Caveolin- and clathrin-independent entry of BKPyV into primary human proximal tubule epithelial cells

Abstract

BK polyomavirus (BKPyV) is a human pathogen that causes polyomavirus-associated nephropathy and hemorrhagic cystitis in transplant patients. Gangliosides and caveolin proteins have previously been reported to be required for BKPyV infection in animal cell models. Recent studies from our lab and others, however, have indicated that the identity of the cells used for infection studies can greatly influence the behavior of the virus. We therefore wished to re-examine BKPyV entry in a physiologically relevant primary cell culture model, human renal proximal tubule epithelial cells. Using siRNA knockdowns, we interfered with expression of UDP-glucose ceramide glucosyltransferase (UGCG), and the endocytic vesicle coat proteins caveolin 1, caveolin 2, and clathrin heavy chain. The results demonstrate that while BKPyV does require gangliosides for efficient infection, it can enter its natural host cells via a caveolin- and clathrin-independent pathway. The results emphasize the importance of studying viruses in a relevant cell culture model.

Sialoglycovirology of Lectins: Sialyl Glycan Binding of Enveloped and Non-enveloped Viruses

“…1). It should be noted that a caveolin/clathrin-independent pathway was observed for some polyomaviruses such as entry of BKPyV into primary human renal proximal tubule epithelial cells [166]. Viral capsid protein 1 (VP1) is responsible for engagement of the host cell receptor, an important step that determines cellular tropism and pathogenesis of the virus.…”

“…JCPyV prefers α2-6Sia neolacto-series on N-linked glycoproteins [167,168] while its binding to the 5-HT 2 serotonin receptor seems to facilitate this endocytosis pathway [169]. SV40, mPy, and BKPyV (either caveolin-dependent or caveolin-independent entry) share a common binding for ganglio-series gangliosides [166,[170][171][172] with differential binding preferences. SV40 prefers the branched α2-3Sia GM1 ganglioside [170], mPyV mainly attaches α2-3Sia on GD1a/GT1b/GT1a [173,174], and BKPyV and MCPyV commonly prefer to bind to α2-8Sia b-series gangliosides including GD3/GD2/GD1b/GT1b for BKPyV [171] and GT1b in cooperative binding with a glycosaminoglycan (GAG) for MCPyV [172].…”

“…(4) It is important to realize that some states of different virus infections may cause the same disease (such as red eyes possibly caused by influenza A viruses, EV70, CAV24v, and HAdV-D). (5) While the entry pathway of Sia-binding enveloped viruses is direct fusion on the host plasma membrane or fusion with the endosomal membrane, the entry pathway of Sia-binding non-enveloped viruses is endocytosis that is followed by endosomal permeabilization/lysis as a result of proteolysis and/or capsid conformational change (picornaviruses [84], caliciviruses [109], reoviruses [111], and adenoviruses [131])/lipolysis (parvoviruses [146,148]) or that is followed by the ERAD pathway (polyomaviruses [166]), except for EMCV, which possibly has direct penetration of its genome through the plasma membrane. (6) Molecular and structural studies have indicated that Sia-binding sites are usually on the heads of viral lectins except for reovirus σ1 capsid protein of T3D strain carrying Sia-binding sites on its body.…”

Establishing RPTE-derived cell lines expressing hTERT for studying BK polyomavirus

“…To test if RPTE-hTERT cells are susceptible to BKPyV infection, RPTE-hTERT cells and RPTE cells were infected with BKPyV (Dunlop) at a MOI of 1 as previous described (10). Protein samples were harvested with E1A buffer (50 mM HEPES [pH 7], 250 mM NaCl, and 0.1% NP-40, with inhibitors: 5 μg/ml PMSF, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 50 mM sodium fluoride and 0.2 mM sodium orthovanadate added right before use).…”

In Vitro and In Vivo Models for the Study of Human Polyomavirus Infection

“…Different entry pathways have been described for PyV depending on both cell type and virus. JCPyV virus enters cells via clathrin-mediated endocytosis, while for BKPyV, caveolae-dependent endocytosis and the caveolin- and clathrin-independent entry pathway have been observed [47,48]. However, further studies are warranted to gain a deeper insight into the cellular receptors and entry pathways of known and newly-discovered PyV.…”