Architecture of the flaviviral replication complex. Protease, nuclease, and detergents reveal encasement within double-layered membrane compartments.
Flavivirus infection causes extensive proliferation and reorganization of host cell membranes to form specialized structures called convoluted membranes/paracrystalline arrays and vesicle packets (VP), the latter of which is believed to harbor flaviviral replication complexes. Using detergents and trypsin and micrococcal nuclease, we provide for the first time biochemical evidence for a double membrane compartment that encloses the replicative form (RF) RNA of the three pathogenic flaviviruses West Nile, Japanese encephalitis, and dengue viruses. The bounding membrane enclosing the VP was readily solubilized with nonionic detergents, rendering the catalytic amounts of enzymatically active protein component(s) of the replicase machinery partially sensitive to trypsin but allowing limited access for nucleases only to the vRNA and single-stranded tails of the replicative intermediate RNA. The RF co-sedimented at high speed from nonionic detergent extracts of virus-induced heavy membrane fractions along with the released individual inner membrane vesicles whose size of 75-100 nm as well as association with viral NS3 was revealed by immunoelectron microscopy. Viral RF remained nuclease-resistant even after ionic detergents solubilized the more refractory inner VP membrane. All of the viral RNA species became nuclease-sensitive following membrane disruption only upon prior trypsin treatment, suggesting that proteins coat the viral genomic RNA as well as RF within these membranous sites of flaviviral replication. These results collectively demonstrated that the newly formed viral genomic RNA associated with the VP are oriented outwards, while the RF is located inside the nonionic detergent-resistant vesicles.
Uchil
Pradeep Devappa
PD
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Satchidanandam
Vijaya
V
D000818:Animals; D002460:Cell Line; D003902:Detergents; D010450:Endopeptidases; D005416:Flavivirus / Q000737:chemistry* / Q000502:physiology / Q000648:ultrastructure; D012367:RNA, Viral / Q000737:chemistry* / Q000648:ultrastructure; D012260:Ribonucleases; D014779:Virus Replication
D003902:Detergents; D012367:RNA, Viral; D012260:Ribonucleases; D010450:Endopeptidases