ARTICLE IN PRESS
Radiation Physics and Chemistry 75 (2006) 359–368
www.elsevier.com/locate/radphyschem
Quantification of RNA in bacteriophage MS2-like viruses in
solution by small-angle X-ray scattering
Deborah A. Kuzmanovica,1, Ilya Elashvilib,2, Charles Wickb,
Catherine O’Connella,3, Susan Kruegerc,
a
Biotechnology Division, NIST, 100 Bureau Drive, Stop 8311, Gaithersburg, MD 20899-8311, USA
Edgewood Chemical Biological Center; 5183 Blackhawk Rd., Aberdeen Proving Ground, MD 21010, USA
c
NIST Center for Neutron Research, NIST, 100 Bureau Drive, Stop 8562, Gaithersburg, MD 20899-8562, USA
b
Received 13 September 2005; accepted 21 November 2005
Abstract
Recombinant forms of bacteriophage MS2 virus particles, wild-type MS2 and MS2 capsids have been examined in
solution using small-angle X-ray scattering (SAXS). SAXS was used to determine the overall size of the virus particles
and to quantify the amount of encapsulated viral RNA. These studies show that analysis of natural and recombinant
forms of MS2 virus by SAXS can be used as both a quantitative measure of nucleic acid content in situ and diagnostic
indicator of sample integrity.
r 2005 Elsevier Ltd. All rights reserved.
Keywords: Bacteriophage MS2; RNA quantification; MS2 capsid; Small-angle X-ray scattering
1. Introduction
Bacteriophage MS2 is an enteric virus of Escherichia
coli. The infective virion contains a 3690 nt singlestranded RNA molecule surrounded by a icosahedral
coat comprised of 90 coat protein dimers and a single
copy of the A (or maturation) protein. The completely
sequenced MS2 genome encodes for four proteins, the
coat protein (M r ¼ 13; 700), A protein (M r ¼ 44; 000),
Corresponding author. Tel.: +1 301 975 6734;
fax: +1 301 921 9847.
E-mail address: [email protected] (S. Krueger).
1
Present Address: Geo-Centers, Inc., Gunpowder Branch,
P.O. Box 68, Aberdeen Proving Ground, MD 21010, USA.
2
Present Address: Chem/Bio Technology Transition Division, Defense Threat Reduction Agency, 8725 John. J. Kingman Rd., Ft. Belvoir, VA 22060, USA.
3
Present Address: Tetracore, Inc., 11 Firstfield Rd., Suite C,
Gaithersburg, MD 20878, USA.
replicase (M r ¼ 60; 700) and a 75 amino acid lyticase
protein (Atkins et al., 1979; Beremand and Blumenthal,
1979; Fiers et al., 1976, 1975; Min Jou et al., 1972).
Whereas the coat and A proteins serve as structural
components of the virus shell, the replicase and lyticase
are needed for replication of the viral genome and lysis
of the bacteria cell, respectively (Atkins et al., 1979;
Beremand and Blumenthal, 1979; Fiers et al., 1976). The
MS2 RNA also contains a 19 nt transcriptional operator
site. Binding of coat protein dimers to this site links
translation of the genomic RNA to the activation of
virus protein shell assembly (Stockley et al., 1994).
The potential for intrastrand base-pairing within MS2
RNA predicts a complex pattern of folding that includes
several stem-loop structures (Fiers et al., 1976). Biochemical and genetic analysis have confirmed that these
stem-loop structures act as binding sites for the coat
protein dimers in vitro and in vivo. Furthermore, cryoEM has revealed that specific structural elements of the
0969-806X/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.radphyschem.2005.11.005
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D.A. Kuzmanovic et al. / Radiation Physics and Chemistry 75 (2006) 359–368
virion attach to the inner surface of the virus protein
shell in an organized network (Konig et al., 2003). This
association between the RNA and coat protein dimers is
presumed to be mediated by the stem-loop motifs.
However, the functional significance of the RNA
network remains obscure. Our group has previously
used small-angle neutron scattering (SANS) to characterize the RNA core. This analysis has revealed that
the genomic RNA in wild-type MS2 exists as a tightly
packed ball, which is confined to a radius of 83 Å. This
indicates that the majority of the viral core is RNA-free
with approximately 70% of the virus core composed of
solvent (Kuzmanovic et al., 2003).
In general, small-angle X-ray scattering (SAXS) is a
process where X-rays are passed through the sample in
solution and the resulting scattering pattern is used to
determine the average size and shape of the sample in
solution (Koch et al., 2003). For simple protein
molecules or large complexes, SAXS has revealed
structural information about a variety of processes
including protein folding dynamics, molecular weight
determination and oligomerization state (Trewhella
et al., 1998). Additionally, SAXS is used to complement
cryo-electron microscopy and crystallography studies by
providing solution structure data (Graille et al., 2005;
Olah et al., 1995; Thuman-Commike et al., 1999). In the
case of complex macromolecular structures like viruses
that contain both nucleic acid and protein components,
the nucleic acids are uniquely resolved by SAXS due to
the fact that X-rays interact more strongly with nucleic
acids than with proteins. Specifically, the phosphate
groups of RNA (or DNA) scatter much more intensely
than other atoms found in proteins, RNA and DNA.
