Herpesvirus nuclear egress: Pseudorabies Virus can simultaneously induce nuclear envelope breakdown and exit the nucleus via the envelopment-deenvelopment-pathway.

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Herpesvirus nuclear egress: Pseudorabies Virus can simultaneously induce nuclear envelope breakdown and exit the nucleus via the envelopment–deenvelopment-pathway

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Katharina S. Schulz a , Barbara G. Klupp a , Harald Granzow b , Lars Paßvogel a , Thomas C. Mettenleiter a,∗ a b

Institutes of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany Institutes of Molecular Virology and Infectology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany

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Keywords: Herpesvirus Pseudorabies virus Nuclear egress Nuclear envelope breakdown pUL34

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1. Introduction

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Herpesvirus replication takes place in the nucleus and in the cytosol. After entering the cell, nucleocapsids are transported to nuclear pores where viral DNA is released into the nucleus. After gene expression and DNA replication new nucleocapsids are assembled which have to exit the nucleus for virion formation in the cytosol. Since nuclear pores are not wide enough to allow passage of the nucleocapsid, nuclear egress occurs by vesicle-mediated transport through the nuclear envelope. To this end, nucleocapsids bud at the inner nuclear membrane (INM) recruiting a primary envelope which then fuses with the outer nuclear membrane (ONM). In the absence of this regulated nuclear egress, mutants of the alphaherpesvirus pseudorabies virus have been described that escape from the nucleus after virus-induced nuclear envelope breakdown. Here we review these exit pathways and demonstrate that both can occur simultaneously under appropriate conditions. © 2015 Published by Elsevier B.V.

Herpesviruses are an extensive family of large DNA-viruses. Within the order Herpesvirales, the family Herpesviridae can be 23 further divided into the three subfamilies Alpha-, Beta- and Gamma24 herpesvirinae (Davison, 2010; Davison et al., 2009), which differ 25 in their host specificity, replication efficiency and target cells for 26 27Q4 establishment of latency (Pellett and Roizman, 2013; Pellett and 28Q5 Roizman, 2007). However, all herpes virions have a similar struc29 ture. The double-stranded DNA-genome, which is enclosed in an 30 icosahedral capsid, is surrounded by proteinaceous tegument, and 31 an envelope, in which viral glycoproteins are embedded (McGeoch 32 et al., 2006). 33 Herpesviruses encode far more genes than many other viruses. 34 Approximately 40 of them are conserved within the Herpesviridae 35 and are designated as “core” genes. These genes encode proteins 36 which are important for general aspects of lytic replication, like 37 DNA-replication, -processing and -encapsidation, capsid assembly 22Q3

∗ Corresponding author at: Friedrich-Loeffler-Institut, Institute of Molecular Virology and Cell Biology, Südufer 10, 17493 Greifswald-Insel Riems, Germany. Tel.: +49 38351 71250; fax: +49 38351 71151. E-mail address: thomas.mettenleiter@fli.bund.de (T.C. Mettenleiter).

and nuclear egress, or for structural proteins of capsid, tegument or envelope (McGeoch et al., 2006). The complex structure of Herpesviruses is also reflected by their replication cycle. The nucleocapsid enters the host cell after fusion of the virion envelope with the plasma membrane, or with the endosomal membrane if endocytosis occurs. For this process the conserved core fusion machinery is essential. It consists of glycoprotein (g)B and the heterodimeric gH/gL complex. Other glycoproteins, which mediate attachment of the virion to cellular surface receptor proteins, enhance the fusion process (Eisenberg et al., 2012). Subsequently the virion is transported along microtubules to the nuclear pore (Sodeik et al., 1997; Zaichick et al., 2013), where the viral genome is released and enters the nucleus through the nuclear pore. Following circularization of linear viral DNA (Strang and Stow, 2005), the viral genome is transcribed and replicated. Protein translation occurs in the cytoplasm and, after transport of capsid proteins into the nucleus, the capsid is assembled around a protein scaffold which is autoproteolytically cleaved and extruded when DNA is packaged to form nucleocapsids (Homa and Brown, 1997). These nucleocapsids exit the nucleus through the nuclear membranes. Final maturation of the virion then takes place in the cytosol. Acquisition of proteins of the inner tegument during or immediately following nuclear egress is followed by addition of an outer

http://dx.doi.org/10.1016/j.virusres.2015.02.001 0168-1702/© 2015 Published by Elsevier B.V.

Please cite this article in press as: Schulz, K.S., et al., Herpesvirus nuclear egress: Pseudorabies Virus can simultaneously induce nuclear envelope breakdown and exit the nucleus via the envelopment–deenvelopment-pathway. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.02.001

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tegument layer in the trans-Golgi region and final envelopment by budding into trans-Golgi vesicles. Interactions between tegument and capsid proteins, among tegument proteins, as well as between tegument proteins and cytoplasmic domains of glycoproteins drive virion formation. Subsequently, the enveloped intravesicular virions are transported to the plasma membrane and released into the extracellular space (Johnson and Baines, 2011; Mettenleiter, 2004, 2006; Mettenleiter et al., 2009).

