Recovery of Recombinant Crimean Congo Hemorrhagic Fever Virus Reveals a Function for Non-structural Glycoproteins Cleavage by Furin

Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family Bunyaviridae (genus: Nairovirus). In humans, CCHFV causes fever, hemorrhage, severe thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV's high pathogenicity...

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Bibliographic Details
Published in:PLoS pathogens 2015-05, Vol.11 (5), p.e1004879-e1004879
Main Authors: Bergeron, Éric, Zivcec, Marko, Chakrabarti, Ayan K, Nichol, Stuart T, Albariño, César G, Spiropoulou, Christina F
Format: Article
Language:English
Subjects:
Amino Acid Motifs
Amino Acid Substitution
Animals
Cell Line
Cercopithecus aethiops
Clone Cells
Cricetulus
Crimean hemorrhagic fever
Experiments
Fatalities
Fever
Furin - genetics
Furin - metabolism
Genomes
Glycoproteins
Glycoproteins - chemistry
Glycoproteins - genetics
Glycoproteins - metabolism
Health aspects
Hemorrhagic Fever Virus, Crimean-Congo - genetics
Hemorrhagic Fever Virus, Crimean-Congo - metabolism
Hepatocytes - enzymology
Hepatocytes - metabolism
Hepatocytes - virology
Host-virus relationships
Humans
Identification and classification
Mesocricetus
Mutation
Peptide Fragments - genetics
Peptide Fragments - metabolism
Physiological aspects
Plasmids
Proprotein Convertases - metabolism
Proteolysis
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
RNA - metabolism
RNA viruses
Serine Endopeptidases - metabolism
Studies
Substrate Specificity
Viral infections
Viral Nonstructural Proteins - chemistry
Viral Nonstructural Proteins - genetics
Viral Nonstructural Proteins - metabolism
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Summary:Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family Bunyaviridae (genus: Nairovirus). In humans, CCHFV causes fever, hemorrhage, severe thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV's high pathogenicity has been the lack of methodology to produce recombinant CCHFV. We developed a reverse genetics system based on transfecting plasmids into BSR-T7/5 and Huh7 cells. In our system, bacteriophage T7 RNA polymerase produced complementary RNA copies of the viral S, M, and L segments that were encapsidated with the support, in trans, of CCHFV nucleoprotein and L polymerase. The system was optimized to systematically recover high yields of infectious CCHFV. Additionally, we tested the ability of the system to produce specifically designed CCHFV mutants. The M segment encodes a polyprotein that is processed by host proprotein convertases (PCs), including the site-1 protease (S1P) and furin-like PCs. S1P and furin cleavages are necessary for producing the non-structural glycoprotein GP38, while S1P cleavage yields structural Gn. We studied the role of furin cleavage by rescuing a recombinant CCHFV encoding a virus glycoprotein precursor lacking a functional furin cleavage motif (RSKR mutated to ASKA). The ASKA mutation blocked glycoprotein precursor's maturation to GP38, and Gn precursor's maturation to Gn was slightly diminished. Furin cleavage was not essential for replication, as blocking furin cleavage resulted only in transient reduction of CCHFV titers, suggesting that either GP38 and/or decreased Gn maturation accounted for the reduced virion production. Our data demonstrate that nairoviruses can be produced by reverse genetics, and the utility of our system uncovered a function for furin cleavage. This viral rescue system could be further used to study the CCHFV replication cycle and facilitate the development of efficacious vaccines to counter this biological and public health threat.
ISSN:1553-7374
1553-7366
1553-7374
DOI:10.1371/journal.ppat.1004879