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Journal of Virology, August 2001, p. 7305-7314, Vol. 75, No. 16
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.16.7305-7314.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
A Human Rotavirus with Rearranged Genes 7 and 11 Encodes a Modified NSP3 Protein and Suggests an Additional Mechanism
for Gene Rearrangement
Elyanne
Gault,1,
Nathalie
Schnepf,1
Didier
Poncet,2
Annabelle
Servant,1
Séverine
Teran,1 and
Antoine
Garbarg-Chenon1,*
Laboratoire de Virologie, Hôpital
Armand Trousseau (EA 2391, UFR Saint-Antoine),
Paris,1 and Unité INRA 1144 Virologie Moléculaire et Cellulaire, 78852 Jouy-en-Josas,2 France
Received 2 February 2001/Accepted 12 May 2001
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ABSTRACT |
A human rotavirus (isolate M) with an atypical electropherotype
with 14 apparent bands of double-stranded RNA was isolated from a
chronically infected immunodeficient child. MA-104 cell culture
adaptation showed that the M isolate was a mixture of viruses
containing standard genes (M0) or rearranged genes: M1 (containing a
rearranged gene 7) and M2 (containing rearranged genes 7 and 11). The
rearranged gene 7 of virus M1 (gene 7R) was very unusual because it
contained two complete open reading frames (ORF). Moreover, serial
propagation of virus M1 in cell culture indicated that gene 7R rapidly
evolved, leading to a virus with a deleted gene 7R (gene 7R
). Gene
7R coded for a modified NSP3 protein (NSP3m) of 599 amino acids (aa)
containing a repetition of aa 8 to 296. The virus M3 (containing gene
7R) was not defective in cell culture and actually produced NSP3m.
The rearranged gene 11 (gene 11R) had a more usual pattern, with a
partial duplication leading to a normal ORF followed by a long 3'
untranslated region. The rearrangement in gene 11R was almost identical
to some of those previously described, suggesting that there is a hot
spot for gene rearrangements at a specific location on the sequence. It
has been suggested that in some cases the existence of short direct
repeats could favor the occurrence of rearrangement at a specific site.
The computer modeling of gene 7 and 11 mRNAs led us to propose a
new mechanism for gene rearrangements in which secondary structures,
besides short direct repeats, might facilitate and direct the transfer
of the RNA polymerase from the 5' to the 3' end of the plus-strand RNA
template during the replication step.
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INTRODUCTION |
Group A rotaviruses are the main
cause of viral gastroenteritis in infants and in the young of many
animal species. Their genome consists of 11 segments of double-stranded
RNA (dsRNA) which can be separated by polyacrylamide gel
electrophoresis (PAGE). Electropherotype profiles of rotavirus dsRNA
typically show four size classes of segments according to their
molecular weight (10). Variations in the mobility of
individual RNA segments allow a genetic characterization of rotavirus
strains. However, group A rotaviruses showing unusual electropherotypes
in which segments of standard size are replaced by rearranged forms of
larger size have been described. Such viruses with a rearranged genome
(for a review, see reference 9) were first isolated from
chronically infected immunodeficient children (30) and
later recovered either from asymptomatically infected immunocompetent
children (5) or from animals (4, 33, 41).
Rotaviruses with genome rearrangements were also generated in vitro by
serial passage at a high multiplicity of infection of animal (16,
38), or human (19, 27) strains. Rotaviruses
carrying rearranged genes are generally not defective, and the
rearranged segments can reassort in vitro and replace their normal
counterparts structurally and functionally (1, 6, 14).
Gene rearrangements in human rotaviruses recovered from stool samples
mostly involve segment 11 and less frequently involve segments 6, 8, 9, and 10. It is not known whether the rearrangements in segment 11 occur
more frequently or if viruses with a rearrangement in segment 11 have
some selective advantage so that they are detected more easily
(10). Gene rearrangements generated in vitro have also
been reported for segment 5 of bovine (16, 42) and segment
7 of human (19, 27) rotaviruses.
Nucleotide sequences of rearranged genes from several group A rotavirus
strains have been described (3, 12, 13, 15, 25, 27, 28, 36, 38,
42). In most cases, the rearrangement resulted from a partial
head-to-tail duplication of the gene: the sequence included a normal 5'
untranslated region (UTR) followed by a normal open reading frame
(ORF). The duplication started from various positions after the stop
codon and extended up to the 3' end, leading to a long 3' UTR
(9). Thus, the rearranged gene expressed a normal protein
product (3, 27, 38). However, Tian et al. described two
bovine rotavirus variants with rearrangements in the gene 5 that
modified the ORF (42). The resulting viruses retained
their capacity to grow in cell culture, although they expressed
modified NSP1 proteins (15, 42). So far, no mosaic structures due to an intermolecular recombination have been described in rearranged genes.
Thus, genome rearrangements have been proposed to play a part in the
evolution of rotaviruses (beside point mutations and gene
reassortments) and to contribute to their diversity (9, 39). Moreover, it has been suggested that rearranged
segments containing a partial duplication of the ORF might be more
efficient templates for dsRNA synthesis than are their homologous
wild-type counterparts and thus may be preferentially selected during
viral replication (29). The mechanism by which genome
rearrangements occur in rotavirus genes has yet not been defined, and
different models have been proposed (see reference 9 for a
review). Current hypotheses suggest that the RNA-dependant RNA
polymerase of the virus may jump back on its template during either the
transcription (plus-strand synthesis) (20) or the
replication (minus-strand synthesis) (9) step. Direct
repeats that might favor the polymerase switch have been found close to
the rearrangement site in some cases (3, 13, 20, 38) but
not in others (25, 36).
In this paper we report the analysis of two rearranged genes (gene 7 and gene 11) in a group A human rotavirus isolated from an
immunodeficient child. The rearrangement in gene 7 was very unusual
because it contained two complete ORFs. The rearranged gene 7 underwent
further evolution in vitro, with a change in the ORF leading to the
expression of a modified NSP3 protein. Furthermore, the comparison of
the two rearranged genes to their normal homologues and the computer
modeling of their mRNAs led us to propose a mechanism for
rearrangements in rotavirus genes based on the existence of secondary
structures between the 3' and 5' ends of the plus-strand RNAs.
Similarly to the model of picornaviruses in which regions of high local
secondary structure such as hairpins or stem-loops have been proposed
as hot spots for RNA recombination (22, 35, 43, 46),
secondary structures in rotavirus mRNAs might correspond to hot
spots for genome rearrangements.
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MATERIALS AND METHODS |
Viruses and cells.
The M isolate was a group A human
rotavirus of genotype G1 isolated in Madagascar from the stool of a
6-month-old infant with severe combined immunodeficiency syndrome.
Virus propagation on cell culture (confluent monolayers of MA-104
cells) was performed as previously described (11).
