The Human Fetal Glial Cell Line SVG p12 Contains Infectious BK Polyomavirus

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e W e thank Michael W. Ferenczy and Eugene O. Major for their informative historical background regarding the origins of various SVG cell lines and subclones. In their letter, the authors also provide independent confirmation of our results that SVG p12 cells (ATCC CRL-8621) are positive for BK polyomavirus (BKPyV) DNA (1). Their letter is particularly important since the laboratory of the authors is one of the pioneers in JC polyomavirus (JCPyV) research and is also the place where the SVG cells originated.We disagree that our recent paper (1) implies that all SVGderived cell lines are BKPyV contaminated. On the contrary, in our paper we concluded that SVG-A cells, derived by limiting dilution of the original SVG cell line, are BKPyV negative, which suggests that the source of the SVG-A cell line was BKPyV negative. This observation was confirmed by Ferenczy and Major.The impact of the BKPyV contamination on SVG research is presently difficult to estimate since many studies utilizing SVGderived cell lines lack a clear specification of the source of the cells (1, 2). This leads us to reemphasize the implications of our recent paper: researchers who have been using or are using SVG-derived cell lines should test their cells for the presence of BKPyV, and reviewers of such work should demand this testing in case this has not been performed.Finally, the evidence provided by Ferenczy and Major showing that the SVG cells in their possession do not contain BKPyV DNA certainly raises concerns about previous and present routines for cell handling at the ATCC.

supporting

confidence: 68%

mentioning

confidence: 99%

Reply to “Contamination of SVG p12 Cells with BK Polyomavirus Occurred after Deposit in the American Type Culture Collection”

Rinaldo

Henriksen

Tylden

et al. 2014

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“…All SV40 experiments were performed with strain 776 and virus with start codon mutations of VP4 (Table 1). All mutants were created by long PCR of pBKV (34-2) (ATCC 45025) (18), pBKV WWT (19), or pWTSV40 JL (20) using overlapping primers that targeted the ATG start codon (Table 1) and the high-fidelity Phusion polymerase (M0530S; New England BioLabs) according to the manufacturer's instructions as previously described (21). Sanger sequencing confirmed successful mutagenesis.…”

Section: Methodsmentioning

confidence: 99%

The Presumed Polyomavirus Viroporin VP4 of Simian Virus 40 or Human BK Polyomavirus Is Not Required for Viral Progeny Release

Henriksen

Hansen

Bruun

et al. 2016

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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...

“…At the core of what distinguishes archetype BKPyV strains with a low replicative capacity from the highly replicative strains found in kidney transplant patients are changes in the noncoding control region (NCCR) of the BKPyV genome (33). Akin to all PyVs, the BKPyV NCCR consists of an approximately 400-bp-long sequence which is sandwiched between the early viral gene region (EVGR) and the late viral gene region (LVGR), altogether yielding the double-stranded BKPyV DNA genome of 5,100 bp (34,35). The NCCR contains cis-acting elements coordinating the timing and consecutive steps of EVGR expression, viral genome replication, and LVGR expression (33,36).…”

mentioning

confidence: 99%

Imperfect Symmetry of Sp1 and Core Promoter Sequences Regulates Early and Late Virus Gene Expression of the Bidirectional BK Polyomavirus Noncoding Control Region

Bethge

Ajuh

Hirsch

2016

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Rearrangements or point mutations in the noncoding control region (NCCR) of BK polyomavirus (BKPyV) have been associated with higher viral loads and more pronounced organ pathology in immunocompromised patients. The respective alterations affect a multitude of transcription factor binding sites (TFBS) but consistently cause increased expression of the early viral gene region (EVGR) at the expense of late viral gene region (LVGR) expression. By mutating TFBS, we identified three phenotypic groups leading to strong, intermediate, or impaired EVGR expression and corresponding BKPyV replication. Unexpectedly, Sp1 TFBS mutants either activated or inhibited EVGR expression when located proximal to the LVGR (sp1-4) or the EVGR (sp1-2), respectively. We now demonstrate that the bidirectional balance of EVGR and LVGR expression is dependent on affinity, strand orientation, and the number of Sp1 sites. Swapping the LVGR-proximal high-affinity SP1-4 with the EVGR-proximal low-affinity SP1-2 in site strand flipping or inserting an additional SP1-2 site caused a rearranged NCCR phenotype of increased EVGR expression and faster BKPyV replication. The 5= rapid amplification of cDNA ends revealed an imperfect symmetry between the EVGR-and LVGR-proximal parts of the NCCR, consisting of TATA and TATA-like elements, initiator elements, and downstream promoter elements. Mutation or deletion of the archetypal LVGR promoter, which is found in activated NCCR variants, abrogated LVGR expression, which could be restored by providing large T antigen (LTag) in trans. Thus, whereas Sp1 sites control the initial EVGR-LVGR expression balance, LTag expression can override inactivation of the LVGR promoter and acts as a key driver of LVGR expression independently of the Sp1 sites and core promoter elements. IMPORTANCEPolyomaviridae currently comprise more than 70 members, including 13 human polyomaviruses (PyVs), all of which share a bidirectional genome organization mediated by the NCCR, which determines species and host cell specificity, persistence, replication, and virulence. Here, we demonstrate that the BKPyV NCCR is fine-tuned by an imperfect symmetry of core promoter elements centered around TATA and TATA-like sequences close to the EVGR and LVGR, respectively, which are governed by the directionality and affinity of two Sp1 sites. The data indicated that the BKPyV NCCR is poised toward EVGR expression, which can be readily unlatched by a simple switch affecting Sp1 binding. The resulting LTag, which is the major EVGR protein, drives viral genome replication, renders subsequent LVGR expression independently of archetypal promoter elements, and can overcome enhancer/promoter mutations and deletions. The data are pivotal for understanding how human PyV NCCRs mediate secondary host cell specificity, reactivation, and virulence in their natural hosts. The Polyomaviridae comprise more than 70 polyomavirus (PyV) members, including at least 13 human PyV (HPyV) species (1). HPyV seroprevalence data obtained using the major capsid prote...