Virology 479-480 (2015) 657–671

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Virology
journal homepage: www.elsevier.com/locate/yviro

Review

Plant virus replication and movement

Manfred Heinlein a,b,n
a
Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, Strasbourg, France
b
Zürich-Basel Plant Science Center, Botany, Department of Environmental Sciences, University of Basel, Basel, Switzerland

art ic l e i nf o a b s t r a c t

Article history: Replication and intercellular spread of viruses depend on host mechanisms supporting the formation,
Received 5 December 2014 transport and turnover of functional complexes between viral genomes, virus-encoded products and
Returned to author for revisions cellular factors. To enhance these processes, viruses assemble and replicate in membrane-associated
19 January 2015
complexes that may develop into “virus factories” or “viroplasms” in which viral components and host
Accepted 28 January 2015
factors required for replication are concentrated. Many plant viruses replicate in association with the
Available online 3 March 2015
cortical ER-actin network that is continuous between cells through plasmodesmata. The replication
Keywords: complexes can be highly organized and supported by network interactions between the viral genome
Plant virus and the virus-encoded proteins. Intracellular PD targeting of replication complexes links the process of
TMV
movement to replication and provides specificity for transport of the viral genome by the virus-encoded
TuMV
movement proteins. The formation and trafficking of replication complexes and also the development
RCNMV
PVX and anchorage of replication factories involves important roles of the cortical cytoskeleton and
Membranes associated motor proteins.
Cytoskeleton & 2015 Elsevier Inc. All rights reserved.
Plasmodesmata
Viral replication complex
Movement protein

Contents

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 657
Organization of Tobacco mosaic virus movement-competent VRCs and virus factories by MP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658
Recruitment of Red clover necrotic mosaic virus MP to VRCs organized by a replicase protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659
VRC formation and movement organized by PVX MPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659
Replication and movement of Turnip mosaic virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 661
Relationship between virus-induced compartments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662
Cellular mechanism of VRC movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662
Cellular mechanisms involved in the formation of VRC at the ER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663
Potential role of aggresomal processes in VRC formation and turnover. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 664
The targeting of plasmodesmata involves additional membrane transport mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665
Relationship of virus movement to the intercellular trafficking of endogenous macromolecules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 666
Conclusions and outlook. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 666
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667

Introduction

Plant viruses replicate and then move between cells through

plasmodesmata (PD), gatable symplasmic channels in the walls of
n
adjoining cells. Although the process of virus movement can be
Correspondence address: Institut de Biologie Moléculaire des Plantes du CNRS,
Université de Strasbourg, Strasbourg, France.
complex and requires support by the coordinated activity of
E-mail address: manfred.heinlein@ibmp-cnrs.unistra.fr several virus-and host-encoded proteins, many viruses achieve

http://dx.doi.org/10.1016/j.virol.2015.01.025
0042-6822/& 2015 Elsevier Inc. All rights reserved.
658 M. Heinlein / Virology 479-480 (2015) 657–671
their movement with the help of classical, virus-encoded ‘move-
ment proteins (MP)’ that bind nucleic acids and target and dilate
PD (Lucas, 2006). Upon introduction into cells by microinjection,
or by transient expression using microparticle bombardment or
agroinfiltration, MPs interact with PD, move between cells and
may co-transport co-injected nucleic acids (Waigmann et al., 1994;
Ding et al., 1995; Fujiwara et al., 1993; Lough et al., 1998), thus
indicating that their function is independent of infection. The
ability of MPs to bind nucleic acids is sequence-independent,
which raises the important question of how these proteins find
their viral RNA cargo in infected cells. Since the MPs are produced
by translation of the viral genome, they may simply attach co-
translationally to viral genomes in their vicinity. Consistently, the
MPs of several RNA viruses, including Tobacco mosaic virus (TMV;
Asurmendi et al., 2004; Heinlein et al., 1998; Kawakami et al.,
2004), Potato virus X (PVX; Bamunusinghe et al., 2009; Tilsner
et al., 2013; Tilsner et al., 2012), Brome mosaic virus (BMV; Dohi et
al., 2001) and Red clover necrotic mosaic virus (RCNMV; Kaido et al.,
2009) colocalize with membrane-associated inclusions that harbor
their viral replication complexes (VRC). As will be discussed below,
VRCs can be highly organized structures involving membranes and
cytoskeletal elements, and in which the binding of MP to viral RNA
involves specific mechanisms, for example interaction with other
viral proteins, as exemplified by VRCs formed by RCNMV. Another
important question is whether MPs transport viral genomes as
part of subcomplexes that are derived from anchored VRCs or
whether infection moves between cells in the form of mobile
VRCs. Studies with TMV and other viruses have shown that VRCs
develop at multiple sites and can be mobile, and that their
subcellular formation and movement are associated with the
spread of the virus (e.g. Boyko et al., 2007). Moreover, VRCs may
associate with PD and load replicated viral genome into PD for
movement (e.g. Tilsner et al., 2013), or move through PD as an
intact VRC to spread infection (Kawakami et al., 2004; Grangeon et
al., 2013). The following paragraphs are aimed to summarize
current knowledge about the mechanisms of VRC formation and
trafficking and thus the membrane- and cytoskeleton-associated
processes through which virus replication and movement are
mechanistically connected. Additional information and alternative
interpretations of findings can be found in several other reviews
published elsewhere (e.g. Laliberté and Zheng, 2014; Liu and
Nelson, 2013; Park et al., 2013; Tilsner and Oparka, 2012; Niehl
and Heinlein, 2011; Heinlein, 2015).
Organization of Tobacco mosaic virus movement-competent
VRCs and virus factories by MP
MP-deficient viruses that replicate but do not move can often be
complemented for movement by virus-specific or functionally
related MPs produced ectopically by a transgene or by transient
expression (Deom et al., 1987; Holt and Beachy, 1991; Morozov et al.,
1997; Niehl et al., 2014). This suggests the existence of active
mechanisms by which MPs and replicated viral genomes are brought
into close proximity despite that the MP is not produced within
replication complexes. One possibility to achieve proximity is by
active targeting of the same subcellular site. During in vivo studies
with TMV the same subcellular localization patterns were shown for
its MP irrespective whether produced during infection or transiently
(Boutant et al., 2010; Heinlein et al., 1998). Thus, this MP has intrinsic
features that allow targeting to sites in the cell at which the virus
replicates. The MP associates with the cytosolic face of the endo-
plasmic reticulum (ER) membrane (Peiro et al., 2013), interacts with
microtubules (Ashby et al., 2006; Boyko et al., 2000a; Heinlein et al.,
1995), and targets microtubule-ER junctions (Sambade et al., 2008)
that may also be targeted by the virus. Moreover, the MP has the
capacity to recruit ER membranes to microtubules (Ferralli et al.,
2006) and may thus contribute to the formation of ER membrane
inclusions that are seen upon ectopic MP expression as well as in the
form of ‘virus factories’ during TMV infection (Heinlein et al., 1998;
Niehl et al., 2013b; Reichel and Beachy, 1998). Experiments using
microinjection of in vitro-coated, Cy5-labeled TMV RNA have demon-
strated that also viral RNA associates with the ER, by virtue of its
5'CAP (Christensen et al., 2009). Interestingly, in the presence of
ectopically expressed MP, the introduced viral genome was capable
to move and spread infection only after producing progeny without
the tag, thus after replication (Christensen et al., 2009). This indicates
that the initially ER-attached viral genome remains at the attachment
site and that only progeny viral RNA is available for the formation of
movement complexes. This observation is remarkable as it demon-
strates that the movement process is linked to replication and that,
therefore, the formation of a ribonucleoprotein (vRNP) consisting
only of MP and viral RNA is not sufficient for movement. Since
replication of the TMV genome depends on the translation of the
183k replicase, it appears plausible that the vRNP complex that
moves between cells is a VRC containing the viral genome, replicase,
MP and host factors. The VRC may also contain the 126k protein,
which is produced by termination of 183k translation at a leaky
amber stop codon within the 183k open reading frame. This shorter
protein acts as a replication co-factor as it lacks the RNA-dependent
RNA polymerase (RdRp) domain present in the full length 183k
protein but interacts with the 183k protein (Goregaoker et al., 2001)
and functions as the viral suppressor of RNA silencing (VSR) (Ding
et al., 2004; Vogler et al., 2007). Supporting evidence for the presence
of the 126k protein in the movement complex comes from domain
swapping experiments indicating a role of the non-conserved region
and the RNA helicase domain of the 126k/183k replicase in TMV
movement (Hirashima and Watanabe, 2001, 2003). Moreover, virus
evolution experiments correlated the efficient movement of evolved
TMV lineages in certain Arabidopsis mutants with adaptive muta-
tions in this protein (Peña et al., 2014). It is clear that the movement
in the form of a VRC rather than as a simple MP:vRNA complex
would represent an efficient means to accelerate the spread of
infection as the viral genome would be already associated with
proteins required to rapidly initiate replication in recipient cells.
