Nuclear entry and egress of parvoviruses

Abstract

Parvoviruses are small non‐enveloped single‐stranded DNA viruses, which depend on host cell nuclear transcriptional and replication machinery. After endosomal exposure of nuclear localization sequence and a phospholipase A2 domain on the capsid surface, and escape into the cytosol, parvovirus capsids enter the nucleus. Due to the small capsid diameter of 18–26 nm, intact capsids can potentially pass into the nucleus through nuclear pore complexes (NPCs). This might be facilitated by active nuclear import, but capsids may also follow an alternative entry pathway that includes activation of mitotic factors and local transient disruption of the nuclear envelope. The nuclear entry is followed by currently undefined events of viral genome uncoating. After genome release, viral replication compartments are initiated and infection proceeds. Parvoviral genomes replicate during cellular S phase followed by nuclear capsid assembly during virus‐induced S/G2 cell cycle arrest. Nuclear egress of capsids occurs upon nuclear envelope degradation during apoptosis and cell lysis. An alternative pathway for nuclear export has been described using active transport through the NPC mediated by the chromosome region maintenance 1 protein, CRM1, which is enhanced by phosphorylation of the N‐terminal domain of VP2. However, other alternative but not yet uncharacterized nuclear export pathways cannot be excluded. Parvovirus capsids enter the nucleus by active import or by an alternative entry pathway that includes local transient disruption of the nuclear envelope. Parvoviral genomes replicate during cellular S phase followed by nuclear capsid assembly. The egress of progeny capsids occurs upon nuclear envelope degradation during cell lysis or by using active transport through the nuclear pore complexes.

Full text

Molecular Microbiology. 2022;118:295–308.
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295wileyonlinelibrary.com/journal/mmi
1 | INTRODUCTION
1.1 | Parvoviruses
Parvovirinae subfamily infects vertebrates including humans. Most
viruses of this subfamily, including minute virus of mice (MVM),
canine parvovirus (CPV), and rat parvovirus (H- 1PV), are autono-
mous, but replication of adeno- associated viruses (AAV) requires the
presence of helper viruses such as adenoviruses or herpesviruses
(dependoparvoviruses) (Cotmore et al., 2019; Pénzes et al., 2020).
While Parvovirinae have an important potential in oncolytic therapy,
AAVs are a major platform in gene therapy. H- 1PV and MVM are
Received: 23 May 2022
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Revised: 10 August 2022
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Accepted: 13 August 2022
DOI : 10.1111/m mi.14974
MICRO REVIEW
Nuclear entry and egress of parvoviruses
Salla Mattola1 | Vesa Aho1 | Luisa F. Bustamante- Jaramillo2 | Edoardo Pizzioli2 |
Michael Kann2,3,4 | Maija Vihinen- Ranta 1
This is an op en access ar ticle under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provide d the original work is properly cited.
© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.
Michael Kann co- last author.
1Department of Biological and
Environmental Science, University of
Jyvaskyla, Jyvaskyla, Finland
2Department of Infectious Diseases,
Institute of Biomedicine, University of
Gothenburg, Gothenburg, Sweden
3Sahlgrenska Academy, Gothenburg,
Sweden
4Department of Clinical Microbiology,
Region Väs tra Götaland, Sahlgrenska
University Hospital, Gothenburg, Sweden
Correspondence
Michael Kann, Department of Infectious
Diseases, Institute of Biomedicine,
University of Gothenburg, Gothenburg ,
Sweden.
Email: michael.kann@gu.se
Maija Vihinen- Ranta, Department of
Biological and Environmental Science,
University of Jyvaskyla, P O Box 35, 40014
Jyvaskyla, Finland.
Email: maija.vihinen-ranta@jyu.fi
Funding information
Academy of Finland, G rant/Award
Number : 330896; Gr aduate School of
the Unive rsity of Jy vaskyla, Grant/Award
Number : 2022; Jane ja A atos Erkon Säätiö,
Grant/Award Number: 2019
Abstract
Parvoviruses are small non- enveloped single- stranded DNA viruses, which depend
on host cell nuclear transcriptional and replication machinery. After endosomal expo-
sure of nuclear localization sequence and a phospholipase A2 domain on the capsid
surface, and escape into the cytosol, parvovirus capsids enter the nucleus. Due to
the small capsid diameter of 18– 26 nm, intact capsids can potentially pass into the
nucleus through nuclear pore complexes (NPCs). This might be facilitated by active
nuclear import, but capsids may also follow an alternative entry pathway that includes
activation of mitotic factors and local transient disruption of the nuclear envelope.
The nuclear entry is followed by currently undefined events of viral genome uncoat-
ing. After genome release, viral replication compartments are initiated and infection
proceeds. Parvoviral genomes replicate during cellular S phase followed by nuclear
capsid assembly during virus- induced S/G2 cell cycle arrest. Nuclear egress of capsids
occurs upon nuclear envelope degradation during apoptosis and cell lysis. An alterna-
tive pathway for nuclear export has been described using active transport through
the NPC mediated by the chromosome region maintenance 1 protein, CRM1, which
is enhanced by phosphorylation of the N- terminal domain of VP2. However, other
alternative but not yet uncharacterized nuclear export pathways cannot be excluded.
