Download Packaging and structural phenotype of brome mosaic virus capsid

Virology 419 (2011) 17–23
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Virology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y v i r o
Packaging and structural phenotype of brome mosaic virus capsid protein with
altered N-terminal β-hexamer structure
Melissanne de Wispelaere a, Sonali Chaturvedi a, Stephan Wilkens b, A.L.N. Rao a,⁎
a
b
Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521-0122, USA
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
a r t i c l e
i n f o
Article history:
Received 19 June 2011
Returned to author for revision 19 July 2011
Accepted 27 July 2011
Available online 23 August 2011
Keywords:
BMV
Genome packaging
Virus assembly
a b s t r a c t
The first 45 amino acid region of brome mosaic virus (BMV) capsid protein (CP) contains RNA binding and
structural domains that are implicated in the assembly of infectious virions. One such important structural
domain encompassing amino acids 28QPVIV32, highly conserved between BMV and cowpea chlorotic mottle
virus (CCMV), exhibits a β-hexamer structure. In this study we report that alteration of the β-hexamer
structure by mutating 28QPVIV 32 to 28AAAAA 32 had no effect either on symptom phenotype, local and
systemic movement in Chenopodium quinoa and RNA profile of in vivo assembled virions. However, sensitivity
to RNase and assembly phenotypes distinguished virions assembled with CP subunits having β-hexamer from
those of wild type. A comparison of 3-D models obtained by cryo electron microscopy revealed overall similar
structural features for wild type and mutant virions, with small but significant differences near the 3-fold axes
of symmetry.
© 2011 Elsevier Inc. All rights reserved.
Introduction
Brome mosaic virus (BMV) is a small RNA plant virus that belongs to
the genus Bromovirus in the family Bromoviridae (Rao, 2001). The
genome of BMV is divided among three single-stranded, messengersense RNAs. Non-structural proteins 1a and 2a, which are encoded
respectively by genomic RNA1 (B1) and 2 (B2), respectively, along with
host proteins constitute the viral replicase (Kao and Sivakumaran, 2000;
Rao, 2001). Genomic RNA3 (B3) is dicistronic and the 5′ ORF encodes
another non-structural movement protein (MP) that promotes cell-tocell spread (Mise et al., 1993). Capsid protein gene (CP) is encoded by
the 3′ ORF and is expressed from a subgenomic RNA (sgB4) that is
synthesized from progeny (−) B3 by internal initiation mechanism
(Miller et al., 1986). Physical and biochemical characterization of BMV
virions suggested that B1 and B2 are packaged independently into two
separate virions whereas B3 and sgB4 are co-packaged into a third virion
(Rao, 2006).
The crystal structure of BMV virion has been determined to a 3.4 Å
resolution revealing a T = 3 icosahedron composed of 180 identical
subunits of a single 19.4-kDa protein (Lucas et al., 2002). In the BMV
virion, amino acid residues 41–189 form the pentameric capsid A
subunits, and residues 25–189 and 1–189 for the B and C subunits
respectively, compose the hexameric capsomeres (Lucas et al., 2002).
Because bromovirus virions are predominately stabilized by RNA–
protein interactions (Rao, 2006), RNA is a prerequisite component for
⁎ Corresponding author. Fax: + 1 951 827 4294.
E-mail address: [email protected] (A.L.N. Rao).
0042-6822/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2011.07.016
the formation of icosahedral capsids in vivo without which no virions are
assembled.
Interaction between amino and carboxyl termini is essential for the
formation of CP dimers, the building blocks for bromovirus assembly
(Zlotnick et al., 2000). Consequently removal of amino acid residues 1–49
and/or 177–189 from amino and carboxyl termini, respectively,
eliminates contact between the two termini and abolishes virion
assembly (Choi et al., 2000; Sacher and Ahlquist, 1989; Zhao et al.,
1995). The first 25 N-terminal amino acids of BMV CP contain an
arginine-rich motif (ARM) and are poorly ordered in the electron density
map, while the remainder of the CP is highly structured (Lucas et al.,
2002; Speir et al., 1995). We have previously engineered a wide range of
deletion and point mutations encompassing the N-terminal 1–50 amino
acid region and characterized their effect on symptom expression, cellto-cell and long distance movement and RNA packaging phenotype in
vitro and in vivo (Choi et al., 2000; Choi and Rao, 2000). These studies
identified two N-proximal motifs, one located between positions 9–19
(i.e. ARM) and the other between positions 39–48 (RNA binding motif),
that play a crucial role in directing RNA packaging into virions.