Consistently, the speed of cell-to-cell movement was reported to
increase significantly after viral exit from the first infected cells
(Kawakami et al., 2004). VRC movement could also explain observa-
tions suggesting a role of replicase (or replicating virus) in contribut-
ing to the degradation of PD-associated callose induced by MP to
facilitate movement (Guenoune-Gelbart et al., 2008). The observation
that the localization of ectopically expressed MP mimics the localiza-
tions of MP produced during infection indicates that the MP, in
addition to being part of the VRC that spreads between cells, provides
the microenvironment for VRC maturation. As already mentioned
and as will be described further below, the MP may employ a
microtubule-dependent aggregation mechanism by which it attracts
and holds together ER membranes and associated factors.
Taken together, the above observations suggest the following
model: Following initial stages of replication by the replicase
produced from the ER-associated viral genome, the MP is produced
from subgenomic RNA. The MP does not interact with the replicase
proteins (Hirashima and Watanabe, 2003) but may remain associated
with the VRC through the binding to the viral RNA and, indirectly,
with the RNA-bound replicase. VRC formation and stability may also
be supported by the ER membrane, to which the viral genome and
the MP, and perhaps also the replicase, have affinity. The spread of
infection has been visually correlated with the occurrence of punc-
tate MP-complexes (Fig. 1A) that show mobility along microtubules
and the cortical ER-actin network in cells at the leading front of
infections sites in leaves. These mobile MP complexes contain RNA
and may represent “early VRCs” that serve to transport infection into
 M. Heinlein / Virology 479-480 (2015) 657–671
adjacent cells (Boyko et al., 2007; Sambade et al., 2008). However, it
remains to be demonstrated whether these mobile, punctate MP
complexes contain the replicase proteins.
Some of the punctate MP-complexes (early VRCs) remain
anchored at the site of their formation and may develop into the
large ER-derived inclusions that are seen at later stages of infection
(Fig. 1B). These inclusions contain replicase, MP, vRNA and produce
CP (Asurmendi et al., 2004; Heinlein et al., 1998; Más and Beachy,
1999) and likely function as “late VRCs” or “virus factories”. The high
level of MP produced in these factories is dispensable for movement
(Boyko et al., 2000c; Heinlein et al., 1998; Niehl et al., 2014; Szécsi et
al., 1999) but may play an important role in the initial formation of
the inclusions (Reichel and Beachy, 1998). Consistent with the
dispensability of MP accumulation in viral factories for movement,
the production and activity of the MP of TMV is usually restricted to
early infection stages whereas the CP (required for virion formation),
replicase (183k) and VSR (126k) continue to accumulate (Lehto et al.,
1990; Oparka et al., 1997; Padgett et al., 1996). Moreover, immuno-
labeling studies indicated that MP disappears from VRCs during late
stages of infection (Szécsi et al., 1999). These observations suggest
that VRCs that are left behind in the infected cell switch function and
grow in size to support the production of virions.
Recruitment of Red clover necrotic mosaic virus MP to VRCs
organized by a replicase protein
Tight linkage between virus replication and movement is
particularly important if more than a single viral RNA must be
moved between cells to spread infection. One such example is Red
clover necrotic mosaic virus (RCNMV). This virus has a bipartite
genome of which the RNA 1 encodes two N-terminally overlapping
replicase subunits (27k and 88k) and the coat protein (CP) and the
RNA 2 encodes the MP (35k). Unlike the MP of TMV, the 35k protein
localizes exclusively to PD in the absence of infection, whereas
during infection it also localizes to VRCs at the ER (Kaido et al.,
2009). This indicates that the presence of the 35k MP in VRCs is
maintained through binding to a VRC component. The p27 and p88
proteins co-localize at the ER and cause membrane restructuring
and proliferation upon expression (Turner et al., 2004). Thus, unlike
TMV, RCNMV can rearrange the ER and produce large ER-associated
complexes without MP. During infection, the two replicase proteins
interact to form a 480 kDa VRC (Mine et al., 2010). In this complex,
p27 is bound to chaperones (heat shock protein (HSP) 70 and
HSP90) and membrane trafficking factors such as Arf1 and Sar1
(Hyodo et al., 2013; Mine et al., 2012) thus potentially linking viral
replication with active interference with the host membrane
trafficking machinery. The p88 protein contains an RdRp motif
required in cis for RNA 1 replication, whereas p27 recruits RNA2
(Hyodo et al., 2011) via binding to a Y-shaped RNA element (YRE)
(Iwakawa et al., 2011) and also binds the VRC to the membrane
(Kusumanegara et al., 2012). The p35 MP associates with VRCs on
the ER through its C-terminal 70 amino acids. Interestingly, deletion
of these 70 amino acids abrogated virus movement and MP
localization to the ER-associated VRCs but did not affect the ability
of this MP to increase PD aperture, to bind RNA or to interact with
wild type MP (Kaido et al., 2011). Since this MP, in contrast to the
MP of TMV, has itself no affinity for the ER (Kaido et al., 2009), the
binding to the VRC at the ER is essential for its function in virus
movement. Binding involves the negative strand and/or replication
of RNA1 but not RNA2 or the replicase proteins, thus indicating that
binding depends on the assembly of a functional VRC. Recruiting
p35 MP to replicated RNA 1 may represent a mechanism to
specifically capture replicated RNA 1 for transport (Kaido et al.,
2009). The formation of transport complexes with replicated RNA
2 may occur in the vicinity without requiring a specific recruitment
659
mechanism. Translation of p35 and the formation of transport
complexes from RNA 2 depend on RNA 2 replication (Mizumoto
et al., 2006), thus ensuring that also RNA 2 is transported only upon
replication. The utilization of specific mechanisms utilized by the
VRC for recruitment of RNA 1 and RNA 2 as replication templates
and for the formation of RNA 1- and RNA 2-specific complexes with
the p35 MP suggests a high degree of organization within the VRC.
A recent study in N. benthamiana indicates that the 35k MP localizes
to small, punctate aggregates on cortical ER during early infection
stages and to larger, rather perinuclear aggregates during later
stages and that both types of inclusions are associated with viral
RNA replication thus representing VRCs. Thus, as infection proceeds,
the VRCs change localization and grow in size. Novel findings
indicate that the stable association of the p35 MP with the cortical
aggregates is facilitated by binding to glyceraldehyde 3-phosphate
dehydrogenase (GAPDH), which also binds p27 and thus appears to
act as an interstitial agent between the p35 MP and the cortical
VRCs. Silencing of GAPDH expression inhibited the accumulation of
p35 in the cortical VRCs and also reduced the efficiency of virus
movement thus indicating that the cortical VRCs are associated
with RCNMV movement whereas the larger, perinuclear VRCs, that
occur at a later time, rather function to maximize virus reproduc-
tion (Kaido et al., 2014). The role of GAPDH in facilitating the
association of the p35 MP with the cortical VRCs adds to the
potential role of chloroplastic proteins in virus replication as already
noted by other studies (Bhat et al., 2013; Cheng et al., 2013; Lin
et al., 2007). It remains to be seen whether the p35 MP associated
with cortical VRCs targets the whole VRC or rather a VRC sub-
compartment to PD to spread infection. Related to this, it will also
be interesting to know whether the RNA 1- and RNA 2 complexes
formed with the 35k MP within VRCs lead to the formation of
separate transport complexes or whether the network interactions
between RNAs and associated proteins within the VRC allow
coordinated RNA 1 and RNA 2 movement by stabilizing a single
transport complex. The formation of cortical VRCs followed by the
formation of larger factories shows resemblance to the processes
observed during TMV infection (Fig. 1). RCNMV certainly represents
an excellent model for addressing the intricate machinery involved
in the coordinated replication and movement of a bipartite genome.
