Virus Research 151 (2010) 109–117

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Virus Research
journal homepage: www.elsevier.com/locate/virusres

Review

Insights into the role of the non-structural protein 2 (NS2) in

Bluetongue virus morphogenesis
Carmen Butan a,∗ , Paul Tucker b
a
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
b
EMBL Hamburg Outstation, c/o DESY, D-22603 Hamburg, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Bluetongue virus (BTV) non-structural protein 2 (NS2) belongs to a class of highly conserved proteins
Received 23 January 2010 found in Orbiviruses of the Reoviridae family. NS2 forms large multimeric complexes and localizes
Received in revised form 25 May 2010 to cytoplasmic inclusions in infected cells. Due to its ability to bind single-stranded RNA (ssRNA), it
Accepted 27 May 2010
has been suggested that the protein participates in the selection and sequestration of the 10 differ-
Available online 4 June 2010
ent BTV-ssRNA segments, prior to their encapsidation and conversion into the BTV double-stranded
RNA (dsRNA) genome. Recent advances in understanding how BTV-NS2 is organized and functions are
Keywords:
largely inferred from structural studies. The X-ray crystal structure of the N-terminal domain of NS2
Non-structural protein 2 (NS2)
Bluetongue virus (BTV)
suggests that the full-length protein could assemble as homomultimers of maximally 10–11 subunits.
Viral inclusion bodies (VIBs) The crystallographic structural information combined with small-angle X-ray scattering experiments on
the C-terminal domain as well as negative-stain electron microscopy on the full-length protein give us a
first glimpse of how the two protein domains associate and function.
Herein, we survey biochemical and recent structural investigations on NS2 important to the under-
standing of the molecular events underlying the process of BTV morphogenesis. We also present a
phylogenetic analysis of the NS2 sequences.
© 2010 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
2. NS2 detected in association with cytoplasmic viral “factories” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
3. Oligomeric characteristics of NS2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
4. Detection of RNA-binding activity of NS2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
5. Functional significance of NS2 phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
6. NS2 enzymatic activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
7. The structure of NS2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
8. The NS2 protein family – sequence and biochemical comparisons of several Orbivirus NS2 proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
9. Similarities of NS2 with the non-structural proteins of other Reoviridae genuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
10. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

1. Introduction
which includes as other family members, viruses of the Orthore-
Bluetongue virus is the prototype of the genus Orbivirus in ovirus and Rotavirus genera.
the Reoviridae family of segmented double-stranded RNA viruses, BTV was initially identified in South Africa, and it has since
spread into other regions of world including Europe, the Middle
East, the South Pacific, North and South America, and parts of Asia.
∗ Corresponding author. Tel.: +1 617 432 4971. Based on serological neutralization of virus particles, there are 25
E-mail address: butan@crystal.harvard.edu (C. Butan). different serotypes identified worldwide. The virus is the causative

0168-1702/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.virusres.2010.05.014
110 C. Butan, P. Tucker / Virus Research 151 (2010) 109–117
agent of bluetongue disease in livestock, which has a high mortal-
ity rate. It is transmitted to its hosts by the bites of midges of the
Culicoides genus.
The infective virion consists of three concentric protein lay-
ers: an outer protein layer composed of structural proteins VP2
and VP5, which surrounds an icosahedral double-layered core con-
taining two major proteins, VP7 and VP3 (Grimes et al., 1998;
Stuart et al., 1998; Stuart and Grimes, 2006). Variations in the outer
layer proteins, especially VP2, determine the virus serotype. The
most inner shell composed of VP3 organizes three minor enzy-
matic proteins, the viral transcriptase complexes: VP1-polymerase,
VP4-capping enzyme, and VP6-helicase, and 10 distinct segments
(S1–S10) of linear, variably sized double-stranded RNA (Roy and
Noad, 2006; Stuart and Grimes, 2006). Three segments, S8–S10,
of the BTV genome encode non-structural proteins (NS1, NS2, and
NS3/NS3A) that are produced in the host cells at different stages
of virus infection. In contrast to the structural proteins (VP1–VP7),
the non-structural proteins (NS1–NS3) have been less well charac-
terized.
The mechanism by which precisely one copy of each of the BTV
viral segments is selectively recruited from the cytoplasmic milieu
of the infected cell and incorporated into the newly assembled viri-
ons remains largely unknown. Biochemical evidence showing that
BTV-NS2 could be associated with the core proteins (VP1, VP3, VP4,
VP6 and VP7) (Kar et al., 2007, 2005; Modrof et al., 2005) as well as
a specific binding towards BTV-ssRNA transcripts (Lymperopoulos
et al., 2006, 2003), are consistent with the idea that NS2 may play
a central role in the selection of BTV messenger RNA (mRNA) seg-
ments for incorporation into the progeny virions and subsequently
for genome replication inside BTV cores.
The aim of this review is to highlight current knowledge on the
structure and on the diverse functions of NS2 relevant to the emerg-
ing role of this protein as a key player in the early morphogenetic
events of BTV assembly.
