Virus Research 101 (2004) 67–81

Emerging themes in rotavirus cell entry, genome organization,

transcription and replication
Hariharan Jayaram a , M.K. Estes b , B.V. Venkataram Prasad a,c,∗
a Program in Structural and Computational Biology and Molecular Biophysics, Houston, TX 77030, USA
b Department of Molecular Virology and Microbiology, Houston, TX 77030, USA
c Verna and Marrs McLean Department of Biochemistry and Molecular Biology,

Baylor College of Medicine, Houston, TX 77030, USA

Abstract

Rotaviruses, causative agents of gastroenteritis in young animals and humans, are large icosahedral viruses with a complex architecture. The
double-stranded RNA (dsRNA) genome composed of 11 segments, which codes for 6 structural and 6 non-structural proteins, is enclosed within
three concentric capsid layers. In addition to facilitating host-specific interactions, the design of the capsid architecture in rotaviruses as in
other dsRNA viruses should also be conducive to the requirement of transcribing the enclosed genome segments repeatedly and simultaneously
within the capsid interior. Several non-structural proteins facilitate the subsequent processes of genome replication and packaging. Electron
cryomicroscopy studies of intact virions, recombinant virus-like particles, functional complexes, together with recent X-ray crystallographic
studies on rotavirus proteins have provided structural insights into the capsid architecture, genome organization, antibody interaction, cell
entry, trypsin-enhanced infectivity, endogenous transcription and replication. These studies underscore contrasting features and unifying
themes between rotavirus and other dsRNA viruses.
© 2003 Published by Elsevier B.V.

Keywords: Rotavirus; dsRNA virus; Cryo-EM; Genome organization; Transcription; Replication

1. Introduction layers yield the complete virus particles (Estes, 2001).

Rotavirus, a member of the family Reoviridae is a non-
enveloped, icosahedral, double-stranded RNA (dsRNA)
virus (reviewed in Lawton et al., 2000). The rotavirus
capsid, like all virus capsids is structured to protect its
genome and deliver it successfully into a suitable host cell,
in which the genome is replicated and the virus particle
makes copies of itself. Several recent structural and bio-
chemical studies have provided crucial insights into how
the different proteins encoded by the virus enable it to
(1) transcribe its genome from within the confines of the
capsid, (2) control the translation of the host genes to en-
hance translation of the rotaviral genes in the cytoplasm
of the infected host cell, (3) enable genome replication
wherein the negative strand is synthesized using the tran-
scribed mRNA as a template, and finally (4) package
the replicated dsRNA genome and assemble the capsid
∗ Corresponding author. Tel.: +1-713-798-5686;
fax: +1-713-798-1625.
E-mail address: vprasad@bcm.tmc.edu (B.V.V. Prasad).
From such studies on rotavirus and other dsRNA viruses
including other members of the family Reoviridae, com-
mon themes are emerging regarding the role of the viral
capsid and non-structural proteins during the virus life
cycle.
The dsRNA viruses, classified into five major groups,
range from the relatively simple viruses with a single
dsRNA segment like in the members of the Totiviridae fam-
ily to more complex viruses in the Reoviridae family, which
contain 10–12 dsRNA segments (Lawton et al., 2000; van
Regenmortel et al., 2000). Other families of dsRNA viruses
like Birnaviridae and Cystoviridae contain two and three
dsRNA segments, respectively. Accordingly, there is a con-
siderable variation in the complexity of the capsid organi-
zation in dsRNA viruses. In the members of Totiviridae and
Birnaviridae, a single-layer capsid encloses the genome,
whereas viruses in Cystoviridae contain a double-layered
capsid. In some genera of Reoviridae such as Rotavirus,
Orbivirus, and Reovirus, viruses exhibit distinct triple-
layered capsid architecture. In all the well-characterized
dsRNA viruses, the capsid structure is based on icosahedral

0168-1702/$ – see front matter © 2003 Published by Elsevier B.V.

doi:10.1016/j.virusres.2003.12.007
68 H. Jayaram et al. / Virus Research 101 (2004) 67–81
symmetry. With the exception of cystoviruses (Mindich,
1988), which use bacteria as hosts, the dsRNA viruses are
non-enveloped.
The dsRNA genome in rotavirus and other dsRNA viruses
presents a unique set of conditions for their survival. Be-
cause the host cells do not possess the enzymatic machinery
to convert dsRNA into a translatable mRNA molecule,
these viruses have to provide for a mechanism to synthesize
mRNA from their genomic dsRNA. Both the transcription
and the subsequent synthesis of progeny dsRNA have to
be carried out in confined environments not only to avoid
degradation of the genome by cellular nucleases but also to
prevent unfavorable antiviral responses in the host cells that
could be triggered by increased concentrations of dsRNA.
All the dsRNA viruses encode their own enzymes necessary
for transcription and are capable of endogenously transcrib-
ing their genomes. Together with this common attribute of
endogenous transcription, the capsid architecture in these
viruses also has to account for host-specific interactions.
The dsRNA viruses are ubiquitous in nature and infect hosts
that range from bacteria and fungi to species throughout
the plant and animal kingdoms (van Regenmortel et al.,
2000). An interesting question is: how does capsid archi-
tecture in these viruses efficiently integrate the requirement
of host-specific cell entry with the common requirement of
endogenous transcription?
Structures of several dsRNA viruses including L-A virus
of yeast origin from Tottiviridae family (Caston et al., 1997),
infectious bursal disease virus from Birnaviridae (Bottcher
et al., 1997), ␾6 from Cystoviridae (Butcher et al., 1997,
2001), and several members of the Reoviridae representing
various genera including rotavirus (Prasad and Estes, 2000;
Tihova et al., 2001), bluetongue virus (BTV) in the Orbivirus
(Grimes et al., 1997; Prasad et al., 1992), orthoreovirus
(Dryden et al., 1993, 1998), aquareovirus (Nason et al., 2000;
Shaw et al., 1996), rice dwarf virus (RDV) in the Phytore-
ovirus genera (Zhou et al., 2001), and cypovirus (Hill et al.,
1999; Xia et al., 2003), have been analyzed by electron cry-
omicroscopy (cryo-EM) techniques. X-ray structures of L-A
virus (Naitow et al., 2002), and transcriptionally competent
cores of BTV (Grimes et al., 1998), orthoreovirus (Reinisch
et al., 2000), and RDV (Naitow et al., 2002), have been de-
termined to near 3 Å resolution. In addition to the structures
of viral capsids, in some cases, individual viral proteins have
been determined by X-ray crystallography (Butcher et al.,
2001; Deo et al., 2002; Dormitzer et al., 2002; Jayaram et al.,
2002; Liemann et al., 2002; Mathieu et al., 2001; Tao et al.,
2002). Together these structural studies have begun to pro-
vide a detailed description of the capsid organization and
molecular insights into the mechanisms of various functional
activities of these dsRNA viruses. This review primarily fo-
cuses on the recent structural findings in rotavirus, and in
relation to other dsRNA viruses, attempts to summarize this
information to underscore unifying principles in capsid ar-
chitecture, genome organization, endogenous transcription
and replication.
2. Rotavirus capsid structure and function
Rotavirus is the major causative agent of infantile diar-
rhea accounting for nearly one million deaths annually in
the world (Cohen, 2001). This fact has led to the virus be-
ing extensively studied to understand its pathogenicity and
discover ways to treat and manage rotavirus infections. The
rotavirus is a relatively large, ∼1000 Å in diameter, icosa-
hedral virus. Its capsid encloses 11 segments of dsRNA
(Fig. 1A), each segment codes for one protein with the ex-
ception of segment 11, which codes for 2 proteins (reviewed
in (Estes, 2001). Of these 12 proteins, 6 are structural (VPs)
and 6 are non-structural (NSPs). The rotavirus capsid is
composed of three concentric protein layers that enclose
the genome (Fig. 1B and C; reviewed in Prasad and Estes,
2000). The complete virions are called triple-layered parti-
cles (TLPs, Fig. 1B), particles that lack the outer layer are
called the double-layered particles (DLPs, Fig. 1E) and are
non-infectious, and particles that lack the outer two layers
are called the single-layered particles (SLPs, Fig. 1D).
2.1. The outer capsid layer and cell entry
The viruses in Reoviridae infect a wide variety of hosts.
Rotaviruses infect the cells of the intestinal epithelium, while
some viruses of the genus Orthoreovirus spread to the cen-
tral nervous system and viruses among the Orbiviruses in-
fect both insect and mammalian hosts. The outermost layers
in Reoviridae are implicated in cell attachment, membrane
penetration and cell entry. In keeping with the wide host
range, the outer capsid layers among the Reoviridae show
a remarkable range of diversity in terms of composition of
proteins, their organization, their associated activities, and
the mechanisms of cell entry.
Cryo-EM reconstructions of rotavirus (Prasad et al.,
1988; Prasad and Estes, 2000; Tihova et al., 2001; Yeager
et al., 1990), orthoreovirus (Dryden et al., 1993, 1998),
BTV (Grimes et al., 1997; Hewat et al., 1992; Schoehn
et al., 1997), rice dwarf virus (Lu et al., 1998) and cy-
povirus (Hill et al., 1999; Xia et al., 2003), have provided
structural insights into the organization of this outermost
capsid layer. Accordingly, members of the Reoviridae can
be classified into two types, those that have a complete
T = 13 outer layer or the non-turreted viruses and those
that have an incomplete T = 13 layer where the normal
T = 13 arrangement is interrupted by the presence of a
turret-like structure at the five-fold axes of symmetry.
In rotavirus, the outermost layer is made up of 780 copies
of VP7 (38 kDa) a glycoprotein, and 120 copies of the
spike protein VP4 (88 kDa) (Prasad et al., 1990) (Fig. 1B).
Cryo-EM studies indicate that the 780 copies of VP7 are ar-
ranged as 260 trimers that are located at the local and strict
three-fold axes of a T = 13 (left-handed) icosahedral lattice.
A distinctive feature of the rotavirus structure is the pres-
ence of 132 aqueous channels, ∼140 Å deep, spanning the
outer two capsid layers, at all the five- and six-coordinated
 H. Jayaram et al. / Virus Research 101 (2004) 67–81 69