VRC formation and movement organized by PVX MPs
PVX is the type member of the economically important potex-
viruses and serves as a model system for viruses encoding three MPs
in overlapping open reading frames, the “triple gene block” (TGB;
Morozov and Solovyev, 2003; Verchot-Lubicz et al., 2010; Verchot-
Lubicz et al., 2007). Similar as the two previous examples, PVX
replicates and moves its genome in association with the ER. TGB2
and TGB3 are ER transmembrane proteins. TGB2 induces the forma-
tion of ER-derived granular vesicles (Ju et al., 2005) to which TGB3 is
recruited during infection (Bamunusinghe et al., 2009; Samuels et al.,
2007; Schepetilnikov et al., 2005). The granular vesicles also contain
the replicase, ribosomes, and virions (Bamunusinghe et al., 2009;
Samuels et al., 2007; Schepetilnikov et al., 2005; Verchot-Lubicz et al.,
2010), and may represent containers into which the VRCs are collected
for movement. The vesicles may form by TGB2/3-mediated recruit-
ment or proliferation of ER membrane around individually replicating
viral genomes or by the recruitment of ER-associated VRCs into
higher-order membrane structures. TGB2 has RNA binding activity
(Verchot-Lubicz et al., 2010), which may be involved in the binding
and stabilization of the VRC(s) within the vesicle. TGB1 is a multi-
functional protein with activities as an RNA helicase (Kalinina et al.,
2002), a suppressor of RNA silencing (Voinnet et al., 2000), and a
translational activator (Atabekov et al., 2000). The protein also gates
PD and localizes to PD in infected cells (Howard et al., 2004; Samuels
660 M. Heinlein / Virology 479-480 (2015) 657–671
 M. Heinlein / Virology 479-480 (2015) 657–671 661

et al., 2007). However, it does not target PD itself but is rather
recruited by TGB2/3, as was shown for other TGB-encoding viruses
(Verchot-Lubicz et al., 2010). TGB1 also interacts with CP, another
protein essential for PVX movement. According to the current model,
the TGB2/3 vesicles that harbor VRCs are motile along the dynamic ER-
actin network and once they encounter PD, the TGB2 and TGB3
proteins interact with TGB1 to insert CP into the channel, probably in
the form a movement complex with viral RNA. This insertion process
may be supported by continuous replication within the PD-associated
VRC, thus directly linking the movement process to viral replication.
Unlike for TMV, the virus does likely not move as a VRC but rather in
the form of a partially CP-encapsidated viral RNA with 50 -associated
TGB1 (Verchot-Lubicz et al., 2010). Consistently, studies with the PVX-
related Potato mop-top virus (PMTV) showed that the TGB2 and TGB3
proteins remain in the infected cell and are recycled by endosomal
membrane trafficking for further rounds of transport (Haupt et al.,
2005). In vitro experiments have shown that the associated TGB1
protein destabilizes the virus coat, thus promoting disassembly and
ribosomal access in newly infected cells (Atabekov et al., 2000;
Rodionova et al., 2003). Thus, following passage through PD, the viral
RNA is set free for association with the ER, where replication may start
as soon as new replicase protein has been produced. Similar to TMV,
the PD-associated VRCs and also the VRCs that remain along the ER
network in the infected cell grow into ER-associated inclusions that act
as viral factories and produce viral progeny.
At late infection stages PVX as well as many other viruses produce
perinuclear inclusions termed “X-bodies” (Esau and Cronshaw, 1967;
Goldstein, 1924; Matthews, 1991). A detailed analysis of the PVX X-
body (Tilsner et al., 2012) revealed the presence of helically arranged
TGB1 aggregates surrounded by recruited membranes that contain
TGB2/3 vesicles and nonencapsidated viral RNA. The observations
suggest an important role of TGB1 in the reorganization of the ER-
actin network, which likely plays a role in X-body formation. The
spatial arrangement of the components and the presence of encapsi-
dated virions at the outside are consistent with the functioning of the
X-body as a viral factory at late stages of infection. The intricate
architecture and compartmentalization of viral gene products of this
late factory as well as a membrane reorganizing function of TGB1 may
reflect important functions in the formation and organization of the
VRC-containing TGB2/3 vesicles occurring during earlier stages or in
the TGB1-related function during viral insertion at PD. However, the X-
body may also be involved in sequestration and degradation of viral
proteins when the stages of viral movement and reproduction have
been accomplished. It has been suggested that the X-body may
sequester TGB1, which may otherwise interfere with the assembly of
stable virions (Tilsner et al., 2012). However, the X-body may also be
related to cellular processes associated with the removal of aggregated
and overexpressed proteins.
Bamboo mosaic virus (BaMV) represents another, currently
intensely studied potexvirus model. Recent studies confirmed that
the role of TGB2/3 in the targeting of TGB1 to PD also applies to
BaMV. Interestingly, the same complex was associated with
virions, thus suggesting that BaMV targets fully encapsidated
virions to PD (Chou et al., 2013). Moreover, BaMV movement
depends on interactions between the helicase domain of the
replicase with CP (Lee et al., 2011). Further studies may reveal
additional features by which the movement of BaMV and PVX can
be distinguished.
Replication and movement of Turnip mosaic virus
Potyviruses represent the largest group of plant viruses and studies
particularly using Turnip mosaic virus (TuMV) have strongly advanced
our knowledge how these important plant viruses spread infection.
The genome of potyviruses consists of a single, roughly 10 kb long,
RNA molecule that codes for a polyprotein, which is processed into ten
mature proteins. In addition, potyviruses encode a protein termed
PIPO (Chung et al., 2008), which is produced as a trans-frame fusion to
the amino terminus of P3 (P3N-PIPO) (Vijayapalani et al., 2012).
Potyvirus replication is associated with the ER (Martin et al., 1995;
Schaad et al., 1997) and involves the formation of motile vesicles that
are formed by the protein 6K2 and which contain viral RNA, viral
replication proteins and host factors (Beauchemin et al., 2007; Cotton
et al., 2009; Dufresne et al., 2008; Huang et al., 2010; Thivierge et al.,
2008). Each of the vesicles originates from a single genome thus
indicating that all viral proteins present in the vesicles are produced by
translation within the vesicles (Cotton et al., 2009). Infection also
involves a perinuclear structure that is not found when 6K2 is
expressed alone, and which contains ER and Golgi membranes, COPII
coatomers and chloroplasts. Using techniques employing photoactiva-
table GFP, it was shown that this structure is functionally linked to the
production of 6K2 vesicles and their trafficking to the cortical ER via
transvacuolar strands (Grangeon et al., 2012). Other viral proteins play
a role in transferring the genome into adjacent cells. Potyvirus
infections are associated with cylindrical inclusions that extend

Fig. 1. Phases of TMV infection. A (c–i) and A0 , early infection (Phase 1, movement, 0–4 h), B and B0 , late infection (Phase 2, virion production, 5–16 h), C and C0 , very late
infection (Phase 3, degradation, 16–30 h). A–C, images derived from TMV-infected cells and tissues. A0 –C0 , Model for TMV infection. Elements of this model may also apply to
other viruses. The drawing in A0 is reproduced with modifications from Amari et al. (2014) and further modified in B0 and C0 (CC BY license). A,a, Infection sites of TMV-MP:
GFP produced in a locally infected N. benthamiana leaf, bar 1 cm; A,b, radially expanding TMV-MP:GFP infection site, bar 500 mm (image reproduced with permission, from
Hofmann et al., 2007, Biochem. Soc. Transact. 35, 142–145); A,c, MP:GFP localized to PD, bar 5 mm; A,d–f, images taken from the subepidermal cell cortex, A,d, MP:GFP
localizes to punctate complexes (early VRCs) aligned as beads on a string along tua:GFP-labeled microtubules, bar 5 mm; A,e, movie frame, MP:RFP punctate complexes (early
VRCs, arrow) are mobile and move along the GFP-tagged ER network, bar 5 mm (image taken from Niehl et al., 2013; author rights); A,f, movie frame, MP:RFP punctate
complexes (early VRCs, arrow) also localize and pause their movements at GFP-labeled microtubules, bar 2.5 mm (image from Niehl et al., 2013, author rights); A,g–h, early
TMV-MP:GFP infection in tobacco BY-2 protoplasts (images from Heinlein, 2002; with permission from Springer Science and Business Media). MP:GFP localizes to
subepidermal punctate complexes (early VRCs) that are aligned like beads on a string. A magnification of the framed area in g (bar 5 mm) is shown in h (bar 5 mm); A, i, Model
of alignment of punctate MP complexes (early VRCs, gray) along microtubules (blue) and the associated ER-actin network (gray). The MP complexes (early VRCs) localize to
microtubule:ER-actin junctions that may represent trafficking hubs associated with linker complexes (red halo) that link microtubules and the ER-actin network to the PM.