The different viruses present in the stool specimen were separated by
cell culture under conditions of limiting dilution. Briefly, after one
passage, serial dilutions of the virus yield (24 replicates for each
dilution) were propagated in 24-well culture plates. After 72 h of
cell culture, rotavirus antigens were detected by indirect
immunofluorescence assay using a polyclonal goat antibody specific for
group A rotaviruses as previously described (11). The
limiting dilution for the viral growth was defined when fewer than 25%
of the inoculated wells were positive for rotavirus culture. The
initial virus yield was then propagated at the defined limiting dilution in 24-well culture plates. Cell lysates from each well were
further propagated in 25-cm2 flasks prior to RNA analysis
by PAGE.
Virus stocks of viruses M0, M1, and M3 (three cell culture-adapted
viruses derived from the M isolate) were subjected to titer
determination in 24-well culture plates. The infected MA104 cells
were
fixed 24 h postinfection and stained for determination of
the
fluorescence-forming units (FFU) using a polyclonal goat antibody
specific for group A rotaviruses. The average titers were 3 ×
10
5 FFU/ml for M0 and M1 and 2 × 10
5
FFU/ml for
M3.
Serial cell culture propagation of viruses was not performed at high
multiplicity of infection (MOI) but using dilutions (1:10)
of the
harvested cell culture fluid for adsorption on MA-104 cells
monolayers.
This corresponded approximately to a MOI of 0.01 FFU/cell.
Nucleic acid analysis.
Rotavirus genomic dsRNA was extracted
either from stool suspensions (~10% [wt/vol] in 150 mM NaCl)
clarified by low-speed centrifugation or from cell culture lysate,
using RNA-PLUS (Bioprobe Systems, Montreuil, France). Prior to reverse
transcription PCR (RT-PCR), dsRNA segments were purified after
electrophoresis in 1% low-melting-point agarose (Sigma-Aldrich Chimie
S.a.r.l, St. Quentin Fallavier, France) using a modified freeze-squeeze
extraction procedure (40). Briefly, the gel slice
containing dsRNA was excised, crushed with a scalpel, vortexed for
~10 s with 400 µl of phenol and frozen at 
80°C for 30 min.
After a rapid thawing, the supernatant was extracted with
phenol-chloroform and precipitated in ethanol by standard procedures.
Rotavirus RNA genomic profiles were determined by PAGE in 10%
polyacrylamide gels (1.5 mm thick) for 16 h at 20 mA at room
temperature. RNA segments were made visible by ethidium bromide
staining of the
gel.
The genomic dsRNA segments were characterized by Northern blotting
after electrophoresis in 1% agarose, using bovine rotavirus
cDNA
probes specific for gene 7, 8, or 9, labeled by random priming
with the
DIG DNA labeling and detection kit (Roche Diagnostics,
Meylan,
France).
Sequencing strategy and nucleotide sequence analysis.
RT-PCR
amplification of genes 7, 7R, 7R, 11, and 11R was performed on
purified dsRNA segments using primers described in Table
1.
Full-length cDNA, including the 3' and 5' ends of genes 7 and 11 of
virus M0 were obtained by the single-primer amplification
strategy
described by Lambden et al. (
23) with some modifications.
Briefly, oligonucleotides P1 (
23) (for gene 7) and ER1
(for
gene 11) were labeled at their 3' ends with digoxigenin-11-ddUTP
(Roche Diagnostics) as specified by the manufacturer and ligated
to
both 3' ends of the dsRNA genome segments. The ligation was
catalyzed
by T4 RNA ligase (Roche Diagnostics). Briefly, dsRNA
and 3'-end-labeled
primers were added to a reaction mixture containing
2 U of T4 RNA
ligase and ligation buffer and incubated for 20
h at 37°C. It
was critical to maintain a ratio of 100 labeled
primer molecules to 1 RNA 3' OH extremity. After ligation, the
enzyme was inactivated by
heating for 2 min at 100°C and eliminated
by ultrafiltration on
polyvinyldiene difluoride membranes (UFC3-IPH
columns; Millipore, St
Quentin, France). Unligated primer molecules
were removed by
ultrafiltration on cellulose membranes (PLTK columns;
Millipore). The
efficiency of the reaction was monitored by Northern
blotting followed
by detection of the digoxigenin-labeled dsRNA
(DIG luminescent
detection kit; Roche Diagnostics). Primer-tailed
dsRNAs were reverse
transcribed and amplified using primer P2
(for gene 7) or ER2 (for gene
11).
Attempts to obtain full-length cDNA copies of the rearranged genes 7R
and 11R by the single-primer amplification strategy
always resulted in
PCR products smaller than the expected size
(data not shown). Such a
phenomenon had previously been observed
in another study of a
rearranged gene (
3) and might be due
to an intermolecular
base pairing between the duplicated sequences
of the rearranged gene,
leading to mispriming and incorrect elongation
during the RT-PCR. To
overcome this problem, the junction regions
of the rearrangements of
genes 7R, 7R
, and 11R were first amplified
and sequenced, allowing
the determination of new direct or reverse
primers, specific for these
regions. These primers used with the
external primers (P2 or ER2)
allowed us to sequence separately,
after RT-PCR, the first and second
halves of the rearranged genes,
including the 3' and 5' ends. For gene
7R
, primers specific for
the 5' and 3' termini were used instead of
external primers (Table
1).
RT-PCR of the purified dsRNA gene segments was performed as follows: 1 to 5 µl of the dsRNA extract was reverse transcribed
in a 50-µl
reaction mixture containing 20 µM EDTA, 10 mM dithiothreitol,
0.5 mM
(each) deoxynucleoside triphosphate, 0.1 µM primer, 10
U of RNase
inhibitor (Life Technologies, Cergy, France), 200 U
of SuperScript II
(Life Technologies), and SuperScript buffer.
After a 45-min incubation
at 45°C the reaction was stopped by
adding 1 µl of 0.5 M EDTA. Then
150 µl of H
2O was added, and 5
µl of cDNA was amplified
by using direct and reverse primers described
in Table
1. PCR was
performed in a 50-µl reaction mixture containing
10 mM Tris-HCl (pH
8.3), 50 mM KCl, 1.5 to 2 mM MgCl
2, 0.2 mM
(each)
deoxynucleoside triphosphate, 0.25 µM (each) primer, and
1.25 U of
AmpliTaq DNA polymerase (Perkin-Elmer, Villebon, France).
Amplification
was performed in a 9700 Perkin Elmer thermocycler
under PCR conditions
of 35 cycles of 94°C for 30 s, 50 to 60°C
for 1 min, and
72°C for 1 min. Concentrations of MgCl
2 and annealing
temperatures were adapted depending on the primers. PCR products
were
analyzed after electrophoresis in 1.5% agarose
gels.
Sequencing was carried out by the dideoxynucleotide chain terminator
method on an ABI Prism 377 automatic sequencer (Applied
Biosystems),
using the ABI PRISM DyeTerminator cycle-sequencing
ready reaction
kit. Nucleotide sequences were determined directly
from the PCR
products, except for gene 7, which was cloned in
the plasmid vector
pBluescript. In all cases the sequence was
determined on both strands,
either from two independent RT-PCR
products or from plasmid DNA of
three independent
clones.
