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.

Keywords: BKPyV; Caveolin; Clathrin; Endocytosis; Entry; Ganglioside; UGCG.

Figure 1

UGCG knockdown and rescue assay. (A) UGCG catalyzes the glycosylation step, transforming ceramide to cerebroside. Cerebroside is then used to synthesize gangliosides, such as GM1, GD1b and GT1b. Sialic acids are α2–3 linked unless labeled otherwise. (B) Normalized expression of UGCG mRNA (mean SEM). RPTE cells were transfected with non-targeting siRNAs or siRNAs targeting UGCG. Total RNA was collected, and reverse transcription and quantitative PCR were performed. UGCG mRNA expression was normalized to GAPDH mRNA. (C) RPTE cells were transfected with non-targeting siRNAs or siRNAs targeting UGCG. Gangliosides were added at 38 hours post transfection. At 48 hours post transfection, cells were infected with BKPyV at an MOI of 0.5 IU/cell. Western blotting was performed on protein samples harvested at 48 hours post infection.

Figure 2

Caveolin protein knockdown and BKPyV entry. (A) Cross-reactivity of siRNAs targeting caveolin 1 and caveolin 2. Individual siRNAs targeting caveolin 1 and an siRNA pool targeting caveolin 2 were transfected into RPTE cells. Caveolin expression at 2 days post infection was examined by Western blotting. (B) Caveolin protein knockdown does not affect BKPyV infection. RPTE cells were transfected with the indicated siRNAs. Viral infection and caveolin protein expression levels were examined by Western blotting at two days post infection. (C) RPTE cells were transfected with the indicated siRNAs. Viral infection and caveolin protein expression levels were examined by Western blotting at one day post infection.

Figure 3

Clathrin heavy chain knockdown and BKPyV entry. (A) RPTE cells were transfected with the indicated siRNAs, and viral infection and clathrin heavy chain expression levels were examined by Western blotting. (B) RPTE cells were transfected with the indicated combinations of siRNAs, and arranged as above.