Observations indicate that some MP complexes are mobile and exhibit stop-and go movements along the ER between the junctions. B,a–c, late MP complexes (late VRCs, viral
factories); B,a, MP:RFP-tagged VRCs/factories (red) entangled within the microtubule network (green), bar 5 mm (image from Niehl et al., 2013; author rights); B,b, MP:RFP-
tagged VRC-factories (red) localizing to microtubule crossovers and branching points (green), bar 1 mm; B,c, MP:RFP-tagged VRC-factories (red) are associated with the ER
and surrounded by ER membranes (green), bar 1 mm; C, a–c, late phase of infection; C, a, epidermal cell showing MP:GFP undergoing redistribution from late VRCs to
microtubules. The size of the VRCs (or the localization of MP:GFP to VRCs) diminishes during this process, bar 5 mm (image from Niehl et al., 2013; author rights); C,b, BY-2
protoplast showing late stage of infection; MP:GFP localized to VRCs and microtubules, bar 10 mm (image from Heinlein, 2008; with permission); C, c, epidermal cells
showing MP:GFP almost exclusively on microtubules, bar 10 mm (image from Ashby et al., 2006; author rights). A0 , during early infection (Phase 1), class XI myosins (Myo XI)
provide motility to the ER and facilitate the concentration of punctate MP complexes/early VRCs at cortical microtubule:ER-actin junctions below the PM (a). Class VIII
myosins (Myo VIII) facilitate the targeting of the early VRCs from the cortical ER sites to the PM and subsequently to PD. This process may involve endocytic recycling (b),
diffusion of the VRC along the PM (c), stabilization of the VRC at PD (d) and active transport through the channel into the adjacent cell (e). B0 , during late infection (Phase 2),
VRCs that remained in the infected cell have grown (with support by class XI myosins (a)) and now function as factories (late VRCs) that produce virions. Factory formation is
associated with a reorganization and proliferation of the surrounding ER and often localized to microtubule crossovers and branches. C0 , at the end of the infection cycle
(Phase 3), the factory is degraded and the ER restores its normal morphology. In TMV infected cells, this process is associated with the extraction of MP from the ER by CDC48
for ERAD. Microtubule-binding factors like MP may transiently accumulate along microtubules (a) before degradation (b).
662 M. Heinlein / Virology 479-480 (2015) 657–671
through PD and are formed by the CI protein (Roberts et al., 1998;
Rodriguez-Cerezo et al., 1997). The targeting of CI to PD is mediated by
P3N-PIPO (Wei et al., 2010b), which itself is targeted to the plasma
membrane (PM) by the ER-Golgi pathway and by interaction with the
PM localized cation-binding protein PCaP1 (Vijayapalani et al., 2012).
Virus movement also depends on the viral CP (Dolja et al., 1994; Dolja
et al., 1995). Studies employing various cellular markers and antibodies
demonstrated that 6K2 vesicles move along actin microfilaments
(Cotton et al., 2009), contain dsRNA (indicative of replicating RNA)
and target PD (Grangeon et al., 2013). Moreover, ectopically expressed
6K2 moved cell-to-cell in the presence but not in the absence of
infection. These observations indicate that TuMV and potentially other
potyviruses move cell-to-cell in a highly intricate replication-compe-
tent, RNA-protein complex delimited by a lipid membrane (Grangeon
et al., 2013). Movement of the 6K2 vesicles may be facilitated by the
potyviral proteins Hc-Pro and CP that were previously shown to
increase the size exclusion limit of PD (Rojas et al., 1997). However, it
seems unclear how a membrane-delimited 6K2 complex would
interact with PD via HC-Pro and CP. Also the relationship of 6K2
vesicle movement and the cylindrical inclusions remains to be
determined. Moreover, it remains to be seen whether the virus moves
in the form of an encapsidated virion. The CI protein of Potato virus A
(PVA) was shown to bind to and copurify with virions (Gabrenaite-
Verkhovskaya et al., 2008) and the CP of TuMV colocalized to PD-
localized CI cones (Wei et al., 2010b). Moreover, mutations in the
conserved region of the Tobacco etch virus (TEV) CP abolished both
virion assembly and virus movement (Dolja et al., 1994; Dolja et al.,
1995). It appears possible that the CI protein recognizes CP-viral
genome complexes produced in 6K2 complexes and that the CI-
associated CP-genome-containing 6K2 complexes are bound by P3N-
PIPO for PD targeting via PCaP1. Since P3N-PIPO targets PD and affects
PD gating (Vijayapalani et al., 2012; Wei et al., 2010b), it may facilitate
the insertion of the CI-associated, CP-genome-containing 6K2 com-
plexes into PD. This process may lead to the accumulation of CI at PD
and the formation of conical CI structures that may facilitate further
virus spread.
Relationship between virus-induced compartments
The above viral systems exemplify the importance of virus-
induced membrane compartments as a recurrent theme. Each virus
encodes one or more proteins to interact with membranes and to
form membrane-protected compartments for virus replication and/
or movement. Since virus movement depends on MPs that facilitate
the spread from infected into adjacent cells, MP-associated com-
plexes were originally believed to solely function in the transport of
replicated viral genomes from localized VRC compartments to PD
for spread. However, as explained in the preceding paragraphs, it
now appears that the mobile compartments that target PD are
associated with the replication machinery and thus likely contain
VRCs themselves. Thus, the VRC-containing compartments occur-
ring during infection are divided into small, mobile compartments
associated with virus movement and larger, sessile compartments
that usually occur later and function as viral factories that replicate
the virus to produce virion progeny (see Fig. 1, for TMV). Although
the two different compartments (viral factory compartment and
mobile VRC compartment) can often be observed in the same cells
and may communicate with each other as suggested for TuMV
(Grangeon et al., 2012), the larger, virion-producing compartments
of TMV and also those of RCNMV and PVX rather mature after virus
movement has occurred. For example, the analysis of leaf infection
sites caused by TMV expressing GFP-tagged MP demonstrated that
cells at the leading front contain only small, punctate MP complexes
as well as MP-labeled PD, whereas larger MP-tagged inclusions
occur later and therefore are present only in cells behind the front
(Boyko et al., 2007; Heinlein et al., 1998; Sambade et al., 2008)
(Fig. 1). The same timing of production of small, punctate MP
complexes in advance of the formation of larger VRCs was also
clearly observed during the time course of infection in protoplasts
(Heinlein et al., 1998). Viral infection may thus be understood as a
rather simple process in which VRCs formed on membranes are
either mobile and target PD to spread infection or remain attached
and over time grow into factories.
Cellular mechanism of VRC movement
Since the highly dynamic ER-actin network is continuous though
PD it provides a direct pathway for the spread of ER-associated VRC
compartments from infected cells into adjacent cells. The ER-
associated actin cytoskeleton and its myosin motors provide moti-
lity to the network (Ueda et al., 2010) and also to network-
associated organelles such as Golgi, peroxisomes and mitochondria
(Peremyslov et al., 2008; Prokhnevsky et al., 2008; Avisar et al.,
2008a; Avisar et al., 2009; Boevink et al., 1998; Stefano et al., 2014).
In addition, the ER network itself is a highly fluid structure in which
lipids, embedded proteins, and protein complexes can move by
diffusion (Avisar et al., 2009; Boevink et al., 1998; Peremyslov et al.,
2008; Prokhnevsky et al., 2008) and only the transport efficiency
and directionality of transport are increased if supported by an
intact actomyosin system (Griffing, 2010; Sparkes et al., 2009).
Consistently, TMV infection continued to spread in the absence of
an intact actin cytoskeleton during a tested period of 24 h
(Hofmann et al., 2009). Interestingly, TMV movement was strongly
inhibited by overexpression of an actin-binding protein. This con-
dition also stalled the trafficking of Golgi complexes and thus is
consistent with a role of myosins in directly or indirectly driving
protein cargo movement along the ER membrane (Hofmann et al.,
2009). In agreement with this, it was found that the direct
inhibition of myosins by either virus-induced gene silencing (VIGS)
or the expression of dominant-negative myosin tails also strongly
affected the intercellular spread of TMV and of several other ER-
associated viruses thus indicating that myosins directly or indirectly
support their movement or the targeting of their MPs to PD (Agbeci
et al., 2013; Amari et al., 2014; Avisar et al., 2008b; Feng et al., 2013;
Harries et al., 2009b). Recently, Amari et al. (2014) showed that
efficient TMV movement involves the myosins XI-2, XI-K, VIII-1,
VIII-2 and VIII-B (Amari et al., 2014). The inhibition of myosin XI-2
or XI-K strongly affected the structure and motility of the ER and
thereby caused the entrapment of MP in large ER aggregates as well
as the inhibition of efficient targeting of MP to PD. The inhibition of
myosin XI-2, but not of myosin XI-K, also caused a reduction in TMV
accumulation. Ectopically expressed 126k replicase protein usually
concentrates into large aggregates that align along actin filaments
and resemble viral factories (Liu et al., 2005). However, when
myosin XI-K was inhibited, this protein dispersed into numerous
small aggregates and the trafficking of these aggregates along actin
strands was impaired (Amari et al., 2014). Inhibition of myosins VIII-
1, VIII-2 and VIII-B had no effect on the structure and motility of the
ER but caused the accumulation of MP along the plasma membrane
(PM) and interfered with its association with PD (Amari et al., 2014),
a finding which is consistent with the localizations of class VIII
myosins, which include PD (Baluska et al., 2001; Golomb et al.,
2008; Reichelt et al., 1999). These observations demonstrate that
different myosins play different roles during specific steps of TMV
movement. Whereas XI-2 and XI-K play a role in the formation and
dynamic behavior of the native ER structure and support the
formation of VRCs and their trafficking along the membrane, class
VIII myosins act at the cell periphery and facilitate directed trans-
port from the ER to PD and into the adjacent cells (Amari et al.,
2014) (Fig. 1A0 ).
