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Cytoplasmic tails of hantavirus glycoproteinsinteract with the nucleocapsid protein

J. Hepojoki,1,23 T. Strandin,1,23 H. Wang,1 O. Vapalahti,1,3 A. Vaheri1

and H. Lankinen1,2

Correspondence

J. Hepojoki

jussi.hepojoki@helsinki.fi

Received 15 February 2010

Accepted 4 May 2010

1Department of Virology, Infection Biology Research Program, Haartman Institute,University of Helsinki, Finland

2Peptide and Protein Laboratory, Infection Biology Research Program, Haartman Institute,University of Helsinki, Finland

3Faculty of Veterinary Medicine, University of Helsinki, Finland

Here we characterize the interaction between the glycoproteins (Gn and Gc) and the

ribonucleoprotein (RNP) of Puumala virus (PUUV; genus Hantavirus, family Bunyaviridae). The

interaction was initially established with native proteins by co-immunoprecipitating PUUV

nucleocapsid (N) protein with the glycoprotein complex. Mapping of the interaction sites revealed

that the N protein has multiple binding sites in the cytoplasmic tail (CT) of Gn and is also able to

bind to the predicted CT of Gc. The importance of Gn- and Gc-CTs to the recognition of RNP

was further verified in pull-down assays using soluble peptides with binding capacity to both

recombinant N protein and the RNPs of PUUV and Tula virus. Additionally, the N protein of PUUV

was demonstrated to interact with peptides of Gn and Gc from a variety of hantavirus species,

suggesting a conserved RNP-recognition mechanism within the genus. Based on these and our

previous results, we suggest that the complete hetero-oligomeric (Gn–Gc)4 spike complex of

hantaviruses mediates the packaging of RNP into virions.

INTRODUCTION

Hantaviruses comprise a rodent- and insectivore-bornegenus in the family Bunyaviridae (Schmaljohn & Hjelle,1997; Vaheri et al., 2008). Bunyaviruses, presently classifiedin five genera (Hanta-, Orthobunya-, Nairo-, Phlebo- andTospovirus), are enveloped, single-stranded RNA viruseswith a trisegmented genome of a negative-sense codingstrategy (Elliott et al., 2000; Nichol et al., 2005; Schmaljohn& Hjelle, 1997). The three genome segments [small (S),middle (M) and large (L)] encode the nucleocapsid (N)protein, envelope proteins (Gn and Gc) and RNA-dependent RNA polymerase (L protein) (Elliott, 1997;Plyusnin & Morzunov, 2001). Unlike other members of thefamily Bunyaviridae that utilize arthropod vectors, hanta-viruses have co-evolved with, and are maintained by,different rodent and insectivore species (Heyman et al.,2009; Klempa, 2009; Vaheri et al., 2008; Vapalahti et al.,2003). Whilst rather benign to the carrier rodents, thetransmission of hantaviruses to man is known to causehaemorrhagic fever with renal syndrome (HFRS) inEurasia and hantavirus cardiopulmonary syndrome(HCPS) in the Americas (Klempa, 2009; Mertz et al.,

2006; Nichol et al., 2005; Pini, 2004; Schonrich et al., 2008;Vaheri et al., 2008; Vapalahti et al., 2003).

Enveloped hantavirus particles are described as pleio-morphic or spherical, measuring 80–120 or 120–160 nm indiameter depending on the method of analysis (Huiskonenet al., 2010; Martin et al., 1985; Nichol et al., 2005). Thelipid bilayer of the virion encloses the ribonucleoprotein(RNP) in which the genomic RNA segments are encapsi-dated, possibly as closed circles organized in helicalstructures with trimers of N protein (Alminaite et al.,2008; Kaukinen et al., 2005; Plyusnin et al., 1996; Wang etal., 2008). Glycoproteins Gn and Gc assemble in virions asextrusions called spikes and arrange to form an unusualfourfold grid-like surface symmetry (Hepojoki et al., 2010;Huiskonen et al., 2010; Martin et al., 1985). By transmem-brane prediction, both hantavirus glycoproteins contain acytoplasmic tail (CT); the one in Gn is approximately 110residues and the one in Gc approximately 10 residues. TheCT of Gn harbours a tandem zinc-finger (ZF) fold, thestructure of which was recently resolved by nuclearmagnetic resonance (Estrada et al., 2009).

Previously, we suggested a functional homology betweenthe glycoproteins of hantaviruses and Semliki Forest virus(SFV) (Hepojoki et al., 2010). It is common for envelopedviruses that the contacts between the RNP and the envelope

3These authors contributed equally to this work.

A supplementary figure showing BacN and RNP binding to Gc-CT ofPUUV is available with the online version of this paper.