This is due, principally, to the fact that the X-ray
scattering length density (SLD) of an atom, which is a
measure of its ability to scatter X-rays, increases with
atomic number. Thus, the X-ray scattering intensity,
which is a function of the SLD squared, is stronger for
materials of higher atomic number. Since phosphate has
the highest atomic number of all of the atoms known to
be found in protein–DNA- or RNA-containing biomolecular molecules, SAXS may be used to provide unique
insight into the physical characteristics of RNA or DNA
in complex biological systems. Although, in theory,
SAXS has been predicted to be an accurate quantitative
method for the quantification of RNA or DNA, in
practice its feasibility has not been demonstrated (Jacrot
and Zaccai, 1981; Koch et al., 2003).
The most sensitive method for the quantification of
RNA in solution is generally thought to be by use of the
real time reverse transcription polymerase chain reaction, generally called RT-PCR (Bustin, 2000; Bustin
et al., 2005). In these experiments, by use of an enzyme
the target RNA in the sample is converted to
fluorescently labeled DNA reporter molecules, which
are amplified and quantified in a single step. Detection
and quantification of the target RNA can be either semiquantitative (by addition of a competitive target RNA
of known concentration) or quantitative (by use of an
internal standard RNA); under the best possible
conditions the uncertainty is estimated to be 10%
(Bustin, 2000). However, routinely the uncertainty is
much greater for a variety of reasons which range from
error due to sample preparation, pipeting, competition
between the target and competitive inhibitor or the
uncertainty of the concentration of the internal standard
or errors related to the use of the fluorescent probes. For
an excellent review see Bustin (2000).
Traditionally, nucleic acids (RNA and DNA) have
been quantified by use of optical density techniques or
absorbance using conventional spectrophotometry
(Sambrook and Russell, 2001). This method is only
useful for samples, which are free from contamination
by proteins, phenol, agarose or other nucleic acids. Since
obtaining pure RNA samples is technically challenging
because RNA is extremely sensitive to the action of
nucleases in the environment, typically RNA quantification is assessed by PCR-based methods like the ones
described above.
Both optical density and PCR-based methods rely on
the analysis of naked RNA in solution. In this study, we
use SAXS to measure the physical characteristics of viral
RNA in its native environment, while it is still
encapsulated in its virus coat. The advantage of being
able to make such measurements is that there is no
uncertainty related to the purification of the RNA, the
addition and activity of enzymes or the need for internal
quantification standards. Specifically, we have examined
two MS2-like virus particles which have been genetically
engineered to contain varying amounts of RNA (MS2HCV and MS2-l) but lack the A protein (Walkerpeach
et al., 1999) (Ambion Diagnostics, personal communication). SAXS was used to compare these samples to
RNA-free MS2 capsids (MS2-Capsid) that contain the
A protein but lack RNA and the wild-type MS2 (WT
MS2), which contains the A protein and normal RNA
levels. These samples were also analyzed by SANS, as
described in Kuzmanovic et al. (2003, in press), as part
of a large series of experiments whose aim is to gain
insight into the physical characteristics of MS2 virus
components in solution under physiological conditions.
2. Materials and methods4
2.1. Bacteriophage, hosts, and medium
MS2 bacteriophage strain 15597-B1 and its E. coli
host 15597 were purchased from the American Type
4
Certain commercial materials, instruments, and equipment
are identified in this manuscript in order to specify the
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Culture Center (Manassas, Va.). E. coli strain 15597
was grown on MS2 broth. MS2 broth contains, per liter:
10 g tryptone, 8 g NaCl and 1 g Bacto-yeast. After
autoclaving, 10 mL of sterile 10% glucose, 2 mL of
1 mol/L (M) CaCl2 and 10 mg/mL of thiamine hydrochloride were added per liter (Davis and Sinsheimer,
1963). WT MS2, MS2-HCV, MS2-l, and MS2Capsid were stored in Tris–salt–magnesium (TSM)
buffer unless otherwise stated. TSM buffer contains
10 mM Tris (pH 7.0), 100 mM NaCl and 1 mM MgCl2.
MS2 phage were grown and purified by ultracentrifugation as described in Kuzmanovic et al. (2003).
MS2-HCV virus particles were produced from the E. coli
expression vector pAR-HCV-2b, which contains a
412 nucleotide sequence from the 50 noncoding core
region of HCV subtype 2b (Genbank Accession
No. M62321) (Walkerpeach et al., 1999). MS2-l virus
particles were produced from the E. coli expression
vector pAR-l-1.0, which contains 908 nucleotides
(1329 to 421) of l sequence (Genbank Accession
No. M17233) (Ambion Diagnostics, personal communication). Both vectors were generous gifts from
Ambion Diagnostics, Inc. (Austin, Texas) and were
grown as described in DuBois et al. (1997) and purified
by cesium gradient ultracentrifugation as described in
Kuzmanovic et al. (2003). The measured density of the
WT MS2 particles was 1.3870.01 g/cm3, which is
the same density value reported by Strauss and
Sinsheimer (1963). The corresponding measured densities for MS2-HCV and MS2-l from these studies were
1.3670.02 and 1.3770.01 g/cm3, respectively.
