title: The 5' untranslated region of a novel infectious molecular clone of the dicistrovirus cricket paralysis virus modulates infection.
abstract: Dicistroviridae are a family of RNA viruses that possesses a single-stranded positive-sense RNA genome containing two distinct open reading frames (ORFs), each preceded by an internal ribosome entry site that drives translation of the viral structural and nonstructural proteins, respectively. The type species, Cricket paralysis virus (CrPV), has served as a model for studying host-virus interactions; however, investigations into the molecular mechanisms of CrPV and other dicistroviruses have been limited as an established infectious clone was elusive. Here, we report the construction of an infectious molecular clone of CrPV. Transfection of in vitro-transcribed RNA from the CrPV clone into Drosophila Schneider line 2 (S2) cells resulted in cytopathic effects, viral RNA accumulation, detection of negative-sense viral RNA, and expression of viral proteins. Transmission electron microscopy, viral titers, and immunofluorescence-coupled transwell assays demonstrated that infectious viral particles are released from transfected cells. In contrast, mutant clones containing stop codons in either ORF decreased virus infectivity. Injection of adult Drosophila flies with virus derived from CrPV clones but not UV-inactivated clones resulted in mortality. Molecular analysis of the CrPV clone revealed a 196-nucleotide duplication within its 5' untranslated region (UTR) that stimulated translation of reporter constructs. In cells infected with the CrPV clone, the duplication inhibited viral infectivity yet did not affect viral translation or RNA accumulation, suggesting an effect on viral packaging or entry. The generation of the CrPV infectious clone provides a powerful tool for investigating the viral life cycle and pathogenesis of dicistroviruses and may further understanding of fundamental host-virus interactions in insect cells.
  
  
  OBJECTIVE
  Dicistroviridae, which are RNA viruses that infect arthropods, have served as a model to gain insights into fundamental host-virus interactions in insect cells. Further insights into the viral molecular mechanisms are hampered due to a lack of an established infectious clone. We report the construction of the first infectious clone of the dicistrovirus, cricket paralysis virus (CrPV). We show that transfection of the CrPV clone RNA into Drosophila cells led to production of infectious particles that resemble natural CrPV virions and result in cytopathic effects and expression of CrPV proteins and RNA in infected cells. The CrPV clone should provide insights into the dicistrovirus life cycle and host-virus interactions in insect cells. Using this clone, we find that a 196-nucleotide duplication within the 5' untranslated region of the CrPV clone increased viral translation in reporter constructs but decreased virus infectivity, thus revealing a balance that interplays between viral translation and replication.
authors: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada Centre for High-Throughput Biology, University of British Columbia, Vancouver, British Columbia, Canada.
authors: Craig H
authors: CH
authors: Kerr
authors: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
authors: Qing S
authors: QS
authors: Wang
authors: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
authors: Kathleen
authors: K
authors: Keatings
authors: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
authors: Anthony
authors: A
authors: Khong
authors: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
authors: Douglas
authors: D
authors: Allan
authors: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
authors: Calvin K
authors: CK
authors: Yip
authors: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada Centre for High-Throughput Biology, University of British Columbia, Vancouver, British Columbia, Canada foster@chibi.ubc.ca ej@mail.ubc.ca.
authors: Leonard J
authors: LJ
authors: Foster
authors: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada foster@chibi.ubc.ca ej@mail.ubc.ca.
authors: Eric
authors: E
authors: Jan
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003588:Cytopathogenic Effect, Viral; D056629:Dicistroviridae / Q000235:genetics* / Q000502:physiology; D004330:Drosophila; D046529:Microscopy, Electron, Transmission; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000235:genetics* / Q000502:physiology; D016019:Survival Analysis; D014158:Transcription, Genetic; D014162:Transfection; D019562:Viral Load; D014771:Virion / Q000648:ultrastructure; D014779:Virus Replication
chemical_list: D020121:5' Untranslated Regions; D012367:RNA, Viral