KEYWORDS
import and export, nuclear envelope, nuclear pore complexes, nucleus, parvoviruses

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known to induce lysis of transformed cells and to activate anticancer
immune responses (Abschuetz et al., 2006; Geletneky et al., 2017;
Gil- Ranedo et al., 2021; Grekova et al., 2012; Hartley et al., 2020;
Marchini et al., 2015). The role of CPV in inducing an antitumor im-
mune response in different tumor models has been discussed (Arora
et al., 2021). The potential of recombinant AAV vectors in gene ther-
apies has been shown by the approval of two AAV therapeutic appli-
cations for the treatment of Leber's congenital amaurosis (Luxturna)
and spinal muscular atrophy (Zolgensma) by the US Food and Drug
Administration (FDA) (Kuzmin et al., 2021; Large et al., 2021). In fact,
the first gene therapy, Glybera medicine, approved in 2012 cor-
rected hereditary lipoprotein lipase deficiency (LPLD). This treat-
ment was stopped in 2018 due to the high cost of c. one million US$
per patient, and only 31 people were treated (Mendell et al., 2021).
Parvoviruses comprise a linear single- stranded DNA of ~4 to
6 kb and an icosahedral capsid of 18– 26 nm in diameter (Cotmore
et al., 198 3; Kaufmann et al., 2004; Mietzsch et al., 2019; Tsao
et al., 1991; Xie et al., 20 02). The viral proteome differs between
members of parvovirus (Cotmore & Tattersall, 2014). Many of the
autonomous parvovirus genome encodes two structural proteins
(VP1 and VP2) and two non- structural proteins (NS1 and NS2)
(Cotmore et al., 1983; Cotmore & Tattersall, 2014), whereas AAV en-
codes at least for three capsid proteins ( VP1, VP2, and VP3) and four
non- structural proteins (Rep40, Rep52, Rep68, and Rep78) (Im &
Muzyczka, 1990; X ie et al., 2002). Capsid p roteins and non- s tructural
proteins are translated from alternatively spliced mRNAs, following
transcription controlled by the early P4 and the late P38 promoter.
While the former guides the expression of NS1 and NS2, the latter
controls the expression of capsid proteins (Christensen et al., 1995;
Cotmore & Tattersall, 1995; Li & Rhode 3rd, 1990). Nonetheless, the
family shows different transcriptional strategies and viruses within
the type species of each genus express a small number of genus-
specific ancillary proteins (Cotmore & Tattersall, 2014).
Parvoviruses use a variety of cell sur face receptors for attach-
ment to their host cells, determining host range and tissue tro-
pism (Govindasamy et al., 2003; Hueffer et al., 2003; Llamas- Saiz
et al., 1996; Michelfelder & Trepel, 2009; Palermo et al., 2006).
CPV uses sialic acid and transferrin receptor (Parker et al., 2001;
Parrish, 199 0), whereas human par vovirus B19V attaches to
erythrocyte P antigen (Brown et al., 1993) and its cellular entry is
facilitated by low pH- mediated interaction with globoside (Bieri
et al., 2021; Bieri & Ros, 2019). The dependoparvovirus AAV2 rec-
ognizes several receptors of target cells including heparan sulfate
proteoglycan, αVβ5 integrin, and basic fibroblast growth factor re-
ceptor 1 (Qing et al., 1999; Summerford et al., 1999; Summerford
& Samulski, 1998). Recently, the AAV receptor (AAVR; KIAA0319L)
was identified as an essential receptor for cell internalization and
trafficking of different AAVs (Meyer & Chapman, 2022; Pillay
et al., 2016). After receptor binding, many parvoviruses enter cells
via clathrin- mediated endocytosis (Bartlett et al., 2000; Cureton
et al., 2012; Parker & Parrish, 2000). The low endosomal pH induces
conformational changes in parvovirus capsid structure, which leads
to exposure of the VP1 N- terminal unique region (VP1u). VP1u of
B19, MVM, and CPV comprises a phospholipase A2 (PLA2) motif,
a nuclear localization sequence (NLS), and three PDZ domains,
which are highly conserved. The PLA2 domain is required for cap-
sid escape from endocytic vesicles (Farr et al., 2005; Popa- Wagner
et al., 2012; Qu et al., 2008; Suikkanen, Antila, et al., 2003b; Zádori
et al., 2001;Ros et al., 2020) presumably by forming holes in the en-
dosomal membrane, while NLS and PDZ domains are implicated in
nuclear import of the capsid.
This review focuses on what has been learned in the past years
about cy toplasmic trafficking, nuclear entry, and exit of parvovirus
capsids.
