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Rotavirus cell entry: not so simple after all
Carlos F Arias and Susana López
Rotaviruses are important agents of severe gastroenteritis in
young children, and show a very selective cell and tissue
tropism, as well as significant age and host restriction. In the
last few years, these properties have been associated with the
initial interaction of the virus with histo-blood group antigens on
the cell surface, although post-attachment interactions have
also been found to define the susceptibility to infection of
human enteroids. These initial interactions seem also to
determine the virus entry pathway, as well as the induction of
signaling cascades that influence the virus intracellular
vesicular traffic and escape from endosomes. Here we review
the current knowledge of the different stages of the virus entry
journey.
Address
Instituto de Biotecnologı́a, Universidad Nacional Autónoma de México,
Av. Universidad 2001, Colonia Chamilpa, Cuernavaca, Morelos, Mexico
Corresponding author: Arias, Carlos F (arias@ibt.unam.mx)
Current Opinion in Virology 2021, 49:42–48
This review comes from a themed issue on Virus entry
Edited by Chelsey Spriggs and Billy Tsai
https://doi.org/10.1016/j.coviro.2021.03.011
1879-6257/ã 2021 Elsevier B.V. All rights reserved.
Introduction
The initial step in a viral infection is the attachment of the
virus to specific receptors on the cell surface, an interac-
tion that frequently triggers cellular signaling cascades
that facilitate either virus entry or replication. In most
instances, regardless of whether the virus is enveloped or
not, virus internalization proceeds through an endocytic
pathway that allows the incoming viral particle to reach an
intracellular compartment where the appropriate cellular
cues that promote virus uncoating and penetration of the
vesicular membrane are present [1]. In this review we will
summarize what is currently known about the different
stages of the rotavirus (RV) entry process to reach its site
of replication, and the cellular counterparts that influence
this process.
RVs, the cause of severe dehydrating diarrhea in many
mammals and an estimated 200,000 human infant deaths
annually, are nonenveloped, segmented double-stranded
Current Opinion in Virology 2021, 48:42–48
RNA (dsRNA) viruses, composed of a triple-layered
protein capsid that surrounds the viral genome [2,3].
The outermost capsid layer is formed by trimers of
VP7 that constitute the smooth surface of the virus,
and by trimers of the spike protein VP4, which functions
as the virus attachment protein. VP4 has to be processed
by trypsin-like proteases in the intestinal lumen for the
virus to become infectious, and this cleavage activation
yields two non-covalently bound subunits, VP8 and VP5
[2]. Cryo-electron microscopy (CryoEM) studies, includ-
ing one at near-atomic resolution, have shown that the C-
terminal domains of the three VP5 subunits interact to
form a trimeric foot that is sandwiched between the
intermediate capsid layer formed by VP6, and the VP7
outer capsid layer [4–6]. The region of VP4 that protrudes
out of the VP7 layer does not maintain the local trimeric
symmetry observed in the foot (Figure 1). One of the VP5
monomers forms the spike stalk, while the other two VP5
subunits build the dimeric spike body that extends away
from the particle surface, and is capped by two VP8-lectin
domains. The third VP8-lectin domain is presumed
to dissociate from the particle after proteolytic activation
[4–6]. VP8 is responsible for the initial attachment of the
virus to the surface of the target cell, while VP5 is
involved in cell membrane penetration [7]. The
uncleaved rotavirus particles show spikes with a structure
indistinguishable from that of trypsin-digested particles,
suggesting that the proteolytical cleavage of VP4 does not
alter the rigid structure of the spike, but allows the
conformational change required for membrane disruption
during virus entry [8].
Receptor binding
RV cell entry is a complex multistep process that begins
with the interaction of the virus particle, through the VP8
galectin domain of VP4, with glycans on the cell surface
[9–11] (Figure 2, A1). During decades, it was believed
that sialic acid (SA), as part of glycosphingolipids (gang-
liosides), or as terminal acidic sugars on saccharide
branches of glycoproteins, was key for RVs attachment
to the cell surface, since the infectivity of several animal
RV strains widely used as model viruses was greatly
reduced by the removal, before infection, of SAs from
the cell surface using sialidases, and were thus considered
to be neuraminidase (NA)-sensitive [7]. In addition,
although it was observed that some animal RV strains
and most human rotaviruses (HRVs) are NA-resistant
[10], some of these strains do recognize SAs, but at
internal positions in the carbohydrate chain, such that
they are not trimmed by the activity of NAs [12], reinfor-
cing the idea of the relevance of SA for RV infection.
However, in the last ten years evidence has highlighted
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 Rotavirus cell entry Arias and López 43

Figure 1

(a) (b)

Current Opinion in Virology

Atomic structure of the VP4 spike.