2.2. Capsid preparations
RNA-free MS2 capsids were prepared using cesium
chloride ultracentrifugation of purified WT MS2 virus
as starting material. In general, WT MS2 viruses were
disrupted and subjected to a series of precipitation steps
to remove the RNA. The RNA-free material was then
permitted to spontaneously assemble into RNA-free
capsids (Ambion Diagnostics Inc., personal communication). These preparations were subjected to cesium
chloride density ultracentrifugation and subsequent
pellet resuspension in TSM as previously described
(Kuzmanovic et al., 2003). A number of similar
purification methods to produce MS2 capsids have been
published by different groups (Mastico et al., 1993;
Thomas and Prescott, 1976).
(footnote continued)
experimental procedure as completely as possible. In no case
does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology
nor does it imply that the materials, instruments, or equipment
identified are necessarily the best available for the purpose.
361
2.3. SDS/polyacrylamide gel electrophoresis
SDS/polyacrylamide gel electrophoresis was performed according to the previously described method
(Laemmli, 1970). Commercially available pre-cast 18%
SDS polyacrylamide gels (Tris–Glycine gels) for the
Novex gel apparatus system were purchased from
Invitrogen (Carlsbad, CA) and used according to the
manufacturer’s
instructions.
The
Tris–Glycine
SDS–PAGE running buffer and sample buffers were
either purchased from Invitrogen (Carlsbad, CA) or
made according to the manufacturer’s instructions.
Before loading on gels, samples were diluted two-fold
with 2 Tris–Glycine sample buffer and incubated at
85 1C for 2 min. Mark12TM Unstained Protein Standard
(Invitrogen, Carlsbad, CA) was used according to
manufacturer’s instructions for the molecular weight
markers. Electrophoresis was carried out for 2–3 h at
30–40 mA/gel. The gels were stained in Brilliant Blue R
solution (Sigma, St. Louis, MO) according to the
manufacturer’s instructions and photographed as previously described (Maniatis et al., 1982).
2.4. SAXS measurements
SAXS data were collected on the BioCAT undulator
beamline 18-ID at the Advanced Photon Source,
Argonne, IL. Samples were exposed to focused X-rays
(12,00072 eV; 2 1013 photons/s) for approximately 1 s
at a sample-to-detector distance of 2.78 m. Two-dimensional (2D) scattering patterns were obtained by using a
5 9-cm charge-coupled device (CCD) detector (Phillips
et al., 2002). Samples were pumped through a 1.5 mm
quartz capillary at 4 mL/s to minimize radiation damage
(Fischetti et al., 2003). Measurements were repeated 5
times on each sample to verify that the scattering
patterns did not change with repeated exposure to the Xrays. Scattered intensities, I(Q), were calculated from
radial averaging of the 2D scattering profiles over the Q
range from 0.005 to 0.193 Å1, with
Q¼
4p sinðyÞ
,
l
(1)
where l ¼ 1:03 Å is the X-ray wavelength and 2y is the
scattering angle, using the routines in the FIT2D data
analysis program (Hammersley, 1998), or using macros
written by the APS staff for IGOR Pro (WaveMetrics,
Inc., Lake Oswego, Oregon). Averaged scattered intensities from sample plus buffer and from buffer alone
were scaled using incident flux values integrated over the
exposure time. Scattered intensities from the MS2 virus
samples alone were obtained by subtracting the average
buffer intensities from the average buffer plus sample
intensities. After buffer subtraction, further analysis of
the scattered intensities from the MS2 virus samples was
performed in the Q range 0.008–0.12 Å1. The SAXS
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362
data were put on an absolute scale, in cm1, by
comparing the scattered intensity of the wild-type MS2
sample at Q ¼ 0 to that calculated using the known Mw,
SLD and volume of the RNA and coat protein
components, as verified by earlier SANS studies
(Kuzmanovic et al., 2003).
2.5. SAXS data analysis
The low-Q portions of the data were analyzed using
the Guinier approximation IðQÞ ¼ Ið0Þ expðQ2 R2g =3Þ
to obtain initial values for the radius of gyration, Rg,
and the forward scattering intensity, Ið0Þ, of the samples
(Guinier and Fournet, 1955). This analysis is valid only
in the region where QRg 1. The GNOM program
(Semenyuk and Svergun, 1991), which makes use of all
of the data, rather than a limited data set at small Q
values, was used to determine the distance distribution
function, P(r), the radius of gyration, Rg, the forward
scattering intensity, I(0) and the maximum dimension,
Dmax. Since all of the data are used, this approach
typically leads to more accurate determinations of Rg
and I(0) that are less influenced by possible aggregation
effects.