Secondary structure of mRNAs.
The mfold
program version 3.1 (24, 47) predicts the possible
secondary structures for RNA sequences. It was made available by
Michael Zuker on his home page, presently located at
http://bioinfo.math.rpi.edu/~zukerm/. The program was established
using the rules set up by Serra and Turner (37) and Walter
et al. (44) concerning the minimal computed free energies.
Protein analysis.
Virus-infected confluent monolayers of
MA-104 cells were washed with 2 ml of phosphate-buffered saline and
scraped. Cell lysis was performed either for 1 h at 4°C with a
buffer containing 50 mM HEPES (pH 7.0), 250 mM NaCl, 5 mM EDTA, 2 mM
sodium pyrophosphate, 1 mM sodium orthovanadate, 5 mM dithiothreitol,
0.1% NP-40, 7.5 µg of aprotinin per ml, 1 µg of leupeptin per ml,
and 100 µg of phenylmethylsulfonyl fluoride per ml or for 5 min at
room temperature with a buffer containing 50 mM Tris-HCl (pH 6.8), 2%
sodium dodecyl sulfate (SDS), and 2% 
-mercaptoethanol. A 40-µg
portion protein extract was boiled in 62.5 mM Tris-HCl (pH 6.8)-2%
SDS-5% -mercaptoethanol-25% glycerol and loaded on a 12%
acrylamide gel for SDS-PAGE. Proteins were blotted on polyvinyldiene
difluoride (Hybond-P; Amersham Pharmacia Biotech, Orsay, France)
membranes by transverse electrophoresis in 250 mM Tris-192 mM
glycine-20% methanol. After a 2-h saturation in 20 mM Tris-HCl (pH
7.6)-137 mM NaCl-0.5% Tween 20 (TBS-T) with 10% skim milk, the
membranes were incubated for 90 min at room temperature with a 1:5
dilution of monoclonal mouse anti-NSP3 ID3 antibody (2),
washed three times with TBS-T, and then incubated for 1 h at room
temperature with a 1:10 000 dilution of a horseradish peroxidase-coupled goat anti-mouse immunoglobulin G (Jackson
Immunoresearch, Interchem, Asnières, France). Finally, the
membranes were washed four times with TBS-T and the proteins were
revealed by enhanced chemiluminescence (ECL Western blotting detection
kit; Amersham).
Nucleotide sequence accession numbers.
The full-length
sequences of genes 11, 11R, 7, 7R, and 7R are available under
GenBank accession no. AF338244 through AF338248.
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RESULTS |
The human rotavirus M isolate is a mixture of viruses with standard
or rearranged genes.
The RNA profile of the human rotavirus M
isolate is shown in Fig. 1. The
electropherotype displayed an unusual pattern of 14 apparent bands of
dsRNA including 10 segments with standard size (segments 1 to 6 and 8 to 11) and 4 extra segments located between segments 3 and 4 (segment
a), 4 and 5 (segments b and c), and 6 and 8 (segment d). In
addition, segment 7 was missing from the profile and the intensity of
segments 6 and 11 was lowered. Such an RNA profile, with additional
high-molecular-weight segments replacing standard-size segments,
strongly suggested that the isolate M genome contained rearranged
genes. Moreover, this profile with 14 apparent bands of dsRNA suggested
that the M isolate was a mixture of several subpopulations of viruses
differing in genotype, as reported for other human rotavirus isolates
with genome rearrangements (17).
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FIG. 1.
RNA profiles of the M isolate and of viruses M0, M1, and
M2. Lanes: M, RNA profile of the M isolate obtained from the original
stool sample; M0, M1, and M2, RNA profiles of viruses M0, M1, and M2
recovered in MA-104 cell culture performed under limiting-dilution
conditions. Numbers indicate the rotavirus gene segments of standard
size. a, b, c, and d represent the extra segments in isolate M. 7R and
11R indicate rearranged genes 7 and 11, respectively.
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Attempts to separate the various viruses contained in the M isolate by
plaque purification in MA-104 cell culture directly
from the stool
sample were unsuccessful. Alternatively, the M
isolate was grown in
MA-104 cell culture from the stool supernatant
under conditions of
limiting dilution. This allowed us to recover
cell culture-adapted
viruses, all of which contained 11 genomic
segments. These viruses were
divided into three species named
M0, M1, and M2 according to their
electropherotypes (Fig.
1).
M0 had a typical electropherotype of group
A rotaviruses, with
11 segments located at their usual position. In M1
and M2, segment
b replaced segment 7. In addition, in virus M2, segment
d replaced
segment 11. Northern blot analysis indicated that segment b
from
M1 and M2 hybridized with a cDNA probe specific for gene 7 and
thus represented a rearranged gene 7. Similarly, segment d in
virus M2
hybridized with a probe specific for gene 11 and represented
a
rearranged gene 11 (data not shown). Segments b and d were subsequently
referred to as genes 7R and 11R, respectively. Although no viruses
containing segments a or c could be recovered in cell culture,
the
existence of these RNA bands in the RNA profile of the M isolate
might
indicate that the M isolate contained more virus species
with different
genome rearrangements than were isolated. Unfortunately,
the gene
origin of RNA bands a and c could not be determined because
there was
not enough of the origin stool sample to perform Northern
blot
experiments on the RNA extracted from the M
isolate.
These results indicated that the M isolate contained a mixture of at
least three nondefective viruses, two of them containing
one or two
rearranged genes. This allowed us to compare the nucleotide
sequences
of rearranged genes with those of the standard genes
from which they
were
derived.
The rearranged segment 7R of virus M1 contains two complete copies
of the NSP3 ORF.
The complete nucleotide sequence of gene 7 from
virus M0 was first determined as a reference. Gene 7 of virus M0 had
96.7% similarity to gene 7 of the human rotavirus strain Wa. However, as opposed to most rotavirus genes 7 deposited in GenBank, which contain two in-frame methionine codons (at nucleotides [nt] 26 and
35), gene 7 of M0 had a CTG in place of the first ATG. Thus, gene 7 of
virus M0 contained 1,074 bp with a 5' UTR of 34 bp and a 3' UTR of 107 bp. The ORF ranged from nt 35 to 967 and coded for a NSP3 protein of
310 amino acids (aa).
As compared to gene 7 of virus M0, gene 7R of virus M1 consisted of an
almost full-length duplication of the gene (Fig.
2).
The rearrangement took place after nt
963, reinitiating the sequence
within the 5' UTR at nt 6. The
rearrangement not only led to the
modification of the last nucleotide
of the first ORF, changing
the last amino acid of the protein
(E-310-D), but also recreated
a new in-frame stop codon TAA at nt 965. Therefore, the rearrangement
of gene 7R was very unusual, since it
resulted in a rearranged
gene containing two complete ORFs. Except for
codon 310 and for
a single silent point mutation at nt 259 in the first
part of
the gene 7R, both ORFs were identical.
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FIG. 2.