In addition to ARM, another structure referred to as β-hexamer
(Fig. 1A)was found in the CP of BMV (Lucas et al., 2002) and CCMV
(Speir et al., 1995). It is defined by the highly conserved 28QPVIV 32
domain in BMV ( 29QPVIV 33 in CCMV; Willits et al., 2003) and is
important for stabilization of the hexameric capsomers during the
assembly process (Speir et al., 1995). Disruption of β-hexamer
structure in CCMV CP modulated symptom expression in cowpea
but there was no effect on the virion assembly from recombinant
proteins (Willits et al., 2003). Here we report that, when an identical
β-hexamer structure was altered in BMV CP, its effect on biological,
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M. de Wispelaere et al. / Virology 419 (2011) 17–23
Fig. 1. Structure of BMV CP. (A) Structure of BMV CP at the icosahedral three-fold axis showing the location of β-hexamer (magenta); (B) Characteristics of T-DNA plasmids used for
Agrobacterium-mediated transient expression in plants. Arrows at the 5′ end represent the location of double 35S promoter (2×35S) whereas squares labeled R and T respectively
denote ribozyme sequence cassette derived from satellite tobacco ring spot virus and the 35S-polyadenylation terminator signals. Bent arrow at the 3′ end represents ribozyme
cleavage site. In B3/β-sub, the boxed region denotes substitution of 28AAAAA32 for 28QPVIV32. In B3/CPKO, methionine start codons located at positions 1 and 9 (italisized in wt) are
mutated to prevent CP translation; (C) Characteristics T-DNA plasmids used for transient ectopic expression of either wt BMV CP (BCPTrans) or β-subTrans. TEV, translation enhancer
sequence from tobacco etch potyvirus.
packaging in vivo and in vitro and virion structural properties were
distinct from those reported for CCMV. Furthermore, this study
determined the 3-D structural characteristics of wild type (wt) and
mutant virions assembled in vivo as well as wt virions assembled from
ectopically expressed CP.
Results and discussion
Biological activity of BMV CP lacking N-proximal β-hexamer structure
Despite conservation in sequence, previous mutational analysis of Nproximal amino acid regions of BMV and CCMV displayed differential
effects on symptom expression and packaging phenotypes (Choi et al.,
2000; Choi and Rao, 2000; Rao, 1997; Rao and Grantham, 1995; Willits
et al., 2003). Thus, to verify whether alteration of amino acids
encompassing the β-hexamer structure would display biological,
packaging and structural phenotypes similar to CCMV (Willits et al.,
2003), N-proximal β-hexamer structure in BMV CP was disrupted by
substituting 28AAAAA32 residues for 28QPVIV32 in a biologically active
agroconstruct of BMV RNA3 (referred to as B3/β-sub in Fig. 1B).
Agroconstructs of wt B1 and B2 (Annamalai and Rao, 2005b) were
coinfiltrated with B3/β-sub into N. benthamiana. Plants infiltrated
with all three BMV wt RNA constructs served as controls. Virions were
purified from the agroinfiltrated leaves and analyzed by TEM and the
levels of BMV replication in total RNAs and the packaging efficiency of
the virions was assessed by Northern blot hybridization. The physical
appearance of purified virions (Fig. 2A), the replication and packaging
profiles of β-sub were indistinguishable from that of wt virus (Fig. 2B).
Since it was shown that mutation of the β-hexamer in CCMV affected
symptom development (Willits et al., 2003), we decided to analyze
the symptomatology of the β-sub virus. As expected N. benthamiana
plants remained symptomless when infected with either wt or β-sub
viruses (data not shown). BMV is known to induce significant visible
symptoms when inoculated to C. quinoa plants (Rao and Grantham,
1995). Consequently, we inoculated equal amounts of virions purified
from the N.benthamiana leaves (Fig. 2) to C.quinoa plants. Throughout
the examination period (7–20 days post infiltration), both the wt and
β-sub viruses induced chlorotic local lesions on inoculated leaves and
produced systemic mottling symptoms in C.quinoa plants (Table 1).