(a) Model of the rotavirus particle structure generated from the atomic coordinates of the triple-layered capsid and the viral RNA polymerase VP1 (
purple). (b) Atomic structure of the VP4 spike. Ribbon representation (top), color-coded to represent the spike foot (green), stalk (red), body (red)
and head (purple). As indicated, each of the VP4 subunits (VP4-A, VP4-B and VP4-C) is highlighted in color. The N-terminal and C-terminal regions
of the three molecules, contacting one another beneath the VP7 layer, form the trimeric foot of the structure. The b-barrel domain of the VP5 C
subunit forms the stalk while the VP5 A and -B subunits build the spike body. The head is constructed by the VP8 lectin domains of the A and -B
VP4 subunits. The VP4-C lectin domain is not present in the structure, and is presumed to be released during proteolysis. The VP6 and VP7 layers
are depicted, as their 3D structure, in blue and yellow, respectively. Diagram of the VP4 domain organization (bottom), color-coded as in top.
Residues delimiting domains and trypsin cleavage sites are indicated. Modified from Rodrı́guez et al. doi: https://doi.org/10.1371/journal.ppat.
1004157.g001.

the relevant role of histo-blood group antigens (HBGA),
non-sialylated glycoconjugates present on the surface of
red blood cells, epithelial cells, and in mucosal secretions
[13], as RV cell attachment and susceptibility factors [11].
The X-ray crystallographic structure of the VP8 subunit
of several NA-sensitive and NA-resistant strains has been
determined, and the crystal structure of some of these
proteins in complex with glycans has been reported
(references in Refs. [10,14]). It has been found that RV
VP8 binds different glycans in a genotype-dependent
manner, and this interaction can be even strain-specific.
In general, the common theme is that glycans interact
with an open-ended, shallow groove on the galectin fold
of VP8 formed by two b-sheets, but variations in its width
and the amino acid residues exposed, as well as the
specific binding site in the groove, determine the type
of glycans that can be accommodated, and thus the
genotype specificity [10,11]. However, it was recently
shown that the P[19] VP8 binds to type 1 glycans at a
novel binding site, completely different from the previ-
ously characterized A-type or precursor binding sites in
RVs [15]. Furthermore, the VP8 domain of the globally
dominant HRV P[4] and P[8] VP4 genotypes was found to
bind to A-HBGA, H-HBGA, and B-HBGA types, and the
crystal structure of the VP8 of a P[6] RV isolated from
neonates, identified a binding site for H-type I glycan
formed by one of the b-sheets and the C-terminal a-helix
of P[6] VP8, similar to the binding site described for the P
[19] VP8, offering clues about the age-restricted tropism
of some neonatal RV strains [16]. More recently, the H-
type 1 binding site in P[8] VP8 was determined by X-ray
crystallography, and variations in affinities in different P
[8] lineages were found, possibly reflecting subtle struc-
tural differences in the binding pocket, and providing a
fine-tuning mechanism for glycan binding in P[8] RV
strains [17,18]. Also, it has been described that [P4] and P
[8] VP8s can recognize Leb HBGA glycans in still an
additional previously undescribed pocket [19]. Alto-
gether, these findings suggest complex and variable
attachment factor binding patterns that may influence
cell susceptibility to infection, as well as age, tissue and
host restriction, and possibly host susceptibility to specific
RV strains [10,16]. In contrast with these observations,
both NA-sensitive and NA-resistant animal and human
RV strains bind to a wide variety of cell lines, although
only a subset of them is efficiently infected [20], suggest-
ing that the initial VP8/glycan interaction of rotavirus with
the cell surface is promiscuous, and that relevant inter-
actions for cell infection specificity may occur at a post-
binding step.