 M. Heinlein / Virology 479-480 (2015) 657–671
Cellular mechanisms involved in the formation of VRC at the
ER
As described in the initial paragraphs, the formation of ER-
associated VRCs involves viral proteins with intrinsic affinity to
the ER membrane. Since infection starts by association of the
viral genome with the ER membrane, as nicely shown for TMV
(Christensen et al., 2009), and the virus-encoded proteins are
translated in the vicinity, VRC complexes for transport and/or
further replication can be formed. Although the complexes may be
kept together through protein:protein and protein:RNA network
interactions, the question arises how these complexes withstand
the pulling and pushing forces of a highly fluid membrane loaded
with other mobile protein complexes. The formation of distinct
replication complexes indicates that the complexes neither dis-
sipate along the network nor occur in endless numbers at random
sites. The inhibition of myosin XI-2 led to the dissipation of
replicase complexes along the ER thus indicating a role in
concentrating replicase and other factors at specific ER sites
(Amari et al., 2014) (Fig. 1A0 ). Moreover, viral replication and
movement depend on interactions with numerous host factors
that must be recruited in some way, and recruitment should occur
to specific sites in order to be efficient. The concentration of VRC
formation to specific cellular sites is illustrated by the subcellular
distribution of MP-tagged TMV VRCs. During early infection in
protoplasts or in epidermal cells at the spreading front of infection
in leaves the VRCs occurred at specific sites that are regularly
spaced, like beads on parallel strings (Heinlein, 2002; Heinlein et
al., 1998). Co-labeling of infected or MP-expressing cells with
specific cellular probes demonstrated that the VRCs and MP
complexes occur at three-way junctions of the ER and aligned to
cortical microtubules (Heinlein et al., 1998; Sambade et al., 2008)
(Fig. 1 A). Moreover, ER–associated viral factories grown from
stationary VRCs at late stages appear to be always associated with
microtubules and sometimes stretched along them (Heinlein et al.,
1998; Niehl et al., 2013b) (Fig. 1 B). These observations suggest that
TMV and potentially other viruses target specific ER sites that are
associated with microtubules.
Such microtubule:ER junctions (cortical microtubule-
associated ER sites (cMERs), cortical landmarks) are indeed known
(Hamada et al., 2012; Hamada et al., 2014) and may play an
important role in the organization of the plant cell cortex (Peña
and Heinlein, 2013). Although the major contributors to the
dynamic ER organization in plant cells are actin filaments, the ER
is also connected to cortical microtubules that provide stable
anchoring points (Sparkes et al., 2009; Hamada et al., 2012;
Hamada et al., 2014). Microtubule-disrupting agents do not disrupt
the ER (Knebel et al., 1990; Quader et al., 1989; Sparkes et al.,
2009) but cause changes in ER mesh size (Hamada et al., 2014)
thus indicating that the microtubule-associated ER anchoring sites
play an important role in determining the native fine mesh
architecture of the network. Accordingly, the density of ER tubule
junctions was found to correlate with microtubule density
(Hamada et al., 2014). Recently, a protein complex that bridges
the ER-actin network and microtubules to the PM at peripheral ER
anchoring points was identified (Wang et al., 2014) and which may
play a structural and regulatory role at these sites. In representing
junctions between ER:actin and microtubules, the ER anchoring
points might provide important intersections of myosin- and
kinesin-driven transport and thus may act as important cortical
trafficking hubs at which organelles and protein complexes
exchange their components (Peña and Heinlein, 2013). Microtu-
bule:ER junctions were indeed correlated with the pausing of
stop-and-go organelle movements along the ER network (Hamada
et al., 2012). Moreover, in being bridged to the PM, these sites may
serve as hubs that link lateral transport along the ER (organelles,
663
protein complexes) with transport towards and from the PM (e.g.
endosomal recycling). Kinesins or other microtubule-associated
proteins (MAP) as well as myosins that are associated with
organelles (Cai and Cresti, 2012; Lee et al., 2001; Liu et al., 1994;
Lu et al., 2005; Ni et al., 2005; Wei et al., 2009) may facilitate
trafficking along the ER-actin network and inter-organelle cargo
exchange at these hubs.
In providing trafficking hubs, the microtubule:ER junctions/ER
anchoring sites should represent ideal targets for viruses that depend
on the recruitment of host factors and membranes to form VRCs and
for the targeting of the VRCs to the PM and PD (Peña and Heinlein,
2013). The association of ER-associated TMV VRCs with microtubules
supports this model. Moreover, the MP of TMV acts as a MAP in vitro
and often accumulates along microtubules in vivo (Ashby et al., 2006;
Boyko et al., 2000a; Boyko et al., 2000b; Ferralli et al., 2006; Heinlein
et al., 1995; Heinlein et al., 1998; Niehl et al., 2014; Padgett et al.,
1996). This microtubule-binding activity may represent an important
mechanism through which the ER-associated MP localizes the devel-
opment of VRCs to microtubule:ER junctions/ER anchoring sites and
thus facilitates the recruitment of host factors and structural ancho-
rage. In addition to targeting microtubules, the MP binds to tubulin
dimers, γ-tubulin and the microtubule end-binding protein 1 (EB1)
(Brandner et al., 2008; Ferralli et al., 2006; Sambade et al., 2008),
suggesting that this protein may interfere with the machinery by
which the dynamic cortical microtubule patterning is regulated.
Previous studies indicated that the MP is capable of interfering with
the recruitment of γ-tubulin to the mammalian centrosome and with
centrosomal microtubule polymerization activity (Boyko et al., 2000a;
Ferralli et al., 2006). Moreover, expression of MP in mammalian cells
caused the polymerization of microtubules at ectopic sites in the
cytoplasm, thus suggesting that the MP hijacks microtubule-
organizing complexes and interferes with their recruitment to cen-
trosomes (Ferralli et al., 2006). Although plants do not have centro-
somes they nucleate microtubules by recruitment of mobile
microtubule nucleating complexes to existing microtubules at dis-
persed sites in the cortical cytoskeleton (Nakamura et al., 2010). In
hijacking such mobile nucleation complexes and causing nucleation
at ectopic sites during infection, the MP may be capable of changing
the local patterning of microtubules, which may be required for
supporting the growth of the ER-associated VRCs. This model is in
agreement with the observation of polymerizing microtubules emer-
ging from MP-expressing VRCs and also with the unusual microtubule
arrangements that are often seen associated with the viral factories
(Niehl et al., 2013b). Moreover, MP complexes that move along the ER
and pause their movements at microtubule-associated ER sites were
shown to interrupt their movements at these sites upon inhibition of
microtubule polymerization (Sambade et al., 2008), thus suggesting
that microtubule nucleation triggers the release of VRCs from their
attachment site for movement. The above considerations are consis-
tent with observations that correlate the binding of MP to micro-
tubules to the formation of VRCs and MP function in virus movement
(Boyko et al., 2000a; Boyko et al., 2007). A role of microtubule
dynamics in virus movement is also consistent with TMV resistance
of tobacco mutants with reduced microtubule polymerization beha-
vior (Ouko et al., 2010). A function of the dynamic microtubule
cytoskeleton in VRC formation and trafficking was recently supported
also by a virus evolution experiment (Peña et al., 2014). In this
experiment it was asked whether TMV would evolve adaptive
mutations in its genome if exposed to Arabidopsis thaliana tortifolia
1/spiral 2 or tortifolia 2 mutants, which differ in dynamic microtubule
behavior from wild type plants. Upon serial passages of TMV through
these plant mutants, several adaptive mutations occurred in the viral
genome that increased the viral fitness and allowed for increased
infectivity and/or accumulation in the mutant A. thaliana environ-
ment. These mutations did not cause an increased viral fitness in wild
type A. thaliana plants or BY-2 Nicotiana tabacum protoplasts. The
664 M. Heinlein / Virology 479-480 (2015) 657–671
ability of TMV to adapt to alterations in the microtubule network
clearly supports the role of dynamic microtubule network functions
during TMV infection. It will be important to determine the mechan-
ism by which the viral genome mutations enhance viral fitness.