Properties of β-sub virions and CP
In BMV and other positive-strand RNA viruses, packaging is functionally coupled to replication (Annamalai and Rao, 2006; Annamalai
et al., 2008; Nugent et al., 1999). Thus to verify whether packaging
phenotype exhibited by the B3/β-sub mutant requires replication
derived CP, B1 + B2 + B3/CPKO (defective in CP translation due
engineered mutations; Fig. 1B) was trans complemented ectopically
with either BCPTrans or BCP/β-subTrans (Fig. 1C). As controls, plants were
ectopically expressed with only with BCPTrans or BCP/β-subTrans inocula.
We characterized virions purified from each inoculum with respect to
Fig. 2. Characteristic features of β-sub virions. (A) TEM images of negatively stained
purified virions from leaves infiltrated with B1 + B2 and either B3 (wt) or B3/β-sub.
Bar = 50 nm. (B) Northern blot analysis of total and virion RNA of indicated samples.
Approximately 10 μg of total RNA or 0.5 μg virion RNA was denatured with
formamide/formaldehyde and subjected to 1.2% agarose electrophoresis prior to
vacuum blotting to a nylon membrane. The blot was then hybridized with 32P-labeled
riboprobes complementary to homologous 3′ TLS present on all four BMV RNAs. The
position of all four BMV RNAs is shown to the left.
M. de Wispelaere et al. / Virology 419 (2011) 17–23
19
Table 1
In vivo and in vitro properties of BMV wild-type and β-sub mutant.
Inoculum
Sequence alteration
Wild type
B3/β-sub
BCPTrans
BCP//β-sub
Δ7aae
28
a
b
c
d
e
QPVIV
QPVIV32 to 28AAAAA32
QPVIV32
28
QPVIV32 to 28AAAAA32
41
KAIKAIA47 deleted
28
28
Trans
32
Symptom phenotype
on C. quinoaa
In vivo
assemblyb
RNAse
sensitivityc
In vitro assembly at pHd
5.8
7.0
CLL, SM
CLL, SM
NT
NT
CLL, No systemic infection
+
+
+
+
+
Resistant
Sensitive
Resistant
Sensitive
Sensitive
+++
−
+++
−
−
+++
+
+++
+
−
Symptom phenotype was examined on C. qunoa; CLL, chlorotic local lesions; NLL, Necrotic local lesions; SM, Systemic mottling; NT, Not tested.
Evaluated by TEM followed by Northern blot hybridization (Figs. 3, 5 and 6).
Evaluated by testing the sensitivity of purified virions to RNAse treatment (Fig. 5).
Evaluated by EM examination; +++, Efficient assembly/packaging of BMV RNAs; +, inefficient assembly/packaging; −, no assembly/packaging.
Data from Calhoun et al. (2007).
physical appaernce by TEM, electrophoretic mobility by agarose gel
electrophoresis and CP by SDS–PAGE (Fig. 3).
Yield of virions obtained from plants infiltrated with either B1 + B2 +
B3/CPKO + BCP Trans or B1 + B2 + B3/CPKO + BCP/β-sub Trans were
significantly lower than wt control and B1 + B2 + B3/β-sub inocula.
TEM analysis of each virion preparation revealed the presence of and
particles with T = 1 or T = 2 symmetry (Figs. 3A–F). Previous work
from our laboratory has shown that expression of BCP Trans results in
the formation of differently sized BMV particles (Annamalai and Rao,
2005b). The appearance of virions formed with BCP/β-subTrans subunits
was however similar to the those formed with the wild-type BCPTrans
subunits (Figs. 3C–F). Likewise the mobility pattern of virions analyzed
by agarose gel electrophoresis was also indistinguishable (Fig. 3G).
However analysis of CP by SDS–PAGE revealed unusual but interesting
features. CP of B3/β-sub expressed from replication-derived mRNA (i.e.