1.1. Nuclear exit

Exit from the nucleus is an essential and highly regulated step in herpesviral replication. In contrast to the plasma membrane, 72 the nuclear envelope is a formidable barrier consisting of two 73 membranes with high complexity. The two membrane layers sur74 round the periplasmic space and are linked at nuclear pores, which 75 allow transport between nucleus and cytoplasm (Beck et al., 2004, 76 77 2007). Although nuclear pores have an outer diameter of ∼125 nm, 78 the open transport canal only averages to a diameter of ∼50 nm 79 (Frenkiel-Krispin et al., 2010; Pante and Kann, 2002). Interestingly, 80 although both the inner (INM) and outer nuclear membrane (ONM) 81 are contiguous, they contain different sets of proteins (Hetzer, 82 2010). Below the INM lies the nuclear lamina, a meshwork of inter83 mediate filaments composed of type A and B lamins. The lamina has 84 been shown to be important for nuclear stability, genome organi85 zation, transcription, DNA repair and signal transduction (Dittmer 86 and Misteli, 2011). 87 It has long been disputed how herpesvirus nucleocap88 sids leave the nucleus, considering their diameter of 125 nm. 89 Initially three different mechanisms were proposed, the single90 envelopment-pathway, egress through dilated pores and the 91 envelopment–deenvelopment-pathway (recently reviewed in 92 Mettenleiter et al. (2013)). 93Q6 For the single-envelopment-pathway, it was assumed that the 94 capsid buds into the INM obtaining a lipid envelope from the INM 95 which are maintained during transport of the enveloped virion 96 through the endoplasmic reticulum and the secretory pathway. 97 However, even though some experimental data was interpreted in 98 favor of this pathway (Johnson and Spear, 1982; Torrisi et al., 1992), 99 it was shown that the composition (Klupp et al., 2000; Kopp et al., 100 2002; Reynolds et al., 2002; Skepper et al., 2001; van Genderen 101 et al., 1994) and the appearance (Granzow et al., 2001; Mettenleiter 102 et al., 2009) of primary and mature virions differs considerably. 103 Moreover, fusion events between primary enveloped virions and 104 the ONM, as well as naked cytoplasmic capsids, were observed 105 for different herpesviruses, contradicting the single-envelopment106 pathway (Granzow et al., 1997, 2001; Stackpole and Mizell, 1968). 107 Therefore, it is highly unlikely that herpesviruses use the single108 envelopment-pathway to exit the nucleus. Alternatively it was 109 proposed that herpesviruses dilate nuclear pores to exit the nucleus 110 (Wild et al., 2005, 2009). However, no solid evidence for herpesvirus 111 transfer through nuclear pores has been provided. In contrast, it 112 has been shown that reorganization of the nuclear pore network is 113 not required for herpesviral nuclear egress (Nagel et al., 2008). The 114 same holds true for the proposed combination of both postulated 115 pathways (Leuzinger et al., 2005). 116 Meanwhile, it is accepted that nuclear egress of herpesviruses 117 proceeds via a two-step mechanism designated as “envelop118 ment–deenvelopment-pathway” (Fig. 1A; Skepper et al., 2001). 119 Here, the nucleocapsid buds at the INM into the perinuclear 120 space followed by fission of the INM-derived vesicle to form a 121 primary, transient envelope. This primary envelope then fuses 122 with the ONM, thereby releasing the nucleocapsid into the cytosol 123 (Johnson and Baines, 2011; Mettenleiter et al., 2009, 2013). Mech124 anistically, this process equals a vesicular (=primary envelope 71

mediated) transport of cargo (=the nucleocapsid) through the nuclear envelope, a process so far unknown in cell biology. 1.2. The nuclear egress complex The conserved heterodimeric nuclear egress complex (NEC) is an essential component of this pathway (Mettenleiter, 2004). It is anchored in the nuclear envelope by its transmembrane protein component which has been designated as pUL34 in herpes simplex virus 1 (HSV-1) (Reynolds et al., 2001; Roller et al., 2000) and pseudorabies virus (PrV) (Klupp et al., 2000), pUL50 in human (HCMV) (Milbradt et al., 2007) and M50 in murine cytomegalovirus (MCMV) (Muranyi et al., 2002) and BFRF1 in Epstein–Barr Virus (Farina et al., 2005; Lake and Hutt-Fletcher, 2004). The transmembrane protein interacts with a second protein designated as pUL31 in HSV-1 and PrV (Fuchs et al., 2002; Reynolds et al., 2001), pUL53 or M53 in HCMV and MCMV (Dal Monte et al., 2002; Muranyi et al., 2002) and BFLF2 in EBV (Gonnella et al., 2005; Lake and Hutt-Fletcher, 2004). When expressed separately, the type II tail-anchored pUL34 localizes intrinsically to the nuclear membrane, whereas pUL31 is distributed diffusely throughout the nucleus and is recruited to the nuclear membrane only by interaction with pUL34 (Mettenleiter, 2004). Until now little is known about the structure of the NEC, since no crystal structure is available. However, HSV-1, PrV, MCMV and HCMV pUL34 homologs consist of a non-conserved carboxyterminal part and a highly conserved amino-terminal part, which can be subdivided in two (Milbradt et al., 2012) or three (Haugo et al., 2011) conserved regions (CR), depending on the selection of sequences used for the comparison and the alignment algorithm. The pUL31 interaction domains have been located within the conserved parts of the pUL34 homologs, although the exact location may differ considerably between the different homologs (Bubeck et al., 2004; Fuchs et al., 2002; Liang and Baines, 2005; Milbradt et al., 2012; Passvogel et al., 2013). For HSV-1 pUL34 the pUL31 interaction domain was localized to amino acids 137 to 181 (Liang and Baines, 2005), whereas in PrV pUL34 it is located between aa 5 and 161 (Passvogel et al., 2013) and the pUL53 interaction domain of HCMV pUL50 was mapped between aa 10 to 169 (Milbradt et al., 2012). However, also residues that are not part of the identified interaction domains may influence NEC formation (Roller et al., 2010). To further examine the pUL31–pUL34 interaction several mutational studies were performed to identify amino acids important for interaction with pUL31 homologs and NEC function. For the MCMV M50 amino acids Glu56 and Tyr57, which are strictly conserved among herpesviruses (EY motif), have been shown by randomtransposon mutagenesis to be important for complex formation (Bubeck et al., 2004). Using site-directed mutagenesis, the same two amino acids were also shown to be required for interaction of HCMV pUL50 with pUL53 (Milbradt et al., 2012) and PrV pUL34 with pUL31 (Passvogel et al., 2013). Remarkably, a charge cluster mutation at that position in HSV-1 pUL34 resulted in a mutant protein which did not localize correctly to the nuclear membrane and was unable to complement replication of a UL34-null virus, despite continuing interaction with pUL31 (Bjerke et al., 2003). Recently, site directed-mutagenesis of PrV pUL34 has been used to identify other amino acids important for interaction with pUL31. In transfection experiments NEC complex formation was impaired after mutation of Asn75 and Gly77. However during infection, pUL34 carrying a mutation in Gly77 was still able to form a complex with pUL31 and largely complemented replication of a UL34 deficient virus mutant, indicating that other viral components might be involved in NEC formation. Both, Asn75 and Gly77 are part of the conserved NTG motif, which, together with the conserved EY motif, may form a structure necessary for pUL31 binding (Passvogel et al., 2014). Interestingly, mutation of Asn103 or a dileucine motif


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Fig. 1. Nuclear egress pathways of PrV. (A) Nuclear egress mediated by envelopment–deenvelopment of nucleocapsids formed in the nucleus. (B) Nuclear escape by nuclear envelope breakdown and leakage of nucleocapsids formed in the nucleus into the cytosol. (C) Nuclear egress and nuclear escape occurring simultaneously as described in this report.