Fig. 1. Architectural features of rotavirus. (A) PAGE gel showing, 11 dsRNA segments comprising the rotavirus genome. The gene segments are numbered
on the left and the proteins they encode are indicated on the right. (B) Cryo-EM reconstruction of the rotavirus triple-layered particle. The spike proteins
VP4 is colored in orange and the outermost VP7 layer in yellow. (C) A cutaway view of the rotavirus TLP showing the inner VP6 (blue) and VP2 (green)
layers and the transcriptional enzymes (shown in red) anchored to the VP2 layer at the five-fold axes. (D) Schematic depiction of genome organization
in rotavirus. The genome segments are represented as inverted conical spirals surrounding the transcription enzymes (shown as red balls) inside the VP2
layer in green. (E and F) Model from Cryo-EM reconstruction of transcribing DLPs. The endogenous transcription results in the simultaneous release of
the transcribed mRNA from channels located at the five-fold vertex of the icosahedral DLP.

positions of the T = 13 lattice. Inserted into the channels
(type II channels) that surround the 12 icosahedral five-fold
vertices are the 60 bilobed spikes, each 120 Å long. The
molecular identity of these spikes as dimers of VP4 was
shown by cryo-EM reconstructions of rotavirus complexed
with anti-VP4 antibodies (Prasad et al., 1990; Tihova et al.,
2001). Subsequent cryo-EM studies discovered that VP4 has
a large globular domain that is buried inside the inner layer,
making the total length of the spike 200 Å (Shaw et al., 1993;
Yeager et al., 1994).
2.1.1. VP4
Although earlier studies implicated VP7 in the cell entry
process (Fukuhara et al., 1988; Sabara et al., 1985), sub-
sequent studies have increasingly indicated that VP4 is the
major player in this process. VP4 is implicated not only in
cell attachment and cell penetration but also in hemagglu-
tination, neutralization and virulence (Estes, 2001). VP4 is
susceptible to proteolysis. Proteolytic cleavage of VP4 en-
hances viral infectivity by several fold (Arias et al., 1996;
Estes et al., 1981) and facilitates virus entry into cells
(Kaljot et al., 1988). During proteolysis, VP4 (88 kDa) is
cleaved into VP8∗ (28 kDa, aa 1–247) and VP5∗ (60 kDa,
aa 248–776) and the cleavage products remain associated in
the virion (Fiore et al., 1991). Trypsinized viruses enter cells
more readily and rapidly than those particles that are not
trypsinized (Kaljot et al., 1988; Keljo et al., 1988). In vitro
experiments have shown that proteolytically activated parti-
cles, as well as recombinant VP5∗ , possess lipophilic activity
(Dowling et al., 2000; Nandi et al., 1992; Ruiz et al., 1994).
It is interesting to note here that VP4 contains a putative
fusion domain similar to that seen in the enveloped viruses
such as alphaviruses and influenza viruses. Proteolysis thus
constitutes a key process in the efficient internalization of
rotaviruses into cells. This is particularly relevant consid-
ering that rotavirus replication takes place in enterocytes in
the small intestine, an environment rich in proteases.
The molecular mechanisms of increased infectivity by
proteolysis and its role in the membrane penetration are not
well understood. Recent studies comparing the biochemical
and structural properties of rotavirus grown in the presence
and absence of trypsin have provided some exciting and
novel insights into this process, and indicated that trypsin
cleavage stabilizes the spike assembly and confers icosahe-
dral ordering (Crawford et al., 2001). These results are con-
sistent with biochemical studies on recombinant VP4, which
show that proteolysis of monomeric VP4 yields dimeric
VP5∗ (Dormitzer et al., 2001). Based on these studies it ap-
pears that that trypsin cleavage has an intracellular role in
ensuring the correct conformation and proper assembly of
70 H. Jayaram et al. / Virus Research 101 (2004) 67–81
the spikes. Further structural and biochemical studies are
required to provide a better understanding of how trypsin
affects intracellular spike assembly.
Several recent studies have shown that rotavirus cell entry
is a multi-step process involving sialic acid (SA)-containing
receptors in the initial cell attachment step and integrins such
as ␣v␤3, ␣4␤1, ␣2␤1 during the subsequent post-attachment
steps (Coulson et al., 1997; Guerrero et al., 2000; Hewish
et al., 2000; Zarate et al., 2000). In this process, the VP8∗
domain is involved in the interactions with SA, whereas
VP5∗ is implicated in interactions with integrins. Involve-
ment of SA during rotavirus infections is not an essential step
for all rotavirus strains. In the majority of rotavirus strains,
including human rotaviruses, cell entry is SA independent
(Ciarlet et al., 2001). In these viruses, the majority of neu-
tralizing monoclonal antibodies that recognize VP4 select
mutations in VP5∗ , suggesting that cell entry is mediated
mainly by the VP5∗ (Kirkwood et al., 1996; Kobayashi et al.,
1990; Padilla-Noriega et al., 1995). Studies with polarized
epithelial cell lines show that with SA-dependent viruses,
the viral entry is restricted to the apical membrane, whereas
SA-independent viruses enter either apically or basolater-
ally thus further documenting variations in the entry mech-
anisms between SA-independent and SA-dependent viruses
(Ciarlet et al., 2001).
Recently, X-ray structure of a portion (aa 64–242) of
the sialic acid binding domain of VP8∗ was determined
and this structure strongly suggests that the distal globular
heads of the spikes are made of VP8∗ (Dormitzer et al.,
2002). Interestingly, the sialic acid binding domain exhibits
a ␤-sandwich fold similar to that seen in galectins, a family
of sugar binding proteins, despite a lack of sequence similar-
ity. Analysis of the crystal structure with bound sialic acid
revealed that although the galectin binding site is blocked
in VP8∗ , the protein binds sialic acid in a shallow groove
on its surface using residues that are conserved in sialic
acid-dependent rotavirus strains. A comparison of this struc-
ture with that of the apo-form of VP8∗ , determined by NMR,
suggests that the ligand binds by inducing a slight conforma-
tional change in the residues involved in binding. The VP8∗
structure further represents one of the first observed cases
of a protein in rotavirus and the dsRNA viruses (other than
the polymerase) taking on a fold seen among host proteins
and unknown thus far among viral proteins whose struc-
tures have been determined. Based on this structural result,
it is proposed that VP4 arose from the insertion of a host
carbohydrate-binding domain into a viral membrane inter-
action protein (Dormitzer et al., 2002).
The proposed sequential interactions with various lig-
ands in the multi-step rotavirus entry processes may in-
volve a series of conformational changes. For instance, in
SA-dependent strains of rotavirus, binding of sialic acid may
induce conformational changes in VP8∗ to enable VP4 to