2. NS2 detected in association with cytoplasmic viral
“factories”
NS2 is regarded as a key player in Bluetongue virus morpho-
genesis because its presence in BTV-infected mammalian cells is
associated with large, electron-dense perinuclear structures, called
‘virus assembly factories’ or ‘viral inclusions bodies’ (VIBs) (Brookes
et al., 1993; Eaton et al., 1990, 1987). The perinuclear inclusion
bodies are the sites in which viral replication and assembly are
thought to occur. The 41 kDa NS2 protein was identified as being
synthesized in large amounts throughout the replication cycle of
BTV. The synthesis can be identified in the first 2–4 h after infection
and is found in large amounts between 12 and 20 h after infection
(Huismans et al., 1987). Immunogold labeling of NS2 antibody and
electron micrographs of infected BHK-21 mammalian cells have
demonstrated that NS2 is associated only with VIBs and not with
virions (Hyatt and Eaton, 1988; Thomas et al., 1990).
NS2 has been successfully expressed in a multitude of hosts,
ranging from bacteria (Taraporewala et al., 2001; Theron et al.,
1994) to mammalian cells (Kar et al., 2007). In insect cells, recombi-
nant baculovirus expressed NS2 has been shown to accumulate as
multimers forming inclusion bodies (IBs) not unlike those observed
in BTV-infected mammalian cells and when NS2 is synthesized
independently of any BTV-encoded proteins the inclusion bodies
are still formed (Thomas et al., 1990), suggesting that the NS2 pro-
teins are the minimal components required to form viral inclusions
(Brookes et al., 1993). A recent study (Kar et al., 2007) concluded
that NS2 of IBs was able to recruit VP3 but not VP7, while the
NS2 proteins of VIBs were able to recruit both inner core pro-
teins, VP3 and VP7, as well as BTV transcripts (ssRNA/mRNA) to
initiate viral genome replication and core assembly. While the
full-length protein was able to generate inclusion bodies resem-
bling VIBs by itself, the capability of amino- (1–115, 1–237) and
carboxyl- (116–357, 238–357) terminal fragments of NS2 to form
inclusion bodies was highly impaired. Also, N-terminal (1–115) and
C-terminal (116–357) deletion mutants expressed in mammalian
cells, which have been infected with BTV, affected the overall virus
replication (Kar et al., 2007).
Viruses are believed to use different cytoskeletal components,
like the microtubule cytoskeleton, for trafficking to the replication
sites inside infected cells or for release from the host cell (Greber
and Way, 2006). By contrast, Kar et al. (2007) reported that a pertur-
bation of the cellular microtubule network, by using microtubule
depolymerizing agents, had no impact on the structure and assem-
bly of IBs in NS2 expressing cells or on VIBs in BTV-infected cells,
suggesting that microtubules are not required for the functional
organization of viral inclusion bodies (Eaton et al., 1987). Moreover,
immunofluorescence microscopy studies with antibodies specific
to NS2 and the intermediate filament vimentin have shown that
the morphogenesis of BTV-VIBs or NS2-IBs (Kar et al., 2007) was
not dependent on the vimentin network. In this context, it is inter-
esting to note that the NS1 protein, the most abundant BTV protein
synthesized in infected cells, forms tubular structures (Huismans
and Els, 1979) that accumulate in bundles similar to the cytoskele-
tal intermediate filaments; however, to date there is no data that
would support for a role of NS1 tubules in BTV trafficking.
3. Oligomeric characteristics of NS2
NS2 exists in heterogeneous oligomeric states. When purified
from the soluble fraction of disrupted recombinant baculovirus
infected cells which were treated with RNase A, NS2 was detected
as ∼7S homomultimers (Uitenweerde et al., 1995). This result is
similar to that obtained by Taraporewala et al. (2001) who further
investigated the quaternary structure of purified NS2, which was
expressed in bacteria with a His-tag at its C-terminus. Sedimenta-
tion through linear sucrose gradients of the bacterially expressed
NS2 followed by SDS-PAGE and Comassie Blue staining of the gra-
dient fractions revealed that the recombinant protein formed large
complexes sedimenting between 18 and 22S. If the protein crude
extract was treated with RNase prior to sedimentation experi-
ments, the protein sedimented between 8 and 10S, and it was
therefore concluded that most of the recombinant protein was
complexed with bacterial RNA (Taraporewala et al., 2001). Based
on the protein molecular mass and sedimentation coefficient, the
8–10S multimer has a molecular mass between 140 and 250 kDa
and assembles from 6 ± 2 subunits (Taraporewala et al., 2001).
4. Detection of RNA-binding activity of NS2
Several studies have linked NS2 with a function in viral mRNA
selection and condensation. The non-specific affinity of NS2 for
ssRNA was initially implied from experiments in which partially
purified protein extracts from mammalian infected cells containing
NS2 were mixed with BTV-mRNA and analyzed by zonal centrifu-
gation in sucrose gradients. NS2 bound the purified BTV-mRNA to
form a complex of ∼22S (Huismans et al., 1987).