www.sciencedirect.com Current Opinion in Virology 2021, 48:42–48

44 Virus entry

Figure 2

(a)
2 VP2 VP7

VP6 VP4

Lipid raft
3
Terminal and HBGAs Co-receptors
(b)
sub-terminal SA Dynamin

Clathrin
Actin
filaments
E-P viruses
4 1
Viral transcripts ACTN4
CDC42
5 EE RhoA

Drebrin

Ca++
ILV ESCRT
EEA1
Rab5
vATPase
2
ME Rab7
M6PR

Cathepsins 3
6
Viral transcripts
LE ILV
M6PR
Rab9

L-P viruses
7 TNG

Current Opinion in Virology

Models for RV cell entry.

(a) Classical endocytic pathway. A1, RV cell entry begins with the interaction of VP8 with glycans on the cell surface. Terminal or subterminal SAs,
as well as HBGAs serve as attachment receptors for the viral particle. A2, after initial attachment, RVs have post-binding interactions with integrins
a2b1, aVb3, and aXb2, and with hsc70 that are embedded in lipid rafts; JAM-A and occludin have also been involved at this step; a novel,
human-specific post-attachment cell molecule has also been proposed. A3, Several RV strains are internalized by clathrin-dependent endocytosis,
in a dynamin-dependent and cholesterol-dependent process. RRV strain enters cells by an endocytic pathway that is clathrin-independent and
caveolin-independent, and depends on RhoA, Cdc42, and actinin-4 (ACTN4), but is independent of endosomal acidification. A4, After
internalization, all RV strains reach early endosomes (EEs), and depend on a functional ESCRT system, including the ESCRT-associated ATPase
VPS4A involved in fission of intralumenal vesicles in maturating endosomes (MEs). A5, Once in the MEs, different RV strains follow different routes
to reach the cytosol. Early penetrating (E-P) viruses exit from ME vesicles, since their infectivity is not dependent on the GTPase Rab7. A6, Other
strains depend on the expression of Rab7 and thus these viruses continue a deeper journey to reach late endosomes (LEs) that provide the
environment for these—Late-penetrating (L-P) strains—to enter the cytosol. A7, Cellular factors (Rab9, M6PR, and cathepsins), transported from
the trans-Golgi network (TGN) to LEs, facilitate RV cell infection. (b) An alternative pathway for virus internalization. B1, RV interacts with
ganglioside receptors and this interaction drives an invagination process that results in virus internalization entry in tight-fitting membrane vesicles.
B2, There is a drop of Ca2+ concentration within the tight-fitting vesicles vesicle, causing the release of the Ca2+-dependent VP7 trimer contacts;
VP5 folds back to a post-cleavage conformation, disrupting the membrane vesicle. B3, The DLP is liberated into the cytosol, where it initiates RNA
transcription. After entering the cells through either of the entry pathways described, the transcribed mRNAs translate into the 12 encoded viral
proteins which, after accumulating to a critical amount, Form the virus factories, known as viroplasms, where the replication of the genomic RNA
and initial assembly of the viral progeny occurs.