Interestingly, most of the mutations cluster in the replicase-encoding
ORF of the virus, thus indicating that the adaptive process may have
caused structural changes in the replicase or in the replicase-encoding
stretch of the viral RNA. This hypothesis is in agreement with the role
of the 126k replicase protein in regulating the size of the VRCs (Liu
et al., 2005) and with a role of microtubules in the anchorage and
assembly of these complexes (Niehl et al., 2013b). It is also interesting
that adaptive mutations were not found in the MP, which interacts
with microtubules in vivo and in vitro (Ashby et al., 2006), or in the
coat protein, which supports the long distance movement of TMV
through the phloem (Osbourn et al., 1990). This suggests that the tor1
and tor2 mutations do not affect the interactions of MP with tubulin
but rather interfere with the important role of local microtubule
patterning and scaffolding in the formation, movement and further
maturation of VRCs. The CP does not interact with microtubules, is
dispensable for virus replication, and accumulates outside the VRCs
(Asurmendi et al., 2004). Thus, although the CP controls VRC growth
by regulating the production of subgenomic RNAs and thus the
amount of MP that accumulates in VRCs (Asurmendi et al., 2004) it
is not involved in the microtubule-related mechanisms that interact
with the VRCs for maturation and movement. tor2 represents an
amino acid exchange mutation in TUA4 (α-tubulin 4) and reduces the
rate of microtubule polymerization (Buschmann et al., 2009). In
contrast, tor1 is a mutation in the microtubule-associated protein
TORTIFOLIA1/SPIRAL2 (TOR1), which inhibits the microtubule-
severing activity of katanin and thus promotes microtubule growth
and stabilization (Wightman et al., 2013; Yao et al., 2008). TOR1
appears to play a central role in regulating local microtubule
patterning within the cortical array, which in turn may be critical
for the efficient attachment and maturation of VRCs.
Although TMV is the primary model for revealing the role of the
microtubule cytoskeleton during infection, there already is growing
evidence for a role of microtubules also during infection of other
viruses. One example is the Ob strain of Tomato mosaic virus. Similar
to TMV this virus encodes a microtubule-interacting MP and forms
VRCs in association with the ER-actin network and microtubules
(Padgett et al., 1996). Another example is PMTV, whose TGB1 protein
interacts with microtubules (Shemyakina et al., 2011; Wright et al.,
2010) and thus may provide anchorage to support VRC formation at
the ER. Microtubule association was also reported for the coat protein
of PVX (Serazev et al., 2003) and the observation that PVX movement
is inhibited by expression of a microtubule-associated protein seems
to support a microtubule-associated movement mechanism (Cho
et al., 2012). Grapevine fanleaf virus (GFLV) is a “tubule-forming” virus,
which moves cell-to-cell in the form of encapsidated virions through
tubules that are assembled by MP within the plasmodesmal pore.
Since the desmotubular ER is displaced by the tubule, this virus
cannot use the ER for movement but rather depends on the secretory
pathway and also on a molecular association between the MP and a
PD-localized PDLP for PD targeting and movement (Amari et al.,
2010). Although the ER is not directly involved in the movement of
this virus, the disruption of microtubules in BY-2 cells expressing the
MP led to the formation of MP tubules at ectopic sites in the
cytoplasm (Laporte et al., 2003) thus suggesting that microtubules
play an important role in the PD targeting of this MP. Microtubules
have also been implicated to have a role during potyvirus infection
since the helper component-protease (HC-pro) of PVA was shown to
interact with HC-pro-interacting protein 2 (HIP2), a SPIRAL2-homolog
of potato (Haikonen et al., 2013). The interaction of potyviral HC-Pro
with microtubules was recently also demonstrated for the HC-Pro of
Potato virus Y (PVY) (Del Toro et al., 2014). Microtubules were also
shown to have important functions during infection and transmission
of the Cauliflower mosaic virus (CaMV). The transactivator protein P6,
a central component of viral factories produced by this virus, forms
complexes that move along actin filaments (the ER-actin network)
and pause their movements at microtubules (Harries et al., 2009a),
which may resemble the stop-and-go movement behavior of com-
plexes formed by the MP of TMV (Sambade et al., 2008), and
suggesting a role of microtubules in the formation of P6-tagged
inclusions. Moreover, the transmission factor P2 of CaMV binds to
microtubules for the formation of transmission bodies from which it
redistributes to microtubules upon aphid feeding to facilitate virion
uptake (Martinière et al., 2013; Martiniere et al., 2009). Also the MPs
of the negative strand RNA ophioviruses interact with microtubules
(Robles Luna et al., 2013).
Potential role of aggresomal processes in VRC formation and
turnover
The above-described processes involving the cytoskeleton-
mediated aggregation of VRCs with host membranes and viral as
well as host proteins supports the important role of the cytoskele-
ton as a scaffold for the anchoring and accumulation of proteins. By
providing such anchored accumulation sites the cytoskeleton may
support virus infection by increasing the local concentration of viral
and host components required for replication, movement, and
assembly, and by shielding the virus and its replication from host
defense. Aggregation of proteins with support of cellular scaffolds
may also facilitate cellular responses that recognize virus compo-
nents and target them for storage and degradation. These processes
are reminiscent of aggresomal mechanisms in mammalian cells that
use microtubules to concentrate protein aggregates at the centro-
some for immobilization in inclusions called aggresomes and for
their subsequent degradation by proteasomes and/or autophagy
(Wileman, 2007). The similarity of pericentriolar inclusions or
“viroplasms” formed by mammalian viruses and aggresomes led
to the suggestion that viruses actually subvert aggresomal processes
for their life cycle. Viruses may have evolved means to make
themselves recognizable as aggregates and thus be targeted to the
microtubule-organizing center (MTOC) to concentrate host and
viral proteins in one place to facilitate replication and assembly.
Moreover, in being part of a pathway that delivers proteins for
degradation, the viroplasms would have means to facilitate their
turnover, thus reducing the toxic effects of accumulated viral
proteins once the production of virus progeny is completed. Unlike
mammalian cells, plant cells do not concentrate their microtubule-
nucleating complexes at one MTOC per cell (the centrosome) but
rather use them in a dispersed manner throughout the cortical
cytoplasm. This may explain why plant viruses usually do not form
a single viroplasm per cell but rather multiple VRCs and factories. As
already discussed, the ER-associated MP of TMV may interact with,
target or even recruit microtubule-nucleation complexes as centers
for cytoskeleton-mediated delivery of host factors to establish VRCs.
This phenomenon may suggest that TMV uses its MP to trigger the
formation of aggresomes for VRC formation and subsequent degra-
dation. The formation of the VRCs around the MTOCs may be
facilitated by the intrinsic ability of the MP to oligomerize (Boutant
et al., 2010) and thus to recruit MP-associated ER membranes to
microtubules. The ER provides an elaborate system of protein
quality control, which involves chaperones and ubiquitin-
mediated degradation by proteasomes to reduce protein aggrega-
tion (Brandizzi et al., 2014). Thus, while MP-mediated ER aggrega-
tion at the MTOC facilitates the formation of the VRC and viral
factories, its accumulation also causes ER stress that subsequently
leads to the activation of the adenosine triphosphatase (ATPase)
CDC48, which removes MP from the membrane for subsequent ER-
associated degradation (ERAD) in the cytoplasm (Niehl et al., 2012;
 M. Heinlein / Virology 479-480 (2015) 657–671
Niehl et al., 2013a). This phenomenon is likely linked to the
unfolded protein response (UPR), a transcriptional program trig-
gered by an increased ER load of misfolded proteins. UPR has been
described for several plant viruses and represents an essential
mechanism to maintain ER homeostasis during infection (Verchot,
2014). In the case of TMV, the accumulation of MP and the
subsequent induction of these ER homeostasis mechanisms are
reflected in the extensive, MP-induced ER membrane remodeling
associated with viral factory formation during mid-stages of infec-
tion and the subsequent restoration of the normal tubular ER
network upon MP clearance during late stages (Heinlein et al.,
1998; Reichel and Beachy, 1998).
The balance between the production of MP and its degradation
likely represents a major determinant for the size of the ER
inclusions. Thus, whereas the high level of MP produced by TMV
may saturate the protein degradation system and thus cause its
accumulation in the ER and the formation of large inclusions, the
MP of ORMV, in contrast, accumulates to low cellular levels and,
accordingly, forms only small VRCs (Niehl et al., 2014). It appears
remarkable that the MP of ORMV allows for faster virus movement
than the MP of TMV despite its lower cellular expression level. This
may indicate that the production of high levels of MP and the
formation of larger factories is inhibitory for movement. Higher MP
levels leading to MP aggregation may thus reduce the pool of MP
available to facilitate the intercellular spread of TMV (Niehl et al.,
2014). However, this hypothesis requires further analysis since a
TMV variant that encodes a mutant MP and showed delayed
degradation and accumulation in large VRCs was not compromised
in its ability to move between cells efficiently (Gillespie et al., 2002).
