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Engelsma, D. H. (2008, October 16). Nuclear export signals: small domains with large impact. Retrieved from https://hdl.handle.net/1887/13258
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63
Chapter 4
Parvoviral NS2 Inhibits mRNA Export from the Nucleus
To be submitted
65
Chapter 4
Dieuwke Engelsma1, Noelia Valle2, Jose Almen- dral2 and Maarten Fornerod1
1Dept. of Tumor Biology, The Netherlands Cancer Instute, Ples- manlaan 121, 1066CX Amsterdam, The Netherlands
2Centro de Biología Molecular “Severo Ochoa” (Consejo Superior de Invesgaciones Cienficas-Universidad Autónoma de Madrid), 28049 Cantoblanco, Madrid, Spain
Abstract
The mouse parvovirus MVM expresses two structural proteins, VP1 and VP2 and two non-structural proteins NS1 and NS2. NS2 is an essenal mulfunconal protein required for virus infecvity. Recently, we have shown that NS2 contains a supraphysiological nuclear export signal (supraNES), which is required for nuclear export of the virus itself. Here we show that in MVM-infected cells, a strong nuclear accumulaon of cellular mRNAs is detected. This accumulaon coincides with reduced translaon efficiency of cellular mRNAs. In cells infected with MVM viruses deficient for NS2, the mRNA export defect is less severe, indicang that NS2 contributes to blocking cellular mRNA export in virus infected cells. In cultured cells, NS2 by itself is sufficient for inhibing mRNA export. mRNA export inhibion
Introduction
Many viruses interfere with host cell gene expression to advance expression of their own genome and/or impair anviral defence mechanisms. In parcular, viruses can hijack the host transport machinery to promote their replicaon and infecvity (reviewed in (Fontoura et al., 2005). Also, viruses can inhibit the host’s transport pathways in order to cause cytotoxicity to the host and/
or to promote their own transport. An example of this is the mRNA inhibion by the Vesicular Stomas Virus Matrix protein, which exerts this funcon by inhibing the mRNA export factor Rae1. It does so by compeng its nuclear pore binding site at Nup98 (Faria et al., 2005, von Kobbe et al., 2000).
Minute Virus of Mice (MVM) is the mouse species of the parvovirus genus. It is a small single stranded DNA virus, composed of an icosahedrical capsid, enrely made up of the two viral proteins, VP1 and VP2, that surround the 5 kb single-stranded DNA molecule. Natural infec-
ons are asymptomac (Kimsey et al., 1986, Rubio et al., 2005), but an immunosupressive variant of MVM, termed MVMi, causes death in newborns and leuko- penia in SCID mice (Ramirez et al., 1996, Segovia et al., 1999). The viral genome contains the informaon of two structural proteins VP1 and VP2 and at least two non- structural proteins NS1 and NS2 (Cotmore et al., 1983).
The two non-structural proteins NS1 (83 kDa) and NS2 (25kDa) are expressed early during infecon (Clemens &
Pintel, 1988) and have a relavely short half-life of less than one hour (Schoborg & Pintel, 1991). Together they are responsible for managing all processes required for viral replicaon. NS1 is a nuclear phosphoprotein that is imported via a classical NLS, composed of at least two lysine-containing domains (Nuesch & Taersall, 1993).
It is required for the viral DNA replicaon (Cotmore &
Taersall, 1987) and for causing cytotoxicity to the host cell (Caillet-Fauquet et al., 1990). NS2 has been shown to funcon in mulple processes like capsid assembly (Cot- more et al., 1997), viral mRNA translaon (Naeger et al., 1993) DNA replicaon (Choi et al., 2005), virus produc-
on (Naeger et al., 1990), cytotoxicity (Legrand et al., 1993) and capsid egress from the nucleus (Eichwald et al., 2002, Miller & Pintel, 2002) though its mode of acon has not been unraveled yet. Thus far, only three interac- tors of NS2 are known: Smn (Survival Motor Neuron), a protein mutated in the neurodegenerave disease spinal muscular atrophy, the phosphothreonine and phospho- serine-binding protein 14-3-3 and CRM1, which is the mostst udied interacon partner.