Schematic diagram of the standard and rearranged genes 7 and of the NSP3m protein. The ORFs are indicated by large boxes, and
the UTRs are indicated by small boxes. Thick lines indicate the stop
codons. The nucleotide positions corresponding to the gene
rearrangement (gene 7R) and to the 91-bp deletion of gene 7R are
indicated. The nucleotide sequence of the junction region of the
rearrangement is detailed. In gene 7R, the duplicated part of the gene
is shaded. In NSP3m, residues are numbered referring to the first
methionine codon (at nt 35) and the repeated residues (aa 8 to 296) are
indicated by a cross-hatched box.
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Gene 7R of virus M1 serially propagated in cell culture undergoes a
further change leading to a modified NSP3 ORF.
Although gene 7R
contained two complete ORFs, it was not possible to ascertain whether
both ORFs were used for protein translation. Considering the hypothesis
that an untranslated ORF was likely to be modified during further
replication cycles, we investigated the evolution of gene 7R during
serial propagation of virus M1 in cell culture as an indirect means of
determining the functionality of the second ORF.
Virus M1 was serially propagated in cell culture at low MOI (
0.01
FFU/cell), and the RNA profiles corresponding to each passage
were
analyzed (Fig.
3). After four passages, a
12th segment (referred
to as 7R
) was detected below segment 7R.
During further passages,
the relative concentration of segment 7R
increased while that
of segment 7R decreased. At passage 10, a
subculture performed
under limiting-dilution conditions allowed us to
recover virus
M3 containing segment 7R
and not segment 7R (Fig.
3).
Nucleotide
sequence analysis of segment 7R
showed that it was
derived from
gene 7R after a 91-bp deletion. The deletion started at nt
922
(within the first ORF of gene 7R) and ended at nucleotide 1013
(within the second ORF), thus connecting the duplicated sequence
of
gene 7R in frame with the first ORF (Fig.
2). Gene 7R
displayed
an
ORF of 1,941 bp coding for a modified NSP3 protein (NSP3m)
of 599 aa
(instead of 310). NSP3m consisted of the first 296 amino-terminal
residues of NSP3 followed by a large repetition of 289 residues
(aa 8 to 296) and the last 14 carboxy-terminal residues of NSP3
(aa 297 to
310). Thus, all but the first 7 and the last 14 aa
of NSP3 were
duplicated in the NSP3m protein. A single change
in the amino acid
sequence was detected in the repeated part of
the protein: A (aa 18)
changed to S (aa 307). As compared to gene
7R, the duplicated part of
gene 7R
also contained an additional
silent point mutation at nt
1207 (A changed to G).
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FIG. 3.
Serial propagation of virus M1 in cell culture. (Left)
Evolution of the virus M1 RNA profile during serial passages in MA-104
cell culture. Pn indicates the number of passages
(n) performed prior to the analysis of the electropherotype.
At P4, segment 7R appeared below segment 7R (arrows), increased in
relative concentration along with further passages, and became a
majority at P9. (Right) RNA profiles of the viruses obtained after
subculture of the P10 viral mixture, allowing us to recover virus M3
containing segment 7R and virus M1 containing segment 7R.
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Taken together, these results indicated that the duplicated coding
sequence contained in gene 7R was not maintained during
further
replication of virus M1 in cell culture and the change
in gene 7R
involving both ORFs led to a rearranged gene 7 with
a single ORF
potentially coding for a modified NSP3
protein.
NSP3 expression in cells infected with M0, M1, or M3.
To
examine the effects of gene 7 rearrangements on the expression of NSP3,
MA-104 cells were infected with virus M0 (gene 7), M1 (gene 7R), or M3
(gene 7R) and cell lysates were analyzed by Western blotting using
an NSP3-specific monoclonal antibody (Fig.
4). As expected, the NSP3 protein
produced by virus M1 had an apparent molecular mass close to 34 kDa,
which was identical to that of the NSP3 protein of virus M0. However,
when equivalent amounts of total proteins (40 µg) extracted from
cells infected with virus M0 or M1 at the same MOI (0.1 FFU/cell) were
analyzed, the signal intensity of NSP3 detected by Western blotting was reproducibly lower for M1 than for M0 (Fig. 4, compare lanes M0 and
M1). This could indicate that virus M1 synthesized NSP3 in smaller
amounts than did virus M0 and might favor the hypothesis that the
second ORF of gene 7R was not used for protein synthesis and even
lowered the expression of the first ORF. The modified protein NSP3m
from virus M3 was clearly detected in Western blots when an
SDS-containing buffer was used for cell lysis and protein extraction
(Fig. 4A). Under these conditions, NSP3m had an apparent molecular mass
of approximately 68 kDa, compared to 34 kDa for NSP3. Interestingly,
when a buffer without SDS was used for cell lysis, the NSP3m protein
was only faintly detected and two bands were observed, one at 68 kDa
and an additional band at approximately 40 kDa (Fig. 4B). This was not
the case for the NSP3 proteins of M0 or M1, which were similarly
detected independent of the buffer used (compare Fig. 4A and B). The
significance of the 40-kDa band observed for NSP3m remains to be
established. This band might have been generated by cleavage during the
extraction procedure, since the duplicated protein which contains two
coiled-coil structures might be less stable and thus more accessible to
proteases. However, no distinct bands corresponding to smaller cleavage
products were detected on the blot, and the possibility of an artifact
should not be excluded. On the other hand, the epitope recognized by the monoclonal antibody ID3, located between aa 206 and 290 (unpublished data), might not be contained in smaller cleavage
products. Taken together, these results indicated that virus M3
actually produced the modified protein NSP3m and suggested that NSP3
and NSP3m might behave differently in MA-104 cells.
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FIG. 4.
Western blot analyses of NSP3 and NSP3m. MA-104 cells
infected by viruses M0, M1, and M3 were recovered 24 h
postinfection, and whole-cell lysates were analyzed by Western blotting
with the monoclonal antibody ID3 specific for NSP3. C indicates
mock-infected cells as a control. Numbers indicate molecular mass
markers in kilodaltons. The position of viral proteins NSP3 and NSP3m
are indicated. (A) Western blotting was performed after cell lysis with
a 2% SDS-containing buffer. The apparent molecular mass of NSP3m
(virus M3) was approximately double the molecular mass of NSP3 (viruses
M0 and M1). (B) Western blot analysis was performed after cell lysis
without SDS. Unlike NSP3, NSP3m was only faintly detected (arrow) and
an additional band of 40 kDa was observed (asterisk).
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The gene 11 rearrangement in virus M2 suggests that gene 11 rearrangements in rotaviruses occur at a definite sequence
location.
The nucleotide sequence of gene 11 from virus M0 was
first determined as a reference. It consisted of 664 nt with a 594-bp ORF flanked by 5' and 3' UTRs of 21 and 49 nt, respectively. Gene 11 of
virus M0 had 98% similarity to the gene 11 of the human rotavirus
strain Wa. The rearrangement of gene 11R of virus M2 consisted of a
partial duplication of gene 11 of virus M0. The rearrangement took
place within the stop codon TAA at nt 614, and the sequence reinitiated
within the ORF at nt 42, re-creating a new in-frame stop codon, TAG, at
the same position (Fig. 5). Thus, gene
11R was 1,237 bp long and contained a normal 594-bp ORF followed by a
long 3' UTR of 622 bp. Compared to gene 11 of virus M0, no mutation was
detected in gene 11R, neither in the first part nor in the duplicated
part of the gene.