B1 + B2 + B3/β-sub) displayed profiles identical to wt and were in
monomeric and dimeric forms (Fig. 3H, left panel). Likewise SDS–PAGE
analysis of CP from either ectopic expression of BCPTrans either in the
presence of replication (i.e. B1 + B2 + B3.CPKO+ BCP Trans; Fig. 3H,
middle panel) or in its absence (as in autonomous ectopic expression
of BCPTrans, Fig. 3H, right panel) also displayed release of monomeric and
dimeric forms. By contrast, ectopic expression of β-sub mutant CP either
in the presence of replication (as in B1 + B2 + B3/CPKO + BCP/βsubTrans; Fig. 3H, middle panel) or in its absence (Fig. 3H, right panel)
failed to release dimers. This suggests that, when expressed via
replication, CP subunits of βsub are more likely to interact and form
dimers than those expressed ectopically. It is also likely that an
interaction between CP and replicase might regulate the dimerization
of CP subunits. Alternatively, maintenance of β-hexamer structure could
be important in optimizing inter subunit interactions (Pappachan et al.,
2008; Satheshkumar et al., 2005).
Stability of β-sub virions
CP profiles shown above prompted us to verify the stability of
virions assembled with CP subunits having β-sub mutation. Therefore,
Fig. 3. Virion properties. (A) TEM images of negatively stained partially purified virions from leaves infiltrated with B1 + B2 and either (A) B3 (wt) or (B) B3/β-sub or (C) B3/CPKO +
BCPTrans or (D) B3/CPKO + BCP/β-subTrans. TEM images shown in panels E and F respectively represent virions from leaves ectopically expressing BCPTrans and BCP/β-subTrans.
(G) Electrophoretic mobility profiles of virions from indicated inocula. Purified virions of were electrophoresed in a 1% agarose/TAE gel containing EtBr. (H) Western blot analysis of
CP. Leaf tissue infiltrated with indicated inocula were prepared in SDS–PAGE sample buffer, denatured by boiling and subjected to 16% SDS–PAGE. After transferring the fractionated
proteins to a nitrocellulose membrane, the blot was probed with antibodies specific for BMV CP. Note the absence of CP dimers (2×) in β-subTrans preparations shown in the middle
and left panels.
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M. de Wispelaere et al. / Virology 419 (2011) 17–23
purified virions of wt, B3/β-sub and B1 + B2 + B3/CPKO + β-sub Trans
were subjected to RNAse treatment. TEM and Northern blot
hybridization respectively assessed the effect of this treatment on
virion morphology and packaged RNA. Results are summarized in
Figs. 4 and 5. RNAse treatment had no visible effect on either the
virion morphology (Fig. 4A) or the encapsidated RNA of control wt
BMV preparations (Fig. 5). Interestingly, RNAse treatment did not
affect the virion morphology of B3/β-sub mutant virions (Fig. 4B) but
lead to degradation of the encapsidated RNA (Fig. 5). Likewise, RNase
treatment of virions assembled with BCP Trans (B1 + B2 + B3/CPKO +
BCP Trans) did not affect their morphology (Fig. 4C) or their contents
(Fig. 5), although virions assembled with BCP/β-sub Trans (B1 + B2 +
B3/CPKO + BCP/β-sub Trans) were susceptible to RNAse treatment
(Figs. 4D, and 5). These observations suggested that virions assembled
with CP subunits harboring β-sub mutation are unstable.
CP subunits having β-sub mutation failed to assemble in vitro
A previous study reported that some CP mutants of BMV (e.g.
Δ7aa) despite efficient packaging in vivo, are assembly defective in
vitro (Calhoun et al., 2007). Thus to verify the in vitro packaging
competence of CP subunits of β-sub mutant in the presence and
absence of RNA, in vitro reassembly assays were performed under two
buffer conditions: Reassembly with buffer A (50 mM NaOAc, pH 4.8,
0.1 M NaCl, 0.2 mM PMSF) would result in the assembly of both empty
and RNA containing virions whereas buffer B (50 mM Tris–HCl, pH
7.2, 50 mM NaCl, 10 mM KCl, 5 mM MgCl2, and 1 mM DTT) would
result in reassembly only RNA containing virions. Results are
summarized in Table 1 and Fig. 6. Irrespective of the buffers used,
efficient in vitro assembly was observed with wt CP subunits (Table 1,
Fig. 6). However, those having β-sub mutation failed to assemble in
vitro into empty particles (Table 1) and inefficiently encapsidated RNA
(Fig. 6). These observations suggest that CP subunits of β-sub mutant
are competent to assemble RNA containing virions in vivo but not in
vitro. These in vitro assembly phenotypes contrast with those reported
for CCMV. For example, under low salt conditions without RNA, CCMV
CP subunits lacking β-hexamer assembled into heterogeneous
mixture of assembly products including T = 3 virions and at pH 7.0
in the presence of viral RNA aberrant assembly products such as tubes
and double-shelled particles were observed (Willits et al., 2003).