bind to the integrins more efficiently. In the SA-independent
strains VP4 may already be in a conformation that is suit-
able for directly interacting with the upstream receptors. In
the multi-step entry process, the conformational adaptability
and flexibility of VP4 may play an important role. Studies
on rotaviruses grown in the absence and presence of trypsin
together with the observation that VP4 can undergo large
pH-induced conformational changes are consistent with such
a notion (Crawford et al., 2001; Pesavento et al., 2001). A
detailed picture will emerge once more intermediates in the
rotavirus entry process are characterized structurally.
2.1.2. VP7
The precise role of VP7 during early interactions of the
virus with the cell is not clear, but its has been postulated that
VP7 may modulate the function of VP4 during the attach-
ment and entry process (Beisner et al., 1998; Mendez et al.,
1996), and may interact with cell surface molecules after the
interaction is initiated by VP4 (Mendez et al., 1999). One of
the first events upon cell entry is the loss of this outer layer
to expose the transcriptionally active DLP to the cytoplasm.
Biochemical studies indicate that the in vivo decapsidation
can be mimicked by treating TLPs with calcium chelators
like EDTA (Cohen et al., 1979). VP7 binds calcium and
the sensitivity of virions to low calcium concentrations is
strain-dependent (Cohen et al., 1979; Gajardo et al., 1997;
Ruiz et al., 1996). Several studies also have suggested
calcium-driven conformational changes in VP7 (Dormitzer
and Greenberg, 1992). Studies on baculovirus-expressed
recombinant VP7 have shown a requirement for calcium
in the formation of VP7 trimers, which crystallize into
hexagonal plates mimicking the arrangement of VP7 on
the capsid (Dormitzer et al., 2000). Thus, while appropriate
levels of calcium help maintain the structural integrity of
the VP7 layer, low calcium concentrations, similar to those
in the cytoplasm, trigger the disassociation of VP7 trimers
leading to uncoating of the VP7 layer. Uncoating of the
outer layer resulting in transcriptionally competent DLPs is
a necessary event in the replication cycle of rotavirus. Some
antibodies directed against VP7 neutralize the virus by in-
hibiting the decapsidation of the TLP (Ludert et al., 2002).
The effect of these neutralizing antibodies is not overcome
by lipofecting virus–antibody complexes into cells. In con-
trast, some antibodies directed against VP4 neutralize by
inhibiting the binding of virus to the cell, an effect that
can be overcome by lipofecting these complexes into cells.
It is possible that neutralizing anti-VP7 antibodies prevent
VP7 from undergoing necessary conformational changes
that facilitate uncoating of the VP7 layer. Thus, VP7 is the
key mediator of the calcium driven uncoating in rotavirus
and the function of the outer layer composed of VP4 and
VP7 is to ensure that the transcriptionally active DLPs are
delivered to the host cytoplasm.
2.2. The intermediate layer and endogenous genome
transcription
The DLP resulting from the removal of the outer layer pro-
teins, VP4 and VP7, is the transcriptionally competent form
 H. Jayaram et al. / Virus Research 101 (2004) 67–81 71

of rotavirus capable of transcribing the enclosed dsRNA seg-
ments within its confines (Fig. 1E). The DLP is composed
of the remaining four structural proteins encoded by the ro-
tavirus genome of which VP6 is the major constituent. In
the DLP, VP6 forms the outer most capsid layer with 260
trimers of VP6 assembled on a T = 13 icosahedral lattice.
In the context of the rotavirus TLP, the VP6 layer, hence-
forth referred to as the intermediate layer, is sandwiched
between the outer T = 13 layer formed by VP7 and VP4,
and the inner T = 1 capsid layer formed by VP2. In the ro-
tavirus structure, the VP6 trimers lie below the VP7 trimers
so that the aqueous channels in the two T = 13 layers are
in register. In the overall organization of the rotavirus, VP6
appears to integrate the two principal functions of the virus,
cell entry and endogenous transcription, through its interac-
tions with the outer layer proteins VP7 and VP4, and the
inner layer protein VP2.
2.2.1. Pseudo-atomic model of the VP6 layer
Although an X-ray structure of the DLP is yet to be de-
termined, docking of the X-ray structure of VP6 into the
cryo-EM density of the DLP has resulted in a pseudo-atomic
model of the T = 13 VP6 layer (Mathieu et al., 2001). VP6
has two domains, the distal domain with eight-stranded
anti-parallel ␤-sandwich fold makes contact with the VP7
layer, and the lower domain consisting of a cluster of
␣-helices makes contact with the inner VP2 layer (Fig. 2).
VP6 trimers interact laterally to form the T = 13 layer and
there appear to be at least two types of contacts between the
trimers. The contacts, across the quasi two-fold axes, closer
to the icosahedral three-fold axes, are similar, whereas as
the trimers approach the icosahedral five-fold axis, the con-
tacts are varied. In contrast to VP7 of BTV, a structural
equivalent of VP6 in rotavirus, the VP6 trimer exhibits
extensive lateral interactions involving charged residues.
Thus, modeling has revealed that all the interacting sur-
faces (VP6–VP4, VP6–VP7, and VP6–VP2) contain the
most conserved residues of VP6. Although the base of the
VP6 layer displays an overall negative surface electrostatic
potential, the VP6 interactions with VP2 are predominantly
hydrophobic. In contrast the interactions with VP7 and VP4
appear to involve several charged residues. In solution, VP6
forms highly stable trimers. The VP6 trimer has a bound
zinc ion that is shown to be necessary for the stability of
the trimer. VP6 trimers easily form 2-D crystalline arrays
and helical tubes with pseudo-hexagonal packing, but rarely
icosahedral shells that are similar to the T = 13 VP6 layer in
the rotavirus structure. The lateral interactions between VP6
trimers do not have all the information to form the closed
shell. In contrast, VP2 has the ability to form native-like
icosahedral shells of appropriate size. Therefore, it is likely
that the VP2 layer provides a proper scaffold for the assem-
bly of VP6 trimers into a T = 13 icosahedral organization.
2.2.2. Role of VP6 in endogenous transcription
Earlier biochemical studies clearly indicated that none of
the components of the DLP alone is capable of transcribing
the dsRNA and that VP6, despite lack of any enzymatic
functions, is essential for endogenous transcription of the
genome. Based on cryo-EM studies of DLPs, Prasad et al.
(1988) were the first to propose that channels in the VP6
layer could be used for mRNA exit. More recent cryo-EM
studies on the actively transcribing DLPs, have shown that
of the three types of the channels in the T = 13 VP6 layer,
the nascent mRNA transcripts exit specifically through the
type I channels located at the five-fold axes (Fig. 1E and F)

