BK Polyoma Virus Allograft Nephropathy: Ultrastructural Features from Viral Cell Entry to Lysis

133

145

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...

Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Infection of Vero Cells by BK Virus Is Dependent on Caveolae

Eash

Querbes

Atwood

2004

133

145

“…Though it is still controversial whether caveolae play a role in endocytosis, SV40 has been reported to internalize through caveolae (4, 5, 15, 24, 26, 28-31, 33, 35) and BKV has also been reported to internalize into HRPTEC through flask-like invaginations similar to caveolae, as shown by morphological analysis (8). Moreover, previous reports indicated that Nlinked glycoprotein with ␣(2,3)-linked sialic acid served as a BKV receptor in monkey-derived Vero cells (9,10), and gangliosides GD1b and GT1b were also reported to work as receptors for BKV in human prostate carcinoma cells (25).…”

Section: Discussionmentioning

confidence: 99%

“…BKV was reported to enter into monkey-derived Vero cells through a caveola-mediated pathway, and internalization depended upon an intact microtubule network (11,12). As revealed by electron microscopy, HRPTEC contains all the ultrastructural features required for caveola-mediated internalization (8). Caveolae (caveolin-containing lipid rafts) are flask-shaped plasma membrane invaginations with a diameter of 50 to 80 nm, enriched in cholesterol and sphingolipids.…”

mentioning

confidence: 99%

Caveolar Endocytosis Is Critical for BK Virus Infection of Human Renal Proximal Tubular Epithelial Cells

Moriyama

Marquez

Wakatsuki

et al. 2007

108

In recent years, BK virus (BKV) nephritis after renal transplantation has become a severe problem. The exact mechanisms of BKV cell entry and subsequent intracellular trafficking remain unknown. Since human renal proximal tubular epithelial cells (HRPTEC) represent a main natural target of BKV nephritis, analysis of BKV infection of HRPTEC is necessary to obtain additional insights into BKV biology and to develop novel strategies for the treatment of BKV nephritis. We coincubated HRPTEC with BKV and the cholesteroldepleting agents methyl beta cyclodextrin (MBCD) and nystatin (Nys), drugs inhibiting caveolar endocytosis. The percentage of infected cells (detected by immunofluorescence) and the cellular levels of BKV large T antigen expression (detected by Western blot analysis) were significantly decreased in both MBCD-and Nys-treated HPRTEC compared to the level in HRPTEC incubated with BKV alone. HRPTEC infection by BKV was also tested after small interfering RNA (siRNA)-dependent depletion of either the caveolar structural protein caveolin-1 (Cav-1) or clathrin, the major structural protein of clathrin-coated pits. BKV infection was inhibited in HRPTEC transfected with Cav-1 siRNA but not in HRPTEC transfected with clathrin siRNA. The colocalization of labeled BKV particles with either Cav-1 or clathrin was investigated by using fluorescent microscopy and image cross-correlation spectroscopy. The rate of colocalization of BKV with Cav-1 peaked at 4 h after incubation. Colocalization with clathrin was insignificant at all time points. These results suggest that BKV entered into HRPTEC via caveolae, not clathrin-coated pits, and that BKV is maximally associated with caveolae at 4 h after infection, prior to relocation to a different intracellular compartment.

“…We and others have previously demonstrated that BKV enters cells through a caveola-mediated internalization mechanism (11,12,38). Images from ultrastructural analysis of renal tubular cells during various stages of infection consistently reveal BK virions inside monopinocytotic vesicles and/or tubuloreticular network as the viral aggregates traverse the cytoplasm en route to the host cell nucleus (11).…”

mentioning

confidence: 83%

Involvement of Cytoskeletal Components in BK Virus Infectious Entry

Eash

Atwood

2005

Posttransplant reactivation of BK virus (BKV) in the renal allograft progresses to polyomavirus-associated nephropathy in 1% to 8% of kidney recipients. Graft dysfunction and loss in 30% to 45% of polyomavirusassociated nephropathy-affected patients are secondary to extensive tubular epithelial cell injury induced by the lytic replication of BKV. The early events in productive BKV infection are not thoroughly understood. We have previously shown that BKV enters cells by caveola-mediated endocytosis. In this report we examine the role of microfilaments and microtubules during early viral infection. Our results show that BKV infection of Vero cells is sensitive to nocodazole-induced disassembly of the microtubule network for the initial 8 hours following virus binding. In contrast, suppression of microtubule turnover with the stabilizing agent paclitaxel has no effect on BKV infectivity. Selective disassembly of the actin filaments with latrunculin A does not impede BKV infection, while inhibition of microfilament dynamics with jasplakinolide results in reduced numbers of viral antigen-positive cells. These data demonstrate that BKV, like other polyomaviruses, relies on an intact microtubule network during early infection. BKV, however, does not share the requirement with the closely related JC virus for an intact actin cytoskeleton during intracellular transport.First discovered in 1970, BK virus (BKV) has recently drawn renewed interest as the causative agent of an infectious complication termed polyomavirus-associated nephropathy in renal transplant recipients (16, 52). Asymptomatic BKV infection is ubiquitous, occurring in 70% to 90% of healthy adults worldwide (41). Surgical injury and potent immunosuppressive therapy following renal transplantation lead to BKV reactivation and progression to polyomavirus-associated nephropathy in 1% to 10% of kidney recipients (24, 51). The clinical concern stems from the extensive virally induced damage to the kidney, which in turn results in severe allograft dysfunction and ultimate graft loss in 45% to 60% of polyomavirus-associated nephropathy-affected patients (32,52).BKV belongs to the family Polyomaviridae, which are small, nonenveloped, double-stranded DNA viruses. Other well-studied polyomaviruses include simian virus 40 (SV40), mouse polyomavirus (PyV), and JC virus (JCV). Despite established differences in receptor specificity (3-5, 9, 13, 14, 27, 28, 47, 48), mode of internalization (1,12,17,33,36,37,39,40,43), and intracellular trafficking mechanisms (2,19,36,37), all the members of the polyomavirus family must deliver their genome to the nucleus of the host cell in order to execute a productive viral life cycle. The nucleus is the site of viral gene transcription, viral DNA replication, and progeny assembly (45).Efforts in our laboratory are focused on understanding the early events during BKV infection, namely host cell entry and subsequent intracellular trafficking to the nucleus. We and others have previously demonstrated that BKV enters cells through a cave...