2 | NUCLEAR ENTRY OF PARVOVIRUS
CAPSIDS
2.1 | Traveling to the nucleus
Subsequent to endosomal escape, the capsids have to reach the
nuclear envelope (NE). Likely, parvoviruses make use of the cellular
microtubule network, as depolymerization of microtubules blocks
CPV infection (Suikkanen, Aaltonen, et al., 2003a), which is also
consistent with their observed velocity toward the nucleus (Mäntylä
et al., 2018). These findings are supported by obser vations on A AV,
showing that their perinuclear accumulation is enhanced by dynein-
and microtubule- mediated transport (Kelkar et al., 2004, 2006; Xiao
& Samulski, 2012). As with CPV, tracking of single AAV particles in
the cytoplasm has demonstrated directed motion of viral capsid to-
ward the nucleus, which is a characteristic of dynein- microtubule
mediated transport (Seisenberger et al., 2001).
Direct transport of the released capsids along microtubules is
likely but it s requirement is not unequivocally proven as microtubule
depolymerization does not affect CPV distribution after microinjec-
tion (Lyi et al., 2 014), which was also obser ved for cells transduced
with recombinant AAV2 vectors (rAAV) (Hirosue et al., 2007). Even
less understood is the observation that the intermediate filament
protein vimentin enhances infection after endosomal escape as
shown for MVM (Fay & Panté, 2013) as intermediate filaments are
not polarized and thus hardly contribute to directed cytoplasmic
transport. The observation showing that vimentin filaments become
disrupted at 14 to 24 h post- MVM infection, well after nuclear entry
should have been completed, indicates an independent phenome-
non that is unrelated to early infection events (Nüesch et al., 2005).
Further, some parvoviruses such as MVM and CPV exploit
ubiquitin- proteasome machinery to enhance their nuclear trans-
location. The presence of a proteosomal inhibitor (MG132) leads
to cytoplasmic perinuclear retainment of capsids. However, the
viral entry, the natural proteolytic cleavage of VP2 to VP3 and
the externalization of the N terminal of VP1 are not affected (Ros
& Kempf, 2004). In contrast, ubiquitination of AAV capsids leads
to their degradation, and treatment with MG132 increases A AV- 2
and AAV- 5 transduction (Ding et al., 2003; Douar et al., 20 01; Yan
et al., 2002; Zhong et al., 2008). It remains an open question if

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the involvement of proteasomes affects cytosolic transpor t or a
subsequent step.
2.2 | Overview of NPC and nuclear import
Many viruses have adapted to replicate in the host's nuclei, allow-
ing exploitation of cellular machiner y like DNA or RNA polymer-
ases. This requires access to the nucleoplasm, which has led to the
evolution of specific mechanisms for reaching this compartment.
Amongst the best- described approaches to enter the nucleus are
interactions with nuclear pore complexes (NPC) (Fay & Panté, 2015;
Guedán et al., 2021).
NPCs are macromolecular structures crossing the NE, allowing
passive dif fusion only of metabolites and proteins smaller than 30–
60 kDa, dependent upon their charge. However, a slow diffusion of
larger molecules up to 230 kDa through the NPC has been observed
(Popken et al., 2015; Timney et al., 2016; Wang & Brat tain, 20 07).
The NPC is composed by approximately 30 proteins termed nucle-
oporins (Nups). The shape of the NPC opening is determined by the
Y complexes (Nup107- Nup160 complex) (Stuwe et al., 2015). These
are crucial for their interactions with the gel- like mesh of highly dis-
ordered nucleoporins present in the NPC channel, which are charac-
terized by high abundance of short stretches of hydrophobic amino
acids comprising phenylalanine (F) and glycine (G) residues. FG-
Nups, which include Nup62, regulate which molecules may traverse
the NE and fix cytoplasmic and nuclear fibers ex truding from the
central part of the NPC (Lyngdoh et al., 2021). Other nucleoporins
like Nup153 and Tpr form a basket- like structure on the nucleoplas-
mic side, which is necessary for impor t and expor t. Many Nups such
as Nup153 and Nup62 are also involved in other, non- transport-
related functions, such as chromosome alignment and binding (Chien
et al., 2020; Hashizume et al., 2013). Nup153 and Nup358 have been
reported to possess conserved zinc finger domains, which are re-
quired for recruitment of coat protein I complex (COPI) coatomers
in the early process of nuclear envelope breakdown (NEBD) during
mitosis (Liu et al., 2003; Prunuske et al., 2006).
Transport of large proteins or nucleoprotein complexes through
the NPC is energy dependent and requires exposure of specific
signaling motifs on cargo surface. Classical nuclear localization
signals (NL Ss) are characterized by shor t stretches of positively
charged amino acids (Arginine and Lysine) exemplified by that of
SV40 (PKKKRKV) (Kalderon et al., 1984). Other signals are proline-
tyrosine NLSs, previously termed M9 domains, which comprise
highly disordered sequences of 20– 30 amino acids interspaced by
hydrophobic or basic residues, as, for example, found in hnRNP A1
(Bradley et al., 2007; Görlich, 1997). Not all NLSs are permanently
exposed. The so- called cryptic NL Ss become exposed only upon
post- translational modifications or protein– protein interactions
(Fagerlund et al., 2002; Gu et al., 2003).