Recombinant NS2 and other ssRNA species (Thomas et al., 1990)
have been shown to interact in vitro in a sequence independent
manner indicating that the binding motif of the ssRNA is non-
specific. Uitenweerde et al. (1995) examined the RNA-binding
affinity of bacterially expressed protein and reported that sucrose
gradient purified NS2 multimers exhibited poly (U)-Sepharose
binding; subsequent investigations by Taraporewala et al. (2001)
reported that Escherichia coli (E. coli) expressed NS2 became radio-
 C. Butan, P. Tucker / Virus Research 151 (2010) 109–117
labeled when exposed to UV light in the presence of phosphate
labeled Rotavirus gene 8 ssRNA. In reactions in which the ratio
recombinant NS2:labeled RNA was 80:1, no free RNA was observed
on a non-denaturating 1% agarose gel, which indicates that mul-
timeric NS2 protein species were required for the RNA binding
(Taraporewala et al., 2001).
Through a series of deletion experiments, three regions within
the NS2 amino acid sequence were shown to be very impor-
tant for the binding of ssRNA in vitro (Fillmore et al., 2002).
These ssRNA binding sites were located at residue positions 2–11
(2–11 ), residue positions 153–166 (153–166 ), and residue posi-
tions 274–286 (274–286 ). Deletion constructs, spanning these
regions, showed significant differences in their abilities to bind to a
ssRNA size ladder and yeast ssRNA, as assessed by electrophoretic
mobility shift assays (EMSA); single region deletions resulted in a
protein that still bound ssRNA but with a weaker affinity than the
intact one. The 153–166 mutant exhibited a slightly greater affin-
ity towards the ssRNA size markers than the 274–286 or 2–11
mutants. The proteins with double-deletions (153–166 274–286 ,
2–11 153–166 , 2–11 274–286 ) displayed poorer binding to ssRNA
than the single region-deletion proteins and the 153-166 274-286
protein displayed a reduced ability to form RNA–protein complexes
in comparison to the 2–11 153–166 or 2–11 274–286 mutants.
These data suggest that sequential domains of NS2 bind ssRNA in
a cooperative manner and pose the question as to whether one
molecule of NS2 simultaneously interacts with multiple ssRNAs, or
whether the full-length protein binds with all three regions to one
ssRNA.
Contrary to the results obtained in earlier work, more recent
research has investigated the possibility of a specific interaction
between NS2 and BTV-RNA segments. Cross-linking studies using
radio-labeled viral RNA and NS2 produced in mammalian infected
cells (Theron and Nel, 1997) as well as in vitro studies using purified
baculovirus expressed protein concluded that NS2 bound prefer-
entially to the BTV-RNA (Lymperopoulos et al., 2006, 2003). The
specificity of interactions between NS2 and four BTV-segments
was investigated. A stem-loop structure between nucleotides 99
and 169 of BTV S10 plus-strand (+)RNA (Lymperopoulos et al.,
2003) was identified to be the interacting region with the protein.
Mutations affecting this structure resulted in a reduced bind-
ing affinity of NS2 towards S10(+)RNA; however, compensatory
mutations within the stem-loop sequence were able to restore
wild-type S10(+)RNA–NS2 binding capabilities. These data indicate
that the NS2–RNA interaction is attributed to the RNA structure and
not to its primary sequence (Lymperopoulos et al., 2006), which
would require that the identification sequences at 5 and 3 ter-
mini of each genomic segment (Markotter et al., 2004) have another
function. RNA structure mapping carried out on three other BTV-
segments (S9, S8 and M6) failed to identify obvious similarity to the
S10(+)RNA stem-loop structure. A subgenomic fragment between
nucleotides 29 and 127 towards the 5 -end of S9(+)RNA was sug-
gested as the recognition site by NS2. In the case of S8, the RNA
secondary structure between nucleotides 736 and 818 towards the
3 -end of RNA was mapped as the interaction region with NS2. In
M6, which is representative for the medium size BTV-RNA seg-
ments, the NS2 interacting region was located in the middle of the
RNA sequence spanning from nucleotide 1163 to nucleotide 1458
(Lymperopoulos et al., 2006). The fact that more than one type of
structure was recognized by NS2 in the BTV-RNA segments and that
S10(+)RNA was poorly competing for binding of NS2 with S9(+)RNA
(Lymperopoulos et al., 2006) could suggest that the structural inter-
actions between NS2 and the different BTV-ssRNA segments are
independent events.
While the above results could lead to proposals for a role of NS2
in the sorting and packaging of BTV-ssRNA segments, the ques-
tion of whether these recognition strategies of the BTV genome
111
segments by NS2 exist in the infected cells remains open, and in
vivo investigations to support this role are further required. In this
context, we note a study (Taraporewala et al., 2002) which used
a Rotavirus mutant to define roles in the viral genome selection
and packaging for the non-structural protein 2 (NSP2), which is a
ssRNA binding protein and a main component of large cytoplas-
mic inclusion structures (viroplasms) in Rotavirus infected cells. In
this respect, Rotavirus-NSP2 is a possible functional equivalent of
BTV-NS2. In cells infected at a non-permissive temperature with a
Rotavirus mutant, tsE(1400), which contains a temperature sensi-
tive lesion in the NSP2 gene, the viroplasms and the replication
intermediates engaged in dsRNA synthesis were fewer and the
progeny virions were mostly empty (Taraporewala et al., 2002).