At the resolution level of the earlier SANS measurements, MS2 has been shown to be approximated very
well by a spherical shell, with inner radius, R1, outer
radius, R2, and shell thickness, t ¼ R2 R1 (Kuzmanovic et al., 2003). Thus, the SAXS data were also fit to
a polydisperse core-shell sphere model (Hayter, 1983) in
order to obtain the average thickness of the protein
shell, t, and the average radius of the core, R1.
In this model, polydispersity is depicted by assuming a
Schulz distribution of the radii, R1 and R2. R2 is related
to R1 by a constant factor. The polydispersity fitting
parameter, p, is defined as p ¼ s=R2, where s2 is the
variance of the Shultz distribution.
The X-ray SLDs of the protein shell and the solvent
were fixed during the fitting procedure. However, the
SLD of the core, assumed to contain both RNA and
solvent, was treated as an additional fitting parameter
that allowed the amount of water, versus RNA, in the
core to be calculated using the relation
rCORE ¼ X rRNA þ ð1 X ÞrSOLVENT ,
3. Results
3.1. A physical model of the shape of MS2 in solution
The SAXS data for the four different MS2 samples
are shown in Fig. 1. Fig. 1a shows that, in general, the
scattered intensities from the MS2-Capsid, MS2-HCV
and MS2-l samples appear similar as a function of Q.
However, Fig. 1b shows that the scattered intensity from
the WT MS2 sample is clearly different from that of
MS2-Capsid, and thus different from MS2-HCV and
MS2-l. Since the MS2-Capsid does not contain RNA,
the scattered intensity is assumed to arise from the coat
protein component only. Therefore, this analysis provides confirmation that, qualitatively, these specific
MS2-HCV and MS2-l samples do not contain as much
RNA as WT MS2.
The amount of RNA in the virion cores, as well as its
spatial distribution, can be quantified by comparing the
SAXS data to that calculated for model (or idealized)
shapes. Fig. 2 shows the SAXS data from the WT MS2
and MS2-Capsid samples, along with the core-shell
model SAXS curves that best fit the data in each case.
The corresponding core-shell model parameters, as well
as those obtained from the fits to the MS2-HCV and
MS2-l virus samples, are listed in Table 1. It can be seen
from the SAXS results in Table 1 that the overall size
(R2) of the virion for the MS2-Capsid and recombinant
samples does not differ from that of wild-type MS2,
considering that there are differences in polydispersity
(or aggregation) between the WT MS2 and the other
three samples. The effects of polydispersity on the SAXS
measurements are described in detail below.
Although the overall size of these samples is not
significantly different, the core (or inner) radius
(R1) values vary significantly between the MS2-Capsid,
(2)
where X is the mass fraction of RNA in the core, defined
as
X¼
M RNA
,
M RNA þ M solvent
(3)
where rCORE is the fitted SLD of the core portion of the
core-shell model and rRNA and rSOLVENT are the known
SLDs of the RNA and the solvent, respectively. Thus, if
there is no RNA in the core, rCORE ¼ rSOLVENT .
Fig. 1. SAXS I(Q) versus Q data from (a) MS2-l (+), MS2HCV (closed diamonds) and MS2-Capsid (open circles)
samples and (b) WT-MS2 (closed squares) and MS2-Capsid
(open circles) samples. The error bars are smaller than the data
points for all curves.
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MS2-HCV and MS2-l samples as compared to the wildtype MS2 virus. In these SAXS experiments, the core
radius (R1) is a measure of the RNA radius in RNAcontaining particles. SAXS intensities are dominated by
scattering from RNA, due to its high X-ray SLD, which
is defined as the sum of the X-ray scattering lengths of
all the atoms in the molecule divided by the total volume
of the molecule. The X-ray scattering length of an atom
is proportional to its number of electrons (atomic
number). Because nucleic acids contain phosphates,
which are higher in atomic number than the atoms
typically found in proteins, i.e., carbon, hydrogen,
nitrogen and oxygen, nucleic acids scatter X-rays more
Fig. 2. SAXS data from WT MS2 (open squares) and MS2Capsid samples (+). The error bars are smaller than the data
points for all curves. Typical best-fit core-shell model scattered
intensities are also shown for WT MS2 (solid line) and MS2Capsid (dashed line) for comparison.
363
strongly than proteins. The X-ray SLD for the coat
2
protein was calculated to be r ¼ 12:3 106 Å , while
2
that of RNA was found to be r ¼ 16:4 106 Å
(Orthaber et al., 2000). The X-ray SLD of the solvent, or
2
water, was found to be rs ¼ 9:4 106 Å (Orthaber et
al., 2000). Thus, the contrast, or the difference between
SLD values between solute and solvent is Dr ¼ 2:9 2
2
106 Å for the coat protein and Dr ¼ 7:0 106 Å
for the RNA. Since the scattered intensity goes as
contrast squared, SAXS is more sensitive to the RNA
component than the coat protein component in the MS2
virion.