Current Opinion in Virology 2021, 48:42–48 www.sciencedirect.com


Post-binding interactions
Several sequential interactions involving VP5 and VP7,
relevant for cell infection, have been reported in the
frequently employed monkey kidney epithelial MA104
cells, and human colonic Caco-2 cells. RVs interact with
integrins a2b1, aVb3, and aXb2 and with the heat shock
cognate protein hsc70, embedded in detergent resistant
membrane domains, and consequently RV entry has been
found to be sensitive to membrane cholesterol depletion
[7,21] (Figure 2, A2). More recently, the tight-junction
proteins JAM-A and occludin were also found to be
involved in cell infection of some RV strains at a post-
attachment step [22]. Of note, gangliosides can also play a
role at a post-binding step, in addition to the role they
have as attachment factors for both NA-sensitive and NA-
resistant RV strains [23]. Interestingly, recent X-ray crys-
tallographic and cryo-EM analyses of RV structural
proteins and double-layered (DLP) and triple-layered
(TLP) particles have shown that the proposed VP7 sites
for interaction with avb3 and aXb2, as well as the
interaction site for hsc70 on VP5, are not exposed on
TLPs, indicating that they could only be accessible after
or during the uncoating process of the virus [14,24].
Furthermore, it was observed that the interaction of VP5
with the a2 subunit of integrin a2b1 depends on a
conformational change of VP5 [6,14,24]. This confor-
mational change was suggested to be present in the RRV
NA-resistant nar3 mutant VP5 domain, since this virus
binds directly to integrin a2b1, obviating the need for the
VP8-glycan interaction [25,26].
More recently, a collection of RV VP8-specific recombinant
mAbs, generated from human, intestinal antibody-secret-
ing B cells, were found to be non-neutralizing for P[4], P[6]
and P[8] HRV strains when assayed in MA104 cells [27].
However, unexpectedly, these ‘non-neutralizing’ antibo-
dies did neutralize P[8] human rotaviruses in enteroid
cultured cells as well as in the human epithelial colonic
cell line HT-29 [28]. The X-ray crystallographic structure
of a P[4] VP8, in complex with a single-chain fragment of
one of these MAbs (MAb9), identified an interaction site
located away from the HBGA binding site [28] recently
described for a P[4] VP8/H type 1 antigen complex [16].
The observation that mAb9 does not block VP8 binding to
HBGA, suggests that it blocks a virus–cell interaction that
occurs only in human intestinal cells (not in MA104 cells),
and points to a specific RV receptor that could be recog-
nized after the initial VP8-glycan interaction [28]. The
existence of a specific post-attachment receptor is addition-
ally supported by the observation that rotavirus infection of
polarized cell monolayers, including human intestinal
enteroids, occurs preferentially through the basolateral
membrane [28,29,30]. The access of virions to putative
coreceptors in the basolateral side could be facilitated by
signaling through the RhoA/ROCK/MLC pathway,
induced by virus attachment to cell receptors on the apical
side, that mediates the disruption of tight junctions [31].
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Rotavirus cell entry Arias and López 45
Despite all these observations, identification of a protein-
aceous receptor or coreceptor essential for the infectivity
of either animal or human RVs has been elusive. These
findings are puzzling and suggest that either a more
relevant entry factor has not been found for RV, the virus
has the plasticity to use more than one route of entry, or
the entry factors for RV are redundant.
Cell internalization
Many animal and human RV strains are internalized into
non-polarized and polarized cells by clathrin-dependent
endocytosis [32,33], in a process that is dependent on
dynamin and on the presence of cholesterol on the cell
surface [29,33,34,35,36,37]. In contrast, the simian RRV
strain, which has been widely used as a model virus to
study RV biology, seems to be an outlier, since it enters
cells by an endocytic pathway that is independent of
clathrin and caveolin, although it is also dependent on
dynamin and cholesterol [33]. RRV entry also depends on
the activity of the small GTPases RhoA and Cdc42, and
on actinin-4 [36,38,39] (Figure 2, A3). Of interest, the
endocytic route employed, whether clathrin-dependent
or clathrin-independent, is determined by the spike pro-
tein VP4 [32], and is not defined by the initial VP8/glycan
interaction since, with exception of RRV, both NA-sen-
sitive and NA-resistant strains, including those that bind
HBGAs, enter cells through clathrin-mediated endocyto-
sis [32]. The VP4/cell factor interaction that defines the
endocytic pathway used has yet to be determined,