Fig. 2. Structure of VP6 trimer. Structure of VP6 trimers showing the upper domain with its seven-strand jelly role topology and the predominantly
helical lower domain (Mathieu et al., 2001). The structure of the upper domain is analogous to the domain in BTV VP7 (Grimes et al., 1995, 1998).
The docking of the VP6 trimers into electron-density from cryo-EM reconstructions has allowed the construction of a pseudo-atomic model for the outer
surface of the DLP in rotavirus.
72 H. Jayaram et al. / Virus Research 101 (2004) 67–81
(Lawton et al., 2000). These studies also confirmed that
DLPs maintain their structural integrity during the process of
transcription. In the pseudo-atomic model of the VP6 layer,
a ␤-hairpin motif of VP6 with a highly conserved sequence
that protrudes into the mRNA exit channel may play a
functional role in the translocation of the nascent transcripts
during endogenous transcription. The net concentration of
negative charges on the walls lining the type I channel may
facilitate the extrusion of the mRNA transcript by increasing
its fluidity because of the electrostatic repulsion between
the interior surface of the channel and the mRNA transcript.
A detailed mutational analysis based on the pseudo-atomic
model of the VP6 layer has helped to elucidate the deter-
minants of VP6 required for assembly on VP2, and how
VP6 may affect endogenous transcription (Charpilienne
et al., 2002). Thirteen site-specific substitution mutations
of amino acids residues that directly contact the VP2 layer,
as identified in the pseudoatomic model, have been stud-
ied in terms of their ability to trimerize, to form virus-like
particles when co-expressed with VP2, and to assemble
onto wild-type cores and form in vitro reconstituted DLPs
that could transcribe the enclosed genomes and produce
rotaviral mRNA. In cases where defects were observed, the
amino acids essential for recovery of transcription or as-
sembly were identified. All the VP6 mutants formed stable
trimers and self assembled into tubular structures similar to
wild-type VP6. This is consistent with the observation from
the X-ray structure of VP6 that the lower ␣-helical domain
of VP6 does not contribute to either the trimeric interactions
or the lateral interactions in the assembly of the tubular
structures. Of the 13 VP6 mutants examined, 3 were unable
to assemble with VP2 and 3 others partially assembled.
These mutants either did not rescue the transcriptase activ-
ity or did so only marginally substantiating further that the
proper assembly of VP6 trimers on VP2 is an absolute re-
quirement for endogenous transcription. Four other mutants
that generally preserved the hydrophobic character of the
contact region, assembled and transcribed well, emphasiz-
ing the importance of hydrophobic interactions in the proper
assembly and stability of VP6 on VP2. An interesting result
was obtained with three of the mutants, which had an extra
charge introduced at the VP6–VP2 interface. These mutants
assembled well on cores, but surprisingly did not rescue
the transcriptase activity in the reconstituted DLPs. This
result prompted the investigators to favor a hypothesis in
which transcript extrusion during endogenous transcription
requires VP6 to undergo subtle conformational changes and
the extra charge in the mutants inhibits such changes.
The idea of conformational changes in VP6 associated
with endogenous transcription is also suggested by the
cryo-EM structural studies on DLP-anti (VP6) MAb com-
plexes. Lawton et al. (1999) in their studies showed that
certain antibodies inhibit transcription as does the presence
of the outermost VP7 layer, which renders the TLP tran-
scriptionally inactive. Biochemical analysis of TLP, and
DLP–antibody complexes in which the antibody inhibited
transcription, revealed that these particles are able to initi-
ate transcription and synthesize capped transcripts of five
to seven nucleotides in length, thereby indicating that the
enzymatic activities of the DLP are not affected by binding
of VP7 or antibody. The short length of transcripts indicates
that elongation is not inhibited by any steric narrowing or
restriction caused by antibody-binding, or analogously VP7
binding, but instead can be attributed to the subtle conforma-
tional changes near the VP6–VP2 interface observed consis-
tently in the structures of transcriptionally inactive TLP and
DLP–antibody complexes. A similar cryo-EM study using
a different set of antibodies with contrasting effects on tran-
scription however concluded that the inhibitory effect of one
of the two antibodies studied may be due to rigidification of
the VP6 trimers upon antibody binding (Thouvenin et al.,
2001). Although no conformational changes in VP6 with
either of the antibodies were seen, there were major differ-
ences in the extent of interaction between VP6 and these
antibodies. With the antibody that inhibited transcription,
five loops from two of the VP6 subunits in the trimer were
involved in the antibody binding, whereas with the other
antibody, which did not inhibit transcription, only one VP6
monomer was involved. From these observations, Thouvenin
et al. (2001) proposed that the inhibitory effect of one of the
antibodies is caused by preventing conformational changes
in the VP6 layer required for transcription. Although the pro-
posed mechanisms of how a VP6-specific antibody inhibits
transcription by these two studies differ, both the studies
clearly indicate that the dynamics of the interaction between
the VP6 and VP2 layers are important for transcription.
The exit of transcripts through the channels at the
five-fold axes appears to be an emerging common theme in
dsRNA viruses. In orthoreovirus, both conventional EM and
cryo-EM studies have shown the transcripts exit through
the turrets at the five-fold axes. In BTV cores, X-ray crys-
tallographic analysis of core crystals soaked with various
substrates and products of the transcription reaction has
shown that the mRNA exit channel in the BTV core is
likely to be at the five-fold vertex (Diprose et al., 2001).
In addition to providing conduits for the exit of transcripts,
another important function of the VP7 layer (equivalent of
VP6 layer in rotavirus), that became evident from these
studies, is that certain regions surrounding the five-fold axes
in this layer function as substrate sinks for the transcription
reaction. Although the dynamic events behind endogenous
transcription are still unclear, the structural and biochem-
ical studies on several dsRNA viruses have brought into
focus the elegance of capsid organization in these viruses
and reveal the fact that capsid architecture and genome or-
ganization are coordinated to ensure multiple and repeated
rounds of transcription.
2.3. The innermost layer
The core of the rotavirus consists of the remaining three
structural proteins VP1, VP2, and VP3 (Fig. 1C and D). Of
 H. Jayaram et al. / Virus Research 101 (2004) 67–81
these three proteins, VP2 is the most abundant and forms the
innermost layer interacting with the VP6 layer on the out-
side and the genomic RNA on the inside. The other two pro-
teins VP1, an RNA-dependent RNA polymerase (Valenzuela
et al., 1991), and VP3, guanylyl and methyl transferase
(Chen et al., 1999; Liu et al., 1992), are present in small
quantities and provide the enzymatic functions required for
producing the capped mRNA transcripts. Of all the structural
proteins of rotavirus, VP2 is the only protein that has the abil-
ity to self-assemble into a native-like icosahedral structure,
when expressed in insect cells. This observation strongly
indicates that VP2 possesses the determinants required to
direct the proper assembly of other rotavirus proteins.
2.3.1. Unique organization of the inner most capsid layer
The VP2 layer consists of 120 molecules organized as 60
dimers on a T = 1 icosahedral lattice. Such an icosahedral
organization with 120 subunits is quite unique and is found
only in dsRNA viruses. This is the most conserved feature
in all structurally characterized members of the Reoviridae
and other dsRNA viruses such as ␾6, a bacterial virus and
L-A virus of yeast origin. The atomic level description of
such an organization with two subunits in the icosahedral
asymmetric units, and hence called a T = 2 icosahedral
structure, is provided by the X-ray crystallographic analy-
ses of BTV and orthoreovirus cores (Grimes et al., 1998;
Reinisch, 2002; Reinisch et al., 2000). In these structures,
one of the two subunits in the icosahedral asymmetric unit
points toward the icosahedral five-fold axis and the other is
slightly offset from the five-fold axis. Each subunit has three
domains with the N-terminal residues facing inward toward
the virus genome. Similar subunit structure and organiza-
tion is also seen in the high-resolution cryo-EM structures
of RDV and CPV and is likely to be replicated in rotavirus
as indicated by the cryo-EM analysis of the recombinant
VP2/6 particles with full-length VP2 and a mutant VP2 with
N-terminal residues deleted. VP2 exhibits RNA-binding
ability through its N-terminal residues. Consistent with this
observation, several points of contact between the VP2 layer
and RNA, presumably through the N-terminal residues of
VP2, are observed in the cryo-EM structure of the DLP.
Similarly, interactions between VP3 and RNA are seen the
X-ray structure of the BTV core. Thus, one of the principal
functions of the innermost layer in these viruses may be
to direct the structural organization of the genome that is
conducive for its endogenous transcription.
2.3.2. Transcription enzyme complex
Where are the transcription enzymes located? Using a
