Ja. Den Boon et al., Identification of sequences in brome mosaic virus replicase protein 1a that mediate association with endoplasmic reticulum membranes, J VIROLOGY, 75(24), 2001, pp. 12370-12381
RNA replication of all positive-strand RNA viruses is closely associated wi
th intracellular membranes. Brome mosaic virus (BMV) RNA replication occurs
on the perinuclear region of the endoplasmic reticulum (ER), both in its n
atural plant host and in the yeast Saccharomyces cerevisiae. The only viral
component in the BMV RNA replication complex that localizes independently
to the ER is la, a multifunctional protein with an N-terminal RNA capping d
omain and a C-terminal helicase-like domain. The other viral replication co
mponents, the RNA polymerase-like protein 2a and the RNA template, depend o
n la for recruitment to the ER. We show here that, in membrane extracts, la
is fully susceptible to proteolytic digestion in the absence of detergent
and thus, a finding consistent with its roles in RNA replication, is wholly
or predominantly on the cytoplasmic face of the ER with no detectable lume
nal protrusions. Nevertheless, la association with membranes is resistant t
o high-salt and high-pH treatments that release most peripheral membrane pr
oteins. Membrane flotation gradient analysis of la deletion variants and la
segments fused to green fluorescent protein (GFP) showed that sequences in
the N-terminal RNA capping module of la mediate membrane association. In p
articular, a region C-terminal to the core methyltransferase homology was s
ufficient for high-affinity ER membrane association. Confocal immunofluores
cence microscopy showed that even though these determinants mediate ER loca
lization, they fail to localize GFP to the narrow region of the perinuclear
ER, where full-length ta normally resides. Instead, they mediate a more gl
obular or convoluted distribution of ER markers. Thus, additional sequences
in la that are distinct from the primary membrane association determinants
contribute to la's normal subcellular distribution, possibly through effec
ts on la conformation, orientation, or multimerization on the membrane.