The polyomavirus BK agnoprotein co-localizes with lipid droplets

Unterstab, Gunhild; Gosert, Rainer; Leuenberger, David; Lorentz, Pascal; Rinaldo, Christine Hanssen; Hirsch, Hans H.

doi:10.1016/j.virol.2010.01.011

Cited by 43 publications

(55 citation statements)

References 48 publications

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“…First, immunofluorescent staining of SVG p12 cells with antiserum directed against BKPyV agnoprotein, previously shown not to cross-react with SV40 or JCPyV agnoprotein, showed the characteristic cytoplasmic staining pattern seen in BKPyV-infected cells (45,57). This result was confirmed in a separate laboratory and by Western blotting.…”

Section: Discussionmentioning

confidence: 55%

The Human Fetal Glial Cell Line SVG p12 Contains Infectious BK Polyomavirus

Henriksen

Tylden

Dumoulin

et al. 2014

J Virol

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The human fetal glial cell line SVG was generated in 1985 by transfecting primary fetal brain cells with a plasmid containing an origin-defective mutant of simian virus 40 (SV40). The cells, which express SV40 large T-antigen, support the replication of human JC polyomavirus (JCPyV) and have been used for JCPyV studies but also for other studies in which cells of neural origin were desirable. We intended to use the SVG p12 cells from ATCC for antiviral drug studies with JCPyV. However, during initial experiments, immunofluorescence microscopy controls unexpectedly revealed cells expressing the late viral proteins VP1, VP2/ VP3, and agno. This was confirmed by Western blotting. Since our agnoprotein antiserum is specific for BKPyV agnoprotein, infection with BKPyV was suspected. Indeed, specific BKPyV PCR of SVG p12 supernatants revealed a viral load of >1 ؋ 10

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Section: Discussionmentioning

confidence: 55%

The Human Fetal Glial Cell Line SVG p12 Contains Infectious BK Polyomavirus

Henriksen

Tylden

Dumoulin

et al. 2014

J Virol

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“…It is also interesting to note that all three Phe residues of agnoprotein (Phe31, Phe35, and Phe39) localize to its hydrophobic region. The functional importance of these Phe residues has recently been evaluated by genetic and biochemical approaches, and it was demonstrated that they positively influence viral DNA replication (1,4) and play regulatory roles in the perinuclear distribution of agnoprotein in infected cells (4,5). Agnoprotein possesses only one highly conserved Cys residue, which is located right after the hydrophobic region of the protein (Cys40).…”

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

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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“…However, two major required features of amino acid sequence have been proposed: one is the amphipathic ␣ -helix (19)(20)(21)(22)(23), and the other is a rather undefi ned hydrophobic region (24)(25)(26). Some LD-targeting proteins, such as caveolin-1 and associated with lipid droplet protein 1 (ALDI) ( 24,42 ), showed dual localization to LDs and ER in relation with cellular lipid dynamics.…”

Section: Discussionmentioning

confidence: 99%

“…Although several cellular and viral proteins localize on the LD membrane, consensus signals for LD targeting are not defi ned ( 1 ). Recent reports have shown that LD-targeting activity resides in amphipathic helices (19)(20)(21)(22)(23) or in hydrophobic regions (24)(25)(26).…”

Section: Immunofl Uorescencementioning

confidence: 99%

“…Both helices are thought to mediate membrane association of NS4B ( 40,41 ). Amphipathic helices are also known to mediate LD association (19)(20)(21)(22)(23); therefore, we hypothesized that the amphipathic helices, especially the hydrophobic amino acids within them, were important for LD targeting of NS4B. From helical wheel projection analysis (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi, provided by D. Armstrong et al), we noticed that 10 hydrophobic residues (43W, 46L, 50W, 57F, 61I, and 64L for the N-terminal amphipathic helix; 242I, 246L, 249L, and 253I for the C-terminal amphipathic helix) were present on the hydrophobic faces of the helices ( Fig.…”

Section: Hydrophobic Amino Acid Residues In Amphipathic ␣ -Helices Inmentioning

confidence: 99%

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