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(L166/167) separating the conserved amino-terminus and the variable carboxy-terminus, impaired viral replication distinctly, while not affecting NEC formation (Passvogel et al., 2014). Remarkably, most of the mutants analyzed in this study, including those that did not affect viral replication, exhibited reduced plaque sizes, indicating that pUL34 is also involved in direct viral cell-to-cell spread (Passvogel et al., 2014). Another study performed on HSV pUL34 showed that also the combined mutation of amino acids Arg158 and Arg161 impaired nuclear envelope localization and interaction with pUL31. This mutant exhibited a defect in cell-to-cell spread as well (Bjerke et al., 2003; Roller et al., 2011), pointing to a function of the NEC beyond nuclear egress. The variable carboxy-termini of HSV-1 and PrV pUL34 containing the transmembrane domain can be substituted by various viral or cellular heterologous sequences without compromising nuclear membrane localization, pUL31 interaction or NEC function, as long as a hydrophobic domain sufficient to anchor the protein in the membrane is available. This could be provided by other viral tail-anchored membrane proteins like HSV-1 pUS9 or pUL56, homologous proteins of other Herpesviruses like HCMV pUL50 or EBV BFRF1, cellular INM proteins like Emerin, Lap2␤ or lamin B receptor or cellular tail-anchored membrane proteins like Bcl-2 (B-cell lymphoma 2) or Vamp (vesicle associated membrane protein) (Ott et al., 2011; Schuster et al., 2012). Even shortening the transmembrane domain to 15 aa did not interfere with membrane insertion (Ott et al., 2011). In general, these results indicate that the carboxy-terminal part of the protein may only be required for anchoring the NEC in the nuclear membrane. At the border between the conserved amino-terminal and the variable carboxy-terminal parts of PrV pUL34 a RQR motif is located between amino acids 173 and 175 (Passvogel et al., 2013). This RXR motif had been identified before in the cytoplasmic domains of HSV-1 and HCMV glycoprotein B, as well as in the cellular lamin B receptor. Fusion of this motif to the membrane protein CD8 caused CD8-relocation to the INM (Meyer et al., 2002; Meyer and Radsak, 2000). Based on these results, it was initially assumed, that the RQR motif could be involved in INM targeting of PrV pUL34. However, mutational studies indicated that it is probably not involved in pUL34 targeting to the INM but serves as a Golgi retrieval signal, relocating Golgi compartment-localized molecules back to the ER, from where they can be transported to the nuclear membrane (Passvogel et al., 2013). For MCMV M50 a potentially essential sequence has been identified between aa 178 and 207. Even though mutants comprising a deletion of this region were able to bind M53, they could not rescue viral replication. Further analysis showed that this region contains a polyproline stretch that is conserved among betaherpesviruses (Bubeck et al., 2004). By comparison of sequences of 36 members of the pUL31 family, an amino-terminal region with low sequence conservation, which is variable in length (aa 1 to 114 in MCMV M53), and a conserved Cterminal region (aa 115 to 333 in MCMV M53) have been identified.

The conserved region can be further subdivided into four conserved regions (CR) (Lotzerich et al., 2006). In many pUL31 homologs, the amino-terminal part contains a classical mono- or bipartite nuclear localization signal (cNLS) (Lotzerich et al., 2006; Schmeiser et al., 2013; Zhu et al., 1999). In PrV pUL31 the NLS was predicted between aa 5 and 20 (Schmeiser et al., 2013) and in HCMV pUL53 it is attributed to aa 18 to 27 (Schmeiser et al., 2013). Presumably, pUL31 is imported into the nucleus via the classical importin mediated pathway (Pemberton and Paschal, 2005; Schmeiser et al., 2013). Alternatively, small molecules like pUL31 should be able to travel through the nuclear pores by passive diffusion (Terry and Wente, 2009). The interaction domain has been located within CR1 in HSV1 pUL31, PrV pUL31, HCMV pUL53, MCMV M53 and EBV BFLF2 by protein complementation assays (Lotzerich et al., 2006; Schnee et al., 2006). It is assumed that an amphipathic helix, which has been found in this region in HCMV pUL53, is responsible for complex formation through hydrophobic and charge–charge-interactions (Sam et al., 2009). Efforts to identify individual amino acids in HCMV pUL53 responsible for binding of pUL50 failed since no single amino acid exchange abolished NEC formation. However, viral replication was reduced after mutation of aa K128, Y129 and L130 to alanine, but only combined mutation of two or three of these critical amino acids resulted in inhibition of NEC formation and suppression of viral replication (Lotzerich et al., 2006). In addition, sequences in CR2 and/or CR3 are possibly involved in NEC interaction (Pogoda et al., 2012; Roller et al., 2010). CR3 of HSV-1 pUL31 was shown to be involved in membrane curvature of the INM (Roller et al., 2010) and mutagenesis of MCMV M53 CR4 resulted in dominant-negative mutants with defects in capsid release (Popa et al., 2010). Yeast-two hybrid studies in PrV indicated that the extreme amino-terminus of pUL31 (up to aa 41) containing the predicted NLS is dispensable for pUL34 interaction (Fuchs et al., 2002). In contrast to pUL31, pUL34 does not cross the nuclear membrane by active nuclear import, but may instead diffuse passively to the INM along peripheral channels of the NPC. There it is retained by NEC formation (Schmeiser et al., 2013). Although a protein complementation assay indicated that PrV pUL31 can be complemented by homologs of the same herpesvirus subfamily, i.e. HSV pUL31 (Schnee et al., 2006), this effect could not be validated in infection experiments (Klupp et al., personal communication). In summary, the structure and function of the two components of the NEC are not clear yet and full understanding of the results of the mutational analyses will have to await elucidation of the NEC crystal structure. 1.3. The envelopment–deenvelopment-pathway For nuclear egress, nucleocapsids bud at the INM into the perinuclear cleft. Below the INM lies the nuclear lamina, which