comparative cryo-EM analysis of DLP and the recombinant
virus-like particles with and without VP1 and VP3, Prasad
et al. (1996) were the first to show that these minor proteins
were incorporated as a heterodimer anchored to the inside
surface of the VP2 layer at each of the 12 five-fold ver-
tices. A similar structural organization of the transcription
enzymes anchored to the inner surface of the T = 2 layer
73
subsequently has been seen in aquareovirus, orthoreovirus,
BTV, RDV, cypovirus, and ␾6 virus. However, one prin-
cipal difference between rotavirus, BTV, or orthoreovirus,
and aquareovirus or cypovirus, is that in the latter group
of viruses the capping enzyme is external to the innermost
layer forming a distinct turret-like structure at the five-fold
axis, although the polymerase is internal. The location of the
transcription enzyme complex at the five-fold axis is con-
sistent with the nascent transcripts exiting through the chan-
nels at the five-fold axes. In rotavirus, biochemical studies
on recombinant VLPs containing VP2 with amino-terminal
deletions co-expressed with VP6, VP1 and VP3 indicate that
removal of 25 N-terminal residues of VP2 completely pre-
vent the incorporation of VP1 and VP3. Thus, in addition to
RNA-binding activity, the N-terminal residues of VP2 are in-
volved in anchoring VP1 and VP3. The N-terminal residues
of the one of the two subunits in the icosahedral asymmetric
unit of the “T = 2” that is close to the five-fold axis may be
involved in anchoring the transcription enzyme, whereas the
N-terminal residues of the other subunit, which is slightly
offset from the five-fold axis may involved in the interac-
tions with the underlying genomic RNA. It is possible that
the unique organization of the innermost layer in the dsRNA
viruses has evolved to serve the dual purpose of properly po-
sitioning the transcription enzyme complex and organizing
the genome to facilitate endogenous transcription.
Although the locations of the transcription enzymes can
be inferred from the structures of the intact virions, the pre-
cise molecular structures of these enzymes inside the virions,
with the exception of the capping enzyme in the “turretted”
dsRNA viruses such as orthoreovirus, are not resolved as
they are present in non-icosahedral amounts. Therefore,
structural understanding of how these enzymes function
has to come from the studies on individual components.
Recently, X-ray crystallographic structures of the ␾6 and
orthoreovirus polymerases have been determined (Butcher
et al., 2001; Tao et al., 2002). These studies have provided
significant mechanistic insights into how these molecules
function both in the transcription and replication processes.
Both the structures have a canonical finger–palm–thumb
core, as seen in several other polymeases, surrounded by
N- and C-terminal elaborations. These elaborations differ
significantly between the two structures perhaps reflect-
ing the differences in the transcription strategies in these
viruses. In ␾6 and birnaviruses, which contain three and
two dsRNA segments, respectively, the transcription is
semi-conservative. In the members of Reoviridae, which
contain 10–12 dsRNA segments, the transcription is conser-
vative. Generally, the polymerase molecules in the members
of Reoviridae are larger than their counterparts in other
dsRNA viruses. The structure of the reovirus polymerase,
perhaps a better representative of the rotavirus polymerase,
shows a cage-like structure with four channels leading to
the central catalytic core (Tao et al., 2002). Further X-ray
crystallographic analyses with appropriate ligands have as-
signed these channels to specific functions such as substrate
74 H. Jayaram et al. / Virus Research 101 (2004) 67–81
and template entry, mRNA or dsRNA exit depending upon
the polymerase involvement during transcription or replica-
tion (Tao et al., 2002). Interestingly, these structural studies
also revealed a binding site for the 5 cap structure seen in
reovirus and all the members of the Reoviridae, suggesting
a potential mechanism for tethering of the non-template
strand during transcription which helps localize the tem-
plate strand and allows for repeated cycling of the template
during the continuous endogenous transcription. One pri-
mary difference between the rotavirus and orthoreovirus is
the location of the capping enzyme. In contrast to orthore-
ovirus, in which the capping enzyme is located on the outer
surface of the innermost layer, in rotavirus, biochemical
and structural studies have shown that the capping enzyme
is in close proximity to the polymerase (Lawton et al.,
1999; Prasad et al., 1996). It remains to be seen how the
polymerase in rotavirus accommodates interaction with the
capping enzyme.
3. Structural organization of the genome
Understanding the structural organization of the genome
particularly in dsRNA viruses assumes a greater impor-
tance because of the intimate involvement of the genome
in the various enzymatic activities within the confines of
the capsid. From all the available biochemical and struc-
tural data to date on several members of the Reoviridae,
including cypovirus, orthoreovirus, BTV and rotavirus, the
emerging consensus is that an independently functioning
transcription enzyme complex, anchored to the inside sur-
face of the innermost capsid layer, transcribes each genome
segment, and that all the genome segments are transcribed
simultaneously (Banerjee and Shatkin, 1970; Bartlett et al.,
1974; Gillies et al., 1971; Skehel and Joklik, 1969; Smith
and Furuichi, 1982). In vitro studies indicate that the tran-
scriptionally competent particles of these viruses are highly
efficient molecular machines capable of repeated cycles
of transcription. During each cycle of transcription, the
dsRNA segment, which has to move around the anchored
polymerase, must be unwound, separated, rejoined, and re-
wound for further cycles of transcription. The transcription
process appears to be impressively fast. In orthoreoviruses,
it is estimated that transcription proceeds at a rate of 50
nucleotides/s. The structural organization of the genome in
these viruses thus must allow for what seems to be a well
orchestrated dynamic process of repeated, simultaneous,
high speed transcription of multiple segments.
3.1. Manifestation of genomic RNA in
icosahedrally-averaged structures
Although the precise organization of the genome in the
members of Reoviridae remains to be elucidated, recent
structural studies on some of these viruses have provided
useful insights. The first visualization of the structural or-
ganization of the genome in a dsRNA virus was provided
by the cryo-EM analysis of rotavirus (Prasad et al., 1996).
These studies indicated that the viral dsRNA forms a do-
decahedral structure in which the RNA double helices,
interacting closely with the VP2 layer, are packed around
the transcription complexes located at the icosahedral ver-
tices. It was suggested that VP2, which is icosahedrally
ordered, with its RNA-binding property is responsible for
the icosahedral ordering of the closely interacting portions
of the RNA, and this ordering is diminished at lower radii.
Subsequent X-ray crystallographic studies on BTV (Gouet
et al., 1999) and orthoreovirus cores (Reinisch et al., 2000)
and cryo-EM studies on RDV, CPV, aquareovirus, have all
consistently shown that a significant portion of the genome
is statistically ordered and manifests as concentric layers
of density in the icosahedrally averaged structures of these
viruses. These concentric layers of RNA are generally
separated by 28–30 Å. Assuming a local hexagonal pack-
ing of the RNA helices, such separation translates into an
inter-strand spacing of 30–32 Å. The observations in these
icosahedrally-averaged structures are consistent with other
studies. A similar inter-strand spacing was deduced from
earlier low angle X-ray scattering studies on orthoreovirus,
which also suggested that the dsRNA genome is tightly
packed as parallel helices in a semicrystalline array (Harvey
et al., 1981). Considering the volume available for the
genome inside the capsid layers and the molecular weight
of the genome in these viruses, typically the concentration
of the RNA inside the capsid layer is around 400 mg/ml
(Gouet et al., 1999). At such concentrations, dsDNA is
known to exhibit a columnar hexagonal liquid crystalline
packing with an inter-strand spacing of about ∼30–32 Å
(Livolant et al., 1989), suggesting thereby the packaged
dsRNA in these viruses behaves like dsDNA. How are the
charges on the condensed dsRNA neutralized? Generally
counter ions like Mg2+ , or organic polyamines like sper-
mines or spermidines are involved in nucleic acid charge
neutralization in viruses. However, Gouet et al. (1999),
found that the levels of metal counter ions in BTV cores,
measured using a scanning proton microprobe, are too low
to be implicated in charge neutralization, and instead have
suggested that the charge neutralization may be through
organic polyamines in this virus.
3.2. Reversible condensation and expansion of the
rotavirus genome
Recent cryo-EM analysis of rotavirus examined under
various chemical conditions unraveled a remarkable ability
of the rotavirus genome to undergo reversible condensation
and expansion within the capsid interior (Pesavento et al.,
2001) (Fig. 3). These studies have provided further insights
into the structural organization of the genome and the na-
ture of interactions between the genome and the internal
proteins. At high pH in the presence of ammonium ions, the
genome condenses to a radius of 180 Å from the original
 H. Jayaram et al. / Virus Research 101 (2004) 67–81 75