Therefore, unlike CCMV, β-hexamer structure plays an important role
in the assembly of BMV in vitro and in vivo. In this respect β-hexamer
Fig. 4. RNase sensitivity of purified virions. (A–D) TEM images of virions purified from
indicated samples were treated with RNase prior to imaging. Approximately 50–
100 μg/ml of purified virions were treated with 1 μg/μl of RNase A for 30 min at 30 °C.
Bar = 50 nm.
Fig. 5. RNA analysis. Following RNase treatment as described under Fig. 4 legend, RNA
was extracted from indicated samples and subjected to Northern blot hybridization as
described under Fig. 2 legend. +, RNase treated; −, untreated. The position of all four
BMV RNAs is shown to the left.
is identical to another BMV CP Δ7aa characterized by lacking 7 amino
acids encompassing an RNA binding domain (Calhoun et al., 2007).
The question that needs to be discussed would be: Why BMV CP
subunits lacking β-hexamer failed to assemble in vitro and what
mechanism regulates their assembly in vivo? As suggested for Δ7aa
(Calhoun et al., 2007), we speculate that during in vivo assembly,
either n host factor or viral replicase could be interacting with mutant
CP subunits to induce a structural alteration such as “molecular
switch” to restore and maintain an optimal conformation amenable
for assembly. This perhaps explains why CP subunits lacking βhexamer are incompetent for assembly in vitro.
Structural analysis of virions lacking β-hexamer by cryo electron
microscopy
The structure of wt and mutant virions was analyzed by cryo electron
microscopy and three-dimensional (3-D) reconstructions were calculated at a resolution of 18 Å (Fig. 7). A comparison of the 3-D models for
wt and mutant virions revealed overall similar features with respect to
the characteristic icosahedral capsid structure and the density packed
against the inside of the capsid most likely representing viral RNA.
However, the difference density calculated by subtracting the mutant
virion from the wild type model revealed a significant positive density
near the icosahedral 3-fold axes of symmetry (see arrow in Fig. 7K). A fit
of the BMV crystal structure (1js9) into the EM density shows that this
Fig. 6. In vitro assembly. CP subunits were isolated from purified virions of the indicated
samples and assembled in vitro either with or without BMV RNA as a substrate.
Encapsidated RNA was isolated from the assembled products and subjected to Northern
blot hybridization as described under Fig. 2 legend. The position of all four BMV RNAs is
shown to the left.
M. de Wispelaere et al. / Virology 419 (2011) 17–23
21
Fig. 7. Structure analysis of wt BMV and its β-sub mutant by cryo electron microscopy. Cryo EM of (A) wt, (B) β-sub (28QPVIV32 → 28AAAAA32) and (C) B1 + B2 + B3CPKO + BCPtrans.
Bar in (A) is 50 nm. 3-D reconstructions calculated of (D) 6247 (wild type), (E) 5623 (β-sub mutant) and (F) 1809 (B1 + B2 + B3CPKO + BCPtrans) from molecular images. The
resolutions of the 3-D models are as determined by the Fourier shell criterion are 15 Å (wild type) and 18 Å (mutant and complemented wild type). (G–I) The reconstruction with
the front half removed to show the interior of the virions of (G) wt, (H), β-sub and (I) B1 + B2 + B3CPKO + BCPtrans. The white line indicates one of the three-fold symmetry axes. Part
of the crystal structure of BMV (1js9) is fitted into the EM density with the QPVIV motifs in spacefill (see arrow). (J, K) The wt reconstruction looking down a three-fold axis with the
crystal structure fitted into the EM density. The arrow in (K) points to the difference density obtained by subtracting the mutant reconstruction from the wt model. Arrowheads
indicate additional minor difference densities, which are likely caused by a reorganization of the capsid proteins due to the mutation in the N-terminus. (L) Close-up view of the betahexamer with difference densities.
difference coincides with the location of the N-terminal CP motif QPVIV
(shown in spacefill in Fig. 7G and K). Additional difference peaks of
lesser density can be seen near the beta sheet domains of the CP (see
arrowheads in Fig. 7K). These minor difference peaks may be caused by a
structural rearrangement of the CP due to the mutation in its Nterminus.
