title: A virus-like particle system identifies the endonuclease domain of Crimean-Congo hemorrhagic fever virus.
abstract: Crimean-Congo hemorrhagic fever virus (CCHFV; genus Nairovirus) is an extremely pathogenic member of the Bunyaviridae family. Since handling of the virus requires a biosafety level 4 (BSL-4) facility, little is known about pathomechanisms and host interactions. Here, we describe the establishment of a transcriptionally competent virus-like particle (tc-VLP) system for CCHFV. Recombinant polymerase (L), nucleocapsid protein (N) and a reporter minigenome expressed in human HuH-7 cells resulted in formation of transcriptionally active nucleocapsids that could be packaged by coexpressed CCHFV glycoproteins into tc-VLPs. The tc-VLPs resembled authentic virus particles in their protein composition and neutralization sensitivity to anti-CCHFV antibodies and could recapitulate all steps of the viral replication cycle. Particle attachment, entry, and primary transcription were modeled by infection of naive cells. The subsequent steps of genome replication, secondary transcription, and particle assembly and release can be obtained upon passaging the tc-VLPs on cells expressing CCHFV structural proteins. The utility of the VLP system was demonstrated by showing that the endonuclease domain of L is located around amino acid D693, as was predicted in silico by B. Morin et al. (PLoS Pathog 6:e1001038, 2010, http://dx.doi.org/10.1371/journal.ppat.1001038). The tc-VLP system will greatly facilitate studies and diagnostics of CCHFV under non-BSL-4 conditions.
  
  
  OBJECTIVE
  Crimean-Congo hemorrhagic fever virus (CCHFV) is an extremely virulent pathogen of humans. Since the virus can be handled only at the highest biosafety level, research is restricted to a few specialized laboratories. We developed a plasmid-based system to produce virus-like particles with the ability to infect cells and transcribe a reporter genome. Due to the absence of viral genes, the virus-like particles are unable to spread or cause disease, thus allowing study of aspects of CCHFV biology under relaxed biosafety conditions.
authors: Institute for Virology, Philipps University Marburg, Marburg, Germany.
authors: Stephanie
authors: S
authors: Devignot
authors: Special Pathogens Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors: Eric
authors: E
authors: Bergeron
authors: Special Pathogens Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors: Stuart
authors: S
authors: Nichol
authors: Public Health Agency of Sweden, Stockholm, Sweden National Veterinary Institute, Uppsala, Sweden Department of Clinical Microbiology, LABMED, Karolinska Institute, Stockholm, Sweden.
authors: Ali
authors: A
authors: Mirazimi
authors: Institute for Virology, Philipps University Marburg, Marburg, Germany friedemann.weber@vetmed.uni-giessen.de.
authors: Friedemann
authors: http://orcid.org/0000-0001-9737-337X
authors: F
authors: Weber
mesh_terms: D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D003225:Hemorrhagic Fever Virus, Crimean-Congo / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D019590:Nucleocapsid Proteins / Q000235:genetics; D017434:Protein Structure, Tertiary; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D022701:Virosomes / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly; D053585:Virus Attachment; D053586:Virus Internalization
chemical_list: D019590:Nucleocapsid Proteins; D014764:Viral Proteins; D022701:Virosomes; D012321:DNA-Directed RNA Polymerases; D004720:Endonucleases