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FIG. 5.
Schematic diagram of the standard and rearranged genes
11. The ORFs are indicated by large open boxes, and the UTRs are
indicated by small open boxes. Thick lines indicate the stop codons.
The nucleotide positions corresponding to the gene rearrangement (gene
11R) are indicated. The nucleotide sequence of the junction region of
the rearrangement is detailed. The duplicated sequence of the gene 11R
is shaded.
|
|
Strikingly, the gene 11R rearrangement was almost identical to the gene
11 rearrangements described for the human strain
Z10262 (
28) and the bovine strain C7/183 (
36). For
both these viruses,
as for virus M2, the rearrangement occurred within
the stop codon
(at nt 614 for virus
Z10262 and nt 615 for virus C7/183)
and
the sequence reinitiated at nt 43 (
Z10262) or 41 (C7/183).
Such a
similarity of the gene 11 rearrangement location in three
unrelated
strains strongly suggested a nonrandom phenomenon. Since
the
rearrangements always involved the 3' and 5' ends of the genes,
we
further investigated the existence of putative secondary structures
in
the single-stranded RNA template for replication which might
bring the
3' and 5' ends close together and thus favor
rearrangements.
Predicted folding of gene 7 and 11 mRNAs.
The nucleotide
sequence of the single-stranded mRNA of gene 11 from virus M0 was
analyzed with the mfold program based on free-energy
minimization developed by Zuker et al. (47). The possible
foldings obtained within 20% of the minimum free energy were analyzed.
All 11 optimal secondary structures predicted by the program showed
that the 3' end (nt 604 to 635) would interact with the 5' end (nt 52 to 18) to form a long panhandle (Fig.
6A). This base pairing placed face to
face the two nucleotides (nt 42 and 614) which mark the boundary of the
rearrangement of gene 11R. Similarly, for gene 7 of virus M0, 26 of the
28 most stable secondary structures predicted by the mfold
program showed that the 3' end (nt 937 to 969) would interact with the
5' end (nt 29 to 1) of the RNA (Fig. 6B). Thus, a base pairing between
nt 6 and 963, the nucleotides involved in the rearrangement of gene 7R,
was predicted. Such secondary structures might favor the occurrence of
rearrangement according to a mechanism that will be discussed below.
View larger version (25K):
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|
FIG. 6.
Predicted optimal secondary structures for gene 7 and 11 plus-strand RNAs. Examples of some optimal folding configurations
predicted by the mfold program for genes 7 (A) and 11 (B) of
virus M0 are shown. dG indicates the minimum free energy values (in
kilocalories per mole). Arrows indicate the positions of the
nucleotides involved in the rearrangements leading to genes 7R and 11R.
On the right, the base pairing between the 3' and 5' ends of the RNA
predicted for the most stable secondary structures is enlarged. The
nucleotides involved in the gene rearrangements are indicated.
|
|
| |
DISCUSSION |
Cell culture recovery of viruses with rearranged genes.
The
RNA profile of the M isolate obtained from the original stool sample
contained 14 gene segments, with 4 segments migrating at unusual
positions. As previously reported (17, 30), such an
electropherotype corresponded to a mixture of virus subpopulations, with some viruses containing rearranged genes. As reported by others
(17), plaque purification of the viruses directly from the
fecal extracts was unsuccessful. However, virus culture under limiting-dilution conditions allowed us to recover viruses with a
standard genome (M0) and viruses with rearranged genes: M1 (containing a rearranged gene 7) and M2 (containing rearranged genes 7 and 11).
Viruses containing the slowly migrating segments a or c that were
observed on the original electropherotype could not be recovered in
cell culture, although 192 RNA profiles were analyzed. This could
hardly be explained by the hypothesis that these viruses were in a
minority in the stool sample compared to M0, M1, and M2 (Fig. 1, lane
M). Indeed, M0 was readily recovered in cell culture although it was
only a minor component of the stool sample as indicated by the absence
of a visible segment 7 on the original electropherotype. This could
indicate that virus M0 had a selective advantage due to the absence of
rearranged genes while the viruses containing segments a or c had a
selective disadvantage, probably because of the rearrangements themselves.
Standard and rearranged genes 7 in viruses M0, M1, and M3.
As
opposed to most rotavirus genes 7 deposited in GenBank
(34), both genes 7 of M0 and 7R of M1 had a CTG instead of
an ATG at nt 26, indicating that the methionine codon at nt 35 was probably used as the initiating codon for NSP3. Consistent with this
hypothesis, the methionine codon at nt 35 is in a better environment
than the one at nt 26 for the initiation of translation according to
Kozak's rules (21). In addition, Piron et al. have shown
that the RNA-binding capacity of NSP3 was conserved when the protein
started at the second methionine (31).
Gene 7 rearrangements had never been described before for human
rotaviruses isolated in vivo, although in vitro-generated
human
rotaviruses with rearranged genes 7 have been reported (
19,
27). The rearranged gene 7R of virus M1 had an unusual pattern,
with two complete ORFs that were identical except for the last
codon of
the first ORF (which was changed because of the rearrangement)
and for
a silent point mutation in the second ORF. To our knowledge,
this is
the first example of a rotavirus RNA segment containing
two copies of
the same gene. Whether both ORFs were used by the
virus M1 for the
translation of NSP3 remains to be established.
However, in the absence
of an internal ribosome entry site on
the mRNA, the second ORF was
most probably not translated. Consistent
with this hypothesis, virus M1
did not overexpress the NSP3 protein,
as indicated by Western blot
analyses.
After serial propagation of virus M1 in cell culture, gene 7R rapidly
underwent a further change, leading to the deleted gene
7R
coding
for the modified protein NSP3m. Virus M3 expressing
the modified NSP3m
protein was not defective and multiplied well
in cell culture. A
fluorescent-focus assay indicated that virus
M3 replicated as
efficiently as virus M0 did (results not shown).
Moreover, when virus
M1 was serially propagated in cell culture,
virus M3 emerged and became
predominant, as indicated in PAGE
by the increasing relative amount of
segment 7R
over segment
7R (Fig.
3). This could indicate that virus
M3 had a selective
advantage over virus M1, perhaps related to the
NSP3m protein.