Journal of General Virology (2010), 91, 2341–2350 DOI 10.1099/vir.0.021006-0

021006 G 2010 SGM Printed in Great Britain 2341

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proteins are mediated by a matrix protein (Flint et al.,2000). However, members of the family Bunyaviridae(Nichol et al., 2005) and genus Alphavirus (Weaver et al.,2005) are devoid of a matrix protein. By analogy toalphaviruses, the envelope glycoproteins of hantavirusesare likely to operate as a surrogate matrix that organizes theinteractions between the envelope and the nucleocapsid.Within members of the family Bunyaviridae, the Gn-CT isrequired for binding to RNP in members of the genusPhlebovirus (Overby et al., 2007b), and the CTs of both Gnand Gc of tospoviruses interact with the N protein (Ribeiroet al., 2009; Snippe et al., 2007). The nucleocapsidinteraction of SFV is mediated by the cytoplasmic domainof the E2 glycoprotein (Metsikko & Garoff, 1990; Vauxet al., 1988). Furthermore, the interaction between the SFVnucleocapsid and the E2 spike protein prefers oligomers ofthe E2 cytoplasmic domain (Metsikko & Garoff, 1990). Thespike complex of SFV is formed of three E1–E2 hetero-dimers (Venien-Bryan & Fuller, 1994; Vogel et al., 1986)and the hetero-oligomerization of E1 and E2 appearscrucial for the binding of nucleocapsid to the full-lengthglycoproteins (Barth & Garoff, 1997). Electron cryo-tomography of Tula virus (TULV) recently revealed thatthe hantavirus RNP associates with the viral envelope(Huiskonen et al., 2010), suggesting that a directinteraction between the RNP and the spike complex alsoexists in hantavirus virions.

In this report, we identify and characterize the hantavirusN–glycoprotein interaction by using well-characterizedneutralizing monoclonal antibodies (mAbs) capable ofco-immunoprecipitating Gn and Gc, and with membrane-bound peptides as an oligomeric mimicry of the cytoplas-mic protein–ligand interphase. Altogether, our results showthat the cytoplasmic domains of the envelope glycoproteinsGn and Gc of hantavirus are essential for binding RNP.

RESULTS

Co-immunoprecipitation (co-IP) of the N proteinwith glycoprotein-specific neutralizing mAbs

We chose to use co-IP in order to establish the glyco-protein and nucleocapsid interaction, and used concen-trated virus preparation for this analysis. Co-IPs of the Nprotein and glycoproteins were first performed fromradiolabelled Puumala virus (PUUV) lysate. As shown inFig. 1(a), neutralizing glycoprotein-specific mAbs to Gn(5A2) and Gc (4G2) were found in co-IP to pull down theN protein along with the previously described Gn–Gccomplex (Hepojoki et al., 2010). The N protein-specificmAb (5E1) precipitated high-molecular-mass complexesthat represent SDS–stable glycoprotein oligomers, recentlydescribed to mostly contain Gn protein (Hepojoki et al.,2010). The presence of N protein in the precipitates ofGn- and Gc-specific mAbs was verified by immunoblott-ing with N protein-specific antiserum (Fig. 1b). Theseimmunoblots revealed double bands, presumably due to

susceptibility of the N protein to proteolysis, as describedpreviously (Vapalahti et al., 1996). As both PUUV-neutralizing mAbs were able to co-immunoprecipitate Nprotein from PUUV extracts and because these mAbs co-immunoprecipitate both Gn and Gc from freshly preparedPUUV lysate, it is likely that the hetero-oligomeric Gn–Gccomplex also mediates the interaction with N protein.

Mapping of N protein-binding sites in the Gn-CT

After establishing the interaction between the glycoproteinsand the RNP, we fine-mapped the interaction sites bySPOT peptide array (Frank, 2002). In virus extractsprepared with detergent, the N protein exists as an RNP(Hepojoki et al., 2010). Therefore, we compared thebinding of N protein, either derived from lysates ofpurified virions or produced via recombinant baculovirusexpression (bacN), to Gn-CT peptides. A cellulosemembrane with 33 overlapping 16-residue peptidesrepresenting the Gn-CT of PUUV was overlaid withPUUV lysate and the binding of N protein was detectedwith the N-specific mAb 5E1. The binding sites of PUUVbacN were instead mapped using 32 overlapping 18-

Fig. 1. Co-IP of PUUV N protein and glycoproteins from viruslysates. (a) IP of radiolabelled proteins of PUUV, lysed afterpelleting through a sucrose cushion, separated by SDS-PAGE anddetected by autoradiography. Co-IPs with neutralizing Gn-specific(5A2), neutralizing Gc-specific (4G2) and N-specific (5E1) mAbsare shown. Viral proteins are identified based on their typicalmobility in SDS-PAGE. (b) N protein co-IP with Gn-specific (5A2)and Gc-specific (4G2) mAbs using non-radiolabelled PUUVlysate, detected by immunoblot using rabbit antiserum to thePUUV N protein. PUUV lysate-induced background is controlledby protein G–Sepharose beads with and without PUUV lysate andmAb-induced background by mAb-loaded beads without additionof PUUV lysate.

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residue peptides. The result shown in Fig. 2(a) indicatesthat N proteins from the two sources share binding regionsin Gn-CT. Because of the globularity of its tandem ZF fold(Estrada et al., 2009), these N protein-binding sites areprobably located close by in the tertiary structure of theGn-CT, thus forming a discontinuous binding domain forthe N protein.

PUUV N binds to Gn- and Gc-CT peptides ofdifferent hantaviruses

Antibodies against the N protein cross-react betweendifferent hantavirus species (Kaukinen et al., 2005) andreassorting of all hantavirus gene segments can occur in co-infection between two different viruses in nature (Razzautiet al., 2009; Rodriguez et al., 1998). Furthermore, the Msegments have been shown to reassort upon cell-culturegene transfections (Plyusnin et al., 2002). Therefore, westudied whether the PUUV bacN and RNP are able torecognize Gn-CT peptides of different hantavirus strains.We compared the binding of PUUV N protein to Gn-CTsof HFRS-causing (PUUV), apathogenic [TULV andProspect Hill virus (PHV)] and HCPS-associated [NewYork virus (NYV)] hantaviruses. The results (Fig. 2b–d)indicate that the PUUV N protein is able to interact withpeptides from Gn-CT of all selected hantaviruses.Furthermore, the binding sites were mapped to practically

the same regions, reacting at varying intensities. Thissuggested that hantavirus N-protein recognition is con-served, and is possibly functionally interchangeable. Inaddition, in the SPOT assay, PUUV bacN was able to bindGc-CTs of HFRS-causing (PUUV, Hantaan virus), apatho-genic (TULV and PHV) and HCPS-associated (LagunaNegra virus, Andes virus, NYV, Sin Nombre virus and ElMoro Canyon virus) hantaviruses (Fig. 2e; SupplementaryFig. S1, available in JGV Online). The SPOT peptide arrayresults are well in line with the co-IP results (Fig. 1) and,together, they suggest that the Gn–Gc complex provides abinding site for RNP, most likely via the N protein (Fig. 2).