although it appears to occur after initial virus binding.
The dynamin-dependent endocytic entry of RV strains is
reinforced by the observation that the actin-binding pro-
tein drebrin inhibits RV entry in a virus strain-indepen-
dent manner through suppressing dynamin-mediated
endocytosis [35] (Figure 1, A3). Dbn1 KO mice infected
with RRV exhibited higher incidence of diarrhea and
more viral antigen shedding compared with the wild-type
littermates, suggesting a negative factor role for Dbn1 in
vivo. Furthermore, pharmacological inhibition of Dbn1
significantly promoted RV infection in human intestinal
enteroids [35,40], suggesting that a dynamin-dependent
endocytic process also occurs in the human gut.
In an alternative pathway for virus internalization, it was
reported that RRV interacts with ganglioside receptors on
the surface of BSC-1 cells and rapidly starts an invagina-
tion process driven by the virus particle itself that results
in its engulfment in tight-fitting membrane endocytic
vesicles (Figure 2, B1). This process was found to be
independent of dynamin and clathrin, and was completed
in about 10 min. [24]. In this study, the uncoating
process of TLPs was followed by live-cell fluorescence
imaging, and was detected as a rapid diffusional motion of
the DLP away from residual outer-layer proteins; this
event was interpreted as release of the DLP from the
endocytic vesicle and productive entry of the virus [24].
Current Opinion in Virology 2021, 48:42–48
46 Virus entry
In contrast with these findings, when the read out was an
infectivity assay, it was found that RRV enters MA104
cells with a half-time of 35 min [33]. The different
observations regarding the use of dynamin for virus
endocytosis, the kinetics of virus internalization, and
the route proposed for productive entry might reflect
the predominance of different pathways depending on
the experimental conditions, the cells, or the virus strains
employed. Alternatively, the biological relevance of the
different proposed models of virus internalization could
be different.
Intracellular vesicular traffic
When infectivity assays have been used as the readout for
productive RV infection, endocytosis through a cholesterol
and dynamin dependent process has been identified as the
route of entry. In this pathway, regardless the cell surface
receptor recognized, and the requirement of clathrin for cell
internalization [32,33,36,37,41], all RV strains reach early
endosomes (EEs) as a first step (Figure 2, A4) [36,41,42]. All
RVs tested also require a functional ESCRT system,
including the activity of the ESCRT-associated ATPase
VPS4A involved in the formation of endosomal intralum-
inal vesicles in maturating endosomes (MEs) [36]. After
reaching MEs, different RV strains follow different routes
to reach the cytosol. Early penetrating (E-P) viruses, like
RRV and SA11-4S, exit from ME vesicles (Figure 2, A5),
since their infectivity does not depend on the GTPase
Rab7 [36,41]. The infectivity of other RV strains tested
depends, at least partially, on the expression of this
GTPase, suggesting that these viruses continue a deeper
journey into the cell to reach late endosomes (LEs) that
likely provide the optimal environment for these strains to
enter the cytosol [41,43] (Figure 2, A6). As noted for cell
internalization, the spike VP4 also determines the differ-
ential trafficking of RVs; a single amino acid change in VP4
can dictate not only the pathway of endocytosis used but
also whether the virus reaches EEs or LEs during the entry
process [41].
Escape from late endosomes
Several conditions have been reported to be important for the
efficient escape of RVs from LEs. Late-penetrating (L-P)
human DS-1 and bovine NCDV RV strains stimulate the
PI3K/Akt and MEK/ERK pathways very early in infection;
specific inhibitors of these signaling pathways do not prevent
virus intracellular trafficking, but trap the viral particles in
LEs, preventing their exit to the cytosol [44]. As a conse-
quence, the infectious viral progeny is reduced 100–1000-
fold. RV-induced phosphorylated PI3K, Akt, and ERK
directly interact with the subunit E of the V-ATPase proton
pump [44], required for endosomal acidification and uncoat-
ing of L-P, but not E-P RV strains such as RRV [36,37,44,45].
In addition to an acidic pH, it has been shown that 25-
hydroxycholesterol and 27-hydroxycholesterol inhibit
human rotavirus infection by sequestering viral particles into
late endosomes [46]. These oxysterols cause the
Current Opinion in Virology 2021, 48:42–48
accumulation of cholesterol within LE compartments alter-
ing their function, and have also been shown to significantly
impair the infection of influenza A virus, vesicular stomatitis
virus, and dengue virus [47–49]. So, it is suggested that the
