Journal of General Virology (1996), 77, 163-165. Printed in Great Britain
163
Do light-induced pH changes within the chloroplast drive
turnip yellow mosaic virus assembly?
J a n R o h o z i n s k i * and J o h n M . H a n c o c k ~
Director's Unit, Research School o f Biological Sciences, Australian National University, Canberra, A C T 0200,
Australia
Turnip yellow mosaic virus (TYMV) induces gross
morphological and biochemical changes in the chloroplasts of infected cells. Viral RNA is synthesized in
vesicles formed by invagination of the outer chloroplast
bilayer. Virion assembly occurs at the neck of these
vesicles and requires illumination. Data collected over
the last three decades are consistent with the hypothesis
that light-induced generation of a low pH drives TYMV
assembly within the intermembrane space of chloroplasts. In a low-pH environment, poly(C) regions within
the genomic RNA of TYMV may interact to form
tertiary structures, and the recognition of these structures by TYMV coat protein initiates virion assembly.
Introduction
at pH 4.5 to 5.4 (Briand et al., 1975). The ability of
poly(C), but not other polyribonucleotides, to interact
specifically with TYMV coat protein at low pH (Briand
et al., 1975) is evidence that some structure unique to
poly(C) is required for this interaction. In their 1975
paper, J. P. Briand and co-workers predicted the existence of poly(C) sequences within TYMV genomic RNA
based on their observations of TYMV coat protein
interacting with TYMV RNA and poly(C). As the
sequence of TYMV is now available (Morch et al., 1988)
we know that there are 14 poly(C) regions between 5 and
11 residues long dispersed throughout the genome.
Recently, there has been excitement over the discovery
of a telomeric DNA structure which forms spontaneously
at low pH (the i-motif: Gehring et al., 1993 ; Patel, 1993).
This structure consists of two pairs of 2-deoxycytidine
duplexes comprising two poly(dC) strands, running
parallel and held together by protonated cytosine base
pairing. The tetramer is formed by two such duplexes,
running antiparallel and held together by inter-digitation
of the base-pairs forming the duplexes (Gehring et al.,
1993 ; Rohozinski et al., 1994). Poly(dC) strands as short
as three bases long can condense into i-motifs (Leroy et
al., 1993). By extension, this structure may also occur in
RNA at low pH. The protonation of cytosine residues
could possibly drive the spontaneous formation of
tetrameric RNA structures between strands of cytidine
similar to those observed in DNA.
Evidence that poly(C) tracts within TYMV RNA can
potentially form complex, acid-pH-dependent structures
comes from early studies on the biophysical properties of
TYMV RNA (Mitkar & Kaesberg, 1965). These studies
demonstrated the presence of extensive secondary struc-
Viruses belonging to the genus Tymovirus are unique
among plant viruses in that virion assembly is associated
with the development of membrane-bound vesicles
within the chloroplasts of infected cells (Matthews, 1991 ;
Hirth & Givord, 1988). This would suggest that
tymoviruses utilize some physiological or structural
feature of the plant chloroplast to facilitate replication
and virion assembly. It follows that the viral genomic
RNA and its gene products must contain features
capable of utilizing the chloroplast and its metabolic
features. Observations of such features are evident in the
literature and when combined with recent developments
in the understanding of nucleic acid structure and
chloroplast physiology, a clear picture of the interactions
between the virus and its host becomes evident.
Requirement o f low p H and p o l y ( C ) for virus
assembly
In vitro studies showed that turnip yellow mosaic
virus (TYMV) assembly from coat protein and nucleic
acid requires low pH (Briand et al., 1975; Bouley et al.,
1975; Jonard et al., 1972). The interaction between RNA
and coat protein involves specific interaction of the
capsid protein with poly(C), in the presence of spermidine
* Author for correspondence.Fax +61 6 279 8056.
e-mail [email protected]
t Present address: MRC Clinical Sciences Centre, Royal Postgraduate MedicalSchool, London,UK.
0001-3582 © 1996SGM
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164
J. Rohozinski and J. M. H a n c o c k
ture in TYMV RNA at low pH, observed as an increased
sedimentation coefficient as the pH was decreased below
6 (Mitkar & Kaesberg, 1965). The temperature-pH
dependence of TYMV RNA secondary structure (Mitkar
& Kaesberg, 1965) mimics the temperature relationship
between formation of tetrameric structures by poly(dC)
and pH, observed by Leroy et al. (1993).
To date there is no direct evidence that poly(C) can
form an i-motif. Discovery of the i-motif formed by
poly(dC) was a surprise as it had previously been
accepted that, at low pH, poly(dC) formed a duplex with
the sugar-phosphate backbones running parallel (Leroy
et al., 1993; Gehring et al., 1993). In the case of poly(C),
there are X-ray fibre diffraction studies that suggest
poly(C) forms a helical structure (possibly a duplex) with
the strands running parallel (Langridge & Rich, 1963).
