PMID- 12208963
OWN - NLM
STAT- MEDLINE
DCOM- 20020930
LR  - 20190508
IS  - 0022-538X (Print)
IS  - 1098-5514 (Electronic)
IS  - 0022-538X (Linking)
VI  - 76
IP  - 19
DP  - 2002 Oct
TI  - Flock house virus RNA polymerase is a transmembrane protein with amino-terminal 
      sequences sufficient for mitochondrial localization and membrane insertion.
PG  - 9856-67
AB  - Localization of RNA replication to intracellular membranes is a universal feature 
      of positive-strand RNA viruses. Replication complexes of flock house virus (FHV), 
      the best-studied alphanodavirus, are located on outer mitochondrial membranes in 
      infected Drosophila melanogaster cells and are associated with the formation of 
      membrane-bound spherules, similar to structures found for many other 
      positive-strand RNA viruses. To further study FHV replication complex formation, 
      we investigated the subcellular localization, membrane association, and membrane 
      topology of protein A, the FHV RNA-dependent RNA polymerase, in the yeast 
      Saccharomyces cerevisiae, a host able to support full FHV RNA replication and 
      virion formation. Confocal immunofluorescence revealed that protein A localized 
      to mitochondria in yeast, as in Drosophila cells, and that this mitochondrial 
      localization was independent of viral RNA synthesis. Nycodenz gradient flotation 
      and dissociation assays showed that protein A behaved as an integral membrane 
      protein, a finding consistent with a predicted N-proximal transmembrane domain. 
      Protease digestion and selective permeabilization after differential epitope 
      tagging demonstrated that protein A was inserted into the outer mitochondrial 
      membrane with the N terminus in the inner membrane space or matrix and that the C 
      terminus was exposed to the cytoplasm. Flotation and immunofluorescence studies 
      with deletion mutants indicated that the N-proximal region of protein A was 
      important for both membrane association and mitochondrial localization. 
      Gain-of-function studies with green fluorescent protein fusions demonstrated that 
      the N-terminal 46 amino acids of protein A were sufficient for mitochondrial 
      localization and membrane insertion. We conclude that protein A targets and 
      anchors FHV RNA replication complexes to outer mitochondrial membranes, in part 
      through an N-proximal mitochondrial localization signal and transmembrane domain.
FAU - Miller, David J
AU  - Miller DJ
AD  - Department of Medicine. Institute for Molecular Virology, University of 
      Wisconsin-Madison, Madison, Wisconsin 53706, USA.
FAU - Ahlquist, Paul
AU  - Ahlquist P
LA  - eng
GR  - R01 GM035072/GM/NIGMS NIH HHS/United States
GR  - R37 GM035072/GM/NIGMS NIH HHS/United States
GR  - GM35072/GM/NIGMS NIH HHS/United States
GR  - K08 AI01770-01/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Virol
JT  - Journal of virology
JID - 0113724
RN  - 0 (Membrane Proteins)
RN  - 0 (RNA, Viral)
RN  - 0 (Viral Proteins)
RN  - EC 2.7.7.6 (DNA-Directed RNA Polymerases)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - DNA-Directed RNA Polymerases/*chemistry
MH  - Drosophila melanogaster/virology
MH  - Membrane Proteins/*chemistry
MH  - Mitochondria/*chemistry
MH  - Molecular Sequence Data
MH  - Nodaviridae/*enzymology
MH  - RNA, Viral/biosynthesis
MH  - Rabbits
MH  - Saccharomyces cerevisiae/chemistry
MH  - Viral Proteins/*chemistry
PMC - PMC136485
EDAT- 2002/09/05 10:00
MHDA- 2002/10/02 04:00
PMCR- 2002/10/01
CRDT- 2002/09/05 10:00
PHST- 2002/09/05 10:00 [pubmed]
PHST- 2002/10/02 04:00 [medline]
PHST- 2002/09/05 10:00 [entrez]
PHST- 2002/10/01 00:00 [pmc-release]
AID - 0403 [pii]
AID - 10.1128/jvi.76.19.9856-9867.2002 [doi]
PST - ppublish
SO  - J Virol. 2002 Oct;76(19):9856-67. doi: 10.1128/jvi.76.19.9856-9867.2002.