Cryo EM reconstructions have been reported for of wt and mutant
CCMV capsids assembled from E. coli expressed subunits that contained
an N-terminal deletion Δβ27-35 in the CP hexamer assembly sequence
(Willits et al., 2003). Despite the deletion, virion assembly appeared
unaffected and consistent with the predicted location of the N-terminal
region of the CP, the reconstruction for the mutant virions showed less
density near the 3-fold axes at the inner surface of the capsid. Contrary
to the CCMV capsids analyzed by Willits et al. (2003) the BMV virions
analyzed in our study were assembled in vivo and contained RNA. The
much larger difference seen in the CCMV reconstructions (Willits et al.,
2003) can be explained based on the fact that the mutation resulted in
the deletion of 6 amino acids corresponding to the loss of about 4 kDa in
the assembled β-hexamer whereas the BMV virions analyzed in our
study contained an amino acid substitutions rather than a deletion.
Furthermore, the CCMV virions were assembled from bacterially
expressed protein and contained no RNA, which may have resulted in
a more localized position of the β-hexamers.
Like BMV and CCMV, CPs of many other plant viruses belonging to
genus Sobemoviruses such as Southern bean mosaic virus (AbadZapatero et al., 1980), sesbania mosaic virus (SeMV) (Pappachan et al.,
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M. de Wispelaere et al. / Virology 419 (2011) 17–23
2008; Satheshkumar et al., 2005), Tombusviridae family — Tomato bushy
stunt virus (Olson et al., 1983) Turnip crinkle virus (Hogle et al., 1986) and
Cowpea mottle virus (Ke et al., 2004) and other viruses such as Tobacco
necrosis virus (Oda et al., 2000) also display a similar structure referred
to as β-annulus. In vitro expressed recombinant CP of SeMV lacking βannulus assembled in vitro (Pappachan et al., 2008) but its effect on in
vivo was not examined. In conclusion, monocot-adapted BMV and dicotadapted CCMV exhibit similar structure, genome organization and
replication mechanisms (Dreher, 1999; Kao and Sivakumaran, 2000).
The CP of BMV and CCMV are composed of 189 and 190 amino acids
respectively and share 70% identity at the amino acid level (Speir et al.,
1995). Exchange of CP between these two viruses exhibited neutral
effects with respect to host range (Osman et al., 1997). However, they do
differ in many properties including the required virus-encoded genes
for cell-to-cell spread (Rao, 1997), electrophoretic mobility patterns of
respective virions and their sensitivity to RNase, the interaction of highly
conserved N-terminal basic arm region of the CP with respective
genomic and subgenomic RNAs during encapsidation (Choi and Rao,
2000) and requirement of 3′ tRNA-like structure in mediating virus
assembly (Annamalai and Rao, 2005a). Data presented here provide
another distinguishing feature for these two viruses, that is, the extent to
which N-proximal β-strand regulates assembly of BMV and CCMV in
vitro and in vivo.
Materials and methods
Agrotransformants and agroinfiltration
The construction and characterization of T-DNA based plasmids for
efficient transient expression of full-length BMV genomic RNAs and
BCPTrans has been described previously (Annamalai and Rao, 2005b). As
shown in Fig. 2A for B3, each plasmid contains, in sequential order, a
double 35S promoter, cDNA complementary to respective full-length
BMV RNAs, a ribozyme sequence of satellite tobacco ring spot virus (Rz)
and a 35S-terminator. CP variants β-sub (28QPVIV32 → 28AAAAA32) and
CPKO ( 1251AUG1253 → 1251AUA1253 and 1275AUG1277 → 1275AUA1277)
were first constructed in the plasmid pT7B3 (Dreher et al., 1989)
using polymerase chain reaction (PCR) with appropriate 5′ and 3′
oligonucleotide primers. The resulting B3 variant sequences were
subcloned into the genetic background of B3 agroplasmid (Fig. 2A) as
decsribed (Annamalai and Rao, 2005b). BMV CP ORF harboring β-sub
mutation was amplified by PCR and subclined into BCPTrans yileding
BCP/β-sub Trans (Fig. 2B). Following transformation into Agrobacterium
strain GV3101, transformants in desired combination were infiltrated
into the abaxial side of N. benthamiana leaves as described (Annamalai
and Rao, 2005b).