On the basis of the specific interactions between NS2 and BTV-
RNA segments, as observed in vitro, a novel paradigm for how
segmented dsRNA viruses package their genome may emerge. The
tentative strategy for the recognition of the BTV genome segments,
based on structure rather than sequence, differs from that of the
better-studied dsRNA bacteriophage phi6, a member of the Cys-
toviridae family, which has a genome of only three dsRNA segments
(Bamford et al., 2002). Three plus-sense ssRNA phi6 segments are
packaged sequentially in a procapsid and involves recognition of
sequence signals located within the 5 non-coding regions of the
ssRNA segments likely by the protein P1, the major structural pro-
tein of the phi6 procapsid (Mindich, 2004).
5. Functional significance of NS2 phosphorylation
A multitude of viral proteins have been reported to be phos-
phorylated, however the actual number of viral non-structural
phosphorylated proteins is much smaller. Examples in this later
category include the Rotavirus non-structural proteins NSP2
(Taraporewala et al., 1999) and NSP5 (Eichwald et al., 2002), which
colocalize in the viroplasms of the infected cells (Fabbretti et al.,
1999). NSP2 undergos autophosphorylation (Taraporewala et al.,
1999) and NSP5 exists in multiple phosphorylated isoforms during
the viral infection (Afrikanova et al., 1996; Welch et al., 1989).
NS2 is the only BTV protein that undergoes phosphorylation.
The phosphorylated mammalian derived NS2 could be resolved as
two closely separated bands on an SDS-PAGE gel and could be iden-
tified by autoradiography (Huismans et al., 1987). An early study
by Devaney et al. (1988) reported that serine residues were the
phosphorylated amino acids in BTV-17 NS2 hydrolysates. Like NS2
isolated from BTV-10 infected mammalian cells, NS2 expressed
in insect cells is post-translationally modified, as the phospho-
rylated forms of NS2 could be detected (Thomas et al., 1990).
Moreover, both NS2 derived from mammalian infected cells and
NS2 expressed in insect cells share the same phosphorylated pep-
tides with the sites of phosphorylation, Ser 249 and 259, located
within the C-terminal segment of the protein (Modrof et al., 2005;
Mumtsidu et al., 2007). There are several plausible hypotheses to
explain the NS2 phosphorylation: (i) the protein could have an
autokinase activity, (ii) another viral protein could phosphorylate
NS2, or (iii) kinases present in the cytoplasm of virus-infected cells
could phosphorylate NS2 (Huismans et al., 1987; Taraporewala et
al., 2001; Theron et al., 1994). Expression of NS2 in E. coli as a
fusion to GST provided the first indication that the protein does
not have an autokinase activity (Theron et al., 1994). Additional
evidence for the absence of any autokinase activity associated with
NS2 was provided when bacterially expressed protein was incu-
bated in kinase buffer containing [␥-32 P] labeled ATP and MgCl2 .
When the protein reaction mixture was resolved by SDS-PAGE and
analyzed by autoradiography, the autoradiograms of the SDS-PAGE
gels showed no phosphate-labeled protein bands (Taraporewala
et al., 2001). It is unlikely that NS2 would be phosphorylated via
112 C. Butan, P. Tucker / Virus Research 151 (2010) 109–117
another viral protein, therefore NS2 ought to be substrate for phos-
phorylation by cellular kinases. Indeed, Taraporewala et al. (2001)
reported that the unphosphorylated recombinant NS2 expressed
in E. coli could be phosphorylated by cellular kinases when mixed
with a cytoplasmic extract from uninfected mammalian cells (fetal
rhesus monkey kidney). The type of the protein kinase responsi-
ble for the NS2 phosphorylation has been recently identified as
the ubiquitous casein kinase 2 (CK2). Using immunofluorescence
analysis and confocal microscopy the intracelullar colocalization of
NS2 with CK2 has been visualized in vivo in BHK-21 cells (Modrof
et al., 2005). This was further confirmed using in vitro pull-down
assays. Wild-type and NS2 mutants, expressed as fusion proteins
with an S-peptide, were incubated with purified CK2 in the pres-
ence of [␥-32 P] labeled ATP. CK2 phosphorylated NS2 and the singly
mutated NS2 (NS2S249A and NS2S259A ), but not the doubly mutated
NS2S249AS259A . In the presence of myricetin, a specific CK2 inhibitor,
NS2 phosphorylation did not occur (Modrof et al., 2005).
The phosphorylation state of BTV-NS2 does not affect its bind-
ing to ssRNA (Modrof et al., 2005). Interactions of wild-type and
two NS2 mutant proteins, NS2S249AS259A (a construct which is
no longer phosphorylated) and NS2S249DS259D (a mutation that is
often used to mimic the phosphorylated state of the protein), with
32 P-labeled BTV-ssRNA were studied using EMSA assays. In the
presence of specific or non-specific unlabeled ssRNA competitors,
the electrophoretic shift patterns of NS2-32 P-labeled BTV-ssRNA
complexes were indistinguishable from each other (Modrof et al.,
2005).