Fig. 3. Reversible condensation and expansion of the rotavirus genome. A cartoon representation of a plausible structural organization of the genome in
normal physiological condition (left) and in the ammonium high pH condition (right). Such a model from several biochemical and structural studies (Gouet
et al., 1999) offers a simple mechanistic explanation of the observed genome condensation in rotavirus (Pesavento et al., 2001). The condensation from a
original radius of 220–180 Å is achieved by simply reducing the inter-strand spacing in the spiral from 31 Å (indicated by pair of arrows in bottom left),
as observed in the normal physiological conditions, to a spacing of 25 Å (pair of arrows bottom right) as observed in the ammonium high pH condition.

radius of 220 Å, and when brought back to physiological pH
the genome expands to its original radius. The genome re-
mains transcriptionally viable after returning to its original
state, thereby indicating that this transformation is merely
structural and the dsRNA is not covalently disrupted by
the ammonium high pH treatment. Apart from VP4, which
is irreversibly altered, the rest of the capsid remains intact
under these conditions. These studies illustrate the remark-
able stability of the capsid and resilience of the genome;
these attributes may be required to carry out the continuous
transcription of multiple segments within the capsid.
Several observations made in this study have direct rel-
evance to the roles of VP2, and the transcription enzyme
complexes in the structural organization of the genome. The
observation that the condensation of the genome is isometric
and concentric with respect to the particle center, suggests
that strong interactions between the genome and the symmet-
rically disposed transcriptional complexes exist, and such
interactions may play a critical role in the structural organi-
zation of the genome. In the condensed state, the reconstruc-
tions show strong density linking the RNA core with the in-
side surface of the VP2 layer at all the five-fold vertices, like
spokes linking a hub and wheel. Without such symmetrically
disposed interactions, the condensed RNA core once disas-
sociated from the VP2 layer would not have been constrained
to remain at the particle center. The isometric condensation
also supports the notion that each dsRNA segment may be
associated with a transcription complex and all the segments
are similarly affected by the ammonium high pH treatment.
The studies by Pesavento et al. (2001) also underscore
the importance of VP2 in the structural organization of the
genome. These studies suggest that the VP2, through its
RNA-binding property, plays an important role in maintain-
ing the appropriate spacing between the RNA strands in the
native expanded state. In the native state the genome exhibits
several points of contact with the VP2 layer. As the genome
proceeds to the condensed state, these contacts are broken
only to be reestablished when the physiological conditions
are restored. The observed condensation is a synergetic ef-
fect of hydroxyl and ammonium ions, as it is not observed
at high pH alone, or with increased concentrations of NH+
or Mg2+ ions at physiological pH. The two inferences that
can be made from these observations are that VP2–RNA in-
teractions are pH-dependent and that the negative charges
on genomic RNA in the native state may only be partially
neutralized. The effect of high pH is to disrupt RNA–VP2
interactions, possibly due to deprotonation of critical VP2
residues, and cause conformational changes in the RNA to
allow further charge neutralization by ammonium ions to
cause condensation. The partial neutralization of the ge-
nomic RNA in the native state may be necessary to maintain
appropriate inter-strand spacing to allow the genomic RNA
to move around the transcription complex during transcrip-
tion. In the condensed state because of the reduced volume
and consequent increase in the concentration of RNA, the
inter-strand spacing is reduced from 30 to 25 Å.
3.3. A model for structural organization of the genome
Any model for the structural organization of the genome
particularly in the members of Reoviridae should allow for
simultaneous, independent and repeated transcription of the
genome segments. Studies by Pesavento et al. (2001) impose
further constrains of reversible condensation and expansion
76 H. Jayaram et al. / Virus Research 101 (2004) 67–81
on such a model. From the elegant X-ray crystallographic
analysis of the BTV cores, which provide a more detailed
and discernible electron density distribution of the genomic
RNA, particularly after including lower resolution diffrac-
tion data and reducing the noise using real space Gaussian
filtering, Gouet et al. (1999) proposed a plausible model
for the arrangement of dsRNA segments in the BTV core.
In this model, each dsRNA segment is spooled around a
transcription enzyme complex at the five-fold vertex. This
model allows for up to 12 independent transcription com-
plexes, each attached to an individual dsRNA segment for
concurrent transcription. Consistent with this idea, to date
no dsRNA virus with more 12 segments has been observed.
In addition to allowing for the observed simultaneous, re-
peated, and independent transcription of the genome, this
model also appears to be consistent with the isometric and
concentric condensation of the genome observed in rotavirus
as shown schematically in Fig. 3.
4. Genome replication and packaging
One of the least understood processes in the rotavirus
replication cycle is the genome replication and packaging.
This also is true with other members of the Reoviridae.
Our understanding of the molecular basis of these processes
particularly in the members of Reoviridae has been limited
mainly because of lack of a reverse genetics system. In two
of the dsRNA viruses, ␾6 (Mindich, 1999) and birnavirus
(Mundt and Vakharia, 1996), which contain three and two
dsRNA segments, respectively, a reverse genetics system has
been successfully established. However, several in vivo and
in vitro studies on rotavirus, reovirus, and BTV have pro-
vided significant information on the downstream events tak-
ing place following endogenous transcription. Subsequent to
endogenous transcription and release of the transcripts, the
rotavirus replication cycle may be viewed as having three
major stages: (1) translation and synthesis of the viral pro-
teins; (2) replication, genome packaging and DLP assem-
bly; (3) budding of the newly formed DLPs into the ER
and assembly of the outer layer to form mature TLPs (re-
viewed in (Estes et al., 2001). The capped positive-stranded
RNA transcripts encode the rotaviral proteins and function
