Phosphorylation Mutants of JC Virus Agnoprotein Are Unable To Sustain the Viral Infection Cycle

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Agnoprotein is a small multifunctional regulatory protein required for sustaining the productive replication of JC virus (JCV). It is a mostly cytoplasmic protein localizing in the perinuclear area and forms highly stable dimers/oligomers through a Leu/Ile/ Phe-rich domain. There have been no three-dimensional structural data available for agnoprotein due to difficulties associated with the dynamic conversion from monomers to oligomers. Here, we report the first nuclear magnetic resonance (NMR) structure of a synthetic agnoprotein peptide spanning amino acids Thr17 to Glu55 where Lys23 to Phe39 encompassing the Leu/Ile/ Phe-rich domain forms an amphipathic ␣-helix. On the basis of these structural data, a number of Ala substitution mutations were made to investigate the role of the ␣-helix in the structure and function of agnoprotein. Single L29A and L36A mutations exhibited a significant negative effect on both protein stability and viral replication, whereas the L32A mutation did not. In addition, the L29A mutant displayed a highly nuclear localization pattern, in contrast to the pattern for the wild type (WT). Interestingly, a triple mutant, the L29A؉L32A؉L36A mutant, yielded no detectable agnoprotein expression, and the replication of this JCV mutant was significantly reduced, suggesting that Leu29 and Leu36 are located at the dimer interface, contributing to the structure and stability of agnoprotein. Two other single mutations, L33A and E34A, did not perturb agnoprotein stability as drastically as that observed with the L29A and L36A mutations, but they negatively affected viral replication, suggesting that the role of these residues is functional rather than structural. Thus, the agnoprotein dimerization domain can be targeted for the development of novel drugs active against JCV infection. IMPORTANCEAgnoprotein is a small regulatory protein of JC virus (JCV) and is required for the successful completion of the viral replication cycle. It forms highly stable dimers and oligomers through its hydrophobic (Leu/Ile/Phe-rich) domain, which has been shown to play essential roles in the stability and function of the protein. In this work, the Leu/Ile/Phe-rich domain has been further characterized by NMR studies using an agnoprotein peptide spanning amino acids T17 to Q54. Those studies revealed that the dimerization domain of the protein forms an amphipathic ␣-helix. Subsequent NMR structure-based mutational analysis of the region highlighted the critical importance of certain amino acids within the ␣-helix for the stability and function of agnoprotein. In conclusion, this study provides a solid foundation for developing effective therapeutic approaches against the dimerization domain of the protein to inhibit its critical roles in JCV infection.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Nuclear Magnetic Resonance Structure Revealed that the Human Polyomavirus JC Virus Agnoprotein Contains an α-Helix Encompassing the Leu/Ile/Phe-Rich Domain

Coric

Sarıbaş

Abou‐Gharbia

et al. 2014

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“…Agnoprotein can be phosphorylated by protein kinase C, and several potential sites including serine at positions 7 and 11 have been implicated experimentally (54,62). Here, we altered serine 7 and serine 11 to alanine and serine 15 to valine by site-directed mutagenesis and developed CG-4 cells that can constitutively express the mutant protein.…”

Section: Resultsmentioning

confidence: 99%

JC Virus Agnoprotein Inhibits In Vitro Differentiation of Oligodendrocytes and Promotes Apoptosis