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stabilizes and shields the nuclear envelope, and prevents direct access of the nucleocapsids to the INM. Therefore, the NEC recruits different kinases to the INM, where they phosphorylate the lamins, resulting in local dissolution of the nuclear lamina to allow interaction of the nucleocapsid with the INM. For HSV-1 and MCMV protein kinase C (PKC) was shown to be involved in nuclear lamina dissolution (Muranyi et al., 2002; Park and Baines, 2006), whereas for HCMV participation of PKC could not be detected (Sharma and Coen, 2014; Sharma et al., 2014). Instead, the pUL13 homolog pUL97 causes nuclear lamina disruption in HCMV infected cells (Hamirally et al., 2009; Krosky et al., 2003; Sharma et al., 2014). Besides pUL13, which is important for lamin phosphorylation in HSV infected cells (Cano-Monreal et al., 2009), the viral kinase pUS3, which is present only in alphaherpesviruses, has been shown to be involved in nuclear lamina disruption (Mou et al., 2007). pUS3 also phosphorylates pUL31 to regulate primary envelopment and fusion (Mou et al., 2009). In addition, HSV-1 tegument protein pUL47 and immediate early protein pUS1 interact with the NEC and/or pUS3 and may also contribute to regulation of efficient primary envelopment (Liu et al., 2014; Maruzuru et al., 2014). A recent proteomic analysis of the HCMV NEC demonstrated that during the course of replication it may associate with different proteins. Especially at late time points of infection, it interacted with several cellular proteins, like Emerin, which possibly serves as a docking molecule to guide the NEC to specific sites of the nuclear lamina. Interestingly, knockdown of Emerin impaired HCMV replication (Milbradt et al., 2014). Other herpesviruses, like KSHV and HSV, have also been shown to phosphorylate Emerin during infection, which may be necessary to induce nuclear lamina disruption (Farina et al., 2013; Leach et al., 2007; Morris et al., 2007). Simultaneous expression of both NEC components is sufficient to induce formation and fission of intraluminal vesicles in the perinuclear space which resemble primary enveloped virions (Desai et al., 2012; Klupp et al., 2007; Lee et al., 2012), indicating that the NEC mediates curvature of the nuclear membrane. For EBV, formation of vesicle-like structures was already observed after expression of only the pUL34-homolog BFRF1, which was inhibited in the presence of dominant-negative forms of the ESCRT (endosomal sorting complex required for transport)-proteins Vps4 and Chmp4b. This may indicate that components of the ESCRT complex, which are important for intracytoplasmic vesicular transport processes, are involved in vesicle formation (Lee et al., 2012). However, these data could not be reproduced in other herpesviruses. Only recently it was shown that the NEC binds to unilamellar model membranes and induces budding and scission (Bigalke et al., 2014). Therefore, it is now clear that the NEC represents the minimal membrane deformation and scission machinery that can function without any other viral or cellular factors. However, these results do not exclude the possibility that herpesviruses recruit other factors to regulate the budding process in vivo (Bigalke et al., 2014). During nuclear egress, mature nucleocapsids are preferred over immature capsid forms (Klupp et al., 2011). This selection is regulated by pUL25 (Klupp et al., 2006), which is part of another conserved heterodimeric complex between the capsid-associated proteins pUL17 and pUL25, called CVSC (capsid vertex specific component) (Cockrell et al., 2011; Toropova et al., 2011; Trus et al., 2007). For some time, the CVSC was called CCSC (C capsid specific component), as it was at first only detected on mature (or C-) capsids (Trus et al., 2007). Only the detection of small amounts of the complex on A- and B-capsids resulted in the change of name (Cockrell et al., 2011). HSV-1 pUL31 directly interacts with the carboxy-terminal 20 amino acids of pUL25 (Yang and Baines, 2011; Yang et al., 2014). However, in the absence of pUL25, pUL31 still interacted with the capsid, presumably via pUL17 (Yang et al., 2014), which is consistent with data obtained for PrV (Leelawong et al., 2011). Nevertheless, in the absence of pUL25 nuclear egress

was inhibited, even though nucleocapsids were closely associated with the INM (Klupp et al., 2006; Kuhn et al., 2008), indicating a functional role for pUL25 in nuclear egress. Moreover, interactions between the NEC and the capsid could be involved in mediating nucleocapsid recruitment to the INM. For instance, interactions between the NEC and pUL33, a conserved protein which is part of the encapsidation machinery (Fossum et al., 2009; Vizoso Pinto et al., 2011) or directly with the core capsid (Leelawong et al., 2011; Ye et al., 2000) have been observed by yeast-two-hybrid analyses. The composition of primary herpes virions within the periplasmic space has not been unraveled yet. However, it was shown that, in addition to the nucleocapsid, also the NEC and the protein kinase pUS3 are present (Granzow et al., 2004; Reynolds et al., 2002). Absence of pUS3 results in accumulations of primary enveloped particles within the perinuclear space, indicating that pUS3 is involved in fusion of the perinuclear virion with the ONM (Klupp et al., 2001; Reynolds et al., 2002; Schumacher et al., 2005) and disruption of the NEC during de-envelopment (Mou et al., 2009). In contrast, it is still disputed whether glycoproteins gB, gD or gM are part of perinuclear virions. While they have been detected in primary HSV virions (reviewed in Johnson and Baines (2011)), they were not detected in PrV (Klupp et al., 2008). Similar results were obtained for tegument proteins pUL49, pUL41, pUL48 and pUL11 (reviewed in Mettenleiter et al. (2013)), as well as pUL47 (Kopp et al., 2002; Liu et al., 2014). Also, there is evidence for (Bucks et al., 2007; Leelawong et al., 2012; Luxton et al., 2006; Morrison et al., 1998) and against (Fuchs et al., 2004; Granzow et al., 2004; Möhl et al., 2009; Trus et al., 2007) presence of the large tegument protein pUL36 in primary enveloped virions. However, none of these proteins have been shown to be essential for nuclear egress. Primary enveloped particles and mature virions differ significantly in their ultrastructure, already indicating major differences in virion composition. Whereas primary enveloped particles lack the surface spikes observed on mature virions, they contain a distinct electron-dense ring closely apposed to the primary envelope (Granzow et al., 1997, 2004; Mettenleiter et al., 2009). To determine the composition of primary enveloped virions in more detail, it will be necessary to purify these particles from the perinuclear cleft for detailed proteomic analysis. Protocols for isolation of primary enveloped virions have been published (Padula et al., 2009; Remillard-Labrosse and Lippe, 2011). The nucleocapsid is released into the cytosol through fusion of the primary envelope with the ONM. This process resembles fusion of the virion envelope with the plasma membrane during entry. The glycoproteins gB, gH, and gL are essential for fusion during entry (reviewed in Eisenberg et al. (2012)), but deletion of either one does not block nuclear egress indicating that both fusion processes are mechanistically different. HSV-1 nuclear egress has been shown to be slightly impaired after simultaneous deletion of gB and gH (Farnsworth et al., 2007), whereas combined deletion of different glycoproteins did not influence PrV nuclear egress (Klupp et al., 2008). Hence, the entry fusion machinery may modulate nuclear egress, but is not essential for this process. Instead, other components of the perinuclear virion could be involved. pUL34 is the only membrane protein proven to be present in perinuclear virions. However, the luminal domain of pUL34 is very short and has been shown to be exchangeable (Ott et al., 2011; Schuster et al., 2012), arguing against a role for pUL34 in deenvelopment. Instead it is more likely that cellular proteins are involved in deenvelopment of perinuclear virions (Maric et al., 2011). Recently, TorsinA, an AAA+ (ATPases associated with diverse cellular activities)-ATPase important for nuclear envelope maintenance (Goodchild et al., 2005; Grundmann et al., 2007; Naismith et al., 2004) has been shown to influence nuclear egress of herpesviruses (Maric et al., 2011). Overexpression of TorsinA reduced HSV-1 replication, and resulted in formation of virus-like-vesicles