The nonstructural protein NSP3 plays an important role
in the
multiplication cycle of rotaviruses. Three domains of activities
have been mapped (
31,
32): the N-terminal part of the
protein
(aa 4 to 150) which binds specifically to the 3' end of the
viral
mRNAs; the middle part of the protein (aa 150 to 240), which
contains
a possible coiled-coil domain and is required for
dimerization;
and the C-terminal domain (aa 206 to 313), which is
responsible
for interaction of NSP3 with the translation initiation
factor
eIF-4GI. NSP3m contained a repetition which included the
entire
dimerization domain and most of the RNA-binding and
the eIF-4GI-binding
domains. This might have resulted in a change of
some of the biological
properties of the protein. For example, the fact
that as opposed
to NSP3, NSP3m was detected in Western blots depending
on the
presence of SDS in the lysis buffer might indicate a stronger
interaction of NSP3m with some cellular components. This deserves
further investigations, since the association of NSP3 with the
cytoskeleton has been previously suggested (
26).
Gene rearrangements are thought to participate in the genetic
variability of rotaviruses (
9). So far, the only case of
a
protein modification induced by a gene rearrangement was reported
for
two in vitro-generated variants of a bovine rotavirus strain
with
rearrangements in gene 5 (
15,
42). For one variant (brvE),
the rearranged gene contained two in-frame partial ORFs, leading
to an
extended NSP1 protein (728 aa instead of 491 aa) (
42).
For
the other variant (brvA), the rearrangement was associated
with several
point mutations, one of which resulted in a new in-frame
stop codon in
the first part of the ORF, truncating the protein
to its first 258 aa
despite partial duplication of the gene (
15).
The
rearranged gene 7R
reported here is another example of a
gene with
extended ORF and confirms that replication-competent
viruses with
modified proteins can be generated by rearrangement
events. However,
rotaviruses with a rearranged gene coding for
a modified protein have
never been isolated in vivo. In the present
case, virus M3 producing a
modified NSP3 protein was obtained
in vitro during cell passages of
virus M1, and it would have been
of interest to know whether virus M1
would have evolved similarly
in
vivo.
Possible mechanisms for rotavirus gene rearrangement.
The
molecular mechanism leading to the rearrangement of rotavirus
genes is not known. A current hypothesis suggests that either during
transcription (plus-strand RNA synthesis) or replication (minus-strand
RNA synthesis), the viral RNA polymerase could interrupt the RNA
synthesis, fall back on its template, and reinitiate RNA synthesis
(9). Direct repeats close to the duplication site have
been proposed as a way for the polymerase to fall back on its template
(9, 20). However, direct repeats do not seem to be an
absolute requirement for genome rearrangements (9). They were found in some rearranged genes (3, 13, 20,
38) but not in others (25, 36), and, in particular,
no direct repeat could be found to explain the rearrangements of genes
7 and 11 reported in this study.
The fact that for both genes 7 and 11 the 5' and 3' ends of the
mRNAs were predicted to form a long panhandle, resulting in
placing
face to face the two nucleotides involved in the rearrangement,
suggested that hot spots for gene rearrangement might also be
related
to such secondary structures. Although the actual existence
of
secondary structures predicted by computer modeling remains
to be
established, previous studies support an interaction between
the 3' and
5' ends of rotavirus mRNAs. The formation of a panhandle
resulting
from an interaction between the 5' and 3' ends and the
single-stranded
nature of at least the two 3'-terminal CC residues
have been reported
as structural constraints required for efficient
minus-strand RNA
synthesis (
7,
8,
45). The predicted secondary
structures
of gene 7 and 11 mRNAs reported here were consistent
with these
constraints. This led us to propose a mechanism for
gene rearrangement
in which secondary structures might facilitate
and direct the transfer
of the RNA polymerase from the 5' to the
neighboring 3' end of the
template during the replication step
(Fig.
7).
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|
FIG. 7.
Possible mechanism for rearrangements of rotavirus
genes. (a) During the replication step, the viral RNA polymerase
initiates the minus-strand synthesis at the 3' end of the plus-strand
RNA. (b) The RNA polymerase reaches nucleotide position x, in the
panhandle formed by the 5' and 3' ends of the RNA, and interrupts RNA
synthesis. (c) Without releasing the nascent minus strand, the RNA
polymerase falls back on its template at nucleotide y in the panhandle
and reinitiates replication. (d) The RNA polymerase completes the
replication up to the 5' end, thus duplicating the sequence x'-y'
(complementary to x-y). Since minus-strand RNAs were predicted to fold
quite differently from plus-strand RNAs (data not shown), a similar
model could not be proposed to explain the occurrence of rearrangement
at the transcription step. In addition, at the transcription stage, the
minus-strand RNA is associated with the plus strand and is probably not
subject to folding.
|
|
RNA recombination has been reported to occur by various mechanisms in a
number of RNA viruses (see reference
22 for a review).
Rotavirus genome rearrangements can be considered intramolecular
recombination events (
9) consistent with the copy choice
mechanism
described for poliovirus, in which the viral RNA polymerase
switches
templates during RNA minus-strand synthesis (
18,
22). Poliovirus
recombination is favored by the presence of
homologous sequences
in the neighborhood of the recombination site and
has been reported
to occur either at random (
18) or at
specific sites (
35,
43).
Indeed, regions of high local
secondary structure such as hairpins
and stem-loops have been reported
as recombinational hot spots
for poliovirus (
35,
43) or
foot-and-mouth disease virus (
46).
Similarly, secondary
structures in rotavirus mRNAs might correspond
to hot spots of
recombination, but, as opposed to the poliovirus
model, rotavirus
genome rearrangements should be considered to
be nonhomologous
recombination events occurring within the same
RNA template. What
initiates the template switch within the 5'-3'
panhandle of rotavirus
mRNA remains to be determined. Neighboring
nucleotides other than
those observed for genes 7R and 11R might
be used for rearrangement,
although some restrictions might exist
because only the viable
resulting viruses will be subsequently
selected, as suggested by Lai
for other RNA viruses (
22). For
instance, rearrangements
occuring upstream of the stop codon and
leading to a modified ORF might
not be
selected.
However, some of the rearrangements previously reported for other genes
11 (
12,
13,
20) or 7 (
27) for which the
nucleotides
involved in the rearrangement are not neighbors in the
predicted
secondary structures could not be directly explained by the
model
we propose. It is of interest that as opposed to genes 7R and
11R, these rearranged genes contain numerous mutations in the
duplicated part of the sequence. Such mutations may account for
a
genetic drift, indicating that after genome rearrangement, the
virus
underwent multiple replication cycles during which deletions
might also
have occurred, concealing the initial location of the
rearrangement.
This was the case for the rearranged gene 7R, which
underwent a
deletion during the cell culture propagation of virus
M1, leading to
gene 7R
, in which the nucleotides involved in
the initial
rearrangement could no longer be
identified.
In summary, the theoretical model we propose, implying the existence of
secondary structures between the 3' and 5' ends of
mRNAs, could
apply to some but not all examples of gene rearrangements.