as templates for production of negative strands to make the
progeny dsRNA. The synthesis of the negative strand and the
subsequent duplex formation is facilitated by the viral poly-
merase VP1. The genome transcription and the replication
are thus complimentary processes both involving the viral
polymerase. Just as transcription takes place in the confined
environment of the capsid interior, the genome replication
also appears to take place inside a protected environment
(Patton and Spencer, 2000). In none of the dsRNA viruses
has free dsRNA been found in infected cells.
Genome replication and packaging in rotavirus and the
viruses in the Reoviridae, take place within the cytoplasmic
inclusions called viroplasms (Petrie et al., 1984). In most of
the dsRNA viruses, each virus particle is thought to contain
a full complement of the genome. The only known excep-
tion is chrysovirus, in which the genome consisting of four
dsRNA segments are packaged separately into four particles
(Wood and Bozarth, 1972). In multi-stranded dsRNA viruses
such as rotavirus, BTV, and reovirus, it remains a mystery
as to how each particle procures a correct set of dsRNA seg-
ments. Although the molecular mechanisms are presently
unclear, it is evident that the virus-encoded non-structural
proteins play a major role in choreographing the entire pro-
cess of genome replication, packaging, and perhaps segment
assortment in these viruses. Recently, structures of two of the
non-structural proteins encoded by rotavirus have been de-
termined. These structural studies have begun to shed some
light into the molecular mechanisms of genome replication
and packaging.
4.1. Structural studies on NSP3
In rotavirus, translation of the viral mRNA transcripts
is facilitated by NSP3, a basic 63 kDa protein that recog-
nizes the consensus 3 sequence on the viral transcripts. As
mentioned above, rotaviral transcripts are capped at their
5 end by the action of the structural protein VP3 during
transcription but their 3 ends are not poly-adenylated. Ro-
taviruses rely on the host translation machinery to produce
the proteins encoded by the genome. In the host cells,
only poly-adenylated and capped messages are efficiently
translated. This is brought about by recognition of the 5
cap by eIF4E and the poly-A tail by PABP (poly-A bind-
ing protein) which then interacts with a cellular factor
eIF4G, a multipurpose adaptor protein that is responsible
for delivering capped and poly-adenylated messages to
the ribosome. Rotaviruses overcome the lack of a poly-A
tail, which would hamper their efficient translation, by us-
ing a consensus sequence at their 3 ends that specifically
binds virus-encoded NSP3. While the N-terminal domain
of NSP3 binds this consensus sequence, the C-terminal half
interacts with eIF4G with an affinity greater than PABP
to give translation of the rotavirus messages a selective
boost following infection. Thus, NSP3 is a protein en-
coded by the virus to subvert the host translation machinery
and selectively enhance the translation of virally encoded
mRNA. How NSP3 achieves this was demonstrated by the
X-ray structure of the two domains of NSP3 bound to the
consensus 3 mRNA sequence (Deo et al., 2002) and the
structure of the other NSP3 domain bound to a peptide cor-
responding to the binding site on eIF4G (Groft and Burley,
2002). The asymmetric NSP3 homo-dimer with an unusu-
ally large dimeric interface binds the 3 consensus sequence
(5 -GUGACC-3 ) and completely buries most of it within a
basic deep cleft on the surface of the homo-dimer creating
a dead end for the 3 terminal nucleotides (Fig. 4). This
tight interaction not only promotes translation of the ro-
tavirus mRNA but also prevents degradation of the rotavirus
message by cellular nucleases. The NSP3 homo-dimer
 H. Jayaram et al. / Virus Research 101 (2004) 67–81
Fig. 4. NSP3 structure. Structure of the rotavirus NSP3 N-terminal domain
in complex with a 3 consensus sequence of rotaviral mRNA (Deo et al.,
2002). A single 5 -GUGACC-3 RNA segment is buried within a basic
deep tunnel formed in the asymmetric dimer. Each subunit of the dimer
participates in different interactions with the mRNA segment.
is thought to co-fold with the 3 consensus end into this
extremely stable complex, which increases the stability of
the NSP3 hetero-dimer. These studies further underscore the
functional significance of the conserved sequences at the ter-
mini of the transcripts in genome translation via their recog-
nition by NSP3 (Poncet et al., 1994; Wentz et al., 1996).
4.2. Structural studies on NSP2
Several in vivo and in vitro studies on rotavirus have
strongly implicated two of the non-structural proteins NSP2
and NSP5 in genome replication and packaging. In vivo
studies have shown that these two proteins along with VP1,
the RNA polymerase, are co-localized in the viroplasms
Fig. 5. NSP2 Structure. Structure of the NSP2 monomer (left), showing a deep cleft (arrow) which may be the site for NTP binding (Jayaram et al., 2002).
Structure of the functional NSP2 octamer showing a view down the four-fold (middle) and a view down one of the two-fold axis in octamer (right). The
deep grooves shown (right, arrows) are lined by basic residues and may be the sites for binding viral mRNA during genome replication and packaging.
77
and are the main constituents of the replication interme-
diates (Aponte et al., 1996; Gallegos and Patton, 1989).
Biochemical studies on recombinant NSP2 have shown
that it readily forms octamers and has NTPase (nucleotide
triphosphatase), RNA-binding and nucleic acid helix desta-
bilizing activities (Taraporewala et al., 1999; Taraporewala
and Patton, 2001). Based on these studies, it is hypothesized
that NSP2 may function as a molecular motor to facilitate
genome packaging using the energy derived from NTP
hydrolysis (Taraporewala et al., 1999).
Recently, the X-ray structure of NSP2 to a resolution of
2.6 Å has been determined (Fig. 5). These studies have pro-
vided a firm ground to begin developing a mechanistic un-
derstanding of how NSP2, in concert with NSP5 and VP1,
may facilitate genome replication and packaging. NSP2
crystallizes as an octamer using the crystallographic 4–2–2
symmetry, with one monomer per asymmetric unit. The
intrinsic ability of NSP2 to form octamers in solution is sup-
ported by other biophysical studies including cryo-EM stud-
ies. In vivo studies also strongly suggest that the oligomeric
structure is the functional form of the NSP2 (Kattoura et al.,
1994).
NSP2 is a two-domain protein, which can be classi-
fied as an ␣/␤ protein based on the observed secondary
structures. A characteristic feature in the NSP2 monomer
is a 25 Å deep cleft between the N- and C-terminal do-