The fitted R1 value for the core radius of WT MS2 is
8572 Å. This is in good agreement with the R1 value of
8471 Å obtained in our earlier SANS study from the
core-shell model fit of the WT MS2 sample in 10% D2O
(90% H2O) TSM buffer (Kuzmanovic et al., 2003). The
SANS data obtained under these conditions, where the
RNA contrast is greater than twice the protein contrast,
are approximately equivalent to the SAXS data. Thus,
both of these R1 values represent the radius of the RNA
in the core of the virion and a direct comparison can be
made between these two data sets, as is shown in
Table 1. Note that the R1 value for the SANS data
obtained in 100% D2O TSM buffer (Kuzmanovic et al.,
2003) in Table 1 is not equivalent to the SAXS result,
since the scattering from the RNA does not dominate
the SANS intensity under these conditions. Table 1 also
shows that the fitted R1 values from SAXS measurements of MS2-HCV, MS2-l and MS2-Capsid are 9472,
9673 and 9072 Å, respectively. This indicates that the
RNA in these particles do not pack as tightly in the core
of the viruses compared to the WT MS2, which has a
radius of 8572 Å. Since the R1 value for the MS2Capsid is smaller than those of the recombinant virus
particles, this would suggest that at least some of the
MS2-Capsid particles contain an amount of RNA that is
measurable by SAXS. Furthermore, the result suggests
Table 1
Parameters from core-shell model fit to SAXS and SANS data
R1 (Å)
Sample
R2 (Å)
Polydispersity
Reference
a
SAXS parameters
WT MS2
MS2 Capsid
MS2-HCV
MS2-l
SANS parameters
WT MS210%D20
WT MS2100%D20
MS2 Capsid 100% D20
a
8572
9072
9472
9673
8471
11571
11571
RNA
RNA
RNA
RNA
13472
13072
13072
13172
Outer
Outer
Outer
Outer
protein
protein
protein
protein
shell
shell
shell
shell
0.01070.005
0.0470.01
0.0670.01
0.0670.01
This
This
This
This
study
study
study
study
RNA
Inner protein shell
Inner protein shell
13671
13672
13971
Outer protein shell
Outer protein shell
Outer protein shell
0.01070.005
0.01070.005
0.01070.005
Kuzmanovic et al., 2003
Kuzmanovic et al., 2003
Kuzmanovic et al., in press
The SLD of the protein shell was fixed at 12.3 106 Å2 and that of the solvent was fixed at 9.4 106 Å2 during all SAXS fitting
procedures.
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Table 2
Percentage of RNA and solvent in the core
Sample
Fitted core SLD %RNAb
( 106 Å2)a
%Solventb
WT MS2
MS2 Capsid
MS2-HCV
MS2-l
11.170.2
9.570.2
9.970.2
10.270.2
7372
9872
9272
8872
2772
272
872
1272
a
The core SLD values were obtained from fits of the SAXS
data to the core-shell model. The SLD of the protein shell was
fixed at 12.3 106 Å2 and that of the solvent was fixed at
9.4 106 Å2 during the fitting procedures.
b
The SLD of RNA was assumed to be 16.4 106 Å2 and
that of the solvent was assumed to be 9.4 106 Å2.
that any residual RNA in the MS2-Capsid particles is
packed more tightly in the core than the RNA contained
in the recombinant particles. This would be expected,
since, unlike the recombinant samples, the MS2-Capsid
sample was made from a WT-MS2 sample.
The amount of RNA in each of the SAXS samples can
be quantified using Eqs. (2) and (3). The X-ray SLD of
the core, obtained by fitting the SAXS data to the coreshell model is compared to the known SLD values of
RNA and water. The calculated amounts of RNA in the
core of all four MS2 samples are listed in Table 2, along
with the fitted core SLD values from the core-shell
model. Note that the 2% error comes from the statistical
counting error in the SAXS intensities. Data of equally
good statistics are being fit to the core-shell structure
model and the percentage of RNA is calculated from
our fitted SLD values. The error on this fitting
parameter is equally good in all cases; so the statistical
error does not vary with RNA density (or amount). The
results confirm that the MS2-Capsid sample is virtually
RNA-free, containing only 2%72% RNA. The MS2HCV sample contains 8%72% RNA in its core and
MS2-l sample contains 12%72% RNA in its core.
These values are in good agreement with the expected
values of 0% RNA for the MS2-Capsid, 5% RNA for
MS2-HCV, and 11% RNA for MS2-l (Walkerpeach
et al., 1999) (Ambion Diagnostics, personal communication). On the other hand, WT MS2 contains
27%72% RNA in its core as measured by SAXS. This
is in good agreement with the expected value of 33.5%
as determined from the MS2 genomic sequence (Fiers
et al., 1976), 31.2% RNA by dry weight (Strauss and
Sinsheimer, 1963) and 33% RNA based on the known
Mw of both the protein and RNA components by SANS
(Kuzmanovic et al., 2003).