The different signals for nuclear transport through the NPC
allow binding of specialized transport receptors, named importins,
which are divided into importin α and β (also known as KPNA, KPNB)
(Cautain et al., 2015). Of the former, seven members, all involved
in nuclear import, are known to serve as adaptor proteins between
the nuclear import signal on the cargo and importin β to which it
binds via an importin- binding domain. Depending on the species,
between 14 and 20 importin βs have been described. Eleven mem-
bers of human importins βs facilitate nuclear import. These include
transportin (TNPO, also called importin β2), six nuclear export, and
three nuclear import and export (Kimura & Imamoto, 2014; Oldrini
et al., 2017). Importin β not only binds to cargos via importin α but
may also directly interact with cargo- exposed importin- binding do-
mains (Lee et al., 2006; Mitrousis et al., 2008). There is, however,
growing evidence that nuclear transport receptor- independent
pathways exist as it was described for, for example, IκBα (Sachdev
et al., 2000).
The import is initiated by binding of importin to its correspond-
ing nuclear import motif. Via multiple interactions, these complexes
pass the hydrophobic mesh in the central pore channel (Yoshimura
et al., 2014). Upon interacting with Nup153, dissociation of the
cargo from the importin occurs through binding of the Ras- like small
GTPase Ran in its GTP- bound form (Walther et al., 2001).
Nuclear export follows a similar principle in which a nuclear ex-
port signal (NES) containing cargo traverses the NPC toward the cy-
toplasm. NESs are characterized by a hydrophobic profile, as is found
on the HIV Rev protein (LPPLERLT) (Fischer et al., 1995). NESs allow
binding of ex portins, such as Chromosomal Maintenance 1 (CRM1) in
complex with RanGTP (Kehlenbach et al., 1999; Petosa et al., 2004).
After translocation through the NPC, export complexes reach cyto-
plasmic filaments where Nup214 binds to CRM1 allowing the closely
localized Nup358- bound RanGAP to trigger the GTPase function of
Ran, catalyzing the hydrolysis of RanGTP to RanGDP. This results in
a conformational change and the dissociation of the transport- cargo
complex (Hutten et al., 2008; Mahadevan et al., 2013; Ritterhoff
et al., 2016; Wälde et al., 2012).
2.3 | Nuclear entry of parvoviruses
through the NPC
Although the molecular details of parvoviral nuclear import remain
controversial, it has been suggested that intact capsids enter the
nucleus followed by genome release at some distance from the NE
(Bernau d et al., 2018; Mänt ylä et al., 2018). Prev ious studies on MV M
demonstrated that the nuclear release of parvoviral genomes occurs
without complete disassembly of the capsids (Cotmore et al., 1999;
Ros et al., 2006; Ros & Kempf, 2004). However, fast diffusion of
intranuclear CPV capsid fragment s demonstrates the presence of
disassembled capsids (Mäntylä et al., 2018). Irrespectively to the
intranuclear fate of capsids, which is linked to the unknown mecha-
nism of genome release, they may have to be primed for genome re-
lease prior to nuclear import as B19V capsid uncoating is enhanced
by cytoplasmic depletion of divalent cations (Caliaro et al., 2019).
Single particle imaging demonstrated that the first nuclear AAV- 2
capsids are detected already 15 min after adding the viral particles to cell

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culture (Seisenberger et al., 2001) although others reported that more
than 2 hours are needed for nuclear capsid arrival (Bartlett et al., 2000;
Sonntag et al., 2006; Zhong et al., 2008). The entry of intact capsids
was also observed for CPV either after infection or af ter cytoplasmic
microinjection of viral particles; the latter observation indicating that
acidification and subsequent structural changes are not essential for
nuclear entry (Harbison et al., 2009; Suikkanen, Antila, et al., 2003b;
Vihinen- Ranta et al., 2002). However, these microinjections were per-
formed using parvovirus- susceptible cells and the technically caused
leakage of capsids from the needle prior to injection leads to exposure
of capsids to the cell exterior thus initiating parallel infections.
Two possible pathways of how parvoviral capsids enter the nu-
cleus have been proposed: a “classical” entr y passing the NPCs using
the NLS on VP1u, which binds to nuclear import factors of the im-
portin family (Table 1). This would allow the capsids to pass the NPC
due to their small diameter which is below the 40 nm size limit of the
NPC (Panté & Kann, 2002) (Figure 1). Alternatively, parvoviral cap-
sids may enter the nucleus through transient holes in the NE, which
are induced by their interaction with Nups (Porwal et al., 2013)
(Figure 2). Due to the low efficiency of all parvoviruses, it remains
not fully evident which pathway leads to progeny infection, and a
combination of both pathways appears possible.