Fine mapping and critical residues inglycoprotein–N interaction sites

To pinpoint the amino acid residues critical to theglycoprotein–N interaction, we selected peptides fromdifferent interaction sites to be analysed by residue-by-residue alanine scanning and N- and C-terminal deletions.The interaction assay was performed using either PUUVbacN (Fig. 3a–d) or RNP (Fig. 3e) as the source of Nprotein. Independently of the protein source, similarresults were obtained. These fine mappings, shown inFig. 3(a–d), revealed short (5–10 aa) regions as minimalinteraction sites in Gn-CT and indicated that only the last(possibly six) C-terminal residues of Gc-CT participate in

Fig. 2. Mapping of hantavirus N protein-binding sites in Gn- and Gc-CTs by peptide scanning. (a–d) Binding of PUUV RNP(left) and bacN (right) to hantavirus Gn-CT peptides on SPOT arrays by protein overlay assay. The Gn-CTs were scanned bythree-residue shift using 16-residue (RNP-binding) or 18-residue (bacN-binding) peptides, sequences of which are separatedby vertical lines. N-protein binding was visualized by enhanced chemiluminescence using an N-specific mAb. (e) Binding ofPUUV bacN to Gc-CTs of different hantaviruses, presented as 18-residue peptides from the C terminus of Gc. Hantavirusabbreviations: PUUV, Puumala virus; TULV, Tula virus; PHV, Prospect Hill virus; LANV, Laguna Negra virus; ANDV, Andes virus;NYV, New York virus; SINV, Sin Nombre virus; ELMCV, El Moro Canyon virus; HTNV, Hantaan virus.

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the binding of the N protein in PUUV (Fig. 3d). Assummarized in Fig. 4, the two N protein-binding sitesadjacent to the ZF in Gn-CT were identified by finemapping to contain two sub-binding sites each. Thebinding site at the N terminus of the ZF domain waslabelled binding site 1 and contains sub-binding sites A(CSKYNTDSKF) and B (RILVEKVK). Binding site 2, at theC terminus of the ZF domain, also contains sub-bindingsites A (KLTSRF) and B (QENLKKS). Alanine scanning ofbinding site 3 (SLFRYRS) at the very C terminus showedthat all residues except for the leucine and phenylalaninecontribute to N protein binding.

Binding of PUUV and TULV N proteins to solublePUUV CT peptides

To compare the ability of CT peptides to bind PUUV andTULV N proteins, we initially employed a peptide pull-downassay using rabbit reticulolysate-expressed, radiolabelled Nproteins. The predicted Gn- and Gc-CTs of PUUV togetherwith shorter peptides GnN, GnM and GnC, representing thebinding sites identified by using the SPOT assay (Fig. 2a),were synthesized. The sequences of the peptides from Gn areshown in Fig. 4. The biotinylated GnN, GnM, GnC and Gc-CT(sequence: CPRRPSYKKDHKP) peptides were coupled at

saturating levels to avidin beads, and the Gn-CT through freethiols to thiopropyl beads. Avidin beads devoid of biotiny-lated ligand and thiopropyl beads coupled with glutathione,respectively, were used as controls for unspecific binding. Allpeptides studied bound PUUV N (Fig. 5), a result inaccordance with the SPOT peptide array results (Fig. 2).However, the binding efficiency of different biotinylatedpeptides varied, increasing in the order GnN,GnM,Gc-CT,GnC. The binding efficiency of thiol-coupled Gn-CTcannot be compared directly with those of the biotinylatedpeptides, due to the different method of peptide immob-ilization. Similar results were obtained with the TULV Nprotein, thereby supporting the possibility of reassortantformation. In conclusion, these results suggested that thedifferent binding sites contribute to the overall N-proteininteraction, possibly in concert either by a shared mechanismor through independent mechanisms.

Our results had shown that the peptides from glycoproteinCTs bind recombinant N protein and also interact with theRNP (Fig. 2). Therefore, we tested the capacity of thesepeptides to pull down the RNP isolated from virion lysatesof either PUUV or TULV. As in the case of in vitro-translated N proteins, the Gn-CT of PUUV bound theRNPs of both PUUV and TULV (Fig. 5). The shorter

Fig. 3. Peptide fine mapping of hantavirus glycoprotein CT interactions with PUUV RNP. (a–e) PUUV N protein binding toalanine-mutated or N- and C-terminally deleted peptides derived from the N-binding peptides shown in Fig. 2 (amino acids arecalculated from the glycoprotein precursor). The peptides were (as in Fig. 2) probed with the PUUV bacN (a–d) and PUUV RNP(e). Individual residues were mapped by 87 alanine replacements to representative 18-residue (a–d) or 15-residue (e) peptides.The resulting binding intensity of a peptide was numerated from SPOT intensities with a ChemiDoc-XRS (Bio-Rad). The graphsabove the SPOT arrays relate each SPOT reactivity (on the y-axis) to residue-by-residue change (on the x-axis). The reactivity ofthe respective parent SPOT peptide was set to 100 %. The bars in the histograms show the binding intensity of alanine-replacement peptides and the scattered lines show the intensity of residue-by-residue N- or C-terminal deletion (13-/18-residue), which are read either starting from the chart mark (N-terminal deletions) or up to the chart mark (C-terminal deletions).