A real space representation of the SAXS data can be
obtained by indirect Fourier transform of I(Q) using
GNOM program. The P(r) function is essentially a
histogram of the distances between all possible pairs of
Fig. 3. Distance distribution functions, P(r) versus r, for WTMS2 (closed squares), MS2-l (+), MS2-HCV (closed diamonds) and MS2-Capsid (open circles) samples. The error bars
are smaller than the data points for all curves.
Table 3
Parameters from distance distribution function determination
Sample
Rg (Å)
I(0) (cm1)
WT MS2
MS2 Capsid
MS2-HCV
MS2-l
106.570.5
115.070.5
113.570.5
112.070.5
2.670.1
0.5770.03
0.8170.04
1.370.1
points in the molecule and it indicates the probability
that a certain distance between two pairs of points in the
molecule will occur. For instance, the most probable
distance in a spherically shaped particle is the radius.
Since a sphere is symmetric, the P(r) function will be
symmetric with a peak at the distance, r, which
represents the radius. Fig. 3 shows the P(r) functions
for the MS2 samples and Table 3 lists the parameters
obtained from the P(r) analysis, namely the radius of
gyration, Rg, of the scattering particle and the forward
scattered intensity at zero angle, I(0). The radius of
gyration, Rg, is a shape-independent geometric measurement of a particle that is defined as the root-mean square
of the distances of all of the electrons of the particle
from its center of electronic mass (Guinier, 1939;
Guinier and Fournet, 1955).
The Rg values seen in Table 3 are consistent with what
would be expected from MS2 particles with differing
amounts of RNA in the core. Since Rg is related to the
moment of inertia of a particle about its center of mass,
the Rg value for a hollow sphere is simply its radius.
However, the Rg value for a solid sphere is smaller, at
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pffiffiffiffiffiffiffiffi
3=5 Rg (Guinier and Fournet, 1955). Thus, it is
expected that Rg would be the largest for the MS2Capsid sample, since its core contains the least amount
of RNA, and the smallest for WT MS2, since its core
contains the most RNA. The other two values would fall
in between, with the Rg value for MS2-HCV being larger
than that for MS2-l, since it has less RNA in its core
than MS2-l. Table 3 shows that the Rg values do indeed
follow this scenario. We note that the Rg of naked MS2
genomic RNA in solution has been shown to be 160 Å
by classical light scattering (Strauss and Sinsheimer,
1963). This is in contrast to the Rg of 106.5 Å that we
detect within the intact virus.
The P(r) functions in Fig. 3 essentially show this result
in graphical form. P(r) for the MS2-Capsid has a peak at
the highest r value (190 Å) because there is virtually no
RNA in the core. Thus, most of the mass lies in the coat
protein shell and there is a very low probability that
there are distances represented by two points in the
interior of the particle. On the other hand, the P(r)
function of WT MS2 has a peak at the lowest r value
(140 Å) since it has the most RNA in its core. Thus, it
is much more probable that there are distances
represented by two points in the interior of the particle.
The peaks in the P(r) functions of MS2-HCV and MS2l occur at r values between those of MS2-Capsid and
WT MS2, since the amounts of RNA in their cores are
between those of MS2-Capsid and WT MS2.
3.2. Sample quality
In scattering experiments, molecular weight (Mw)
determination often serves as a measure of sample
quality. This is possible because when scattering data are
obtained on an absolute scale then the Mw of the particle
is proportional to the scattered intensity at zero angle,
I(0), when the concentration of the particles is known
(Jacrot and Zaccai, 1981). Analysis of the scattered
intensity, I(Q), measured over a range of Q values yields
the molecular weight, the degree of oligomerization (or
polydispersity), the overall radius of gyration and the
maximum dimensions of the macromolecule (Perkins,
1994)U The I(Q) versus Q data for both MS2-HCV and
MS2-l in Fig. 1a show shallow secondary maxima which
is an indication of polydispersity. The MS2 Capsid I(Q)
versus Q data show sharper secondary maxima, indicating less polydispersity. However, Fig. 2 shows that the
MS2-Capsid data do not fit the core-shell model well as
Q approaches zero. Rather, I(Q) increases more sharply
than the model curve as Q approaches zero, indicating
some aggregation in the MS2-Capsid sample. The WT
MS2 sample, in contrast, shows no signs of either
aggregation or polydispersity, as indicated by the sharp
secondary maxima in the I(Q) versus Q data, as seen in
Fig. 1b, and the good fit of the core-shell model to I(Q)
as Q approaches zero, as seen in Fig. 2.