While TMV and ORMV may use MTOCs as targets for the formation
of ER-associated VRCs and derived viral factories, and many other
viruses associate with the ER for replication (Bamunusinghe et al.,
2011; Carette et al., 2000; Grangeon et al., 2012; Han and Sanfacon,
2003; Huang and Zhang, 1999; Restropo-Hartwig and Ahlquist, 1996;
Ribeiro et al., 2008; Ritzenthaler et al., 2002; Schaad et al., 1997;
Turner et al., 2004), other viruses may use different locations to
establish their VRCs. For example, Carnation Italian ringspot virus
(CIRV) and Melon necrotic spot virus (MNSV) form factories from the
outer membrane of mitochondria (Mochizuki et al., 2009; Weber-Lotfi
et al., 2002), whereas the tonoplast is implied to play a role in the
replication of Alfalfa mosaic virus (AlMV) and Tomato mosaic virus
(ToMV) (Hagiwara et al., 2003; Ibrahim et al., 2012; Van Der Heijden et
al., 2001). Tomato bushy stunt virus (TBSV) induces the formation of
multivesicular bodies from peroxisomes (McCartney et al., 2005) and
several other viruses show associations with chloroplasts (Angel et al.,
2013; Bhat et al., 2013; Cheng et al., 2013; Cowan et al., 2012; Jang et
al., 2013; Prod’homme et al., 2003; Wei et al., 2010a). However, since
Golgi membranes, chloroplasts, peroxisomes, and also the tonoplast
are tightly associated with the ER (Stefano et al., 2014; Viotti, 2014), ER
membranes and associated vesicle trafficking as well as the above-
described roles of the cytoskeleton in supporting membrane traffick-
ing and anchorage may represent basic principles that also play a role
in membrane recruitment and the association of viruses with these
organelles.
The targeting of plasmodesmata involves additional membrane
transport mechanisms
Although the ER is linked to the desmotubular ER and thereby
provides a direct pathway for the targeting of ER-associated VRCs to
PD, additional mechanisms may play a role. As already mentioned,
the TGB2 and TGB3 proteins of PMTV associate with endocytic
vesicles proposed to be involved in recycling TGB2 and TGB3 back
to the ER once they have delivered the VRC to PD. Both proteins
were found in association with vesicles stained with FM4–64, a
665
widely used marker of the endocytic pathway. TGB2 also coloca-
lized in vesicles with an Arabidopsis homolog of Rab5 (Ara7), a
regulator of vesicle fusions in early endosomes, and interacted with
RME8, an endocytic DNAJ chaperone (Haupt et al., 2005). The MP of
TMV and also that of the DNA virus Cabbage leaf curl virus (CaLCuV)
interact with the Arabidopsis synaptotagmin SYTA, which regulates
endosome recycling. This observation indicates that these MPs
reach the PM and that SYTA may support virus movement by
allowing these MPs to reach PD through an endosomal recapture
pathway (Lewis and Lazarowitz, 2010). That the MP of TMV
interacts with the PM was recently confirmed by dominant negative
inhibition of class VIII myosins, which led to accumulation of MP in
the PM rather than at PD, thus indicating that class VIII myosins
play a role in redirecting the MP from the PM to PD (Amari et al.,
2014). Since class VIII myosins have been reported to play a role in
endocytotic processes (Golomb et al., 2008; Haraguchi et al., 2014;
Sattarzadeh et al., 2008), it appears possible that these myosins
support a PD-localized endosomal recycling pathway that delivers
and retrieves the MP between the ER and the PM at PD and thereby
maintains a high concentration of MP at PD (Fig. 1 A0 ). A role of MP
associations with the PM in supporting the movement of ER-
associated VRCs is also indicated by inhibition of PVX movement
upon overexpression of Remorin, a protein that interacts with TGB1
and clusters in the PM at PD (Raffaele et al., 2009). Taken together,
these observations may suggest that PD are linked to underlying
cortical microtubule:ER junctions/ER-anchoring sites at which VRCs
and developing viral factories are formed. Although the ER-
desmotubule continuum may target VRCs and associated MP from
these junctions into adjacent cells, the MP may target PD also by an
endosomal pathway that delivers MP to the PM around PD. This
pathway may allow MP to interact with the PD-localized and
glycosylphosphatidylinositol (GPI)-anchored beta-glucanase AtBG_-
pap, which is proposed to control callose levels in PD and to play a
role in virus movement (Zavaliev et al., 2013). The association of PD
with underlying viral factors at microtubule:ER junctions/ER-
anchoring sites is consistent with the often-observed formation of
‘paired bodies’ or ‘caps’, thus the accumulation of VRCs and viral
proteins on both sides of PD behind the virus front (Kotlizky et al.,
2001; Szécsi et al., 1999; Tilsner et al., 2013).
An association with endosomal compartments also plays a role
for the movement of CaMV. Like the already mentioned MP of GFLV,
also the MP of this virus (P1) is a “tubule-forming MP”, which
assembles into tubules within PD and allows the intercellular
transport of encapsidated virion particles. P1 carries three Tyr-
based sorting motifs (YXXΦ) that mediate binding to a yet unchar-
acterized m-like plant adaptin. m2 adaptins are subunits of AP
adapter complexes that recognize and recruit cargo proteins into
clathrin-coated vesicles. These observations are again consistent
with an endocytic pathway that retrieves MPs from the PM and
concentrates their localization to the PD. Mutation of the binding
motifs in MP abolished its accumulation in PD and its assembly into
tubules but not its targeting to the PM, suggesting a role of
endosomal trafficking in a pathway that concentrates MP at PD.
The role of endosomal trafficking was confirmed by colocalizing P1
vesicles with the endosomal marker FM6-64 and by inhibition of PD
targeting as well as the presence of P1 in FM6-64-tagged vesicles by
treatment with Tyrphostin A23, an inhibitor of recognition of Tyr
signals by m2 (Carluccio et al., 2014). Tyr-based sorting motifs
(YXXΦ) were also found in the TGB3 of PMTV and the MP of GFLV
and other nepoviruses (Haupt et al., 2005; Laporte et al., 2003), thus
supporting a widespread role of endoscytic retrieval pathways that
direct the MPs from the PM to PD.
The MPs of TMV, Turnip vein clearing virus (TVCV) and CaMV
were reported to interact with the cell wall protein pectin-
methylesterase (PME), which is targeted to the PM by the
secretory pathway (Chen et al., 2000; Dorokhov et al., 1999).
666 M. Heinlein / Virology 479-480 (2015) 657–671
However, the TMV MP did not colocalize with PME in vivo
(Hofmann et al., 2007). Moreover, neither lower concentrations
of the secretory pathway inhibitor Brefeldin A (BFA, 10 mg/ml), nor
the inhibition of the COPII coat-activating GTPase SAR 1 abolished
virus movement and the targeting of the TMV MP to PD thus
indicating that the ER-to-Golgi pathway is not involved in TMV
movement (Tagami and Watanabe, 2007). Although the PD target-
ing of MP was inhibited by high concentrations of BFA ( 450 mg/
ml), such higher concentrations also disrupted the structural
integrity of the ER (Wright et al., 2007), which is in agreement
with the role of the ER-actin network in the PD targeting by MP.
Nevertheless, the secretory pathway plays an important role for
tubule-forming viruses. Since the formation of the MP tubule inside
the PD channel displaces the desmotubule, these viruses cannot
rely on the ER for PD targeting. The P1 MP of CaMV failed to target
PD and formed typical BFA compartments upon BFA treatment
(Carluccio et al., 2014). The compartment contained internalized
FM4-64, thus indicating that also downstream endosomal P1
trafficking was affected. Moreover, as for several other tubule-
forming MPs, the assembly of P1 tubules in PD depends on the
presence of PD-targeted PDLP, which itself requires an intact COPII-
dependent ER-to-Golgi secretory pathway to reach PD (Amari et al.,
2010; Thomas et al., 2008). Thus, while P1 and other tubule-
forming MPs use an endosomal retrieval pathway to target PD from
the PM, PDLPs are required as docking proteins to maintain these
MPs at the channel for tubule formation. Unfortunately, it still
remains unclear how these proteins reach the PM in the first place
and also how the virions are targeted from replication sites to the
tubule for movement. A more direct association with the secretory
pathway was shown for the membrane-associated MP of Melon
necrotic spot virus (MNSV). Transient expression of this MP (p7B) led
to its localization to the ER, Golgi complexes, and PD suggesting that
this MP targets PD via a COPII-mediated ER-to-Golgi pathway.