mains of the protein. The N-terminal domain, predomi-
nantly made of ␣-helices, exhibits a novel fold, whereas
the C-terminal domain, surprisingly, despite any notice-
able sequence homology, exhibits a fold that is observed
in the Histidine Triad (HIT) proteins, a family of ubiqui-
tous cellular proteins that hydrolyze nucleotides (Brenner,
2002; Lima et al., 1997; Lima et al., 1996). This struc-
tural similarity with the HIT proteins led to the proposal
that the cleft might correspond to the active site for NTP
hydrolysis.
78 H. Jayaram et al. / Virus Research 101 (2004) 67–81
The nucleic acid binding activity of the NSP2 is inde-
pendent of the NTPase activity unlike in a classical helicase
where the two activities are coupled. In that respect, NSP2
cannot be described as a helicase. In many of the oligomeric
helicases, nucleic acid binds inside a large central hole (Yu
et al., 1996). The donut-shaped NSP2 octamer also displays
a central hole with a diameter large enough for binding
nucleic acid. However, both the rim and interior surface of
the hole are mostly hydrophobic, and thus it is unlikely that
the nucleic acid binds inside the hole in the NSP2 octamer.
Instead, the prominent grooves, lined by positively-charged
residues at the sides of the octamer are possible locations
for nucleic acid binding. The proposed NTP binding sites
in the monomeric subunits are located on either side of
this groove. Although NTP hydrolysis is not directly linked
either to the RNA binding or to the helix destabilizing ac-
tivity of NSP2, it is possible that binding of nucleotide into
the cleft alters the conformation of the monomer and affects
octamer–RNA interactions. Such allosteric changes may
facilitate translocation of the bound RNA through the poly-
merase during replication resulting in concomitant synthesis
and packaging of duplex RNA into assembling particles.
Indeed the octameric structure of NSP2 makes it a tempt-
ing platform or a scaffold around which the replication
complex is organized. It is possible that the hydrophobic
side of the octamer, around the four-fold axis, may bind
to the VP1; given that NSP5 is an acidic protein, the basic
grooves of the NSP2 octamer may be the sites for binding
NSP5. Although the role of NSP5 in the overall process of
replication remains to be elucidated, it is plausible, that by
having its binding site on NSP2 overlap with that of its RNA
binding site, the function of NSP5 is to regulate the binding
of nucleic acid by NSP2 during replication and packaging.
In other members of Reoviridae, the existing biochem-
ical data suggest that NSP2 may be functionally homol-
ogous to NS2 of BTV and sNS of reovirus (Fillmore
et al., 2002; Gillian and Nibert, 1998; Gillian et al., 2000;
Taraporewala et al., 2001). Another protein with which ro-
tavirus NSP2 may have similarities is P4 of ␾6, which is an
NTPase (Gottlieb et al., 1992). However, one critical differ-
ence is that, unlike rotavirus NSP2 and its putative counter-
parts in BTV and reovirus, P4 is a structural protein. It is an
integral part of the transcriptionally competent ␾6 core struc-
ture, located at each of the five-fold vertex as a hexamer (de
Haas et al., 1999). Because of the well-established in vitro
replication and packaging system, ␾6 is the only dsRNA
virus for which there is more definitive understanding of
how the genomic dsRNA segments are packaged inside
(Mindich, 1999). Assisted by the packaging protein P4, the
three dsRNA segments in ␾6 are packaged sequentially into
a preformed core, which undergoes significant conforma-
tional change upon packaging. It remains to be seen whether
such a model for packaging RNA into preformed cores is ap-
plicable to dsRNA viruses with larger numbers of segments.
Based on the existing biochemical and structural data on
Reoviridae members, alternative packaging models assisted
by non-structural proteins including co-assembly of core
proteins and genome segments remain a distinct possibility.
5. Concluding remarks
Despite the lack of a reverse genetics system, in recent
years significant progress has been made in our understand-
ing of the structure–function relationships in rotavirus. This
has been primarily due to the advances in molecular bi-
ology of rotaviruses that have resulted in cloned rotaviral
genes and purification of protein complexes and virus-like
particles. Starting with the first cryo-EM reconstruction of
rotavirus in 1988, which provided a low resolution picture
of the virus architecture (Prasad et al., 1988) subsequent
cryo-EM studies on virus–antibody complexes, recombi-
nant virus particles (Prasad et al., 1990, 1996), and more
recent X-ray crystallographic analyses of several rotaviral
proteins have enabled a better understanding of the molec-
ular mechanisms underlying cell entry (Dormitzer et al.,
2002), antibody neutralization (Prasad et al., 1990; Tihova
et al., 2001), trypsin-enhanced infectivity (Crawford et al.,
2001), assembly (Mathieu et al., 2001), genome organiza-
tion (Pesavento et al., 2001; Prasad et al., 1996), endogenous
transcription (Lawton et al., 1997, 1999), mRNA translation
(Deo et al., 2002), and genome replication and packaging
(Jayaram et al., 2002). The outcome of such studies should
be potentially useful in the development of vaccines and
identification of suitable targets for rational drug design to
counteract rotavirus.
In parallel, cryo-EM and X-ray crystallographic studies
on other dsRNA viruses have underscored how the mod-
ular architecture of the virus can integrate disparate host
specificities with the common requirement of transcribing
the dsRNA segments within the capsid interior. Despite the
lack of extensive sequence identity, the structural and bio-
chemical studies thus far indicate a remarkable convergence
in both form and function necessitated by the endogenous
transcription. Further biochemical and structural studies are
required to establish whether these viruses share any com-
mon themes in the events downstream of transcription. A
major achievement in the near future may be the establish-
ment of reverse genetics systems for some of the members
of Reoviridae. Recent developments in the use of RNA in-
terference techniques are exciting and likely to provide fur-
ther insights into the specific role of each of the virally
encoded protein in the pathogenesis and morphogenesis of
these viruses (Dector et al., 2002).
Acknowledgements
This work is supported by grants from the Robert Welch
Foundation (BVVP), National Institutes of Health AI 36040
(BVVP) and DK 31044 (MKE). We thank Drs. J.A. Lawton
and J.B. Pesavento for help with the figures.
 H. Jayaram et al. / Virus Research 101 (2004) 67–81
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