As mentioned before, acidification leads to exposure of VP1u,
which comprises a NLS with basic residues as shown for CPV
(Cotmore et al., 1999, 2010 ; Vihinen- Ranta et al., 2002). Similarly, the
externalized N- terminus of AAV2 VP1 and VP2 proteins comprise
three NLS- like motifs which are essential for the progression of in-
fection (Grieger et al., 2006; Hoque et al., 1999; Johnson et al., 2010;
Sonntag et al., 2006), as well as three PDZ- motifs crucial for nuclear
entry and infection (Popa- Wagner et al., 2012). These NLSs may not
only contribute to the nuclear transport of the capsids but also to
the transport of capsid proteins, required for nuclear assembly of
TABLE 1 Key facts on nuclear entry of parvovirus capsids
Nuclear entry
requirements Active transpor t NPC
Interac tion with
Nups NEBD
Autonomous
parvovirus
B19V: Potential depletion
of capsid- associated
divalent cations for
uncoating (Caliaro
et al., 2019)
CPV and H - 1 P V : Ca2+
release for NE
disruption (Porwal
et al., 2013)
CPV: Capsids recruit
importin β to form
capsid- importin β
complex
(Mäntylä et al., 2020)
capsid- importin
β complex is
transported
into the nucleus
(Mäntylä
et al., 2018)
H - 1 P V :
Coprecipitation
with Nup358,
Nup153 and
Nup62 (Porwal
et al., 2013)
Interaction with
Nups may
trigger PLA2
exposure
to induce
initial Ca2+
release for NE
disintegration
(Porwal
et al., 2013)
MVM: NE invagination and redistribution of
lamin A/C (Cohen et al., 2006)
H - 1 P V : Ca2+ release triggers activation of
mitotic factors (PKC, cdk2/cdk1 and
caspase 3) for NE disintegration by local
lamin B depolymerization. No soluble
cytosolic factors needed in permeabilized
cells (Por wal et al., 2013)
CPV failed to infect cells preloaded with
hepatitis B capsids by microinjection
(Porwal et al., 2013)
Dependo-
parvovirus
AAV 2: Capsid
acidification (pH 5. 2)
and Ca2+ release
was required for NE
disruption (Porwal
et al., 2013)
AAV 2: Three NLS- like
motifs in VP1 and
VP2 essential for
infection (Johnson
et al., 2010).
Three PDZ- motifs on
VP1u essential
for nuclear entry
and infec tion
(Popa- Wagner
et al., 2012).
Labeled capsids pass-
through NPC, no
evidence of NE
disintegration:
(Kelich et al., 2015)
rAAV2: Interaction
with importin β
with or without
interaction
with importin
α (Nicolson &
Samulski, 2014)
AAV 2
coprecipitates
with Nup358,
Nup153 and
Nup62 (Porwal
et al., 2013)
NEBD limited to microinjected AAV2 capsid
exposed to pH 5.2 (Porwal et al., 2013)
NE invagination showed by EM (Cohen
et al., 2006; Cohen & Panté, 20 05)

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progeny par voviruses. In fact, VP1/VP2 trimers of MVM are actively
imported although using importin β - independent pathway (Riolobos
et al., 2006). Of note, the nuclear import of these trimers requires
VP1/VP2 phosphorylation by Raf- 1 kinase during entr y of the cell
into S phase, which may contribute to MVM specificity for trans-
formed cells (Lombardo et al., 2000; Riolobos et al., 2006).
More direct evidence for importin β- recruitment on assembled
capsids was found in time- lapse microscopy of CPV after infection,
showing that the formation of importin β- CPV- complexes slows the
diffusion of cytoplasmic CPV capsids (Mäntylä et al., 2018, 2020).
Further, importin β- CPV capsid complexes are transported simulta-
neously through the NE (Mäntylä et al., 2018), which is consistent
with data on rAAV- 2 nuclear import, which depends on interac-
tion with importin β alone or in complex with importin α (Nicolson
& Samulski, 2014). However, at least some nuclear capsids remain
decorated with importin β (Mäntylä et al., 2018) arguing against a
classical nuclear import of at least a fraction of capsids, as importin
β becomes removed from the cargo within the nuclear basket during
classical NLS- dependent nuclear import.
However, the number of detected intranuclear capsids is very
low but in agreement with the small number of nuclear replica-
tion compartment foci detected in early stages of MVM and CPV
infection (Ihalainen et al., 2007; Ruiz et al., 2006). It can be thus not
excluded that only a minor fraction of parvoviral capsids initiates
infection.
2.4 | Entry through the NE by increased nuclear
envelope permeability
Various parvoviruses exhibit a unique feature in that they permea-
bilize transiently the NE, as it was shown for H- 1PV, CPV, and AAV2
(Cohen et al., 2006; Cohen & Panté, 2005; Popa- Wagner et al., 2012;
Porwal et al., 2013) (Table 1). This nuclear envelope break- down
(NEBD) occurs within minutes after capsid exposure to nuclei, being
in agreement with a rapid passage of the capsid into the nucleus ob-
served in infection. This led to the hypothesis that these holes in
the nuclear envelope allow nuclear entr y of intact capsids (Figure 2).