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peptides of Gn-CT (GnN, GnM and GnC) were also able tomediate the binding of PUUV RNP. The relative bindingintensity of the Gn-derived peptides to RNP wasGnM,GnN5GnC, being rather different from the bindingintensity of in vitro-translated N protein. In contrast to thebinding of in vitro-translated TULV N, the binding of GnM

to the RNP of TULV was undetectable in this assay. Thisinconsistency obtained using N protein from differentsources is in line with the SPOT assay results (Fig. 2a–d),where weak (if any) RNP binding, but reasonable bacNreactivity, to this fairly conserved GnM peptide was seen.

Taken together, the binding of PUUV and TULV RNPs toPUUV-derived Gn-CT peptides was found to be verysimilar. The PUUV Gc-CT also reacted with the PUUVRNP, but the binding to TULV RNP was not tested in thepull-down assay.

Inhibition of glycoprotein–RNP interaction bypeptides from glycoprotein CTs

We were finally interested to see whether the RNP-bindingpeptides could interfere with the glycoprotein–RNPinteraction. Co-IP of glycoproteins and RNP fromradiolabelled PUUV lysate was performed using eitherGn-specific mAb 5A2 or Gc-specific mAb 4G2 in thepresence of Gn-CT or Gc-CT peptides, respectively. Thefull-length Gn-CT was able to block the interactionbetween RNP and glycoproteins, as seen at 30 mg ml21

(Fig. 6). This result demonstrates the involvement of Gn-CT in the interaction between Gn and N protein. The shortGnM peptide was selected as a control, as this peptideshowed only moderate binding to RNP (Fig. 5). As shownin Fig. 6, the full-length Gc-CT was unable to block theinteraction between glycoproteins and N protein. Also, wedid not observe any interference with the glycoprotein–RNP interaction by the shorter Gn-CT-derived peptidesGnN and GnC (data not shown). Together, this indicatesthat the complete Gn-CT forms a multivalent interactionsurface for the binding of RNP.

DISCUSSION

In members of the family Bunyaviridae, the segmented,tripartite genome is packaged by the N protein to form ahelical RNP structure (Eifan & Elliott, 2009). In the case ofhantaviruses, it is assumed that the large cytoplasmic/intraviral domain of Gn (Gn-CT) mediates the interactionbetween the RNP and the envelope; in this report, we have

Fig. 4. Summary of PUUV N protein- and RNP-binding sites in Gn-CT. Three binding sites were identified for the N protein byalanine-scanning and deletion analyses and, of these, sites 1 and 2 were found to contain sub-binding sites A and B. Residuescritical to the interaction are highlighted in black and conserved functionality between strains is highlighted in light grey(Arg : Lys, Asp : Glu and Ser : Thr). The PUUV Gn-CT peptides used in further N-binding studies were CT (cytoplasmic tail;Gn aa 526–637), GnN (amino-terminal binding site; Gn aa 541–558), GnM (middle-part binding site; Gn aa 594–613) andGnC (carboxyl-terminal binding site; Gn aa 619–637). Amino acid sequences of these synthetic peptides are depicted. Thepeptide analyses are shown in a sequence alignment of nine hantavirus strains; abbreviations are as in the legend to Fig. 2.

Fig. 5. Pull-down of PUUV and TULV N proteins and RNPs withPUUV glycoprotein CTs. In vitro-translated and radiolabelled Nproteins or purified RNPs of PUUV and TULV were precipitatedwith bead-immobilized Gn- or Gc-CT peptides. The Gn-CTpeptide encompassing aa 526–637 in the PUUV glycoproteinprecursor was immobilized to thiopropyl beads through free thiols.Biotin-conjugated peptides Gc-CT (aa 1135–1148 of the gly-coprotein precursor) and GnN, GnM and GnC (representing Nprotein-binding sites 1, 2 and 3, respectively; see Fig. 4) wereimmobilized via biotin to monomeric avidin beads at saturatinglevels. Thiopropyl beads coupled with glutathione or empty avidinbeads were used as controls, respectively. The peptide-boundproteins were separated by SDS-PAGE (10 % gel) and detectedby autoradiography (in vitro-translated N) or immunoblotting usingrabbit antiserum to PUUV N protein (RNP).

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characterized this crucial interaction for the first time. Thehantavirus glycoprotein–RNP interaction was verified by co-IP and mapped by SPOT peptide scanning to the amino acidlevel. The mapping results and previous findings lead us topropose a model in which both Gn and Gc, by theircytoplasmic tails, mediate binding to the RNP. Additionally,our data suggest that the N protein of PUUV interacts withGn- and Gc-CTs of several hantavirus types, thus in theoryenabling generation of reassortants via CT recognition ofclosely related viruses (Plyusnin et al., 2002; Razzauti et al.,2009).

