Human polyomavirus JC small regulatory agnoprotein forms highly stable dimers and oligomers: Implications for their roles in agnoprotein function

Sarıbaş, A. Sami; Arachea, Buenafe T.; White, Martyn K.; Viola, Ronald E.; Safak, Mahmut

doi:10.1016/j.virol.2011.08.015

Cited by 22 publications

(85 citation statements)

References 72 publications

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“…To support this notion in functional assays, we demonstrated that agnoprotein enhances the DNA binding activity of LT-Ag to the viral origin (Ori) without itself directly interacting with DNA (4). We also demonstrated that agnoproteins of BKV, SV40, and JCV form highly stable, SDS-resistant homodimers and oligomers (7,18). In particular, the Leu/Ile/Phe-rich region of the protein plays a direct role in forming such structures and is required for the stable expression of the protein (1).…”

mentioning

confidence: 84%

“…As we previously demonstrated, the stable dimer and oligomer formation by agnoprotein can be monitored by employing SDS-PAGE under reducing conditions, followed by Coomassie blue staining in vitro (1,7). As shown in Fig.…”

Section: Agnoprotein Is Predicted To Contain a Hydrophobic ␣-Helixmentioning

confidence: 98%

“…Plasmid constructs. The construction of the pMAL-c5x-Agno and Bluescript KS(ϩ)-JCV Mad-1 WT plasmids was previously described (7). Various alanine (Ala) substitution mutations on agnoprotein (Agno) in a viral background (JCV Mad-1) were created using a QuikChange site-directed mutagenesis kit (catalog no.…”

Section: Fig 2 Comparison Of the 1dmentioning

confidence: 99%

“…However, previous computer modeling studies revealed that the Leu/Ile/Phe-rich domain of the protein is involved in forming an amphipathic ␣-helix (1,6,7) and is highly conserved in the agnoproteins of JCV, SV40, and BKV (1). These 3D modeling studies also suggest that a small portion of the amino-terminal region and carboxy-terminal half of the protein may adopt an intrinsically disordered conformation.…”

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confidence: 99%

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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

J Virol

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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.

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mentioning

confidence: 84%

Section: Agnoprotein Is Predicted To Contain a Hydrophobic ␣-Helixmentioning

confidence: 98%

Section: Fig 2 Comparison Of the 1dmentioning

confidence: 99%

mentioning

confidence: 99%

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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

J Virol

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“…Agnoprotein is a 71 aa multi-functional protein with numerous reported protein-protein interactions (Darbinyan et al, 2004;Endo et al, 2003;Johannessen et al, 2008;Safak et al, 2002;Suzuki et al, 2005). It also retains a central hydrophobic TMD which, along with an N-terminal region, is required for ER/plasma membrane localization and membrane integration (Suzuki et al, 2010), and forms stable oligomers within infected cells (Coric et al, 2014;Saribas et al, 2011Saribas et al, , 2013Suzuki et al, 2010). Agnoprotein expression both increases plasma membrane permeability and elevates cytosolic calcium, resulting in enhanced virion release (Suzuki et al, 2010).…”

Section: Dna Virus Viroporinsmentioning

confidence: 99%

Viroporins: structure, function and potential as antiviral targets

Scott

Griffin

2015

Journal of General Virology

120

146

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The channel-forming activity of a family of small, hydrophobic integral membrane proteins termed 'viroporins' is essential to the life cycles of an increasingly diverse range of RNA and DNA viruses, generating significant interest in targeting these proteins for antiviral development. Viroporins vary greatly in terms of their atomic structure and can perform multiple functions during the virus life cycle, including those distinct from their role as oligomeric membrane channels. Recent progress has seen an explosion in both the identification and understanding of many such proteins encoded by highly significant pathogens, yet the prototypic M2 proton channel of influenza A virus remains the only example of a viroporin with provenance as an antiviral drug target. This review attempts to summarize our current understanding of the channel-forming functions for key members of this growing family, including recent progress in structural studies and drug discovery research, as well as novel insights into the life cycles of many viruses revealed by a requirement for viroporin activity. Ultimately, given the successes of drugs targeting ion channels in other areas of medicine, unlocking the therapeutic potential of viroporins represents a valuable goal for many of the most significant viral challenges to human and animal health.

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Nuclear Magnetic Resonance Structure of the Human Polyoma JC Virus Agnoprotein

Coric

Abou‐Gharbia

et al. 2017

J of Cellular Biochemistry

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Agnoprotein is an important regulatory protein of the human polyoma JC virus (JCV) and plays critical roles during the viral replication cycle. It forms highly stable dimers and oligomers through its Leu/Ile/Phe-rich domain, which is important for the stability and function of the protein. We recently resolved the partial 3D structure of this protein by NMR using a synthetic peptide encompassing amino acids Thr17 to Gln52, where the Leu/Ile/Phe- rich domain was found to adopt a major alpha-helix conformation spanning amino acids 23 to 39. Here, we report the resolution of the 3D structure of full-length JCV agnoprotein by NMR, which not only confirmed the existence of the previously reported major α-helix domain at the same position but also revealed the presence of an additional minor α-helix region spanning amino acid residues Leu6 to Ala10. The remaining regions of the protein adopt an intrinsically unstructured conformation.

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Human polyomavirus JC small regulatory agnoprotein forms highly stable dimers and oligomers: Implications for their roles in agnoprotein function

Cited by 22 publications

References 72 publications

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

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

Viroporins: structure, function and potential as antiviral targets

Nuclear Magnetic Resonance Structure of the Human Polyoma JC Virus Agnoprotein

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