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in the cytoplasm and the perinuclear space of the host cell. They resembled primary enveloped virions ultrastructurally and in their composition, e.g. they contained pUL34, an intrinsic factor of perinuclear virions (Granzow et al., 2004). The presence of TorsinA in many of the cytoplasmic vesicles supports a functional role for TorsinA in nuclear egress (Maric et al., 2011). Possibly, overexpression of TorsinA impairs fusion of perinuclear virions with the ONM and, therefore, results in accumulation of capsids in the perinuclear space. The virions then translocate within the perinuclear space to the ER, where they are trapped. Alternatively, nuclear egress may proceed normally but cytoplasmic capsids become enveloped in a membrane containing TorsinA and pUL34 (Maric et al., 2011).

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1.4. Nuclear exit of ribonucleoprotein-complexes

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For a long time it has been thought that the herpesvirusmediated envelopment–deenvelopment-pathway is the only 435 vesicular transport mechanism across the nuclear envelope and, 436 Q7 thus, unique in cellular biology (Mettenleiter, 2004, 2006, 2009, 437 2013; Johnson and Baines, 2011; Roller, 2008). However, recently 438 it was shown for Drosophila that large ribonucleoprotein com439 plexes (RNPs) are transported out of the nucleus in a similar process 440 (Speese et al., 2012). Previously, it had been assumed that large 441 RNPs, which exceed the nuclear pore diameter, also exit through the 442 nuclear pore complexes, if necessary after structural remodeling 443 (Grunwald et al., 2011). It is unclear why large ribonucleopro444 tein complexes are transported through the nuclear membrane, as 445 transport through nuclear pores is very efficient and compromises 446 cellular processes to a lesser extend. The choice of the transport 447 mechanism could simply be a matter of size of the cargo, but it could 448 also be advantageous to co-transport functionally related mRNAs 449 or to transport mRNAs in a translationally repressed state. Alter450 nately, transport through the nuclear membrane could also serve 451 as an additional, independently regulated transport mechanism, to 452 allow export of RNP complexes when general transport is inhibited 453 (Montpetit and Weis, 2012). 454 Nuclear exit of RNP complexes and nuclear egress of her455 pesviruses appear very similar. Both processes depend on the 456 phosphorylation of cellular lamins by kinases, like PKC, resulting 457 in lamina disruption (Muranyi et al., 2002; Speese et al., 2012). In 458 addition, herpesviral nuclear egress, as well as vesicular transport 459 of RNP complexes across the nuclear envelope, might involve the 460 AAA+ -ATPase Torsin (Jokhi et al., 2013; Maric et al., 2011). Knock461 down of the Torsin gene in Drosophila resulted in the accumulation 462 of large RNP complexes within the perinuclear space. These RNP 463 complexes were abnormally shaped and remained attached to the 464 INM. In addition, fewer large RNP complexes could be observed 465 in Torsin-deficient cells, indicating that in Drosophila Torsin is 466 involved in regulating RNP complex scission of the INM (Jokhi et al., 467 2013). 468 434

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1.5. Nuclear exit via the fragmented nuclear envelope Nuclear egress is a highly regulated process, allowing Herpesviruses to exit the nucleus without impairing nuclear envelope integrity. Although the NEC is essential for this pathway, deletion of either pUL34 (Klupp et al., 2000) or pUL31 (Fuchs et al., 2002) drastically reduced but did not abolish PrV replication. To analyze whether PrV nucleocapsids can use an alternative exit pathway, the residual infectivity of UL34- (Klupp et al., 2011), and UL31deleted PrV (Grimm et al., 2012), was used for reversion analysis. After several passages on rabbit kidney (RK13) cells, the mutant viruses PrV-UL34Pass and PrV-UL31Pass replicated to titers comparable to wild type virus. Ultrastructural analyses showed that both passaged viruses did not exit the nucleus via the envelopment–deenvelopment-pathway, but induced nuclear envelope

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breakdown (NEBD) in ca. 50% of infected cells (Fig. 1B). Nucleocapsids leaked through the fragmented nuclear envelope in the cytosol, where final maturation proceeded (Grimm et al., 2012; Klupp et al., 2011). As a consequence the selection of mature capsids, which occurs during nuclear egress, was impaired, resulting in a larger amount of immature capsids in the cytosol as well as in the extracellular space. In general, after infection with different herpesviruses the nuclear membrane remains intact even at late times of infection or when pseudomitosis is induced as shown for HCMV (Hertel et al., 2007; Hertel and Mocarski, 2004). Only recently it was shown that HSV infection can also cause NEBD in cells deficient in TorsinA. Interestingly, nuclear envelope fragmentation caused a reduction in virus replication. gB and gH, as well as NEC component pUL34, enhanced NEBD, whereas pUS3 had an inhibitory effect on NE breakdown. However, in contrast to NEBD induced by the PrV mutants, HSV NEBD does not rescue replication of an UL34 deficient virus (Maric et al., 2014). Congruent mutations were detected in both passaged PrV virus mutants in seven genes, encoding for tegument and envelope proteins, as well as the maturational protease (Grimm et al., 2012). Parallel, inhibitor studies were performed to determine, which cellular pathway might be manipulated. Roscovitine, an inhibitor of cdk1, cdk2 and cdk5, and, in higher concentrations, ERK1/2, as well as U0126, a MEK1/2 inhibitor, specifically impaired replication of both passaged viruses (Grimm et al., 2012) indicating involvement of these pathways in NEBD. The VZV pUL46 homolog ORF12 induces ERK1/2 phosphorylation (Liu et al., 2012). Since PrV pUL46 was found to be mutated in and inhibitors of the ERK signaling cascade selectively impaired replication of both passaged viruses, the involvement of the ERK signaling cascade in NEBD induction was analyzed in more detail. However, although wild type and mutated pUL46 were able to induce ERK phosphorylation and activate ERK target genes, the mutated pUL46 was not responsible for NEBD. In contrast to expectations, the mutations present in pUL46 of both passaged viruses restricted the extent of NEBD (Schulz et al., 2014). Since pUL46 did not target the ERK signaling cascade to induce NEBD, other factors possibly involved in NEBD induction were analyzed. Both passaged virus mutants exhibit an enhanced fusion activity, resulting in formation of large syncytia. To analyze whether the fragmentation of the nuclear envelope is the result of deregulated fusion processes, core components of the Herpesvirus fusion machinery were deleted from the genomes of both passaged viruses. However, neither deletion of gB, nor of gH had an influence on nuclear envelope fragmentation (Schulz et al., 2013).pUL34 is part of the nuclear egress complex and therefore essential for the nuclear exit of Herpesviruses via the envelopment–deenvelopment pathway. However, it is not clear if NEBD is induced only in the absence of the NEC. To determine whether and how pUL34 influences fragmentation of the nuclear envelope, pUL34 was reintroduced into its original locus in PrV-UL34Pass. The resulting PrV-UL34Pass/UL34wt was analyzed for mechanisms of nuclear exit.