However,
since rearranged genes seem to be likely to evolve rapidly
with time,
it should be considered that in addition to the initial
event of
rearrangement, a rearranged gene may be the result of
a multiple-step
process involving different mechanisms. Analyses
of additional
sequences of rotavirus gene rearrangements will
help us gain a better
understanding of this process. The finding
that the rearranged gene
7R
had a modified ORF confirms that
gene rearrangements not only
participate in the genetic variability
of rotaviruses but also can lead
to the synthesis of modified
proteins. In the absence of a reverse
genetic system for rotaviruses,
the isolation of nondefective
rotaviruses with rearranged genes
coding for modified nonstructural
proteins may be a means of specifying
some of their
functions.
| |
ACKNOWLEDGMENTS |
We are grateful to Anne-Marie Cassel-Béraud for providing
the clinical sample containing the rotavirus M isolate. We thank Jean
Cohen for helpful discussions and critical reading of the manuscript.
We thank Paul Dény for constructive comments. We thank Laurence
Albiges for excellent technical assistance and Christophe Goujon for
advice on computer modeling.
This work was supported in part by the MESRT grant Programme de
Recherches Fondamentales en Microbiologie, Maladies Infectieuses et
Parasitologie "Réseau de Recherche sur les
Gastro-Entérites à Rotavirus."
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Virologie, Hôpital Armand Trousseau, 26 Ave. du Dr. Arnold
Netter, 75571 Paris Cedex 12, France. Phone: 33 1 44 73 62 81. Fax: 33 1 44 73 62 88. E-mail: a.chenon{at}trs.ap-hop-paris.fr.
Present address: Laboratoire de Bactériologie, Virologie,
Hygiène, Hôpital Avicenne, UFR Santé, Médecine,
Biologie Humaine, Université Paris 13, 93009 Bobigny Cedex, France.
| |
REFERENCES |
| 1.
|
Allen, A. M., and U. Desselberger.
1985.
Reassortment of human rotaviruses carrying rearranged genomes with bovine rotavirus.
J. Gen. Virol.
66:2703-2714[Abstract/Free Full Text].
|
| 2.
|
Aponte, C.,
N. M. Mattion,
M. K. Estes,
A. Charpilienne, and J. Cohen.
1993.
Expression of two bovine rotavirus non-structural proteins (NSP2, NSP3) in the baculovirus system and production of monoclonal antibodies directed against the expressed proteins.
Arch. Virol.
133:85-95[CrossRef][Medline].
|
| 3.
|
Ballard, A.,
M. A. McCrae, and U. Desselberger.
1992.
Nucleotide sequences of normal and rearranged RNA segments 10 of human rotaviruses.
J. Gen. Virol.
73:633-638[Abstract/Free Full Text].
|
| 4.
|
Bellinzoni, R. C.,
N. M. Mattion,
O. Burrone,
A. Gonzalez,
J. L. La Torre, and E. A. Scodeller.
1987.
Isolation of group A swine rotaviruses displaying atypical electropherotypes.
J. Clin. Microbiol.
25:952-954[Abstract/Free Full Text].
|
| 5.
|
Besselaar, T. G.,
A. Rosenblatt, and A. H. Kidd.
1986.
Atypical rotavirus from South African neonates.
Arch. Virol.
87:327-330[CrossRef][Medline].
|
| 6.
|
Biryahwaho, B.,
F. Hundley, and U. Desselberger.
1987.
Bovine rotavirus with rearranged genome reassorts with human rotavirus.
Arch. Virol.
96:257-264[CrossRef][Medline].
|
| 7.
|
Chen, D., and J. T. Patton.
2000.
De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation.
RNA
6:1455-1467[Abstract].
|
| 8.
|
Chen, D., and J. T. Patton.
1998.
Rotavirus RNA replication requires a single-stranded 3' end for efficient minus-strand synthesis.
J. Virol.
72:7387-7396[Abstract/Free Full Text].
|
| 9.
|
Desselberger, U.
1996.
Genome rearrangements of rotaviruses.
Adv. Virus Res.
46:69-95[Medline].
|
| 10.
|
Estes, M.
1996.
Rotaviruses and their replication, p. 1625-1655.
In
B. Fields, D. Knipe, and P. Howley (ed.), Fields virology, 3rd ed. Raven Press, New York, N.Y.
|
| 11.
|
Garbarg-Chenon, A.,
F. Bricout, and J. C. Nicolas.
1984.
Study of genetic reassortment between two human rotaviruses.
Virology
139:358-365[CrossRef][Medline].
|
| 12.
|
Gonzalez, S. A.,
N. M. Mattion,
R. Bellinzoni, and O. R. Burrone.
1989.
Structure of rearranged genome segment 11 in two different rotavirus strains generated by a similar mechanism.
J. Gen. Virol.
70:1329-1336[Abstract/Free Full Text].
|
| 13.
|
Gorziglia, M.,
K. Nishikawa, and N. Fukuhara.
1989.
Evidence of duplication and deletion in super short segment 11 of rabbit rotavirus Alabama strain.
Virology
170:587-590[CrossRef][Medline].
|
| 14.
|
Graham, A.,
G. Kudesia,
A. M. Allen, and U. Desselberger.
1987.
Reassortment of human rotavirus possessing genome rearrangements with bovine rotavirus: evidence for host cell selection.
J. Gen. Virol.
68:115-122[Abstract/Free Full Text].
|
| 15.
|
Hua, J., and J. T. Patton.
1994.
The carboxyl-half of the rotavirus nonstructural protein NS53 (NSP1) is not required for virus replication.
Virology
198:567-576[CrossRef][Medline].
|
| 16.
|
Hundley, F.,
B. Biryahwaho,
M. Gow, and U. Desselberger.
1985.
Genome rearrangements of bovine rotavirus after serial passage at high multiplicity of infection.
Virology
143:88-103[CrossRef][Medline].
|
| 17.
|
Hundley, F.,
M. McIntyre,
B. Clark,
G. Beards,
D. Wood,
I. Chrystie, and U. Desselberger.
1987.
Heterogeneity of genome rearrangements in rotaviruses isolated from a chronically infected immunodeficient child.
J. Virol.
61:3365-3372[Abstract/Free Full Text].
|
| 18.
|
Kirkegaard, K., and D. Baltimore.
1986.
The mechanism of RNA recombination in poliovirus.
Cell
47:433-443[CrossRef][Medline].
|
| 19.
|
Kojima, K.,
K. Taniguchi,
M. Kawagishi-Kobayashi,
S. Matsuno, and S. Urasawa.
2000.
Rearrangement generated in double genes, NSP1 and NSP3, of viable progenies from a human rotavirus strain.
Virus Res.
67:163-171[CrossRef][Medline].
|
| 20.
|
Kojima, K.,
K. Taniguchi,
T. Urasawa, and S. Urasawa.
1996.
Sequence analysis of normal and rearranged nsp5 genes from human rotavirus strains isolated in nature implications for the occurrence of the rearrangement at the step of plus strand synthesis.
Virology
224:446-452[CrossRef][Medline].
|
| 21.
|
Kozak, M.
1996.
Interpreting cDNA sequences: some insights from studies on translation.
Mamm. Genome
7:563-574[CrossRef][Medline].
|
| 22.
|
Lai, M. M.
1992.
RNA recombination in animal and plant viruses.
Microbiol. Rev.