The presence of ssRNA is reported to trigger and enhance
the oligomerization state of phosphorylated BTV-NS2 (Modrof et
al., 2005). When wild-type protein as well as the unphosphory-
lated construct, NS2S249AS259A , both carrying a FLAG epitope at
their C-terminus, were co-immunoprecipitated by their respective
untagged proteins in the presence of RNA, the amount of untagged
protein that sedimented during the co-immunoprecipiation inter-
actions was noticeably lower in the case of the unphosphorylated
versus phosphorylated protein complexes. This suggests that the
wild-type phosphorylated complexes were bigger in size or more
stable than the mutant unphosphorylated ones. However, in the
presence of an RNase A treatment, no significant difference in the
level of interactions between the phosphorylated versus unphos-
phorylated complexes was found. Since the RNA-binding activity of
the BTV-NS2 is not dependent on its phosphorylation status, these
results support a model in which the oligomerization of phospho-
rylated NS2 proteins might be triggered by the presence of RNA
molecules (Modrof et al., 2005).
Notably, NS2 phosphorylation is essential for the protein func-
tion in virus assembly and replication. The unphosphorylated
FLAG-tagged NS2S249AS259A construct altered the formation of viral
inclusion bodies in BHK cells that were initially transfected with
the protein mutant and 24 h later infected with BTV-10, which
would imply that VIBs formation is NS2 phosphorylation depen-
dent (Modrof et al., 2005).
6. NS2 enzymatic activities
Purified bacterially expressed NS2 protein could bind to
nucleotides and non-specifically hydrolyse the ␣, ␤, and ␥ phos-
phodiester bonds of nucleotide triphosphates (NTPs) to their
corresponding nucleotide monophosphates (NMPs) (Taraporewala
et al., 2001). From the four substrates, purine and pyrimidine, [␣-
32 P] labeled NTPs, recombinant NS2 displayed higher efficiency
for the hydrolysis of ATP as compared with the other nucleotides
for which NS2 has a preference in the order GTP > UTP/CTP.
Taraporewala et al. (2001) reported that the phosphatase activity
of recombinant NS2 could be inhibited in vitro by the presence of
the unlabeled competitor ATP over [␣-32 P] labeled ATP. In earlier
studies (Horscroft and Roy, 2000), the ATP as well as GTP hydrolysis
by NS2 were found to be dependent on divalent ions, such as Ca2+ ,
Mg2+ , Mn2+ . Furthermore, ouabain, which is an inhibitor of cellular
ATPases, did not abolish the ATPase activity of NS2 (Horscroft and
Roy, 2000).
BTV genome transportation and packaging are energy demand-
ing processes in which the NTP hydrolysis by NS2 may provide the
required energy. In this respect, NS2 may act like a molecular pack-
aging engine in a manner similar to the hexameric ATPase P4 of
dsRNA bacteriophage phi12 (Mancini et al., 2004). It is also possi-
ble that the nucleotidyl phosphatase activity of NS2 might fuel the
unwinding of the recognizable loop structures of the BTV-ssRNA
substrates, prior to their packaging, in a function similar to that
of an RNA helicase (Yao et al., 1997). The regions of NS2 responsi-
ble for the nucleotidyl phosphatase activity are currently unclear,
although a construct, which lacked the first 177 amino acids at
the N-terminus, was still able to hydrolyse NTPs (Mumtsidu et al.,
2007).
7. The structure of NS2
High-resolution structural information is difficult to obtain for
the Orbivirus NS1 and NS2 proteins because of their tendency to
form higher oligomers of variable size. Electron microscopy has
been used extensively to localize NS2 in infected cells (Brookes et
al., 1993; Eaton et al., 1990; Huismans et al., 1987) but studies aimed
at elucidating the structure of NS2 oligomers by higher resolution
imaging in VIBs have, to the best of our knowledge, not been per-
formed. Mumtsidu et al. (2007) showed electron micrographs of a
recombinant protein with an 8-residue insertion between the N-
and C-terminal domains. The images showed a number of donut-
like structures with an external diameter of around 100 Å and
an internal hole with a diameter of around 20 Å. It is uncertain
how representative these structures are of the in vivo situation for
three reasons. Firstly this protein, being expressed in E. coli, is not
phosphorylated, secondly the effect of the insertion on the struc-
ture, although not likely to be significant, is unknown and thirdly,
although the preparation was treated with RNase 1, it is impossible
to be sure that all RNA is removed.
The high-resolution structure of the 182 amino acid N-terminal
domain of NS2 from BTV has been reported (Butan et al., 2004)
(Fig. 1A). The crystal is composed of infinite chains of the pro-
tein domain that form a spiral. Suggestively the spiral (Fig. 1B)
has the approximate outer dimensions of the donut-like structures
observed in the EM images of the (modified) full-length protein.