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2. Materials and methods

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Rabbit kidney (RK13), African green monkey kidney (Vero) and porcine kidney (PSEK) cells were used. Isolation of PrV-UL34Pass has been described (Klupp et al., 2011). For substitution of gfp, present in the UL34 gene locus of PrV-UL34Pass, by wild type UL34, the 1.3 kbp XhoI fragment containing UL34 was ligated into plasmid pcDNA and cotransfected with PrV-UL34Pass genomic DNA, followed by screening for non-fluorescent plaques. Correct deletion of gfp sequences and insertion of UL34 was verified by


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type PrV-Ka, PrV-UL34Pass and PrV-UL34Pass/UL34wt at a MOI of 3 or left uninfected. After 18 h the cells were scarped into the supernatant and cell lysates were separated on sodium dodecyl sulfate 12% polyacrylamide gels. After Western blotting, membranes were blocked with 6% skim milk in TBST, and subsequently incubated with monospecific rabbit sera against pUL31 (Fuchs et al., 2002), pUL34 (Klupp et al., 2000), gI (Brack et al., 2000) and pUL38 (unpublished). Bound antibody was detected with peroxidase-conjugated goat anti-rabbit secondary antibodies (Dianova, Hamburg, Germany). After incubation with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific), the blots were analyzed using an imager (VersaDoc; Bio-Rad). 2.3. One-step-replication kinetics and plaque size determination

Fig. 2. Protein expression of PrV-UL34Pass/UL34wt. RK13 cells were infected with wild type PrV-Ka, PrV-UL34Pass, PrV-UL34Pass/UL34wt at a MOI of 3 or were left uninfected. 18 h post infection cell lysates were harvested, separated on 12% SDS-polyacryamide gels, blotted and incubated with antisera as indicated on the right. Molecular masses (in kDa) of marker proteins are indicated on the left.

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sequencing after PCR amplification of the corresponding genomic region.

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RK13, Vero and PSEK cells were infected with PrV-Ka, PrVUL34Pass and PrV-UL34Pass/UL34wt at an MOI of 3. At 0, 4, 8, 12, 24 and 36 h after low pH treatment (Mettenleiter, 1989), cells were scraped into the supernatant and lysed by freezing at −80 ◦ C and thawing. Cellular debris was removed by centrifugation, and titers were determined on RK13 cells. Mean values of three independent experiments and corresponding standard deviations were calculated and plotted. For determination of plaque sizes, RK13 cells, which had been used for isolation of PrV-UL34Pass, were infected under plaque assay conditions with 200 plaque forming units (pfu) in 1 ml with PrV-Ka, PrV-UL34Pass and PrV-UL34Pass/UL34wt. After 48 h of incubation, the cells were fixed and stained with 1% crystal violet. For each virus 30 plaques were measured microscopically. The plaque diameter of PrV-Ka was set as 100%, and the corresponding plaques sizes of PrV-UL34Pass and PrV-UL34Pass/UL34wt were calculated. Mean values of three independent experiments and corresponding standard deviations are given. For statistical analysis, the repeated-measures analysis of variance (ANOVA) with a Tukey posttest was used (***P < 0.001). 2.4. Electron microscopy

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Fig. 3. Replication kinetics of PrV-UL34Pass/UL34wt. RK13 (A), Vero (B) or PSEK (C) cells were infected with wild type PrV-Ka (diamond), PrV-UL34Pass (square) and PrV-UL34Pass/UL34wt (triangle) at a MOI of 3. At the time points indicated, cells were scraped into the supernatant, and virus progeny titers were determined on RK13 cells. Mean values of three independent assays and the corresponding standard deviations are indicated.


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p.i. the cells were fixed and processed for electron microscopy as described before (Klupp et al., 2000).

Fig. 4. Plaque formation of PrV-UL34Pass/UL34wt. RK13 cells were infected with 200 plaque forming units (pfu) of PrV-Ka, PrV-UL34Pass and PrVUL34Pass/UL34wt. Two days post infection cells were fixed and stained with 1% crystal violet. For each virus diameters of 30 plaques were measured microscopically. Plaque diameters are given in ␮m (left) and as percentage of the PrV-Ka plaque size which was set as 100% (right). Mean values of three independent experiments and corresponding standard deviations are given. Repeated measurement ANOVA with a Tukey posttest was used for statistical analysis (***P < 0.001).

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PrV-UL34Pass was isolated after multiple passaging of a UL34 deficient PrV in RK13 cells (Klupp et al., 2011). Although PrVUL34Pass replicates comparable to wild type PrV, it does not exit the nucleus via the NEC but induces NEBD. To analyze whether the absence of UL34 is necessary for NEBD induction and to determine the nuclear exit strategy of PrV-UL34Pass in the presence of a functional nuclear egress complex, wild type UL34 was reinserted in the PrV-UL34Pass genome. To this end, genomic DNA of PrV-UL34Pass containing a gfp expression cassette within the partially deleted UL34 locus was cotransfected with a recombination plasmid encompassing a wild-type PrV-derived 1.3 kbp XhoI fragment comprising the UL34 gene. Non-fluorescent plaques were selected, purified to homogeneity and further analyzed. Viral DNA was isolated and the corresponding region was sequenced to verify correct deletion of gfp sequences and insertion of wild type UL34 (data not shown).

Fig. 5. Electron microscopic analysis of cells infected with PrV-Ka, PrV-UL34, or PrV-UL34Pass. RK13 cells were infected with PrV-Ka (A), PrV-UL34 (B) or PrV- UL34Pass (C) at a MOI of 1 for 14 h, and subsequently processed for electron microscopy. Arrowheads mark extracellular virions, arrows indicate primary virions. White stars denote intact nuclei, filled star indicates a disrupted nucleus. Bars represent 500 nm in (A), and 3 ␮m in (B) and (C).