Consistently, PD localization of this protein as well as MNSV
movement was strongly reduced by inhibition of either the actin
cytoskeleton or the ER-to-Golgi route with either BFA or by over-
expression of a dominant-negative SAR1 mutant (Genoves et al.,
2010). Another MP that appears to target PD via the COPII-
dependent secretory pathway route is the MP17 of Potato leaf roll
virus (PLRV; Vogel et al., 2007). However, in this case an association
with Golgi complexes has not been demonstrated.
Relationship of virus movement to the intercellular trafficking
of endogenous macromolecules
To allow for their movement between cells viruses must have
adapted to existing mechanisms for macromolecular trafficking
through PD. Numerous reports indicate that plant development
critically depends on the ability of cells to support the intercellular
transport of peptides and hormones, as well as of transcription factors
and RNA molecules (small RNAs, mRNAs) (Gallagher et al., 2014; Han
et al., 2014; Hisanaga et al., 2014; Sparks et al., 2013; Stahl and Simon,
2013). The first transcription factor that was known to act non-cell-
autonomously and move between cells through PD is the maize
homeodomain transcription factor KNOTTED1 (KN1) (Kim et al.,
2002; Lucas et al., 1995). The mechanism of intercellular movement
of KN1 and of a subclass of KN1-related proteins may share features
with the movement of tobamoviral MPs, since both appear to depend
on CCT8, a type II chaperonin complex, for movement (Fichtenbauer
et al., 2012; Xu et al., 2011). Interestingly, both classes of proteins also
interact with the microtubule-associated protein MPB2C, which
negatively regulates PD targeting of these proteins and their transport
(Kragler et al., 2003; Ruggenthaler et al., 2009; Winter et al., 2007),
which is consistent with the association of VRCs and other macro-
molecular transport complexes with microtubules. Several studies
support the association of cortical microtubules with endosomal
transport pathways involved in the targeting of the PM and PD. For
example, the microtubule-associated protein CLASP interacts with
SORTING NEXIN 1 (SNX1), a component of the retromer that localizes
to early endosomes and sorts transmembrane proteins, such as PIN2,
from the trans-Golgi network (TGN) back to the PM (Ambrose et al.,
2013). It has been proposed that the binding of SNX endosomes to
cortical microtubules by CLASP enhances their recycling near the PM,
a model that is highly consistent with the proposed role of endosomal
recycling in viral trafficking through PD. Microtubule-associated
endosomal recycling appears to play a role also in the PD-mediated
trafficking of transcription factors. Evidence comes from studies
addressing the mechanism of PD-mediated SHORTROOT (SHR) traf-
ficking in the developing Arabidopsis root playing a fundamental role
in the determination of endodermal cell fate (Vaten et al., 2011). SHR
trafficking is facilitated by interaction with the endosome-associated
protein SHR-INTERACTING EMBRYONIC LETHAL (SIEL) (Koizumi
et al., 2011). Importantly, SHR movement as well as the association
of SIEL with endosomes is microtubule-dependent, thus linking the
endosome-mediated trafficking of SHR to microtubules (Wu and
Gallagher, 2013). SIEL interacts also with other non-cell-autonomous
transcription factors, such as AGAMOUS-LIKE21, CAPRICE, and TAR-
GET OF MONOPTEROUS7 (Koizumi et al., 2011), thus suggesting a
general role of microtubule-associated endosomal trafficking and
recycling in PD-mediated macromolecular movement.
Conclusions and outlook
Viruses depend on interactions with cellular proteins and
membranes to support the formation of membrane-associated,
multifactorial protein:nucleic acid complexes for replication and
movement. RNA virus infection in plants is initiated with the
association of the viral RNA genome with the ER, or with mem-
branes of organelles connected to the ER, and local translation
produces virus factors that form interaction networks among
themselves as well as with host proteins to form a VRC. Viruses
encode one or more proteins that specifically link the VRC to
membrane and cause the encapsidation of the VRC into protective
membrane sheaths or vesicles. Viruses also have evolved mechan-
isms to interact with the cytoskeleton for local anchorage and
transport. For many years the mechanisms involved in virus
replication and virus movement have been studied separately, as
if these are separate events each requiring its own specialized
mechanisms. However, the picture now emerges that viruses
develop VRCs for replication as well as for movement. Movement
to PD or even between cells as a VRC ensures the vicinity of MPs to
the viral genome and thus may explain the apparent lack of
evolution of any sequence specificity in the binding of MPs to
nucleic acids. Time-resolved in vivo studies suggest that VRCs
develop at specific cellular sites of anchorage from which they
may be released for intra-and intercellular transport or at which
they may remain anchored and form virion-producing viral fac-
tories while infection may already spread further (Fig. 1). As
exemplified by PVX, VRCs released from their original ER sites
may get again anchored at the ER at PD to feed partially encapsi-
dated viral RNA into the channel for movement, whereas other
viruses like TMV may spread between cells in the form of wholly
intact VRCs. The membrane-associated mechanisms of VRC forma-
tion and movement are tightly associated with the cytoskeleton,
thus reflecting the viral exploitation of the microtubule- and actin-
dependent functions in plant endomembrane organization
(Brandizzi and Wasteneys, 2013). TMV, and perhaps also other
viruses, appears to interact with distinct microtubule-ER junctions
that may attach the ER-actin network to the PM (ER-anchoring
sites) and which may provide a cortical nexus at which actin- and
 M. Heinlein / Virology 479-480 (2015) 657–671
microtubule-driven transport pathways for the trafficking of protein
complexes and organelles converge (Peña and Heinlein, 2013). TMV
is proposed to interact with these sites for the recruitment of host
factors and the MP may enhance this process by controlling
microtubule nucleation events that facilitate VRC attachment and
detachment as well as aggresomal viral factory formation and
degradation processes. Since MP binds to the viral genome as well
as membrane, its additional activity to interact with tubulin dimers,
assembled microtubules, and microtubule-associated factors may
recruit and regulate the association of the factory with the micro-
tubule system. Whereas the microtubule system may provide a
mechanism for VRC anchorage and release, the ER-associated actin
network and myosin XI motors facilitate the formation and growth
of the VRC as well as VRC trafficking along the ER network, and class
VIII myosins support MP transport from the VRC to the PM by
endosomal cycling (Amari et al., 2014). Microtubule:ER junctions/
ER-anchoring sites may act as general platforms for endosomal
trafficking and recycling between the ER and the PM and thus for
ER-to-PM transport of MP and of other proteins destined to specific
PM domains and PD (Peña and Heinlein, 2013). In providing an
exchange platform for endosomal transport, these sites may also
provide a pathway for the recruitment of replicase-interacting
TOM1 and TOM2A proteins that are required for TMV replication
but usually localize to the vacuolar membrane in the absence of
infection (Hagiwara et al., 2003).
The analysis of virus replication and movement in plants has
come a long way. However, future studies are needed to further
substantiate the role of microtubule:ER junctions/ER-anchoring sites
during tobamoviral infection and to indicate which other viruses may
interact with this pathway. Moreover, although viral factors involved
in the formation of replication sites and/or in movement have been
characterized, we are still far from attaining a complete list of host
proteins involved in these processes. It will be important to further
determine the composition of viral complexes that move between
cells and the role of the constituent proteins in the movement
process. It would also be highly interesting to determine whether
and to which extent viruses deliberately trigger host defenses to
induce specific mechanisms required for replication and spread. For
example, the formation of viral factories may reflect a virus-triggered
host defense mechanism that aims to compartmentalize the VRC and
which the virus exploits to protect the growing factory. The same
process may attract the whole machinery the virus needs for efficient
replication. Moreover, viruses may deliberately overexpress some of
their proteins to trigger ER homeostasis and maintenance mechan-
isms that provide important degradation pathways required for the
turnover of viral products at the end of the viral life cycle. Another
important goal for future studies is to reveal host gene expression
changes that may orchestrate the processes involved in virus
replication and movement. Interaction with host RNA silencing
pathways (Burgyan and Havelda, 2011; Ding and Voinnet, 2007;
Pumplin and Voinnet, 2013) may allow viruses to manipulate host
gene expression in order to weaken plant defences or to induce
genes for the delivery of host factors required for infection. Moreover,
the tight regulation of gene expression by both the virus and the host
during early stages of infection may represent a critical determinant
for the outcome of infection with respect to disease.
Acknowledgment
I thank Annette Niehl and Khalid Amari for reading the manu-
script before submission. Current research is supported by the
University of Strasbourg Institute of Advance Study (USIAS), the
French Agence National de la Recherche Scientifique (ANR, Plant-
KBBE 2012), and the Swiss National Science Foundation (Grant
31003A_140694).
667
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