Mechanistically, parvoviral NEBD shows similarities to mitosis in
that Ca++, released from the lumen between inner and outer nuclear
membrane, initiates activation of PKCα, which activates Cdk2 and/
or Cdk1, followed by activation of caspase 3 (Figure 2b). The acti-
vation of the kinases allows the hyper- phosphorylation of lamin B,
which was described to cause local lamin (Cohen et al., 2006). Such
depolymerization is required for open holes of up to 190 nm (Porwal
et al., 2013), which are large enough to allow entry of the capsids
or even larger complexes as capsid- importin complexes. The role of
caspase 3 is in the proteolytic cleavage of lamin B and not in the di-
rect disruption of the nuclear membranes (Cohen et al., 2006, 2011;
Cohen & Panté, 2005) (Figure 2c). Caspase 3 is upregulated and ac-
tivated just prior to mitosis (Hsu et al., 2006), being in concordance
with its function during parvoviral- mediated NEBD.
Pore formation depends on interaction with the NPC in par-
ticular by capsid binding to at least three Nups (Nup358, Nup153,
and Nup62). Blocking AAV2 or H- 1PV interaction with Nup153
by hepatitis B virus capsids, which specifically interacts with
Nup153 (Schmitz et al., 2010) inhibited NEBD. The relevance of
this finding for infection was later confirmed by CPV, which failed
to infect cells preloaded with hepatitis B virus capsids by micro-
injection (Mäntylä et al., 2020). As Nup153 is localized in the
nuclear basket close to the inner ring of the NPC, these obser-
vations indicate that the parvoviruses should be associated with
importins in order to reach the nuclear side of the NPC (Figure 2a).
Furthermore, NEBD was accelerated when the capsids were pre-
acidified and neutralized implying the need of VP1u exposure.
In fact, PLA2 exposure could also be achieved by direct interac-
tion of parvoviral capsids (AAV2 and H- 1PV) with Nups leading
to the hypothesis that the accessible PLA2 domain triggers the
initial Ca++ efflux. However, PLA2 activity on MVM capsids has
not been reported to be involved in causing NE disruption (Cohen
et al., 2011) and other mechanisms causing permeabilization can-
not be excluded. This includes amphipathic helices identified on
VP1u (Leisi et al., 2016) as they permeabilize membranes, which
was as shown for endosomal escape of adenoviruses (Wiethoff &
Nemerow, 2015), or the PDZ domains, which exhibit membrane
FIGURE 1 Nuclear entry of parvoviruses through the NPC.
Cytoplasmic parvoviruses (PVs) that have undergone structural
changes within the endosome bind to importin α (KPNA) /
importin β (KPNB). This allows transport through cellular nuclear
pore complexes (NPCs). Upon reaching the nuclear basket, the
PV- importin complex dissociates, releasing the capsid into the
nucleoplasm. Figure created with BioRender.

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affinity (Fanning & Anderson, 1999) and induce membrane curva-
ture (Herlo et al., 2018).
In summar y, there are two seemingly contradictory models of nu-
clear import of parvoviral capsids but both rely on interaction with
the NPC, which was also demonstrated by single particle tracking of
rAAV (Junod et al., 2021; Kelich et al., 2015). Numerous data support
that this interaction is mediated by importin β, however, the interac-
tion between importin α and the NLS exposed on VP1u on capsid sur-
face is not well understood. Nuclear import of microinjected capsids
(Harbison et al., 2009; Suikkanen, Aaltonen, et al., 20 03a) suggests that
a sub- fraction of capsids might expose their VP1us without acidifica-
tion. Fur ther, it cannot be totally excluded that the nuclear c apsids after
microinjection are derived from infection occurring in parallel.
Differences between the models comprise later events once the
capsids arrive on the nuclear side of the NPC. While the classical im-
port model favors dissociation of the importins from the capsids and
diffusion of the latter deeper into the nucleus, the NEBD model sup-
ports interaction with Nup153 possibly after importin β dissociation,
FIGURE 2 Entry through the NE by increased nuclear envelope permeability. (a) Parvovirus capsids bound to importins (KPNA: Importin
α/KPNB: Importin β) bind to Nups. The binding triggers exposure of PLA2 on VP1u inducing calcium efflux. (b) the release of calcium
activates PKCα, which activates cdk2/cdk1. Caspase 3 is also activated. (c) Hyper- phosphorylation of Lamin B by kinases as well as L amin
B- cleavage by caspase 3 leads to its local degradation. (d) The formation of transient holes allows entry of NPC- bound or cytosolic capsids or
capsid- importin complexes. Figures created with BioRender.

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disintegration of the NE and entry of the capsid. These could be either
cytosolic capsids (eventually importin β- bound) or the capsids that have
initiated the NEBD, likely after NPC- dissociation which is mediated
by cdk- 1 (Kutay et al., 2021). However, as long as it remains unknown
which capsids initiate infection, none of the models can be excluded.
