title: The Presumed Polyomavirus Viroporin VP4 of Simian Virus 40 or Human BK Polyomavirus Is Not Required for Viral Progeny Release.
abstract: 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.
  
  
  OBJECTIVE
  Almost 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.
authors.affiliation: Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway.|Department of Clinical Medicine, UiT The Arctic University of Norway, Tromsø, Norway.
authors.forename: Stian
authors.initials: S
authors.lastname: Henriksen
authors.affiliation: Department of Pharmacy, UiT The Arctic University of Norway, Tromsø, Norway.
authors.forename: Terkel
authors.initials: T
authors.lastname: Hansen
authors.affiliation: Department of Medical Biology, UiT The Arctic University of Norway, Tromsø, Norway.
authors.forename: Jack-Ansgar
authors.initials: JA
authors.lastname: Bruun
authors.affiliation: Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway christine.rinaldo@unn.no.|Metabolic and Renal Research Group, UiT The Arctic University of Norway, Tromsø, Norway.
authors.forename: Christine Hanssen
authors.identifier: http://orcid.org/0000-0002-6970-6728
authors.initials: CH
authors.lastname: Rinaldo
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001739:BK Virus / Q000235:genetics*; D019556:COS Cells; D036022:Capsid Proteins / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004261:DNA Replication / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D011120:Polyomavirus / Q000235:genetics*; D027601:Polyomavirus Infections / Q000821:virology*; D013539:Simian virus 40 / Q000235:genetics*; D014709:Vero Cells; D053586:Virus Internalization; D014779:Virus Replication / Q000235:genetics
chemical_list: D036022:Capsid Proteins