Recovery of an arenavirus entirely from RNA polymerase I/II-driven cDNA.
The prototypic arenavirus lymphocytic choriomeningitis virus has been a primary workhorse of viral immunologists for almost a century, and it has served as an important model for studying basic principles of arenavirus molecular biology. Its negative-stranded bisegmented RNA genome has, however, posed a major obstacle to attempts at manipulating the infectious virus by reverse genetic techniques. Here, we report the recovery of infectious lymphocytic choriomeningitis virus (the immunosuppressive strain clone 13) entirely from cDNA. Intracellular transcription of the short and the long viral genome segment from polymerase (pol) I-driven vectors and coexpression of the minimal viral-transacting factors NP and L from pol II-driven plasmids resulted in the efficient formation of infectious virus with genetic tags in both genome segments. The cDNA-derived viruses behaved identically to wild-type virus in both cell culture and infected mice. Importantly, they caused a chronic infection and suppressed the adaptive immune response to an unrelated third-party virus. This technology provides an important basis for investigating viral determinants of persistent infection and immunosuppression. In addition, our findings demonstrate that pol I/II-based vector systems may represent an efficient alternative strategy for the recovery of cytoplasmic negative-strand RNA viruses from cDNA.
Flatz
Lukas
L
Institute of Experimental Immunology, Department of Pathology, University Hospital, Schmelzbergstrasse 12, 8091 Zurich, Switzerland.
Bergthaler
Andreas
A
de la Torre
Juan Carlos
JC
Pinschewer
Daniel D
DD
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D016679:Genome, Viral / Q000235:genetics; D008217:Lymphocytic choriomeningitis virus / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D012318:RNA Polymerase I / Q000737:chemistry*; D012319:RNA Polymerase II / Q000737:chemistry*; D012367:RNA, Viral / Q000096:biosynthesis*
D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral; D012319:RNA Polymerase II; D012318:RNA Polymerase I