Similar studies have also indicated that at low pH,
poly(A) forms a helical structure consisting of a duplex
with the strands running parallel (Rich et al., 1961). This
poly(A) structure is almost identical to the one postulated
to be formed by poly(C) at acid pH (Langridge & Rich,
1963).
As TYMV coat protein does not interact with poly(A)
at low pH in the presence of spermidine (Briand et al.,
1975), it can be concluded that the TYMV coat protein
recognizes a structure more complex than a polyribonucleotide duplex with the strands running parallel. The
possibility of poly(C) forming a higher order structure at
low pH in the presence of a polyamine cannot be
dismissed. Cations are known to stabilize higher order,
acid-pH-dependent triple-stranded RNA structures
(Klinck et al., 1994) and polyamines can induce
conformational changes when bound to nucleic acids
(Manning, 1978). Our argument is reinforced by the
observation that chemical modification of C residues
within TYMV RNA results in loss of binding by coat
protein (Bouley et al., 1975).
It is the formation of higher level tertiary structures at
low pH (possibly the unique i-motif) by RNA containing
poly(C) regions that could explain the specific binding of
TYMV coat protein to poly(C) or TYMV RNA.
The role of light-induced low pH in Tymovirus
assembly in vivo
Using TYMV-infected Brassica protoplasts Renaudin et
al. (19.76) demonstrated that virion assembly requires
illumination, whereas light is not required for viral RNA
replication (Boyer et al., 1993) or, presumably, protein
synthesis. During photosynthesis protons are pumped
into the lumen of the grana and stroma lamellae (Dilley
et al., 1987), which is connected to the space between the
inner and outer chloroplast membranes (Heber & Heldt,
1981). This yields a light-induced pH gradient between
the lumen (which is contiguous with both the grana and
stroma lamellae) and outer chloroplast stroma (Heber &
Krause, 1971). This pH gradient is normally used for
ATP synthesis in the illuminated chloroplast. The pH
within the lumen can be as low as pH 4.5.
TYMV RNA replicates within invaginations of the
outer lipid bilayer of chloroplasts, and virion assembly is
probably initiated at the necks of these vesicles
(Matthews, 1991). Thus, the replication and assembly
sites may be dictated by the requirement for a low-pH
environment for virion assembly. If the formation of an
RNA tetrameric structure induces virion assembly, the
virus must utilize a site where the generation of a low pH
is possible. Thus the position of the TYMV assembly site
within the cell is clearly consistent with the hypothesis,
and previously cited literature, that low pH is required
for RNA tetramer formation and subsequent assembly
into virions.
There has been a report of TYMV assembly in
etiolated cabbage hypocotyls (Feranandez-Gonzalez et
al., 1980), which do not contain chlorophyll. Nonphotosynthetic chloroplasts could potentially support
assembly of TYMV by generating a low pH in the
intermembrane space with NADPH dehydrogenase. This
is a chloroplast-encoded complex homologous to mitochondrial NADH dehydrogenase (Complex I) (Dyer et
al., 1993), the function of which is yet to be elucidated.
In mitochondria, the NADH dehydrogenase complex
generates a low pH in the mitochondrial intermembrane
space and thus drives ATP synthesis (Douce &
Neuburger, 1989). The chloroplast NADPH dehydrogenase may serve an analogous role in developing
chloroplasts, generating a low intermembrane pH for
ATP synthesis within the developing plastid. NADPH
dehydrogenase could thus generate the low pH necessary
to assemble TYMV in plastids that do not contain
chlorophyll.
Discussion
The currently accepted model that Matthews (1991) has
proposed for TYMV assembly in vivo suggests that viral
coat protein becomes inserted into the outer chloroplast
membrane in an oriented fashion, exposing part of the
molecule to the intermembrane space. This model can be
extended so that the viral RNA synthesized within the
vesicle moves into the intermembrane space or neck of
the vesicle where, under conditions of light-induced low
pH, the poly(C) regions form tetrameric structures,
associate with coat protein and initiate virion assembly.
Interaction between i-motifs and coat protein in vivo
could form a stable aggregate consisting of the viral
RNA and a pentamer or hexamer of coat protein, such
as those observed in vivo (Matthews & Witz, 1985), or by
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Is T Y M V assembly chloroplast-pH-dependent?
high pH dissociation of virus in vitro (Keeling et al.,
1979). This stable structure could initiate virion assembly
in vivo. The possibility of a low pH occurring throughout
the whole vesicle cannot be overlooked and in the case of
Poinsettia mosaic virus (PoiMV), a relative of TYMV,
the replicative vesicles are bound by the inner chloroplast
membrane alone, so that PoiMV replication effectively
takes place in the intermembrane space (Lesemann et al.,
1983).
The model proposed here is consistent with all the
known physical and chemical interactions between
TYMV coat protein and viral RNA. It explains the
poly(C) regions within the viral RNA, the reason why
TYMV replicates in chloroplast structures, and why light
is required for virion assembly in photosynthetically
competent tissue.
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