365
It was shown in Kuzmanovic et al. (2003) that, while
polydispersity and aggregation both can have large
affects on the fitted I(0) value, resulting in large errors in
the Mw determinations, they have little influence on the
best fit values of the core-shell model parameters, R1
and t ¼ R2 R1, which are measures of the overall
shape of the particles, and the core SLD, which is a
measure of the amount of RNA in the core of the
particles. Although the polydispersity prevents a determination of Mw for the MS2-Capsid, MS2-HCV and
MS2-l samples, the polydispersity does not affect the
measurement of the overall physical dimensions of these
particles that are described by the fitted parameters of
the core-shell model. The fact that the SAXS fitted coreshell model parameters agree well with those obtained in
previous SANS studies (in the case of WT MS2) and
with the expected amount of RNA in each sample is an
indication that these samples produced sufficiently good
data to obtain these parameters reliably. The level of
polydispersity clearly reduces the observed measurement
of the overall size (R2) of the recombinant and MS2Capsid samples as compared to WT MS2 sample, as
shown in Table 1.
In general, the recombinant samples (MS2-HCV and
MS2-l) and MS2-Capsid contain polydispersity or
aggregation compared to the WT MS2 sample. Polydispersity can be caused by a number of different factors
such as sample contamination, radiation damage due to
exposure to high-energy X-rays, or sample degradation
(Lindner and Glatter, 2000). Fig. 4 shows a SDS–PAGE
gel of the MS2-HCV and MS2-l samples used in this
study. There are no detectable contaminating proteins in
these samples or in the WT MS2 and MS2-Capsid
Fig. 4. SDS–PAGE gel of the MS2-HCV and MS2-l samples
used in this study. Lanes 1, 2, and 3 contain the molecular
weight sizing marker, MS2-HCV and MS2-l samples,
respectively.
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D.A. Kuzmanovic et al. / Radiation Physics and Chemistry 75 (2006) 359–368
samples analyzed from stocks prepared for SANS
analysis (Kuzmanovic et al., 2003, in press). Therefore,
contamination is an unlikely cause of the polydispersity
described here. These samples were part of a large batch
prepared for a series of experiments using both SAXS
and SANS. Due to beamtime availability, the samples
prepared for SANS were made fresh and measured
within 24 h while the SAXS samples described here were
analyzed several months later. A comparison of the
SAXS and SANS results (Table 1) indicates that there is
little or no polydispersity in the WT MS2 samples,
independent of the time elapsed from sample preparation to measurement. However, the MS2-Capsid sample
seems to have become more polydisperse during the
several month waiting period prior to the SAXS
measurements. A similar level of polydispersity was also
found in both the MS2-HCV and MS2-l samples.
4. Discussion
Due to the high-intensity X-rays now available at
synchrotron sources, SAXS is often used to study kinetic
processes such as the maturation of the Nudaurelia
capenses o virus, the folding of tobacco mosaic virus
RNA or Tetrahymena ribozyme and the enzyme–
substrate interactions of phenylalanyl-tRNA synthetase
with tRNAphe (Canady et al., 2001; Muroga et al., 1999;
Russell et al., 2000; Tuzikov et al., 1988). This study
takes advantage of the fact that SAXS can provide
general structural information about a virus protein
shell as well as unique resolution of the nucleic acid
component of virus protein–nucleic acid complexes.
These properties of SAXS have permitted a detailed and
quantitative analysis of the RNA content, structure and
spatial distribution of the RNA in recombinant and
wild-type forms of bacteriophage MS2 and its RNA-free
capsid. Using SAXS, the quantity of RNA inside of the
virus particles was measured. The MS2-Capsid samples
were found to be essentially RNA free with a RNA
content of 2%72%. The MS2-HCV and MS2-l and
WT MS2 contained 8%72%, 12%72%, and 27%7
2% of RNA, respectively. These results are in good
agreement with the minimum amount of RNA expected
based on the sequence contained on the expression
vectors that were used to generate these recombinant
particles (Walkerpeach et al., 1999) and on the published
sequence information for the WT MS2 virus (Fiers
et al., 1976).
Also, it is remarkable that the SAXS measurements
are sensitive to the relatively small amounts of RNA in
the recombinant MS2 particles. It was expected that the
amount of RNA in the WT MS2 core (30%) would be
detectable, since the contrast difference between the
protein shell and the RNA core in a SAXS experiment is
similar to that in a SANS experiment performed on WT
MS2 in 10% D2O (90% H2O) TSM buffer (Kuzmanovic
et al., 2003). However, it is clear from our results that
the SAXS technique is sensitive enough to detect
significantly smaller amounts of RNA in the virus core.
In fact, the fitted X-ray SLD of the MS2 core is sensitive
to the amount of RNA in the core to within 2%. This
would suggest that as little as 2–4% RNA in the virus
core is detectable using SAXS. Table 1 shows that this is
true, since the fitted R1 value for the MS2-Capsid is
9072 Å. This compares with a value of 11571 Å for
MS2-Capsid measured in 100% D2O TSM buffer by
SANS (Kuzmanovic et al., in press), where the neutron
SLD for the protein shell is much larger than that of the
RNA core. Thus, a small amount of RNA like that in
the MS2-Capsid core would not be detectable in the
100% D2O SANS data. Moreover, Table 1 shows that
even 30% RNA in the core of the WT MS2 sample is
not detectable by SANS in 100% D2O TSM buffer,
since R1 ¼ 115 1 Å in this case as well (Kuzmanovic
et al., 2003).
