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The human fetal glial cell line SVG was generated in 1985 by transfecting primary fetal brain cells with a plasmid containing an origin-defective mutant of simian virus 40 (SV40). The cells, which express SV40 large T-antigen, support the replication of human JC polyomavirus (JCPyV) and have been used for JCPyV studies but also for other studies in which cells of neural origin were desirable. We intended to use the SVG p12 cells from ATCC for antiviral drug studies with JCPyV. However, during initial experiments, immunofluorescence microscopy controls unexpectedly revealed cells expressing the late viral proteins VP1, VP2/ VP3, and agno. This was confirmed by Western blotting. Since our agnoprotein antiserum is specific for BKPyV agnoprotein, infection with BKPyV was suspected. Indeed, specific BKPyV PCR of SVG p12 supernatants revealed a viral load of >1 ؋ 10

dPolyomavirus BK (BKV) causes polyomavirus-associated nephropathy (PyVAN) and hemorrhagic cystitis (PyVHC) in renal and bone marrow transplant patients, respectively. Antiviral drugs with targeted activity against BKV are lacking. Since the antimalarial drug artesunate was recently demonstrated to have antiviral activity, the possible effects of artesunate on BKV replication in human primary renal proximal tubular epithelial cells (RPTECs), the host cells in PyVAN, were explored. At 2 h postinfection (hpi), RPTECs were treated with artesunate at concentrations ranging from 0.3 to 80 M. After one viral replication cycle (approximately 72 hpi), the loads of extracellular BKV DNA, reflecting viral progeny production, were reduced in a concentrationdependent manner. Artesunate at 10 M reduced the extracellular BKV load by 65%; early large T antigen mRNA and protein expression by 30% and 75%, respectively; DNA replication by 73%; and late VP1 mRNA and protein expression by 47% and 64%, respectively. Importantly, the proliferation of RPTECs was also inhibited in a concentration-dependent manner. At 72 hpi, artesunate at 10 M reduced cellular DNA replication by 68% and total metabolic activity by 47%. Cell impedance and lactate dehydrogenase measurements indicated a cytostatic but not a cytotoxic mechanism. Flow cytometry and 5-ethynyl-2=-deoxyuridine incorporation revealed a decreased number of cells in S phase and suggested cell cycle arrest in G 0 or G 2 phase. Both the antiproliferative and antiviral effects of artesunate at 10 M were reversible. Thus, artesunate inhibits BKV replication in RPTECs in a concentration-dependent manner by inhibiting BKV gene expression and genome replication. The antiviral mechanism appears to be closely connected to cytostatic effects on the host cell, underscoring the dependence of BKV on host cell proliferative functions.

The minor capsid protein of human BK polyomavirus (BKPyV), VP2, and its N-terminally truncated form, VP3, are both important for viral entry. The closely related simian virus 40 (SV40) reportedly produces an additional truncated form of VP2/3, denoted VP4, apparently functioning as a viroporin promoting progeny release. The VP4 open reading frame is conserved in some polyomaviruses, including BKPyV. In this study, we investigated the role of VP4 in BKPyV replication. By transfecting viral genomes into primary human renal proximal tubule epithelial cells, we demonstrated that unaltered BKPyV and mutants with start codon substitutions in VP4 (VP2M229I and VP2M229A) abolishing putative VP4 production were released at the same level to supernatants. However, during infection studies, VP2M229I and VP2M229A exhibited 90% and 65% reduced infectivity, respectively, indicating that isoleucine substitution inadvertently disrupted VP2/3 function to the detriment of viral entry, while inhibition of VP4 production during late infection was well tolerated. Unexpectedly, and similarly to BKPyV, wild-type SV40 and the corresponding VP4 start codon mutants (VP2M228I and VP2M228A) transfected into monkey kidney cell lines were also released at equal levels. Upon infection, only the VP2M228I mutant exhibited reduced infectivity, a 43% reduction, which also subsequently led to delayed host cell lysis. Mass spectrometry analysis of nuclear extracts from SV40-infected cells failed to identify VP4. Our results suggest that neither BKPyV nor SV40 require VP4 for progeny release. Moreover, our results reveal an important role in viral entry for the amino acid in VP2/VP3 unavoidably changed by VP4 start codon mutagenesis. IMPORTANCEAlmost a decade ago, SV40 was reported to produce a late nonstructural protein, VP4, which forms pores in the nuclear membrane, facilitating progeny release. By performing transfection studies with unaltered BKPyV and SV40 and their respective VP4-deficient mutants, we found that VP4 is dispensable for progeny release, contrary to the original findings. However, infection studies demonstrated a counterintuitive reduction of infectivity of certain VP4-deficient mutants. In addition to the isoleucine-substituted SV40 mutant of the original study, we included alanine-substituted VP4-deficient mutants of BKPyV (VP2M229A) and SV40 (VP2M228A). These revealed that the reduction in infectivity was not caused by a lack of VP4 but rather depended on the identity of the single amino acid substituted within VP2/3 for VP4 start codon mutagenesis. Hopefully, our results will correct the longstanding misconception of VP4's role during infection and stimulate continued work on unraveling the mechanism for release of polyomavirus progeny. Currently there are 13 known species of human polyomaviruses, and of these at least four are associated with diseases mainly affecting immunocompromised patients. BK polyomavirus (BKPyV) is the chief agent of polyomavirus-associated nephropathy (PyVAN) and polyomavirus-associated hemo...