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Fig. 6. Electron microscopic analysis of cells infected with PrV-UL34Pass/UL34wt. RK13 cells were infected with PrV-UL34Pass/UL34wt at a MOI of 1 for 10 h (A) or 12 h (B), and subsequently processed for electron microscopy. Arrowheads mark extracellular virions, arrows indicate primary virions. The primary enveloped virions marked in (A) are enlarged in the inset. Bars represent 3 ␮m in (A) and (B), and 500 nm in (A), inset.

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3.2. Protein expression of PrV-UL34Pass/UL34wt To analyze protein expression, RK13 cells were infected with PrV-Ka, PrV-UL34Pass and PrV-UL34Pass/UL34wt at a MOI of 3 and harvested 18 h p.i. Western blots were incubated with polyclonal sera against pUL31, pUL34, gI and pUL38. As shown in Fig. 2, PrV-UL34Pass/UL34wt expresses pUL34 analogous to PrVKa. Presence of pUL34 resulted in an enhanced level of pUL31 compared to parental PrV-UL34Pass. Detection of pUL38 was used as a loading control and gI expression was analyzed to verify the acceptor virus, since PrV-UL34Pass contains a mutation in gI resulting in a smaller expression product (Grimm et al., 2012). 3.3. Replication of PrV-UL34Pass/UL34wt RK13, Vero or PSEK cells were infected with PrV-Ka, PrVUL34Pass and PrV-UL34Pass/UL34wt at a MOI of 3. After different times post infection cells and supernatant were harvested, and viral titers were determined. As shown in Fig. 3A, replication of PrV-Ka, PrV-UL34Pass and PrV-UL34Pass/UL34wt was comparable in RK13 cells. However in Vero (Fig. 3B) and PSEK (Fig. 3C) cells PrV-UL34Pass replicated to distinctly lower titers than PrV-Ka. This defect was compensated in PrVUL34Pass/UL34wt. It has been suggested that pUL34 is not only involved in nuclear egress, but also in direct viral cell-to-cell-spread (Haugo et al., 2011; Passvogel et al., 2013, 2014; Schuster et al., 2012). To examine whether cell-to-cell spread is increased after introduction of pUL34 into the PrV-UL34Pass genome, RK13 cells were infected with PrV-Ka, PrV-UL34Pass and PrV-UL34Pass/UL34wt at a low MOI and plaque diameters were determined. Fig. 4 shows that PrV-UL34Pass plaque sizes average ca. 60% of PrV-Ka, whereas PrV-UL34Pass/UL34wt plaques in RK13 cells reach approximately 80%.

Fig. 7. Electron microscopic analysis of cells infected with PrV-UL34Pass/UL34wt. RK13 cells were infected with PrV-UL34Pass/UL34wt at a MOI of 1 for 14 h (A) or 16 h (B), and subsequently processed for electron microscopy. Arrowheads mark extracellular virions, filled stars indicate disrupted nuclei, white star denotes an intact nucleus. Inset in (B) shows a nucleocapsid budding into a fragment of the nuclear envelope. The second fragment visible contains a nuclear pore. Bars represent 3 ␮m in (A) and (B), and 200 nm in (B), inset.

3.4. Ultrastructural analyses The ultrastructural phenotype of infected cells was analyzed by electron microscopy. RK13 cells were infected with PrV-Ka (Fig. 5A), PrV-UL34 (Fig. 5B) or PrV-UL34Pass (Fig. 5C) at a MOI of 1 for 14 h and subsequently processed for ultrastructural analysis. As demonstrated previously (Klupp et al., 2000, 2011), in PrV-Ka infected cells primary enveloped virions within the perinuclear space as well as numerous extracellular virions lining the plasma membrane were observed, whereas only intranuclear capsids were found in intact nuclei after infection with PrV-UL34. In contrast, after infection with PrV-UL34Pass, besides nucleocapsids in intact nuclei also nuclei with disrupted nuclear envelope and adjacent extracellular virions were found. After infection with PrV-UL34Pass/UL34wt the nuclear envelope was intact at 10 h post infection and virions were observed to exit the nucleus via the envelopment–deenvelopment pathway (Fig. 6A). 2 h later, intact nuclei still predominated (Fig. 6B), although single nuclei with fragmented envelope were also observed. The amount of nuclei showing NEBD increased further by 14 h p.i. (Fig. 7A) and at 16 h p.i. all of the nuclei had a fragmented nuclear envelope (Fig. 7 B). Thus, the envelopment–deenvelopment pathway seems to be advantageous early in infection, whereas at later times nuclear


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envelope fragmentation occurs. Further, the results show that absence of pUL34 is not necessary for NEBD induction and indicate that PrV-UL34Pass/UL34wt is able to leave the nucleus via the envelopment–deenvelopment pathway, as well as by inducing nuclear envelope fragmentation (Fig. 1C). This is further illustrated by the fact that PrV-UL34Pass/UL34wt virions bud into fragments of the nuclear envelope even after NEBD had occurred (Fig. 7, inset).

4. Discussion In this study we analyzed whether absence of pUL34 and inability to use the NEC for nuclear egress is necessary for NEBD induction by PrV. We found that fragmentation of the nuclear envelope is still induced by PrV-UL34Pass/UL34wt, which expresses wildtype pUL34 from its original gene locus. However, this mutant virus was not only able to induce NEBD, but apparently also used the envelopment–deenvelopment-pathway. Apparently, the vesicular transport through the nuclear membrane was beneficial early in the replication cycle when the nuclear membrane still appeared intact, whereas at later times nuclear envelope fragmentation predominates. In ultrastructural kinetic analyses NEBD was first observed 12 h post infection and increased until 16 h p.i., when most nuclei showed a fragmented envelope. Recently, NEBD has been observed after infection of TorsinA deficient mouse embryonic fibroblasts (MEF) with wild type HSV-1 (Maric et al., 2014). However, induction of NEBD by HSV-1 reduced viral replication by ca. 8-fold and, therefore, differed from viral NEBD induced by the passaged PrV mutants, which replicate comparable to wild type PrV (Grimm et al., 2012; Klupp et al., 2011). In addition, in HSV-1 infected TorsinA knockout MEFs, NEBD was reduced by deletion of gB or gH, indicating that fusion promoting factors enhance breakdown (Maric et al., 2014). However, deletion of either glycoprotein from the passaged PrV mutants did not influence NEBD (Schulz et al., 2013). This difference in the function of fusion proteins gB and gH on NEBD induction correlates with their difference in function during nuclear egress in HSV-1 and PrV. While simultaneous deletion of both proteins did not influence PrV nuclear egress, HSV-1 nuclear egress was impaired (Farnsworth et al., 2007; Klupp et al., 2008). Unlike for the passaged PrV mutants, during infection of TorsinA knockout MEFs with UL34 deficient HSV-1 NEBD does not compensate for nuclear egress. This indicates that certain processes taking place during the nuclear egress of HSV-1 via the envelopment–deenvelopment-pathway may be necessary for virus maturation. These mechanisms are either not necessary during NEBD induced by passaged PrV mutants or compensated for by the mutations present in the genomes of the passaged viruses. Moreover, HSV-1 induced NEBD was enhanced after deletion of the US3 gene. This observation correlates with data showing that the nuclear lamina is severely impaired after infection with a US3 deficient or a US3-kinase dead virus (Bjerke and Roller, 2006). A UL34 null HSV-1 mutant caused less lamina disruption than infection with the wild type and failed to induce NEBD (Maric et al., 2014). The fact that NEBD has only been observed after herpesvirus wild type infection in cells lacking TorsinA, could indicate a functional role for TorsinA in maintaining nuclear envelope structure during herpesvirus infection. It is conceivable that PrV-UL31Pass and PrV-UL34Pass also manipulate TorsinA to induce nuclear envelope fragmentation. We show here that PrVUL34Pass/UL34wt is still able to induce NEBD and, thus, may use both exit pathways simultaneously and/or in temporal order, indicating that other mutations in the genome of the passaged mutants are responsible for the induction of NEBD and the ability of the virus to exit the nucleus via this pathway.