The WT MS2 genomic RNA has been shown to be
tightly packed within the MS2 virus core with a radius of
8472 Å by SANS (Kuzmanovic et al., 2003). The
corresponding value for WT MS2, as determined in this
SAXS study, is 8572 Å. In addition, an individual MS2Capsid particle may contain up to 4% RNA in the core,
and this RNA is packed into a core radius only slightly
larger than that for the WT MS2 particles. On the other
hand, the radii of the RNA cores contained in the
recombinant MS2-HCV and MS2-l virus particles are
9472 and 9672 Å, respectively. This indicates that the
RNA in the MS2-HCV and MS2-l particles is not
packed as tightly in the core of the recombinant viruses
as compared to the WT MS2.
A number of factors may be responsible for the
loosely packed RNA seen in the recombinant virus
particles. The MS2-HCV and MS2-l particles, unlike
the WT MS2 and MS2-Capsid particles, contain no A
(or maturation) protein. The A protein has been shown
to be necessary for RNA packing on the basis of genetic
and sedimentation gradient experiments (Argetsinger
and Gussin, 1966; Heisenberg, 1966). Another possibility is the amount or type of RNA is important for RNA
packing. The WT MS2 sample contains a relatively high
percentage of RNA compared to the recombinant and
MS2-Capsid samples (Fiers et al., 1976). Also, the native
MS2 genomic RNA has been shown to form a variety of
secondary structures that may play a role in RNA
packing (Fiers et al., 1976). These structures are not
present in the small, foreign RNA molecules contained
in the MS2-HCV and MS2-l particles. However, it is
not clear from these studies if one or all of these factors
are responsible for the observations described here.
Finally, our analysis of these MS2 samples was the
result of a large-scale effort to gain insight into the
physical characteristics of MS2 virus structure in situ.
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D.A. Kuzmanovic et al. / Radiation Physics and Chemistry 75 (2006) 359–368
Large stocks of each of these samples were generated
and analyzed by both SANS and SAXS techniques. Due
to the availability of the beamtime, the SANS experiments were performed almost immediately. However,
the SAXS experiments were performed several months
later. The WT MS2 samples show no evidence of
polydispersity, independent of elapsed time from sample
preparation to measurement. But the MS2-HCV, MS2-l
and MS2-Capsid samples show a measurable and
significant level of polydispersity compared to the WT
MS2 sample. Since the SAXS measurements were
recorded five times to verify that the scattered intensity
curves did not vary with continued exposure to the
X-rays, and since the samples were in motion during
each measurement, it is unlikely that the polydispersity
described here is due to exposure to X-rays. Rather, it is
more likely that the aggregation or polydispersity
described herein is due to a low level of sample
degradation. This degradation is not sufficient to greatly
affect the measurements of the structural properties of
the virus protein shell or the RNA contained therein.
But, it may be an important quality benchmark for these
recombinant virus particles, which can be modified for
use as quantitative diagnostic standard carriers, and for
use in vaccine development and gene delivery systems
(Heal et al., 2000; Legendre and Fastrez, 2005; Mastico
et al., 1993; Pasloske et al., 1998; Stockley and Mastico,
2000; Walkerpeach et al., 1999). An elevated amount of
polydispersity from these experiments suggests that
recombinant MS2 particles and RNA-free capsids can
degrade or become unstable over a period of several
months and, as a result, may have a decreased
concentration or potency.
To our knowledge, these experimental results are the
first to indicate that SAXS, in addition to providing
structural information about the virus protein shell, can
also be used as a reliable quantitative measure of viral
RNA content in situ and can provide information about
overall sample quantity and quality of intact virus
samples in solution. Therefore, SAXS analysis may be
used as a sensitive quantitative and quality assurance
tool. Laboratory-based SAXS instruments are commercially available and are suitable for experiments of this
type, making the application of these instruments
feasible for quality assurance measurements of viral
particles.
Acknowledgments
We acknowledge the support of the National Institute
of Standards and Technology (NIST), US Department
of Commerce, and the US Army Aberdeen Proving
Ground in providing facilities used in this work. This
material is based upon activities supported by the
National Science Foundation under Agreement No.
367
DMR-9986442. DAK was supported by the NISTNational Research Council Research Associateship
during her postdoctoral fellowship at the NIST Biotechnology Division. Use of the Advanced Photon
Source was supported by the US Department of Energy,
Basic Energy Sciences, Office of Science under contract
no. W-31-109-ENG-38. BioCAT is a National Institutes
of Health-supported Research Center RR-08630. SK
gratefully acknowledges and thanks Dr. Elena Kondrashkina for her assistance in making the SAXS
measurements and for reducing the raw data. We would
also like to thank Dr. Breck Byers, Dr. Janet Huie
and Dr. Thomas Silhavy for careful reading of this
manuscript.
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