T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus

• Published in: Journal of general virology Vol. 89; no. 9; pp. 2157 - 2166
• Main Authors: Habjan, Matthias, Penski, Nicola, Spiegel, Martin, Weber, Friedemann
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.09.2008; Society for General Microbiology
• Subjects: Animals; Base Sequence; Biological and medical sciences; Bunyaviridae; Cell Line; Clone Cells; Cricetinae; DNA, Complementary - genetics; DNA, Viral - genetics; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Fundamental and applied biological sciences. Psychology; Humans; Interferons - antagonists & inhibitors; Microbiology; Miscellaneous; Mutation; Phlebovirus; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Rift Valley fever virus; Rift Valley fever virus - genetics; Rift Valley fever virus - pathogenicity; Rift Valley fever virus - physiology; RNA Polymerase I - genetics; RNA Polymerase I - metabolism; RNA Polymerase II - genetics; RNA Polymerase II - metabolism; Transfection; Viral Nonstructural Proteins - genetics; Viral Nonstructural Proteins - physiology; Viral Proteins - genetics; Viral Proteins - metabolism; Virology; Virus Cultivation - methods; Virus Replication; Virus; Bunyaviridae; Complementary DNA; Gene; Microbiology; Sandfly fever group virus; Rift valley fever virus; Phlebovirus
• ISSN: 0022-1317; 1465-2099
• DOI: 10.1099/vir.0.2008/002097-0

Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, D-79008 Freiburg, Germany Correspondence Friedemann Weber friedemann.weber{at}uniklinik-freiburg.de Rift Valley fever virus (RVFV) is responsible for large and recurrent outbreaks of acute febrile illness among humans and domesticated animals in Africa. It belongs to the family Bunyaviridae , genus Phlebovirus , and its negative-stranded RNA genome consists of three segments. Here, we report the establishment and characterization of two different systems to rescue the RVFV wild-type strain ZH548. The first system is based on the BHK-21 cell clone BSR-T7/5, which stably expresses T7 RNA polymerase (T7 pol). Rescue of wild-type RVFV was achieved with three T7 pol-driven cDNA plasmids representing the viral RNA segments in the antigenomic sense. The second system involves 293T cells transfected with three RNA pol I-driven plasmids for the viral segments and two RNA pol II-driven support plasmids to express the viral polymerase components L and N. It is known that the 5' triphosphate group of T7 pol transcripts strongly activates the antiviral interferon system via the intracellular RNA receptor RIG-I. Nonetheless, both the T7 pol and the pol I/II system were of similar efficiency. This was even true for the rescue of a RVFV mutant lacking the interferon antagonist nonstructural proteins. Further experiments demonstrated that the unresponsiveness of BHK-21 and BSR-T7/5 cells to T7 pol transcripts is most probably due to a deficiency in the RIG-I pathway. Our reverse genetics systems now enable us to manipulate the genome of RVFV and study its virulence mechanisms. Moreover, the finding that BHK-derived cell lines have a compromised RIG-I pathway may explain their suitability for propagating and rescuing a wide variety of viruses. Present address: Institut für Virologie, Universitätsmedizin Göttingen, D-37075 Göttingen, Germany. Supplementary material is available with the online version of this paper.