Merabova

Kaniowska

Kaminski

et al. 2008

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Productive infection of oligodendrocytes, which are responsible for the formation of myelin sheath in the central nervous system, with the human neurotropic virus JC virus (JCV) causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). In addition to encoding T antigen and the capsid proteins, which are produced at the early and late phases of the infection cycle, respectively, JCV encodes a small regulatory protein named agnoprotein that is important for successful completion of the virus life cycle. Here we used bipotential CG-4 cells to examine the impact of agnoprotein on oligodendrocyte differentiation and survival in the absence of JCV lytic infection. We demonstrate that the expression of agnoprotein delayed the formation of complex outgrowth networks of the cells during oligodendrocyte differentiation. These alterations were accompanied by high levels of DNA damage, induction of proapoptotic proteins, and suppression of prosurvival signaling. Accordingly, apoptosis was significantly increased upon the induction of CG-4 cells toward differentiation in cells expressing agnoprotein. These observations provide the first evidence for the possible involvement of agnoprotein, independent from its role in viral replication, in a series of biological events that may contribute to the pathological features seen in PML lesions.First exposure to the human polyomavirus JC virus (JCV) commonly occurs during childhood and results in a subclinical outcome. Reactivation of the initially latent virus in individuals with impaired immune systems results in the development of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) (9,31,35). PML manifests clinically with signs of neurological deficits of various degrees, including weakness, visual deficits, and cognitive abnormalities. PML is mainly associated with advanced human immunodeficiency virus (HIV)-induced diseases and AIDS (8, 9), lymphoproliferative or myeloproliferative diseases, malignancies, and rheumatic disorders treated with corticosteroids and cytotoxic drugs (1,16,25,34,39,52,58,71,75,77). Recently, PML has also been diagnosed in patients with multiple sclerosis and Crohn's disease receiving natalizumab (37,40,42,74), a humanized monoclonal antibody that binds to the ␣4 subunit of ␣4␤1 and ␣4␤7 integrins and inhibits the trafficking of leukocytes across the blood-brain barrier.The histological hallmarks of white matter from PML patients include the appearance of oligodendrocytes with enlarged hyperchromatic nuclei that contain inclusion bodies shaped by crystalline arrays of JCV particles and astrocytes with bizarre nuclei and mitotic figures (21, 35). The presence of virions has also been demonstrated among lamellae of the myelin sheath of a viable axon (47). In cell culture systems, JCV efficiently infects primary cultures of human brain containing oligodendrocytes and astrocytes. Recent studies have shown that JCV can infect multipotential human central nervous system progenitor cells isolated fro...

“…In vitro and in vivo kinase assays demonstrated that agnoprotein is a target for phosphorylation by PKC. When each of the PKC phosphorylation sites was mutated to Ala singly and in combination, virus containing each of these mutations failed to propagate (104). Thus, phosphorylated forms of agnoprotein may have essential functions in the viral life cycle.…”

Section: Recent Studies Of Primate Polyomavirusesmentioning

confidence: 99%

Regulation of Gene Expression in Primate Polyomaviruses

White

Safak

Khalili

2009

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Polyomaviruses are a growing family of small DNA viruses with a narrow tropism for both the host species and the cell type in which they productively replicate. Species host range may be constrained by requirements for precise molecular interactions between the viral T antigen, host replication proteins, including DNA polymerase, and the viral origin of replication, which are required for viral DNA replication. Cell type specificity involves, at least in part, transcription factors that are necessary for viral gene expression and restricted in their tissue distribution. In the case of the human polyomaviruses, BK virus (BKV) replication occurs in the tubular epithelial cells of the kidney, causing nephropathy in kidney allograft recipients, while JC virus (JCV) replication occurs in the glial cells of the central nervous system, where it causes progressive multifocal leukoencephalopathy. Three new human polyomaviruses have recently been discovered: MCV was found in Merkel cell carcinoma samples, while Karolinska Institute Virus and Washington University Virus were isolated from the respiratory tract. We discuss control mechanisms for gene expression in primate polyomaviruses, including simian vacuolating virus 40, BKV, and JCV. These mechanisms include not only modulation of promoter activities by transcription factor binding but also enhancer rearrangements, restriction of DNA methylation, alternate early mRNA splicing, cis-acting elements in the late mRNA leader sequence, and the production of viral microRNA.Polyomaviruses comprise a family of small nonenveloped DNA tumor viruses which have small, circular, doublestranded DNA genomes, have been isolated from many species of mammals and birds, and are characterized by a very limited host range with respect to the species that they can productively infect (48). We will focus mainly on three primate viruses, simian vacuolating virus 40 (SV40), BK virus (BKV), and JC virus (JCV), not only because they have taught us important lessons about eukaryotic molecular biology but also because BKV and JCV cause important human diseases. SV40 was discovered almost 50 years ago (115) and was the first primate polyomavirus to be described. SV40 differs significantly from the previously discovered mouse polyoma virus (111) in that it does not possess a middle T antigen in the early region and it expresses an accessory regulatory protein, agnoprotein, which is encoded in the late region. The species of origin of SV40 is the rhesus macaque, and the virus was discovered as a contaminant in early batches of polio vaccine. While the polio vaccinations at that time likely infected many people, the prevalence of SV40 infections in humans today has been debated (36,122). In 1971, two bona fide human polyomaviruses were discovered. BKV, also known as polyomavirus BK, was first isolated by Gardner et al. (37) by culture in Vero cells from the urine of a patient receiving immunosuppressive therapy following kidney transplantation. BKV is widespread throughout the human population around...