Comparison of centrifugation methods for molecular and morphological analysis of membranes associated with RNA replication of the flavivirus Kunjin
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Cited by (16)
Chapter 2 New Insights into Flavivirus Nonstructural Protein 5
2009, Advances in Virus ResearchCitation Excerpt :Fractionation of WNV-infected cells to enrich for RdRp activity led to progressive depletion of NS5 from membrane fractions but had little effect on the RdRp activity exhibited by these fractions (Grun and Brinton, 1986, 1987, 1988). Further studies demonstrated that WNVKUN RdRp activity was predominantly associated with cytoplasmic “heavy” membrane fractions which could be sedimented at >16,000g (termed the “16K fraction”) (Chu and Westaway, 1987, 1992; Chu et al., 1992). The heavy membrane fraction was found to be enriched for NS3, NS2A, NS2B, and NS4A.
Spatial and temporal organization of tick-borne encephalitis flavivirus replicated RNA in living cells
2008, VirologyCitation Excerpt :Further relevant pathogens within the family Flaviviridae are hepatitis C virus (genus Hepacivirus) and the pestiviruses (genus Pestivirus). All Flaviviridae share common replication features including also cytoplasmic localization of RNA replication in association with virus-induced membrane structures that co-sediment with RNA-dependent RNA polymerase (RdRp) activity (Chu, Westaway, and Coia, 1992; Grun and Brinton, 1988). Hence, molecular studies exploiting the well-characterizedTBEV replicons may provide a reference for the molecular biology of other important viruses within this group of pathogens.
The non-structural protein 4A of dengue virus is an integral membrane protein inducing membrane alterations in a 2K-regulated manner
2007, Journal of Biological ChemistryCitation Excerpt :The structures are induced by the viral infection and seem to originate from different cellular organelles (for review see Ref. 34). So called convoluted membranes and paracrystalline structures are putative sites of viral polyprotein processing, whereas proliferating ER and vesicles of about 100 nm in diameter (designated smooth membrane structures) may represent the sites of viral RNA replication (10, 35–38). Earlier studies revealed that KUNV NS4A localizes within these virus-induced membranes and interacts with most of the other viral non-structural proteins, including NS3 and NS5 (9).
Subcellular localization and membrane topology of the dengue virus type 2 non-structural protein 4B
2006, Journal of Biological ChemistryCitation Excerpt :This virus induces membrane structures called convoluted membranes and paracrystalline structures, representing the putative sites of viral polyprotein processing. In addition, proliferating ER and vesicles of about 100 nm in diameter described as small, spherical smooth membrane structures that seem to harbor the viral RCs were observed (9, 29, 43-46). It is possible that the cytoplasmic foci observed in our DV studies represent one or several of these membrane structures.
Comparisons of physical separation methods of Kunjin virus-induced membranes
2004, Journal of Virological MethodsArchitecture of the flaviviral replication complex: Protease, nuclease, and detergents reveal encasement within double-layered membrane compartments
2003, Journal of Biological ChemistryCitation Excerpt :Thus, our results obtained by analyses of both the replicase proteins and the viral RNAs are in agreement with the presence of RC within vesicle packets as shown for KUNV (15, 23) and additionally suggest that the CM/PC and VP with its bounding ER form a closed compartment. These membranes are sufficiently heavy to sediment at 16,000 × g (24, 25). The differential solubility of the outer ER-like membranes alone to nonionic detergent should as a result release all of the inner vesicles, which being smaller would no longer be expected to sediment at 16,000 × g. Indeed, treatment of P16 fractions with 1% TX100 followed by sedimentation at 16,000 × g showed that ∼60–80% of labeled viral RNA species remained in the supernatant fraction (Fig. 4C, lanes 2 and 3).
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