies have identified the multifunctional role of the gammaherpesvirus small capsid proteins. Epstein-Barr virus (EBV) is a gammaherpesvirus that infectsgreater than 90% of the general population and causes several lymphoproliferative diseases. Primary infection in adolescents with EBV results in infectious mononucleosis (46). EBV is also oncogenic and is associated with malignancies such as Burkitt's lymphoma (4), nasopharyngeal carcinoma, and posttransplant lymphoproliferative disease (47).Herpesviruses assemble icosahedral capsid structures in the nuclei of infected cells; six proteins are required for assembly of these structures. The capsid shell is largely composed of multimers of the major capsid protein, which are linked by the triplex structure (trimer of the two triplex proteins) and decorated by the small capsid protein. The internal scaffold protein directs the closure of the shell into an icosahedral structure (reviewed in references 17, 35, and 41). The EBV serine protease is synthesized as a 605-amino-acid polyprotein, which cleaves itself between amino acids Ala235 and Ser236 (release [R] site) and also between amino acids Ala568 and Ser569 (maturation [M] site). The catalytic domain resides in the N-terminal 235 amino acids (13). The six EBV capsid proteins are BcLF1 (major capsid protein), BORF1 (triplex 1), BDLF1 (triplex 2), BdRF1 (scaffold protein), BVRF2 (protease), and BFRF3...

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Assembly Protein Precursor (pUL80.5 Homolog) of Simian Cytomegalovirus Is Phosphorylated at a Glycogen Synthase Kinase 3 Site and Its Downstream “Priming” Site: Phosphorylation Affects Interactions of Protein with Itself and with Major Capsid Protein

Cited by 19 publications

References 59 publications

Small Capsid Protein pORF65 Is Essential for Assembly of Kaposi's Sarcoma-Associated Herpesvirus Capsids

Small Capsid Protein pORF65 Is Essential for Assembly of Kaposi's Sarcoma-Associated Herpesvirus Capsids

PPARgamma agonists rescue increased phosphorylation of FGF14 at S226 in the Tg2576 mouse model of Alzheimer's disease

Self-Assembly of Epstein-Barr Virus Capsids

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