56:61-79[Abstract/Free Full Text].
|
| 23.
|
Lambden, P. R.,
S. J. Cooke,
E. O. Caul, and I. N. Clarke.
1992.
Cloning of noncultivatable human rotavirus by single primer amplification.
J. Virol.
66:1817-1822[Abstract/Free Full Text].
|
| 24.
|
Mathews, D. H.,
J. Sabina,
M. Zuker, and D. H. Turner.
1999.
Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure.
J. Mol. Biol.
288:911-940[CrossRef][Medline].
|
| 25.
|
Matsui, S. M.,
E. R. Mackow,
S. Matsuno,
P. S. Paul, and H. B. Greenberg.
1990.
Sequence analysis of gene 11 equivalents from "short" and "super short" strains of rotavirus.
J. Virol.
64:120-124[Abstract/Free Full Text].
|
| 26.
|
Mattion, N. M.,
J. Cohen,
C. Aponte, and M. K. Estes.
1992.
Characterization of an oligomerization domain and RNA-binding properties on rotavirus nonstructural protein NS34.
Virology
190:68-83[CrossRef][Medline].
|
| 27.
|
Mendez, E.,
C. F. Arias, and S. Lopez.
1992.
Genomic rearrangements in human rotavirus strain Wa; analysis of rearranged RNA segment 7.
Arch. Virol.
125:331-338[CrossRef][Medline].
|
| 28.
|
Palombo, E. A.,
H. C. Bugg, and R. F. Bishop.
1998.
Characterisation of rearranged NSP5 gene of a human rotavirus.
Acta Virol.
42:55-59[Medline].
|
| 29.
|
Patton, J. T.,
J. Chnaiderman, and E. Spencer.
1999.
Open reading frame in rotavirus mRNA specifically promotes synthesis of double-stranded RNA: template size also affects replication efficiency.
Virology
264:167-180[CrossRef][Medline].
|
| 30.
|
Pedley, S.,
F. Hundley,
I. Chrystie,
M. A. McCrae, and U. Desselberger.
1984.
The genomes of rotaviruses isolated from chronically infected immunodeficient children.
J. Gen. Virol.
65:1141-1150[Abstract/Free Full Text].
|
| 31.
|
Piron, M.,
T. Delaunay,
J. Grosclaude, and D. Poncet.
1999.
Identification of the RNA-binding, dimerization, and eIF4G1-binding domains of rotavirus nonstructural protein NSP3.
J. Virol.
73:5411-5421[Abstract/Free Full Text].
|
| 32.
|
Piron, M.,
P. Vende,
J. Cohen, and D. Poncet.
1998.
Rotavirus RNA-binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F.
EMBO J.
17:5811-5821[CrossRef][Medline].
|
| 33.
|
Pocock, D. H.
1987.
Isolation and characterization of two group A rotaviruses with unusual genome profiles.
J. Gen. Virol.
68:653-660[Abstract/Free Full Text].
|
| 34.
|
Rao, C. D.,
M. Das,
P. Ilango,
R. Lalwani,
B. S. Rao, and K. Gowda.
1995.
Comparative nucleotide and amino acid sequence analysis of the sequence-specific RNA-binding rotavirus nonstructural protein NSP3.
Virology
207:327-333[CrossRef][Medline].
|
| 35.
|
Romanova, L. I.,
V. M. Blinov,
E. A. Tolskaya,
E. G. Viktorova,
M. S. Kolesnikova,
E. A. Guseva, and V. I. Agol.
1986.
The primary structure of crossover regions of intertypic poliovirus recombinants: a model of recombination between RNA genomes.
Virology
155:202-213[CrossRef][Medline].
|
| 36.
|
Scott, G. E.,
O. Tarlow, and M. A. McCrae.
1989.
Detailed structural analysis of a genome rearrangement in bovine rotavirus.
Virus Res.
14:119-127[CrossRef][Medline].
|
| 37.
|
Serra, M. J., and D. H. Turner.
1995.
Predicting thermodynamic properties of RNA.
Methods Enzymol.
259:242-261[Medline].
|
| 38.
|
Shen, S.,
B. Burke, and U. Desselberger.
1994.
Rearrangement of the VP6 gene of a group A rotavirus in combination with a point mutation affecting trimer stability.
J. Virol.
68:1682-1688[Abstract/Free Full Text].
|
| 39.
|
Taniguchi, K., and S. Urasawa.
1995.
Diversity in rotavirus genomes.
Semin. Virol.
6:123-131.
|
| 40.
|
Tautz, D., and M. Renz.
1983.
An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels.
Anal. Biochem.
132:14-19[CrossRef][Medline].
|
| 41.
|
Thouless, M. E.,
R. F. DiGiacomo, and D. S. Neuman.
1986.
Isolation of two lapine rotaviruses: characterization of their subgroup, serotype and RNA electropherotypes.
Arch. Virol.
89:161-170[CrossRef][Medline].
|
| 42.
|
Tian, Y.,
O. Tarlow,
A. Ballard,
U. Desselberger, and M. A. McCrae.
1993.
Genomic concatemerization/deletion in rotaviruses: a new mechanism for generating rapid genetic change of potential epidemiological importance.
J. Virol.
67:6625-6632[Abstract/Free Full Text].
|
| 43.
|
Tolskaya, E. A.,
L. I. Romanova,
V. M. Blinov,
E. G. Viktorova,
A. N. Sinyakov,
M. S. Kolesnikova, and V. I. Agol.
1987.
Studies on the recombination between RNA genomes of poliovirus: the primary structure and nonrandom distribution of crossover regions in the genomes of intertypic poliovirus recombinants.
Virology
161:54-61[CrossRef][Medline].
|
| 44.
|
Walter, A. E.,
D. H. Turner,
J. Kim,
M. H. Lyttle,
P. Muller,
D. H. Mathews, and M. Zuker.
1994.
Coaxial stacking of helixes enhances binding of oligoribonucleotides and improves predictions of RNA folding.
Proc. Natl. Acad. Sci. USA
91:9218-9222[Abstract/Free Full Text].
|
| 45.
|
Wentz, M. J.,
J. T. Patton, and R. F. Ramig.
1996.
The 3'-terminal consensus sequence of rotavirus mRNA is the minimal promoter of negative-strand RNA synthesis.
J. Virol.
70:7833-7841[Abstract].
|
| 46.
|
Wilson, V.,
P. Taylor, and U. Desselberger.
1988.
Crossover regions in foot-and-mouth disease virus (FMDV) recombinants correspond to regions of high local secondary structure.
Arch. Virol.
102:131-139[CrossRef][Medline].
|
| 47.
|
Zuker, M.,
D. H. Mathews, and D. H. Turner.
1999.
Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide, p. 11-43.
In
J. Barciszewski, and B. F. C. Clark (ed.), RNA biochemistry and biotechnology. Kluwer Academic NATO ASI, Dordrecht, The Netherlands.
|
Journal of Virology, August 2001, p. 7305-7314, Vol. 75, No. 16
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.16.7305-7314.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