CRM1 exports NS2 out of the nucleus by binding the NS2 NES (82-MTKKFGTLTI-91) (Askjaer et al., 1999, Eichwald et al., 2002). This NES been used in many studies because of its potent binding to CRM1 (Askjaer et al., 1999, Pe- tosa et al., 2004). The CRM1-NS2 interacon has been implicated in ssDNA replicaon (Choi et al., 2005) and vi- ral egress from the nucleus (Eichwald et al., 2002, Miller
& Pintel, 2002). Early in infecon, NS2 is mainly localized in the cytoplasm, while at later me points, NS2 accu- mulates in the nucleus. Furthermore, an enhancement of the CRM1-NS2 interacon has been shown to improve viral fitness poinng to a role for nuclear export path- ways in MVM pathogenicity (Lopez-Bueno et al., 2004).
Through screening a phage library, we have previously idenfied a novel type of NES termed supra- physiological NESs or supraNESs. These NESs displayed 100-fold higher affinity towards CRM1 as compared to regular NESs (Engelsma et al., 2004) and as a conse- quence they bind CRM1 stably in the cytoplasm in the absence of RanGTP. Furthermore, these NESs arrest at the NPC at Nup358, revealing a dissociaon step in nu- clear export in vivo.
We recently idenfied NS2 as a supraNES-containing protein that is required for nuclear export of the viral capsid prior to cell lysis. This funcon of NS2 was strictly dependent on the NS2 NES and in parcular on its sup- raphysiological affinity for CRM1 (Engelsma et al., 2008).
In order to detect possible other funcons of NS2 that are not dependent on this supraNES we studied nucleo- cytoplasmic transport processes in viruses lacking the NS2 supraNES. We find that cellular mRNAs accumulate in the nucleus of MVM-infected cells, dependent on NS2 but independent of the NS2 NES. NS2 lacking its NES is sufficient to cause this effect, indicang novel funcon of
Parvoviral NS2 Inhibits mRNA Export from the Nucleus
the NS2 protein that may be important for MVM fitness.
Results and discussion
NS2 is required for the inhibion of mRNA export in MVM-infected cells
Viruses may interfere with host-cell mRNA export (Fon- toura et al., 2005). We therefore determined whether we could detect an mRNA export defect in MVM-infected cells. To determine if mRNA export was blocked we per- formed FISH with a poly(dT) probe (Fig. 1A). In wild-type MVM-infected cells, we observed a clear nuclear mRNA accumulaon indicang that mRNA export was reduced.
In contrast, a MVM strain not expressing the NS2 gene (MVM-ΔNS2 (Miller & Pintel, 2002)) did not show poly(A) accumulaon, even though infecon efficiencies were equally efficient (Fig 1B). The MVM-ΔNS2 strain is prone to revesion of the NS2-negave phenotype (NS, JMA, unpublished observaons). Indeed, an occasional cell
was observed that did express NS2 (Fig. 1A, right and B), which correlated with poly(A) accumulaon. We con- clude that NS2 is required for the poly(A) accumulaon phenotype. To invesgate whether the NS2 supraNES is involved in the poly(A) accumulaon, we infected cells with an MVM strain expressing a mutant NS2 protein ((NS2ΔNES)(Miller & Pintel, 2002)). As shown in Figure 1C, mRNA export was inhibited to the same extent as wild-type MVM.
To test if this block in mRNA export also caused a defect in protein translaon, cells were infected with MVM and incubated with 35S to label newly synthesised proteins.
Already six hours aer infecon a decrease in protein ex- pression could be observed, that pursued up to 18 hours post infecon (Figure 1D). Conclusively, MVM infecon causes both a defect in mRNA export and a defect in pro- tein synthesis, which depends on the NS2 protein.
NS2 is sufficient for the inhibion of mRNA export Figure 1. NS2 is required for the inhibition of mRNA export in MVM-infected cells.
A9 cells were infected for 24 h with MVMp (A), MVMΔNS2 (A,B) or MVMΔNES (C). Cells were incubated with Cy5-la- belled polydT probes to visualize polyA RNA and stained with an NS2 or an NS1 anbodies. Bar, 10 μm. (D) A9 cells were mock-infected or infected with MVMp and simltaneously incubated with 35S. Aer indicated me points cells were harvested and analysed by autoradiography. Protein synthe- sis has been reduced in cell lysated from MVM-infected cells.
