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The polyomavirus BK agnoprotein co-localizes with lipid droplets

Transplantation Virology, Institute for Medical Microbiology, Department of Biomedicine, University of Basel, CH-4003 Basel, Switzerland.
Virology (Impact Factor: 3.28). 02/2010; 399(2):322-31. DOI: 10.1016/j.virol.2010.01.011
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ABSTRACT Agnoprotein encoded by human polyomavirus BK (BKV) is a late cytoplasmic protein of 66 amino acids (aa) of unknown function. Immunofluorescence microscopy revealed a fine granular and a vesicular distribution in donut-like structures. Using BKV(Dunlop)-infected or agnoprotein-transfected cells, we investigated agnoprotein co-localization with subcellular structures. We found that agnoprotein co-localizes with lipid droplets (LD) in primary human renal tubular epithelial cells as well as in other cells supporting BKV replication in vitro (UTA, Vero cells). Using agnoprotein-enhanced green fluorescent protein (EGFP) fusion constructs, we demonstrate that agnoprotein aa 20-42 are required for targeting LD, whereas aa 1-20 or aa 42-66 were not. Agnoprotein aa 22-40 are predicted to form an amphipathic helix, and mutations A25D and F39E, disrupting its hydrophobic domain, prevented LD targeting. However, changing the phosphorylation site serine-11 to alanine or aspartic acid did not alter LD co-localization. Our findings provide new clues to unravel agnoprotein function.

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Available from: Rainer Gosert, Jul 31, 2015
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    • "In that study, we detected transiently expressed His-tagged BKV agnoprotein (agno-His) in Vero cells using a monoclonal His-tag antibody (DIA900, Dianova, Hamburg, Germany) together with a rabbit antiserum against the ER marker protein calnexin (ADI-SPA860, Enzo Life Sciences, Lausen, Switzerland). In confocal microscopy analysis, we were unable to detect significant overlapping signals of BKV agnoprotein and calnexin (see Supplementary figure 1a in Unterstab et al., 2010). "
    Virology 07/2013; 441(2):197–199. DOI:10.1016/j.virol.2013.03.018 · 3.28 Impact Factor
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    • "The Phe residues of JCV agnoprotein appear to be involved in viral DNA replication (Saribas et al., 2012). With respect to the function of Phe residues, the protein distribution studies with confocal microscopy showed that Phe39 residue of BKV agnoprotein may play a role in localization of the protein to the lipid droplets in infected cells (Unterstab et al., 2010). In addition to in vitro stable dimer/oligomer formation, we have recently reported to dimer formation in vivo in the infected cells (Saribas et al., 2011). "
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    ABSTRACT: Agnoprotein is one of the key regulatory proteins of polyomaviruses, including JCV, BKV and SV40 and is required for a productive viral life cycle. We have recently reported that agnoprotein forms stable dimer/oligomers mediated by a predicted amphipathic α-helix, spanning amino acids (aa), 17 to 42. Deletion of the α-helix renders a replication incompetent virus. Here, we have further characterized this region by a systematic deletion and substitution mutagenesis and demonstrated that a Leu/Ile/Phe-rich domain, (spanning aa 28-39) within α-helix is indispensable for agnoprotein structure and function. Deletion of aa 30-37 severely affects the dimer/oligomer formation and stable expression of the protein. Mutagenesis data also indicate that the residues, 34-36, may be involved in regulation of the splicing events of JCV transcripts. Collectively, these data suggest that the Leu/Ile/Phe-rich domain plays critical roles in agnoprotein function and thus represents a potential target for developing novel therapeutics against JCV infections.
    Virology 06/2013; 443(1). DOI:10.1016/j.virol.2013.05.003 · 3.28 Impact Factor
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    • "Agnoprotein is abundantly expressed late in the viral life cycle, and localizes predominantly to the cytoplasm both in vitro [9] [15] and also in vivo as shown in biopsies of BKV-associated nephropathy [16]. In the cytoplasm, BKV agnoprotein shows a reticular distribution but also colocalizes with lipid droplets, the latter being mediated by an amphipathic helix of 20 aa in the central part of the 66 aa BKV agnoprotein [15]. Despite its abundant expression of BKV agnoprotein in vivo, agnoprotein-specific humoral and cellular immune responses were low or undetectable in individuals exposed "
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    ABSTRACT: Background. Among human polyomaviruses, only BK virus (BKV) and JC virus (JCV) encode an agnoprotein upstream of VP1 on the viral late transcript. BKV agnoprotein is abundantly expressed late in the viral life cycle, but specific cellular and humoral immune responses are low or absent. We hypothesized that agnoprotein might contribute to BKV immune evasion by downregulating HLA expression, similar to Herpes simplex virus-1 ICP47. Methods UTA-6 or primary human renal proximal tubular epithelial cells (RPTEC) were co-transfected with plasmids constitutively expressing agnoprotein, or ICP47, and enhanced green-fluorescent protein (EGFP). EGFP-gated cells were analyzed for HLA-ABC and HLA-DR expression by flow cytometry. HLA-ABC and HLA-DR expression was also analyzed on UTA-6 bearing tetracycline-regulated agnoprotein or ICP47. Effects of agnoprotein on viral peptide-dependent T-cell killing were investigated using (51)Cr release. Results. ICP47 downregulated HLA-ABC without affecting HLA-DR, whereas agnoprotein did not affect HLA-ABC or HLA-DR expression. Interferon- γ treatment increased HLA-ABC in a dose-dependent manner, which was antagonized by ICP47, but not by agnoprotein. In UTA-6 cells, agnoprotein expression did neither impair HLA-ABC or -DR expression nor peptide-specific killing impaired by HLA-matched T-cells. Conclusion. Unlike the HSV-1 ICP47, BKV agnoprotein does not contribute to viral immune evasion by down-regulating HLA-ABC, or interfere with HLA-DR expression or peptide-dependent T-cell cytotoxicity.
    Clinical and Developmental Immunology 03/2013; 2013:626823. DOI:10.1155/2013/626823 · 2.93 Impact Factor
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