The structure (Fig. 1A) is a ␤-sandwich. Two extensive interfaces
are described, one of which involves the extension of the ␤-sheet
and the second that involves an arm that links one protein molecule
to the next, thus generating oligomers that span the crystal. The
residues that from mutational work (Fillmore et al., 2002) are
implicated in RNA binding are in disordered regions at the C- and N-
termini of the X-ray solved domain but can, nonetheless, be located
on the inside of the spiral structure. The N-terminal domain, when
expressed alone is insoluble and consequently RNA-binding exper-
iments are not possible. There are no reported attempts to produce
soluble constructs of this domain which ought to be possible based
on the structural information.
No high-resolution structure of the C-terminal domain of NS2
is available, however Mumtsidu et al. (2007) reported a low-
resolution envelope (Fig. 2A) obtained from small-angle X-ray
scattering measurements (SAXS) of the carboxyl-terminal segment
(residues 178–354) of NS2. Size exclusion chromatography and
the SAXS work indicated this domain to be dimeric in solution.
These authors went on to construct a model of the full-length pro-
 C. Butan, P. Tucker / Virus Research 151 (2010) 109–117
Fig. 1. (A) A ribbon representation of three molecules of the N-terminal domain
of NS2, the central one being colored blue through red from N- to C-terminus. Two
other domains are colored light green and magenta. (B) Two surface representations,
with underlying ribbon representation, of a decamer of the spiral formed by N-
terminal domains (residues 8–160) in the crystal structure (Butan et al., 2004). Two
distinct twofold axes relate the monomers. These are between the white and pink
molecules and between the pink and cyan molecules.
tein (Fig. 2B) assuming that the C-terminal domain must reside
inside the torus formed by the N-terminal domain, that the twofold
of the C-terminal dimer would coincide with a twofold of the
N-terminal domain, and that the domains would not change con-
formation substantially in the full-length protein. The second and
third assumptions are clearly no more than reasonable hypothe-
ses. However speculative, the model does match the diameter of
the central hole observed in the EM images. It also has some pre-
dictive power in that the RNA is required to be bound between the
two protein domains.
The structural work throws little light on the proposed catalytic
activity of NS2 since the N-terminal domain contains no recogniz-
able part of an NTPase. It is intriguing to note that EM images of the
recombinant molecule with the insertion between domains in the
presence of the non-hydrolysable ATP analog ATP-␥-S show donut
structures with the same external diameter but a central aperture
reduced in size. The implication is that there is a conformational
change in the C-terminal domain upon ATP binding, but does not
preclude both domains being involved in nucleotidyl phosphatase
activity.
Fig. 2. (A) The low-resolution shape of the C-terminal NS2 dimer. (B) The N-terminal
dodecamer with four molecules shown in surface representation (left) showing how
the C-terminal dimer may be docked into this protein surface (right) (Mumtsidu et
al., 2007).
113
8. The NS2 protein family – sequence and biochemical
comparisons of several Orbivirus NS2 proteins
NS2 is highly conserved within the BTV strains and there
is a significant degree of sequence conservation between the
NS2 proteins from BTV strains and the NS2 proteins from
diverse groups of Orbiviruses (Epizootic Hemorrhagic Disease Virus
(EHDV), African Horsesickness Virus (AHSV), Peruvian Horsesick-
ness Virus (PHSV), Chuzan virus, and Yunnan virus). Sequence
data for 38 Orbivirus NS2 proteins, pulled out from the Swis-
sProt/GenBank/NCBI databases, were aligned using the program
Multalin (http://bioinfo.genotoul.fr/multalin/multalin.html). Mul-
tiple alignments reveal a high degree of conservation throughout
the protein sequence with the highest amino acid conservation
clustering in the N-terminal domain of the protein (Fig. 3). The con-
served residues, particularly those with high level of similarity and
identity greater than 90%, have been mapped on the spiral surface
generated from the protein N-terminal domain solved structure
(Fig. 4). We carried out a phylogenetic analysis of the NS2 sequences
using the neighbor-joining method and found that NS2 from Yun-
nan virus forms a distinct group with PHSV-NS2, whereas NS2 from
Chuzan virus is most closely related to AHSV-NS2 proteins. NS2
proteins from EHDV of different serotypes grouped together in a
separate cluster (Fig. 5).
Sedimentation analysis and non-reducing SDS-PAGE indicated
that like NS2 from BTV, NS2 proteins from EHDV, and AHSV are
homomultimers of 7S consisting of six or more NS2 monomers
(Theron et al., 1994). Both BTV and AHSV-NS2 have been shown to
be phosphoproteins in vivo (Devaney et al., 1988; Huismans et al.,
1987). By contrast, NS2 from EHDV appeared not to be phophory-
lated when over-expressed in insect cells using a recombinant
baculovirus (Theron et al., 1994). However, the unphosphorylated
form of EHDV-NS2 expressed in bacteria as a GST fusion protein
could be phosphorylated when mixed with a cytoplasmic extract
prepared from uninfected insect cells (Theron et al., 1994). The
kinase associated with the cytoplasm of uninfected Spodoptera
frugiperda cells, presumably CK2 (Modrof et al., 2005), showed
specificity for the fused protein (Theron et al., 1994). Both BTV-
NS2 and AHSV-NS2 are phosphorylated only at serine residues
(Devaney et al., 1988; Modrof et al., 2005; Mumtsidu et al., 2007;
Thomas et al., 1990). Theron et al. (1994) reported that it is likely
that the phosphorylation site of EHDV-NS2 is a serine rather than a
threonine residue. Analysis of sequence conservation of NS2 from
different Orbiviruses indicates that Ser249 is at least 80% identical
in BTV-NS2 sequences and conserved in all AHSV and EHDV-NS2
analyzed sequences but not in PHSV-NS2. The second phospho-
rylation site, Ser259, is less conserved among Orbiviruses: it is
conserved within all BTV-NS2 analyzed sequences but not in the
other Orbivirus NS2 sequences (Fig. 3).