Viral proteins were idenfied by immunoprecipitaon with an VP and an pS1 anbodies (lanes 9-10).
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Chapter 4
To test whether NS2 is sufficient for inhibing mRNA ex- port, we expressed GFP-tagged NS2 in mouse fibroblasts, and probed these cells for nuclear poly(A) accumulaon 24 h post transfecon. As shown in Figure 2, we observed a clear nuclear poly(A) accumuon in cells expressing either wild-type GFP-NS2 or GFP-NS2AAAA proteins, but none in cells expressing GFP only. We conclude that NS2 expression is sufficient for the mRNA accumulaon phenotype, and that the CRM1/NES interacon is not re- quired for this.
In conclusion we report a novel funcon of the NS2 pro- tein that is independent of its supraphysiological associa-
on with CRM1. We find that inhibion of mRNA export coincides with a recucon of cellular protein synthesis.
Interesngly, NS2 mutants viruses have a strongly re- duced translaonal efficiency of their viral mRNAs (Nae- ger et al., 1993). We suggest that this decrease may be caused by an increase of cellular mRNAs that compete with the viral mRNAs for translaon factors in the cyto- plasm. In is of note that NS2 mutant viruses do not show a reduced nuclear export of their own mRNAs (Naeger et al., 1993). It will be interesng to know how these viral mRNAs escape the cellular mRNA export inhibion.
Whereas much progress has been made in understand- ing the role of NS2 in viral nuclear egress ((Eichwald et al., 2002, Lopez-Bueno et al., 2004, Miller & Pintel, 2002) {Engelsma, 2008} and DNA replicaon (Choi et al., 2005), the role of the NS2 protein in cytotoxicity (Legrand et al., 1993) has so far not been mechaniscally explained.
Recently, mRNA export has been idenfied as a crucial factor in protecng cells against influenza-mediated cell death (Saerly et al., 2007). It is plausible that inhibion of mRNA export by MVM may also be important for NS2-
mediated cytotoxicity.
Materials and methods Anbodies
Anbodies were diluted in PBS/ 1% milk and used as fol- lows; rabbit an-serum against CRM1 was used (1:250) (Fornerod et al., 1997), rabbit an-serum against VP (1:200) (Maroto et al., 2004), mAb against NS1 (1:10 for IF) (Segovia et al., 2003) and NS2 rabbit an-serum against NS2 was used 1:200 (Faisst et al., 1995).
Plasmids and plasmid construcon
NS2-GFP and NS2AAAA-GFP have been described in En- gelsma et al. (2008).
Cell culture and transfecons
A9 cells were cultured in DMEM, containing 10% fetal bo- vine serum (Invitrogen) and anbiocs. MCF7 cells were transfected using Fugene 6 (Roche). Images were taken using Leica TCS-NT2, SP2 and AOBS confocal microscopes and analysed using Image J soware. 100 nM of LMB was used when indicated.
MVM infecons
Infecons and virus producon was performed as de- scribed previously (Eichwald et al., 2002)
Immunofluorescence stainings and image analysis Staining were done as described previously (Bernad et al., 2004).
FISH
Cells were fixed and permeabilized as described previ- Figure 2. NS2 is sufficient for the inhibition of mRNA export. NS2-GFP, NS2AAAA-GFP or GFP-transfected cells (upper panels) were incubated with Cy5-labelled polydT probes to visualize polyA RNA (lower panels). Bar, 10 μm.
ously and subsequently equilibrated in 2 x SSC (300mM NaCl, 30mM sodium citrate, pH7.0), containing 15% for- mamide, for 5 min at RT. The hybribridizaon was per- formed with 2.5ng/ul Cy5-labelled 50-mer polydT probe in 10% dextran sulphate, 1x Denharts soluon, 10 μg/
μl yeast tRNA, 2 x SSC and 15% formamide for 1h at 37
°C. Aer two washes of 30 ‘ with 2 x SSC containing 15%
formamide, cell were mounted in Vectashield (Vector Laboratories).
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