For a full infectious cycle of a virus, one of the most criticalsteps after entering the host cell and reproduction ofprogeny genomes is the egress of newly synthesized virionsbearing the genetic material. Several enveloped virusesencode a matrix protein that bridges between the nucleo-capsid and the viral envelope by interacting with both RNPor capsid and the membrane protein(s). To mediate thepackaging of viral RNA, an enveloped virus that lacks amatrix protein instead requires a direct interaction betweenthe intraviral domains of the envelope-embedded glycopro-teins and the RNP. The most thoroughly characterizedexamples of this type of direct glycoprotein–RNP inter-action have been described for the families Coronaviridae,

Flaviviridae and Togaviridae (Lo et al., 1996; Narayananet al., 2000; Sturman et al., 1980) and, while this is the firstreport of glycoprotein–RNP interaction for a virus in thegenus Hantavirus, it has already been reported for othermembers of the family Bunyaviridae (Liu et al., 2008;Overby et al., 2007a; Ribeiro et al., 2009; Snippe et al., 2007).In addition, this interaction has recently been identifiedwith the TULV Gn-CT and N protein using recombinantproteins (Wang et al., 2010). The homo-oligomerization ofthe hantavirus N protein (Kaukinen et al., 2005) along withreports of nucleic acids co-purifying with recombinant Nprotein (Gott et al., 1993) suggest that, in the case ofhantaviruses, formation of the RNP complex precedes theinteraction between the N protein and CTs. Indeed, for bothsevere acute respiratory syndrome-related coronavirus andhepatitis C virus (a flavivirus), oligomerization of the Nprotein through packaging of the viral genome has beenshown to be crucial for the interaction with the envelopeprotein(s) (He et al., 2004; Nakai et al., 2006).

In a recent report (Hepojoki et al., 2010), we described thespike of hantaviruses to be formed of a tetrameric Gncomplex and the spikes to interconnect through Gchomodimers. Furthermore, the present results suggest byseveral approaches that the hantavirus Gn–Gc complexmediates RNP interaction. Thus, we were encouraged topropose a model for the initial steps in the assembly ofhantaviruses, in which, similarly to alphaviruses (Barth &Garoff, 1997; Metsikko & Garoff, 1990; Overby et al.,2007b), we suggest that both hetero-oligomeric complexformation and polyvalence of Gn and Gc are essential forthe spike–RNP interaction. In the hantavirus assemblymodel (Fig. 7), we consider, to begin with, that theinteraction between the spike (Gn–Gc oligomer) and theRNP requires oligomerization of both components. Theimportance of Gn oligomerization is supported by ourprevious study (Hepojoki et al., 2010) in which we showedthat Gn forms a homotetrameric complex, where four Gn-CTs come into very close proximity, possibly by assistanceof transmembrane segments. The spike complex of TULVwas recently studied by electron cryo-tomography(Huiskonen et al., 2010), with results in accordance withthe reported biochemical data of virion glycoproteininteractions (Hepojoki et al., 2010).

Our model, depicted in Fig. 7, describes two alternativeroutes for the formation of the spike complex. In theliterature, both Gn and Gc are shown to be essential forproper folding of these proteins (Pensiero & Hay, 1992;Persson & Pettersson, 1991; Shi & Elliott, 2002;Spiropoulou, 2001). The minimal interaction complex ofGn and Gc has, however, not been demonstrated. Thus, wesuggest two alternative models to form the (Gn–Gc)4 spikecomplex. In model (a), Gn proteins associate via homo-oligomerization to gradually form a tetrameric Gncomplex. This would be supported by observations thatthe Gn protein of Uukuniemi virus is incorporated into thevirions with more rapid kinetics than the Gc protein(Kuismanen, 1984). In model (b), the heterodimeric Gn–

Fig. 6. Blocking of glycoprotein–RNP interaction by synthetic CTpeptides. (a, b) Co-IP of PUUV N protein from purified andradiolabelled PUUV lysate with Gn-specific (a) or Gc-specific (b)mAbs was done in the presence of Gn-CT (GnM as control) or Gc-CT peptides, respectively. Immunoprecipitated proteins separatedby SDS-PAGE were detected by autoradiography (Gn-specific IP;a) or by immunoblotting using rabbit antiserum to PUUV N protein(Gc-specific IP; b). SDS-PAGE bands of N, Gn and Gc andglycoprotein complexes (labelled on top of the gel as Gn) areindicated.

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Gc is formed directly after translation and oligomerizeswith similar units to create the (Gn–Gc)4 spike complex (asillustrated in step 4). Whatever the route to spike complexformation, we suggest that only after the formation of thiscomplex would the Gn-CT become available to the RNP.Upon the formation of the final spike complex, theorientation of the four Gn-CTs would alter and bringGc-CTs to the complex. This would then form a dockingplatform for the complementary surface created of Nprotein and viral RNA. After the attachment of RNP to thefirst spike, the budding of virions would be initiated viatransient interactions between Gn-CTs and RNP. Thiswould bring neighbouring spikes into close contact toenable the Gc homodimer-mediated interactions betweenspikes to stabilize.

METHODS

Cultivation, purification and lysates of PUUV. Vero E6 green

monkey kidney epithelial cells (ATCC: 94 CRL-1586) were grown in

Eagle’s minimal essential medium supplemented with 10 % heat-

inactivated fetal bovine serum (Gibco), 2 mM L-glutamine (Sigma

Aldrich), 100 IU penicillin ml21 (Orion Pharma) and 100 mg

streptomycin ml21 (Fluka), at 37 uC in a humidified atmosphere

containing 5 % CO2. PUUV Sotkamo strain infections and sub-sequent purification were done as described previously (Hepojokiet al., 2010). To radiolabel the viral proteins, infected Vero E6 cellcultures were starved for 1 h at 37 uC in medium depleted ofmethionine and cysteine, and propagated with a 1 mCi (37 MBq)mixture of [35S]cysteine and [35S]methionine (Wallac Perkin-Elmer)for 3 days at 37 uC. Pelleted virus was suspended in TBS (50 mMTris, 150 mM NaCl) or HBS (25 mM HEPES, 150 mM NaCl), storedas aliquots at 270 uC and lysed in 1 % Triton X-100 upon thawing.

