Polyomavirus-associated nephropathy occurs in ϳ5% of renal transplant recipients and results in loss of graft function in 50 to 70% of these patients. The disease is caused by reactivation of the common human polyomavirus BK (BKV) in the transplanted kidney. The early events in productive BKV infection are unknown. In this report, we focus on elucidating the mechanisms of BKV internalization in its target cell. Our data reveal that BKV entry into permissive Vero cells is slow, is independent of clathrin-coated-pit assembly, is dependent on an intact caveolin-1 scaffolding domain, is sensitive to tyrosine kinase inhibition, and requires cholesterol. BKV colocalizes with the caveola-mediated endocytic marker cholera toxin subunit B but not with the clathrin-dependent endocytic marker transferrin. In addition, BKV infectious entry is sensitive to elevation in intracellular pH. These findings indicate that BKV entry into Vero cells occurs by caveola-mediated endocytosis involving a pH-dependent step.Humans are the natural host species for two members of the polyomavirus family, BK virus (BKV) and JC virus (JCV) (22). Both viruses establish persistent subclinical infections in the kidneys and peripheral blood in 85% of the population worldwide (10, 36). The urotheliotropic nature of BKV is characterized by infection of the epithelial lining of the collective ducts, the transitional epithelial cells of the renal calyces, the parietal epithelium of the Bowman's capsule, and the transitional epithelium of the renal pelvis and the urinary tract (11, 56). Sporadic reactivation of BKV resulting in limited viral replication is seen in 0.5 to 20% of healthy seropositive individuals, yet renal function is left unaffected (20,23,62). A dramatic increase in viral activity leading to disease progression occurs predominantly in the context of greatly impaired T-cell-mediated immunity (26,27).Kidney transplant recipients are subjected to a potent immunosuppressive therapy and are thus at a high risk for exacerbated BKV replication (1). Active BKV infection in the renal allograft, known as polyomavirus nephropathy, has been linked to progressive graft dysfunction and ultimate graft loss (13). Sustained high levels of viral replication result in lytic destruction of the host tubular cells and release of BKV progeny to perpetuate the infection (1, 33, 34). BKV, the causative agent of polyomavirus nephropathy, is drawing increasing attention as a significant factor in the failure of renal transplants (48).Similar to those of the other polyomaviruses, BK virions are small (40.5 to 45 nm in diameter) and consist of a superhelical circular double-stranded DNA genome contained within a nonenveloped icosahedral capsid (18). Though the exact identity of the BK receptor(s) is unknown, evidence suggests that gangliosides type II, GD1a, and GT play important roles in the initial interaction between BKV and the permissive monkey kidney (Vero) cell line, as well as in BKV hemagglutination of human type O red blood cells (54,59,60). Following pen...