3 | NUCLEAR EGRESS
Once parvovirus genome has entered the nucleus, successful replica-
tion depends on cell entry into the S phase. The S phase- dependent
activation of DNA replication machinery is needed to provide the re-
sources necessary for viral replication. These cellular factors include
DNA polymerase δ required for conversion of ssDNA to dsDNA tem-
plate for viral gene transcription (Cotmore & Tattersall, 2013). The
progression to the S phase is accompanied by virus- induced cellular
DNA damage, ataxia telangiectasia mutated (ATM)- dependent DNA
damage response (DDR) and pre- mitotic cell cycle arrest in MVM
infection (Adeyemi et al., 2010; Cotmore & Tattersall, 2013; Ruiz
et al., 2011). In AAV infection, cytotoxic viral Rep proteins induce
S- phase arrest (Berthet et al., 2005; Saudan et al., 2000), and UV-
treated A AV particles evoke ATM- and Rad3- related kinase (ATR)-
dependent DDR charac terized by accumulation of cells in the late S
and/or G2 phases (Jurvansuu et al., 2005; Raj et al., 2001; Schwartz
et al., 2009; Winocour et al., 1988). Preventing cell entry from G2
phase to mitosis maintains nuclear structure thereby allowing the
prolonged assembly of new virions (Adeyemi & Pintel, 2014; Chen
et al., 2010; Morita et al., 2003). The empty capsids are formed in the
nucleus, and they mature into DNA- filled capsids at the late S/G2
phase (Gil- Ranedo et al., 2015). Af ter AAV capsid assembly, involv-
ing capsid accumulation in nucleoli (Sonntag et al., 2010; Wistuba
et al., 1997), targeting of viral ssDNA to viral capsid is mediated by
Rep proteins (Bleker et al., 2006; Dubielzig et al., 1999).
Viral infection elicits various responses in the host cell which
can lead to plasma membrane ruptures, formation of membrane
vesicles, nuclear fragmentation, and finally to cell lysis (Labbé &
Saleh, 2008). The cellular egress of many non- enveloped viruses is a
passive process which relies on cell lysis to release viral progeny into
the extracellular space (Daeffler et al., 2003; Georgi & Greber, 2020;
Tollefson et al., 1996). The major form of cell death described for
parvoviruses is apoptosis, however, also necrosis has been detected
(Chen & Qiu, 2010; Nykky et al., 2010).
In apoptotic cells, the NE permeability is regulated by caspase-
dependent and - independent alterations of NPCs and caspase- dependent
cleavage of la mins and other NE prote ins (Ferrando- May, 2005; Kihlmark
et al., 2004; Roehrig et al., 2003; Strasser et al., 2012). As described ear-
lier, nuclear entry of parvovirus capsids is accompanied by the NE dis-
integration and activation of the key enzymes of mitosis (Porwal et al.,
2013). However, nuclear microinjection of H- 1PV capsids does not in-
duce NEBD making it unlikely that this entry- related mechanism is re-
quired for capsid egress from the nucleus.
During parvovirus infection, the disintegration of host DNA
is followed by DNA damage response and activation of apoptosis
TABLE 2 Key facts on nuclear egress of parvovirus capsids
NS2- CRM1 interaction Active tr ansport NPC Phosphorylation Apoptosis
Autonomous
parvoviruses
MVM: CRM1 interacts with the NES in
NS2 (Bodendorf et al., 1999; Eichwald
et al., 2002; Engelsma et al., 20 08; Fornerod
et al., 1997; Maroto et al., 2004; Miller &
Pintel, 2002)
MVM: NS2 NES is required
for active nuclear
export of progeny
viruses (Engelsma
et al., 2008)
MVM: Phosphorylation of serine residues
in the exposed N - terminal end of
VP2 functions as a NES contributing
to active export (Maroto et al., 2004).
Phosphorylation of the capsid surface
enhances nuclear export capacity. VP2
N- terminal phosphorylation is involved in
passive release, but not required for active
transport (Wolfisberg et al., 2016)
CPV: Infection activates caspases 9,8, 3/7.
(Nykky et al., 2010)
Minute virus of canines (MVC): Infection-
induced DNA damage leads to p53-
dependent cell death (Chen & Qiu, 2010)
Dependo-
parvovirus
AAV: Activities of helper virus leads to cell lysis
and viral exit
(Meier et al., 2020, Smith & Enquist, 2002)

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(Adeyemi et al., 2010; Chen & Qiu, 2010). The cell death events are
mediated by apoptotic caspases (Roos & Kaina, 2006). CPV- infected
cells have a relatively long lifespan even thou gh the initiator cas pases
8 and 9, and effector caspases 3 and 7 are activated early in infection
and remain active until very late in infection, until 48– 72 hpi (Nykky
et al., 2010). Analysis of infected cells has indicated that capsids are
released from host cells already at 12 hpi (Zhao et al., 2016). These
observations support the model that viral capsids egress the nucleus
and the host cell prior to apoptosis- induced cell lysis. After nuclear
exit cytoplasmic MVM progeny capsids are transported through
COPII- vesicles of ER and cisternae of Golgi and continue toward
the cellular periphery in lysosomal/late endosomal vesicles. The ve-
sicular capsid transport and cellular exocytosis depend on gelsolin-
induced degradation of actin (Bär et al., 2008).