Progeny analysis and packaging assays
Total RNA from agroinfiltrated leaves were extracted as described
(de Wispelaere and Rao, 2009) and the RNA pellet was suspended in
RNAse-free water. Virions were purified from agroinfiltrated leaves
using standard procedure (Rao and Grantham, 1995). For Northern
blot analysis samples of virion RNA (0.5 μg) or plant total RNA (5 μg)
were dried in a microfuge and suspended in 10 μl of sample buffer
(10× MOPSbuffer/formamide/formaldehyde/H2 O in a ratio of
1:1.8:5:2.2 respectively), heated at 65° for 10 min and electrophoresed in 1.2% agarose-formaldehyde gel (de Wispelaere and Rao,
2009). Following a 3 h electrophoresis, fractionated RNA was
transferred to a nylon membrane with a VacuGene XL blotting unit
(Phramacia Biotech). The blot was then processed for prehybridization and hybridization using 32P-labeled riboprobes corresponding to
either 3′ conserved tRNA-ilke structure (TLS) (Rao et al., 1989). CP
samples were analyzed by Western blots as described previously
(Osman et al., 1997).
Coat protein preparation, in vitro assembly assays and electron
microscopy
Preparation of CP subunits and in vitro assembly assays using either
in buffer A (50 mM NaOAc, pH 4.8, 0.1 M NaCl, 0.2 mM PMSF) or buffer B
(50 mM Tris–HCl, pH 7.2, 50 mM NaCl, 10 mM KCl, 5 mM MgCl2, and
1 mM DTT) was as described previously (Choi et al., 2000). Following
purification of assembled virions by Centricon-100, each in vitro
assembled virion preparation was spread on glow discharged grids
and negatively stained with 1% uranyl acetate prior to examining with a
FEI Tecnai12 transmission electron microscope (Bamunusinghe et al.,
2011).
RNase sensitivity of wt and mutant virions
Purified virions preparations of wt BMV and mutants were treated
with 1 μg/μl of RNase A for 30 min at 30 °C. Following RNase treatment
each preparation was divided into two lots. One lot was subjected to
EM examination and the other lot was used to extract viral RNA and
subjected to Northern blot hybridization.
EM reconstruction
Wild type and mutant BMV preparations were diluted to between 1
and 0.5 mg/ml, spread over glow discharged holey carbon film (C-flat)
and vitrified by plunging into liquid ethane. Grids were mounted in a
Gatan 626 cryo holder and examined in a JEM-2100 transmission
electron microscope operating at 200 kV. Electron micrographs were
recorded with a minimum dose kit on a 4096 × 4096 pixel charge
coupled device (CCD) camera (TVIPS F415MP) at an electron optical
magnification of 40,000×. The pixel size on the specimen scale was
determined to 2.82 Å using catalase as standard. Images were recorded
at an underfocus of −1.5 μm, placing the 1st zero of the contrast transfer
function (CTF) at around 1/(20 Å). Data sets of 6247 (wild type), 5623
(β-sub mutant) and 1809 (B1 + B2+ B3CPKO+ BCPtrans) images were
selected with the BOXER program in the EMAN image analysis software
package (Ludtke et al., 1999). All subsequent image analyses were
performed with the IMAGIC 5 package of programs(van Heel et al.,
1996) as described(Annamalai et al., 2005). Briefly, single images were
bandpass filtered to remove low- and high spatial frequencies, contrast
inverted and centered by three iterations of translational alignment
using the total average of the dataset as reference. The data set was then
subjected to the ‘alignment-by-classification’ procedure and 5 of the
resulting 48 class sums were selected as references for the first round of
multi reference alignment (MRA). MRA was iterated until stable class
sums were obtained. Projection angles were determined with the
angular reconstitution procedure as implemented in IMAGIC 5.
Icosahedral symmetry was assumed during the angle assignment and
the subsequent reconstruction- and refinement procedures. The
reconstruction was refined by using back projections distributed
randomly in the asymmetric triangle of the Euler sphere to align the
data set of raw images. The resolution of the final reconstructions was
estimated via Fourier shell correlation using a cut off of 0.5.
Acknowledgments
Research in this laboratory is supported in part by a grant from
University of California, Riverside.
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