Antibodies. PUUV-neutralizing bank vole mAbs 5A2 (Gn-specific)and 4G2 (Gc-specific), human mAb 1C9 (Gc-specific) and bank volemAb 5E1 (N-specific) have been described previously (Lundkvist et al.,1991, 1993; Lundkvist & Niklasson, 1992) and were kindly provided byProfessor Ake Lundkvist, Swedish Institute for Infectious DiseaseControl and Karolinska Institutet, Stockholm, Sweden. A cross-reactive polyclonal serum, recognizing N proteins of a variety ofhantaviruses, has also been described elsewhere (Vapalahti et al., 1995).

Co-IP of PUUV Gn–Gc and RNP. Co-IP of RNP with Gn and Gcproteins from the radiolabelled and purified PUUV lysates in HBSwas performed by adding 10 mg of either mAb 5A2 (Gn-specific),mAb 4G2 (Gc-specific) or mAb 5E1 (N-specific) to the lysate,followed by overnight incubation at 4 uC. The formed immunocom-plexes were precipitated with 20 ml GammaBind G Sepharose(Amersham, GE Healthcare) by 1 h end-over-end shaking at roomtemperature (RT). The Sepharose-bound material was washed threetimes in HBS, boiled in Laemmli sample buffer for 5 min and

Fig. 7. Model of glycoprotein spike and RNP interaction at the site of virion assembly. Two alternative routes to form a hetero-oligomeric spike complex are illustrated. (a) Two Gn monomers (1) form a Gn homodimer (2) that interacts either sequentiallywith two Gn monomers or with a Gn homodimer to form a Gn tetramer (3). (b) Newly synthesized Gn monomer (1) interactswith Gc to form a Gn–Gc heterodimer (2) that oligomerizes to a heterotetramer (3). (a, b) Eventually, the formation of a hetero-oligomeric complex (4) results in exposure of the previously unexposed Gn-CT (in 1–3), which allows the binding of RNP (5).The assembly of virions would thus begin via concurrent binding of RNP and oligomerization of spike complexes throughhomodimeric Gc contacts (6).

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separated by SDS-PAGE (10 % gel). The gels were dried and proteinswere detected by autoradiography.

Co-IP from non-labelled PUUV lysate, precleared (in TBS supple-mented with 0.5 % TX-100) with approximately 50 ml GammaBind GSepharose beads for 1 h at RT, was done using beads preloaded with

glycoprotein-specific mAbs 1C9, 4G2 and 5A2 (approx. 10 mgmAb : 30 ml Sepharose). The precleared lysate diluted with TBS to

approximately 0.1 % TX-100 was aliquotted onto the mAb-preloadedbeads, followed by overnight incubation at 4 uC in an end-over-endshaker. The controls were unloaded Sepharose and mAb-preloaded

Sepharose without the virus lysate. Samples were washed thoroughly,eluted in Laemmli sample buffer by boiling for 5 min, separated by

SDS-PAGE, transferred onto nitrocellulose and immunoblotted withthe anti-2/3N serum.

Mapping of PUUV N protein-binding sites in CTs of Gn and Gc

by SPOT peptide scanning. MultiPep (Intavis AG) was used forsynthesis of SPOT peptide arrays by Fmoc chemistry according to the

manufacturer’s instructions. The Gn-CTs of four different hantavirusstrains were synthesized on an amino-functionalized cellulosemembrane (Frank, 2002) as 16- or 18-residue long overlapping

peptides with a three-residue shift as described previously (Hepojokiet al., 2010). The 18 carboxyl-terminal residues containing the

predicted Gc-CT were synthesized from nine different hantavirusstrains. Binding of PUUV bacN and RNP in the SPOT peptide assaywas detected with N protein-specific mAb 5E1, following our

previous description for interaction-site mapping using SPOT peptidearrays (Hepojoki et al., 2010). After blocking [3 % skimmed milk inTBS plus 0.05 % Tween 20 (T-TBS)], the membrane was overlaid

with 1 mg ml21 (in T-TBS with 5 mg BSA ml21) of baculovirus-expressed PUUV bacN (a kind gift from Reagena Ltd, Finland),

prepared as described previously (Vapalahti et al., 1996), and reactedovernight at 4 uC. All SPOT peptide reactions were recorded on X-rayfilm (SuperRX; Fuji Medical) by enhanced chemiluminescence.

Probing of the SPOT membrane with virus lysate followed ourprevious assay protocol (Hepojoki et al., 2010) and binding was

detected by using mAb 5E1, as with PUUV bacN.