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Acknowledgments We thank C. Meinke, M. Sell and P. Meyer for expert technical assistance, M. Jörn for photographic help and M. Ziller for help with statistical analysis. This work was supported by the Deutsche Q8 Forschungsgemeinschaft (DFG Me 854/12-1). References Beck, M., Forster, F., Ecke, M., Plitzko, J.M., Melchior, F., Gerisch, G., Baumeister, W., Medalia, O., 2004. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 306 (5700), 1387–1390. Beck, M., Lucic, V., Forster, F., Baumeister, W., Medalia, O., 2007. Snapshots of nuclear pore complexes in action captured by cryo-electron tomography. Nature 449 (7162), 611–615. Bigalke, J.M., Heuser, T., Nicastro, D., Heldwein, E.E., 2014. Membrane deformation and scission by the HSV-1 nuclear egress complex. Nat. Commun. 5, 4131. Bjerke, S.L., Cowan, J.M., Kerr, J.K., Reynolds, A.E., Baines, J.D., Roller, R.J., 2003. 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KSHV ORF67 encoded lytic protein localizes on the nuclear membrane and alters emerin distribution. Virus Res. 175 (2), 143–150. Farnsworth, A., Wisner, T.W., Webb, M., Roller, R., Cohen, G., Eisenberg, R., Johnson, D.C., 2007. Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane. Proc. Natl. Acad. Sci. U. S. A. 104 (24), 10187–10192. Fossum, E., Friedel, C.C., Rajagopala, S.V., Titz, B., Baiker, A., Schmidt, T., Kraus, T., Stellberger, T., Rutenberg, C., Suthram, S., Bandyopadhyay, S., Rose, D., von Brunn, A., Uhlmann, M., Zeretzke, C., Dong, Y.A., Boulet, H., Koegl, M., Bailer, S.M., Koszinowski, U., Ideker, T., Uetz, P., Zimmer, R., Haas, J., 2009. Evolutionarily conserved herpesviral protein interaction networks. PLoS Pathog. 5 (9), e1000570. Frenkiel-Krispin, D., Maco, B., Aebi, U., Medalia, O., 2010. Structural analysis of a metazoan nuclear pore complex reveals a fused concentric ring architecture. J. Mol. Biol. 395 (3), 578–586. Fuchs, W., Klupp, B.G., Granzow, H., Mettenleiter, T.C., 2004. Essential function of the pseudorabies virus UL36 gene product is independent of its interaction with the UL37 protein. J. Virol. 78 (21), 11879–11889. Fuchs, W., Klupp, B.G., Granzow, H., Osterrieder, N., Mettenleiter, T.C., 2002. The interacting UL31 and UL34 gene products of pseudorabies virus are involved in egress from the host-cell nucleus and represent components of primary enveloped but not mature virions. J. Virol. 76 (1), 364–378. Gonnella, R., Farina, A., Santarelli, R., Raffa, S., Feederle, R., Bei, R., Granato, M., Modesti, A., Frati, L., Delecluse, H.J., Torrisi, M.R., Angeloni, A., Faggioni, A., 2005.


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2 and regulates herpesvirus-induced nuclear envelope breakdown.

Crystal Structure of the Herpesvirus Nuclear Egress Complex Provides Insights into Inner Nuclear Membrane Remodeling.

Nuclear envelope breakdown: actin' quick to tear down the wall.

A single herpesvirus protein can mediate vesicle formation in the nuclear envelope.

A cellular function is required for pseudorabies virus envelope glycoprotein processing and virus egress.

Large scale RNAi reveals the requirement of nuclear envelope breakdown for nuclear import of human papillomaviruses.

Identification of conserved amino acids in pUL34 which are critical for function of the pseudorabies virus nuclear egress complex.

Nuclear envelope-associated endosomes deliver surface proteins to the nucleus.

The Herpesvirus Nuclear Egress Complex Component, UL31, Can Be Recruited to Sites of DNA Damage Through Poly-ADP Ribose Binding.

Nuclear pore basket proteins are tethered to the nuclear envelope and can regulate membrane curvature.

Cellular mechanisms of alpha herpesvirus egress: live cell fluorescence microscopy of pseudorabies virus exocytosis.

Fibrils attached to the nuclear pore prevent egress of SV40 particles from the infected nucleus.

Unexpected features and mechanism of heterodimer formation of a herpesvirus nuclear egress complex.

Polypeptides of the nuclear envelope.

Nuclear envelope rupture is induced by actin-based nucleus confinement.

The Great (Nuclear) Escape: New Insights into the Role of the Nuclear Egress Complex of Herpesviruses.

Nuclear envelope: Curving out a nuclear pore.

SUN proteins facilitate the removal of membranes from chromatin during nuclear envelope breakdown.

The Role of the Equine Herpesvirus Type 1 (EHV-1) US3-Encoded Protein Kinase in Actin Reorganization and Nuclear Egress.

The Prolyl Isomerase Pin1 Promotes the Herpesvirus-Induced Phosphorylation-Dependent Disassembly of the Nuclear Lamina Required for Nucleocytoplasmic Egress.

Nuclear and nuclear envelope binding proteins of the glucocorticoid receptor nuclear localization peptide identified by crosslinking.

Deletion of the ORF9p acidic cluster impairs the nuclear egress of varicella-zoster virus capsids.

The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the Holoenzyme.

Nuclear envelope permeability.