Organization of flaviviral replicase proteins in virus-induced membranes: a role for NS1' in Japanese encephalitis virus RNA synthesis.

The organization of flaviviral replicase proteins within the membrane-bound replication complexes of West Nile (WNV), dengue (DENV) and Japanese encephalitis viruses (JEV) was probed by investigating the combined effect of detergents and trypsin on both viral replicase activity and profile of metabolically labelled viral proteins. While trypsin treatment of virus-induced membrane fractions degraded the vast majority of replicase proteins, viral RNA-dependent RNA polymerase (RdRp) activity remained completely unaffected. Solubilization of the membranes with deoxycholate (DOC) however rendered the replicase accessible to trypsin.

Nuclear localization of flavivirus RNA synthesis in infected cells.

Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus.

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.

Characterization of RNA synthesis, replication mechanism, and in vitro RNA-dependent RNA polymerase activity of Japanese encephalitis virus.

In vitro RNA-dependent RNA polymerase assays revealed that the JEV replication complex (RC) synthesized viral RNA utilizing a semiconservative and asymmetric mechanism. Peak viral replicase activity and levels of viral RNA observed 15-18 h postinfection (h p.i.

The regulatory elements of the Mycobacterium tuberculosis gene Rv3881c function efficiently in Escherichia coli.

We report efficient expression of the Mycobacterium tuberculosis gene Rv3881c in Escherichia coli from its M. tuberculosis promoter, attributable to an E. coli consensus Pribnow box and ribosome binding site.