Peptide synthesis. N-terminally biotinylated GnN, GnM and GnC

peptides (sequences shown in Fig. 4b) and Gc-CT (Biot-CPRRPSYKKDHKP-OH) were synthesized on a 25 mmol scale on aMultiPep synthesizer according to the manufacturer’s protocol

(Intavis AG) for standard Fmoc chemistry. Wang resin preloadedwith C-terminal amino acid was used for Gn-derived peptides and

preloaded 2-chlorotrityl chloride resin for the synthesis of Gc-CT, toprevent autocleavage by diketopiperazine formation. Double cou-plings were done by activating amino acids bearing standard side-

chain-protecting groups (Novabiochem) in a 1 : 1 : 2 ratio of aminoacid : O-(6-chlorobenzotriazol-1-yl)-N,N,N9,N9-tetramethyluroniumhexafluorophosphate (HCTU) : 4-methylmorpholine in N,N-

dimethylformamide (DMF). After coupling and subsequent DMFwash cycles, the unreacted peptide chains were acetylated (50 mM

acetic anhydride, 130 mM N,N-di-isopropylethylamine in DMF).Fmoc deprotection after each amino acid addition was done twicewith 25 % piperidine in DMF.

The Gn-CT peptide (526Cys–Phe638 in PUUV Gn; see Fig. 4b) wassynthesized on an Applied Biosystems peptide synthesizer 433A with

Fmoc chemistry. HCTU in DMF at a 1 : 1 molar ratio to amino acidwas used for the activation. Reaction times for deprotection andcoupling were extended and triple coupling was performed for each

amino acid addition at a 3 : 1 ratio of amino acid to resin. After eachcoupling, acetylation was used to block non-reacted amino groups.

The synthesis products of GnN, GnM, GnC and Gc-CT peptides weredried on resin and cleaved for 2 h at RT in cleavage mix 1 [95 %trifluoroacetic acid (TFA), 2.5 % tri-isoprolylsilane, 2.5 % H2O] to

remove the side-chain-protecting groups and to detach the peptide

from the resin. The resin-dried Gn-CT peptide was cleaved twice for

3 h at RT in cleavage mix 2 (92.5 % TFA, 2.5 % thioanisole, 2.5 %

ethanedithiol, 2.5 % H2O). The cleavage mixture containing the

peptide and resin was filtered and the peptide was precipitated by

adding 10 vols ice-cold tert-butyl methyl ether on ice. Precipitated

peptide was washed once with ether, solubilized in water and

lyophilized. Salt removal from peptides was done in a C18 reversed-

phase column (Supelco Discovery; Wide Pore) using a steep 0–70 %

acetonitrile–H2O gradient in 0.1 % TFA, and by collecting the main

peak. The fraction containing the peptide of interest was verified by

mass spectrometry (MALDI-TOF; AutoFlex III, Bruker) for GnN,

GnM, GnC and Gc-CT. The Gn-CT peptide was analysed by Tris/

Tricine SDS-PAGE and by reactivity with polyclonal antiserum

against glutathione S-transferase-fused TULV Gn-CT (result not

shown).

Pull-down assays. The Gn-CT peptide was coupled to thiopropyl

Sepharose 6B (Amersham Pharmacia) through free thiol groups in

HBS according to the manufacturer’s protocol. Residual reactive

groups on peptide-coupled or control beads were reacted with

reduced glutathione. Peptides GnN, GnM, GnC and Gc-CT were

coupled to monomeric avidin beads (Pierce Biotechnology) in HBS

for 1 h at RT (2 mg peptides : 1 ml beads) and uncoupled peptides

were washed out. The N protein of PUUV or TULV was expressed

and labelled radioactively ([35S]methionine; Wallac, Perkin-Elmer) in

rabbit reticulolysates (TnT Quick; Promega) using plasmids harbour-

ing the S segment under the T7 transcriptional promoter (Li et al.,

2002; Vapalahti et al., 1996). The RNPs of PUUV and TULV were

purified by gel permeation from virions pelleted through a sucrose

cushion as described recently (Hepojoki et al., 2010). The recombin-

ant N or RNP was incubated overnight with end-over-end shaking at

4 uC with 10 ml peptide-coupled or control beads in 500 ml HBS.

Laemmli sample buffer was added to beads after three HBS washes,

then samples were boiled for 5 min and separated by SDS-PAGE

(10 % gel). The N protein was detected by either immunoblotting or

autoradiography.

ACKNOWLEDGEMENTS

This work was supported by following resources: the Helsinki

University Nanoscience Training and Research Programme and the

Finnish Funding Agency for Technology and Innovation (TEKES

40264/07) to H. L.; the Magnus Ehrnrooth Foundation, the Finnish

Culture Foundation and the Paulo Foundation to T. S.; an EU grant

(QLK2-CT-2002-01358) and the Sigrid Juselius Foundation to A. V.

We thank Drs Ilpo Kuronen and Helena Sirola of Reagena Ltd for

supplying purified baculovirus-expressed PUUV N protein.

REFERENCES

Alminaite, A., Backstrom, V., Vaheri, A. & Plyusnin, A. (2008).Oligomerization of hantaviral nucleocapsid protein: charged residues

in the N-terminal coiled-coil domain contribute to intermolecular

interactions. J Gen Virol 89, 2167–2174.

Barth, B. U. & Garoff, H. (1997). The nucleocapsid-binding spike

subunit E2 of Semliki Forest virus requires complex formation with

the E1 subunit for activity. J Virol 71, 7857–7865.

Eifan, S. A. & Elliott, R. M. (2009). Mutational analysis of the

Bunyamwera orthobunyavirus nucleocapsid protein gene. J Virol 83,

11307–11317.

Elliott, R. M. (1997). Emerging viruses: the Bunyaviridae. Mol Med 3,

572–577.