BTV-NS2 and homologues from EHDV and AHSV have a high
content of charged residues. Theoretical estimates of charge as a
function of pH, from the protein primary sequences, indicate differ-
ent charge distributions between these NS2 proteins. Variation in
the NS2 surface charge, to which phosphorylation also contributes,
may explain the different binding affinities of the NS2 proteins
towards ssRNA. Unlike NS2 from BTV, whose phosphorylation in
vitro had no impact on the ssRNA binding ability of the protein,
the phosphorylated EHDV-NS2 protein was significantly less effi-
cient in binding ssRNA than the unphosphorylated form over a wide
range of ionic strengths (Theron et al., 1994). ssRNA binding of NS2
tested over a broad range of salt concentrations indicated that there
is a difference in the ability of NS2 of BTV, EHDV, and AHSV to bind
non-specifically to ssRNA. Irrespective of whether an artificial RNA
substrate or viral mRNA was used, at physiological salt concentra-
tions, BTV-NS2 bound ssRNA very efficiently, AHSV-NS2 only had a
low efficiency for binding ssRNA, whereas EHDV-NS2 ssRNA bind-
114 C. Butan, P. Tucker / Virus Research 151 (2010) 109–117

ing was between those of AHSV-NS2 and BTV-NS2 (Uitenweerde et

al., 1995).

9. Similarities of NS2 with the non-structural proteins of

other Reoviridae genuses

BTV-NS2 shares a number of functional similarities with non-

structural proteins of other Reoviridae members. As already
mentioned in the previous paragraphs, the NSP2-NSP5 protein
pair plays a major role in the formation and structure of the
viroplasms in the Rotavirus infected cells and is a possible func-
tional homologue of BTV-NS2. NSP2 (35 kDa) self-assembles into

Fig. 3. Sequence alignment of 28 BTV-NS2 protein sequences, representing 13

serotypes, and 10 other Orbivirus NS2 protein sequences from Epizootic Hem-
Fig. 3. (Continued ).
orrhagic Disease Virus (EHDV), African Horsesickness Virus (AHSV), Peruvian
Horsesickness Virus (PHSV), Chuzan virus, and Yunnan virus colored by residue con-
servation. Absolutely conserved residues are shaded red, and residues conserved in
at least 50% of all sequences are colored red. Green asterisks mark the location of the
phosphorylation sites and a green line underlines the CK2 recognition site. For the
accession codes of the above protein sequences see the Supplementary information.
 C. Butan, P. Tucker / Virus Research 151 (2010) 109–117
Fig. 4. Mapping of residue conservation on two representations (A and B) of a decamer surface of the crystallographic spiral generated from the solved structure of the NS2
amino-terminal domain. The color code is as follows: fully conserved residues are colored in magenta, regions of sequence conservation greater than 90% are colored in
green, and residues which are similar and conserved in at least 50% of the sequences are colored in cyan.
octameric structures (Taraporewala et al., 2002) and like BTV-NS2
has non-specific ssRNA binding activity, forming large RNA-protein
complexes. Unlike BTV-NS2, which has nucleotidyl phosphatase
activity, NSP2 has only an NTPase activity (Taraporewala et al.,
1999). In addition, NSP2 has RTPase and NDP-kinase activities
(Kumar et al., 2007; Vasquez-Del Carpio et al., 2006). NS2 and
NSP2 have some similarity to single-stranded DNA binding proteins
in that they have Mg2+ and ATP independent helix-destabilizing
activity (Taraporewala et al., 2001), but this is unlikely to be sig-
nificant in vivo for proteins located in the host cytoplasm. The
NSP2 octamer has been solved by X-ray crystallography (Jayaram
et al., 2002) and no structural similarity with the X-ray structure
of the N-terminal one-half residues of NS2 has been detected. The
structure of monomeric NSP2 reveals two domains separated by a
deep cleft, which is predicted to be the active site for the NTPase
and NDP-kinase activities. Four basic grooves, next to the catalytic
sites of the NSP2 crystallographic octamer, may correspond to the
Fig. 5. A phylogenetic tree for all 38 NS2 sequences was built using the
neighbor-joining algorithm as implemented in the software package PHYLIP
(http://evolution.genetics.washington.edu/phylip.html). The phylogenetic tree fig-
ure was prepared using http://tree.bio.ed.ac.uk/software/figtree/. The tree has a
midpoint rooting, an increasing order of the nodes, and a cladogram representa-
tion of the branches. Numbers are assigned to the different nodes and the scale bar
represents 0.3 amino acid substitution per site.
