Flock house virus RNA replicates on outer mitochondrial membranes in Drosophila cells

被引:177
作者
Miller, DJ
Schwartz, MD
Ahlquist, P
机构
[1] Univ Wisconsin, Inst Mol Virol, Madison, WI 53706 USA
[2] Univ Wisconsin, Dept Med, Madison, WI 53706 USA
[3] Univ Wisconsin, Howard Hughes Med Inst, Madison, WI 53706 USA
关键词
D O I
10.1128/JVI.75.23.11664-11676.2001
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection of Drosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses. Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40- to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombus-virus- and togavirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.
引用
收藏
页码:11664 / 11676
页数:13
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