Trans-complementation of a genetic defect in the coxsackie B3 virus 2B protein.
The enterovirus 2B protein contains a putative amphipathic alpha-helix that includes three positively charged and one negatively charged residue. Previously, we observed that replacement of the glutamic acid-40 residue with a lysine residue (mutation 2B-E[40]K) in the amphipathic alpha-helix of the coxsackie B3 virus 2B protein resulted in a quasi-infectious phenotype. On one occasion, however, transfection of 2B-E[40]K RNA transcripts gave rise to a virus stock in which the mutation was retained. This study was aimed at elucidating the molecular mechanism underlying this observation. Sequence analysis of the viral RNA provided no evidence for a second-site suppression mutation that rescued the defect of the 2B-E[40]K mutation in cis. Therefore, the possibility was considered that the defect caused by the 2B-E[40]K mutation was complemented in trans by viable revertants that had emerged in the virus population. The transfection-derived virus stock indeed contained a small fraction of (pseudo)revertant viruses, carrying the original glutamic acid-40, threonine-40 or asparagine-40, rather than the introduced lysine-40. Consistent with the idea that the 2B-E[40]K virus is unable to grow without the aid of trans-acting wild-type(-like) proteins, only the (pseudo)revertant viruses were able to produce individual plaques. Further support for the idea of trans-rescue was obtained using a genetic complementation assay, which revealed the occurrence of a low level of trans-complementation of the 2B-E[40]K mutation by wild-type virus. This is the first report that provides evidence that a genetic defect in the enterovirus 2B protein can be complemented in trans.
van Kuppeveld
Frank J M
FJM
Department of Medical Microbiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands1.
van den Hurk
Patrick J J C
PJJC
Department of Medical Microbiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands1.
Schrama
Ina W J
IWJ
Department of Medical Microbiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands1.
Galama
Jochem M D
JMD
Department of Medical Microbiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands1.
Melchers
Willem J G
WJG
Department of Medical Microbiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands1.
D000818:Animals; D002460:Cell Line; D029822:Enterovirus B, Human / Q000235:genetics* / Q000502:physiology*; D004769:Enterovirus Infections / Q000821:virology; D005816:Genetic Complementation Test; D006801:Humans; D009154:Mutation; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014162:Transfection; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000235:genetics*
D012367:RNA, Viral; D014764:Viral Proteins; C097017:coxsackie B3 virus protein 2B