In contrast to these cell- destruction- based exit mechanisms,
which were previously thought to be the main pathway for progeny
parvoviral egress, more recent evidence supports that parvoviruses
are also able to actively egress the nucleus into cytosol before pas-
sive release through cell lysis at the final stage of the infection occur s
(Table 2). Active translocation has been previously shown for MVM,
which utilizes the CRM1- mediated active nuclear export pathway
for nuclear exit of capsids through the NPC (Eichwald et al., 2002;
FIGURE 3 Nuclear egress of progeny capsids. Packaging of viral genomes inside capsids causes a conformational change exposing the
VP2 N- terminal on the capsid surface. (a) MVM capsids are actively exported out of the nucleus through NPCs mediated by the interaction
between NS2 NES with CRM1 (Bodendorf et al., 1999; Eichwald et al., 2002; Engelsma et al., 2008; Fornerod et al., 1997; Maroto
et al., 2004; Miller & Pintel, 2002). (b) The phosphorylation of the exposed VP2 N- terminal end on the capsid surface acts as a nuclear
export signal enhancing capsid export out of the nucleus (Maroto et al., 2004). (c) Phosphorylation of the capsid surface enhances capsid
export (Wolfisberg et al., 2016). (d) Activation of apoptosis and necrosis affect the structure of the nuclear lamina, and capsids are released
to the cytoplasm in late infection (Chen & Qiu, 2010; Nykky et al., 2010, (Wolfisberg et al., 2016). Figures created with BioRender.

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Engelsma et al., 2008) (Figure 3a). CRM1, also called exportin 1, is
a versatile nuclear export receptor which shuttles between the nu-
cleus and cytoplasm (Fornerod et al., 1997) and translocates mul-
tiple cargoes including ribosomal subunits (Ho et al., 2000; Moy &
Silve r, 1999; Thomas & Kutay, 2003). The binding of CRM1 to the
cargo is promoted by RanGTP (Koyama & Matsuura, 2010) and me-
diated by NES. For nuclear export of MVM capsids, CRM1 interacts
with the NES in NS2 in a RanGTP- independent manner (Bodendorf
et al., 1999; Eichwald et al., 2002; Engelsma et al., 2008; Fornerod
et al., 1997; Maroto et al., 2004; Miller & Pintel, 2002).
Several findings, however, support CRM1- independent nuclear
capsid export, being thus most likely NS2- independent. Similar to
nuclear import of MVM VP1/VP2 trimers, nuclear egress of MVM
capsids is enhanced by Raf- 1 kinase- mediated phosphorylation of
three serine residues in the N- terminus of VP2 on capsid surface
(Maroto et al., 2004) (Figure 3b). This pathway relies on exposure of
the N- terminal domain of VP2, which is exposed in DNA- containing
parvovirus capsids during their maturation (Agbandje- McKenna
et al., 1998; Kaufmann et al., 2008; Kontou et al., 2005; Sánchez-
Martínez et al., 2012; Tsao et al., 19 91). Moreover, the phosphoryla-
tion of the capsid surface residues has been linked to nuclear export
capacity prior to the passive release by cell lysis. Although confor-
mational change of the VP2 N- terminus on the capsid surface was
required for phosphorylation, the VP2 N- terminus was dispensable
for nuclear capsid egress (Wolfisberg et al., 2016) (Figure 3c.) Non-
phosphorylated capsids exit the nucleus passively upon NE damage
during apoptosis (Figure 3d). The cellular and nuclear egress of AAV
was earlier thought to rely on cell lysis caused by overexpression of
helper virus, adenovirus or herpesvirus, proteins (Meier et al., 2020;
Smith & Enquist, 2002). Recently, the presence of viral membrane-
associated accessory protein (MAAP) was observed for AAV at the
late stages of infection. MAAP is located in the plasma membrane
and in the n uclear periphe ry (Galiber t et al., 2021; Ogden et al., 2019).
This protein is a viral egress factor, which also promotes AAV capsid
association with extracellular vesicles (Elmore et al., 2021).
4 | CONCLUDING REMARKS
The versatile therapeutic potential of parvovirus has researchers
focused on understanding the full mechanism of infection. In gene
therapy, for which an efficient delivery of modified parvoviral vec-
tors (mostly AAV) is crucial, nuclear entry seems to be a bottleneck
and detailed knowledge may help improving their clinical administra-
tion. Similar to nuclear entry, the studies of viral egress have shown
also controversial result s. Improving the knowledge on export may
assist oncolytic therapy using autonomous parvoviruses, as their po-
tential depends upon efficient spread.
ACKNOWLEDGMENTS
This work was financed by the Jane and Aatos Erkko Foundation
(MVR), Academy of Finland under the award numbers 330896 (MVR),
and the Graduate School of the University of Jyvaskyla (SM).
DATA AVAIL ABILI TY STATEMENT
Data sharing not applicable - no new data generated.
ETHICS STATEMENT
The work presented here did not include human or animal subjects
nor human or animal material or data. Thus, no formal consent or
approval was necessary.
ORCID
Maija Vihinen- Ranta https://orcid.org/0000-0003-0959-1153
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How to cite this article: Mattola, S., Aho, V., Bustamante-
Jaramillo, L. F., Pizzioli, E., Kann, M. & Vihinen- Ranta, M. (2022)
Nuclear entry and egress of parvoviruses. Molecular
Microbiology, 118, 295–308. ht tps://doi.org /10.1111/
mmi.14974