substantial accumulation of cholesterol in the LE compart-
ment decreases the exit of L-P RVs from these vesicles,
thereby arresting cytoplasmic virus replication [46].
The transport of cellular factors from the trans-Golgi
network (TGN) to LEs also seem to facilitate RV cell
infection. Newly synthesized lysosomal acid hydrolases
are delivered from the TGN to endosomes by mannose-6-
phosphate receptors (M6PRs), and recycling of M6PRs
back to the Golgi network depends on the small GTPase
Rab9 (18). In this regard, most L-P viruses depend on the
activities of Rab9, the cation-dependent M6PR, and
cathepsins B, L, and S, suggesting that these acidic
hydrolases facilitate the exit of transcriptionally active
L-P viruses into the cytoplasm (Figure 2, A7). Endoly-
sosomal cysteine cathepsins are also required for proces-
sing the capsid or the surface proteins of other viruses,
such as reovirus, Ebola virus, severe acute respiratory
syndrome (SARS) coronavirus, and Nipah virus (see
references in Ref. [41]).
Membrane penetration
To reach the cytosol and initiate replication within the
cell, RVs must traverse a lipid membrane, whether this
being the plasma membrane or the membrane of an
endocytic vesicle. Based on a detailed structure of infec-
tious RRV, a mechanistic model for RV entry has been
proposed [6,24,50]. In this model, after a trypsin-acti-
vated virus particle is endocytosed in tight-fitting vesi-
cles, there is a drop of Ca2+ concentration within the
vesicle, possibly mediated by the interaction of hydro-
phobic loops on VP5 with the vesicular membrane, that
causes the loosening of the Ca2+-dependent VP7 trimer
contacts, releasing this protein from the viral particle and
allowing VP5 to fold back to its most stable, post-cleavage
conformation (Figure 2, B2), rearrangement that disrupts
the vesicular membrane and liberates the DLPs into the
cytosol to begin transcription [24] (Figure 2, B3). Using
reconstituted RRV TLPs with VP4 labeled with a fluo-
rescent Ca2+ sensor, it was observed that after engulf-
ment of the virus particle is completed, a slow (1-min
duration) loss of Ca2+ precedes the onset of VP7 dissoci-
ation by about 2 min and DLP release by about 7 min,
supporting the model for the molecular mechanism of
membrane disruption by RV [50]. In a related study, it
was described that viral RNA synthesis can be detected as
early as 15 min after virus adsorption [51]. Regardless the
factors that facilitate RV membrane penetration previ-
ously discussed for different rotavirus strains, and the
vesicular compartment from which DLPs are liberated
into the cytosol, the mechanism of membrane disruption
driven by a conformational change of VP5 explains all
available biochemical, microscopy and structural data.
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Conclusions
Advances in the complex process of RV cell entry have
been challenging, and many questions remain. Is there a
critical cellular receptor for virus entry? To what extent
post-attachment interactions are required for RV infec-
tion? What is the VP4/cell interaction that determines the
route of virus entry and intracellular trafficking? What are
the cellular cues required for the escape of E-P and L-P
viruses from endocytic vesicles? Are there more than one
pathway of virus productive entry? Do RV strains other
than RRV are internalized through the tight-fitting vesi-
cle model? To what extent the VP8/glycan interaction
defines the host susceptibility to RV infection? Are other
viral or host factors involved? Does genotype-dependent
variation of glycan recognition have implications for tis-
sue and host restriction? Virus tracking by live-cell imag-
ing systems coupled with novel superresolution micros-
copy techniques, application of the recently implemented
reverse genetic system for manipulating the rotavirus
genome, as well as the availability of human intestinal
enteroids that represent a system that more faithfully
mimics virus infection of enterocytes in the human gut,
should allow us to gain insight into some of these ques-
tions. The continuous use of RNAi and CRISPR)/Cas9
technology, high resolution structural information of the
rotavirus capsid proteins in complex with glycans or other
cell surface molecules, cryo-electron microscopy (Cryo-
EM) and cryo-electron tomography (Cryo-ET) analyses
should also contribute to this end.
Conflict of interest statement
Nothing declared.
Acknowledgements
The authors would like to acknowledge support from grants A1-S-15356 and
Fordecyt 302965 from the National Council for Science and Technology,
CONACyT-Mexico (SL). We thank Daniel Luque and Javier M. Rodrı́guez
for kindly providing Figure 1.
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