115
ssRNA and NSP5 binding sites (Jiang et al., 2006). While the func-
tional significance of the central hole, 20 Å in diameter, observed
in the NS2 electron micrographs remains unclear (Mumtsidu et al.,
2007), recent investigations have concluded that the 37 Å diameter
inner cavity observed in the donut-like structure of octameric NSP2
provides a protective environment for the newly synthesized dsR-
NAs (Jiang et al., 2006). Intriguingly, Martin et al. (2010) recently
reported that tubulin granules are components of Rotavirus viro-
plasms, interacting with NSP2. A similar interaction has not been
detected in NS2 viral inclusion bodies in BTV-infected cells (Modrof
et al., 2005).
Cellular kinases (Eichwald et al., 2004) as well as the binding
partner NSP2 (Afrikanova et al., 1998) are responsible for the vari-
ation in phosphorylation of NSP5 (molecular weight 26–35 kDa)
in vivo. Unlike BTV-NS2, NSP5 phosphorylation is not important
for viroplasms formation (Contin et al., 2010) and the functional
significance of NSP5 phosphorylation in the Rotavirus life cycle
is unknown. NSP5 is reported to have both ss and dsRNA bind-
ing affinities (Vende et al., 2002) and an ATP-specific nucleotidyl
hydrolysis activity, which currently is unique among the Reoviri-
dae proteins (Bar-Magen et al., 2007). It is suggested that the energy
released upon ATP hydrolyis would be used for genome replica-
tion and packaging but also to sequester the viral RNA, that might
otherwise trigger innate defense (Bar-Magen et al., 2007).
In the Reovirus genera, the ␴NS (41 kDa)–␮NS (80 kDa) pro-
teins in combination appear to be the functional homologues of
Rotavirus NSP2–NSP5 and BTV-NS2. ␴NS associates with ␮NS in
Reovirus viral factories (Miller et al., 2003). When expressed alone
in cells, ␮NS forms structures that resemble the viral factories in
the infected cells, as assesed by light microscopy (Becker et al.,
2003; Broering et al., 2002; Touris-Otero et al., 2004). Like NS2, ␴NS
isolated from Reovirus infected cells is found in large complexes,
between 40S and 60S, which dissociate upon treatment with RNase
A (Gomatos et al., 1981) to 4 ± 2 monomers (Huismans and Joklik,
1976). Furthermore, in in vitro experiments ␴NS bound ssRNA in a
multimeric, cooperative fashion (Gillian et al., 2000). Similar to NS2
and NSP2, ␴NS has ATP and Mg2+ independent strand displacement
activity (Gillian et al., 2000), but again the significance of this is
unclear unless it is involved in siRNA metabolism and suppression
of host response.
However, there are some important distinctions between NS2
and ␴NS–␮NS. ␴NS does not undergo phosphorylation and does
not hydrolyze NTPs (Gillian et al., 2000). It may modulate an
NTPase activity via its interaction inside the viral factories with the
Reovirus core protein ␮2, which has ssRNA binding affinity and is a
determinant of the NTPase activity associated with Reovirus cores
(Brentano et al., 1998). Currently, there are no reports of investi-
gations concerning the ␮NS phosphorylation status or any NTPs
hydrolysis activity. Similarly to NSP2, ␮NS also colocalizes with
cytoskeletal elements in infected cells (Mora et al., 1987).
116 C. Butan, P. Tucker / Virus Research 151 (2010) 109–117
In conclusion each of the Reoviridae genuses, Orthoreoviruses,
Orbiviruses and Rotaviruses, has one or two non-structural pro-
teins that are the morphological basis for viral progeny generation.
They provide ssRNA binding functions together with some abil-
ity to hydrolyse nucleotide triphosphates. Despite the functional
similarities there are no structural similarities described to date.
10. Concluding remarks
It is difficult to deduce information about function from high-
resolution structural information, e.g. from X-ray crystallography
or NMR, of heterogeneous oligomeric proteins such as NS2. Great
care must also be taken interpreting biophysical results obtained
with modified proteins containing bulky tags since these might also
affect the oligomeric state. In addition, because NS2 has been shown
to bind ssRNA non-specifically it is impossible to be certain that
preparations, even when treated with RNase, are devoid of bound
RNA. It seems to us that high-resolution EM comparisons between
recombinant and “native” inclusion bodies to establish the similar-
ity (or difference) between the in vivo and recombinant situations
would be very useful – in particular to check if the type of struc-
tures observed in (Mumtsidu et al., 2007) do actually occur in vivo.
It might also be possible to design solvent contrast studies using
neutron scattering to try to establish the location of bound RNA
relative to the models described in Section 7.
A further puzzle is the reported nucleotidyl phosphatase activity
but the inability to locate potential catalytic residues in the aligned
sequences. Those mutational analyses that have been performed
have not typically been coupled with assays on the nucleotidyl
phosphatase activity, and it is not even certain that this activity
is necessary for virus viability.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.virusres.2010.05.014.
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