J. Hepojoki and others

2348 Journal of General Virology 91

Downloaded from www.microbiologyresearch.org by

IP: 54.162.133.179

On: Wed, 24 Feb 2016 13:30:39

Elliott, R. M., Bouloy, M., Calisher, C. H., Goldbach, R., Moyer, J. T.,Nichol, S. T., Pettersson, R., Plyusnin, A. & Schmaljohn, C. (2000).Family Bunyaviridae. In Virus Taxonomy: Seventh Report of the

International Committee on Taxonomy of Viruses, pp. 599–621. Edited

by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B.Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J.

McGeoch, C. R. Pringle & R. B. Wickner. San Diego, CA: Academic

Press.

Estrada, D. F., Boudreaux, D. M., Zhong, D., St Jeor, S. C. & DeGuzman, R. N. (2009). The hantavirus glycoprotein G1 tail contains

dual CCHC-type classical zinc fingers. J Biol Chem 284, 8654–8660.

Flint, S. J., Enquist, L. W., Krug, R. M., Racaniello, V. R. & Skalka, A. M.(2000). Principles of Virology: Molecular Biology, Pathogenesis, andControl. Washington, DC: American Society for Microbiology.

Frank, R. (2002). The SPOT-synthesis technique. Synthetic peptide

arrays on membrane supports – principles and applications.J Immunol Methods 267, 13–26.

Gott, P., Stohwasser, R., Schnitzler, P., Darai, G. & Bautz, E. K.(1993). RNA binding of recombinant nucleocapsid proteins of

hantaviruses. Virology 194, 332–337.

He, R., Leeson, A., Ballantine, M., Andonov, A., Baker, L., Dobie, F.,Li, Y., Bastien, N., Feldmann, H. & other authors (2004).Characterization of protein–protein interactions between the nucleo-

capsid protein and membrane protein of the SARS coronavirus. VirusRes 105, 121–125.

Hepojoki, J., Strandin, T., Vaheri, A. & Lankinen, H. (2010).Interactions and oligomerization of hantavirus glycoproteins. J Virol

84, 227–242.

Heyman, P., Vaheri, A., Lundkvist, A. & Avsic-Zupanc, T. (2009).Hantavirus infections in Europe: from virus carriers to a major

public-health problem. Expert Rev Anti Infect Ther 7, 205–217.

Huiskonen, J. T., Hepojoki, J., Laurinmaki, P., Vaheri, A., Lankinen,H., Butcher, S. J. & Grunewald, K. (2010). Electron cryo-tomography

of Tula hantavirus suggests a unique assembly paradigm forenveloped viruses. J Virol 84, 4889–4897.

Kaukinen, P., Vaheri, A. & Plyusnin, A. (2005). Hantavirus

nucleocapsid protein: a multifunctional molecule with both house-keeping and ambassadorial duties. Arch Virol 150, 1693–1713.

Klempa, B. (2009). Hantaviruses and climate change. Clin MicrobiolInfect 15, 518–523.

Kuismanen, E. (1984). Posttranslational processing of Uukuniemi

virus glycoproteins G1 and G2. J Virol 51, 806–812.

Li, X. D., Makela, T. P., Guo, D., Soliymani, R., Koistinen, V., Vapalahti,O., Vaheri, A. & Lankinen, H. (2002). Hantavirus nucleocapsid proteininteracts with the Fas-mediated apoptosis enhancer Daxx. J Gen Virol

83, 759–766.

Liu, L., Celma, C. C. & Roy, P. (2008). Rift Valley fever virus structuralproteins: expression, characterization and assembly of recombinant

proteins. Virol J 5, 82.

Lo, S. Y., Selby, M. J. & Ou, J. H. (1996). Interaction between hepatitis

C virus core protein and E1 envelope protein. J Virol 70, 5177–5182.

Lundkvist, A. & Niklasson, B. (1992). Bank vole monoclonalantibodies against Puumala virus envelope glycoproteins: identifica-

tion of epitopes involved in neutralization. Arch Virol 126, 93–105.

Lundkvist, A., Fatouros, A. & Niklasson, B. (1991). Antigenic

variation of European haemorrhagic fever with renal syndrome virus

strains characterized using bank vole monoclonal antibodies. J GenVirol 72, 2097–2103.

Lundkvist, A., Horling, J., Athlin, L., Rosen, A. & Niklasson, B. (1993).Neutralizing human monoclonal antibodies against Puumala virus,causative agent of nephropathia epidemica: a novel method using

antigen-coated magnetic beads for specific B cell isolation. J Gen Virol

74, 1303–1310.

Martin, M. L., Lindsey-Regnery, H., Sasso, D. R., McCormick, J. B. &Palmer, E. (1985). Distinction between Bunyaviridae genera bysurface structure and comparison with Hantaan virus using negative

stain electron microscopy. Arch Virol 86, 17–28.

Mertz, G. J., Hjelle, B., Crowley, M., Iwamoto, G., Tomicic, V. & Vial,P. A. (2006). Diagnosis and treatment of new world hantavirus

infections. Curr Opin Infect Dis 19, 437–442.

Metsikko, K. & Garoff, H. (1990). Oligomers of the cytoplasmic

domain of the p62/E2 membrane protein of Semliki Forest virus bind

to the nucleocapsid in vitro. J Virol 64, 4678–4683.

Nakai, K., Okamoto, T., Kimura-Someya, T., Ishii, K., Lim, C. K., Tani,H., Matsuo, E., Abe, T., Mori, Y. & other authors (2006).Oligomerization of hepatitis C virus core protein is crucial forinteraction with the cytoplasmic domain of E1 envelope protein.

J Virol 80, 11265–11273.

Narayanan, K., Maeda, A., Maeda, J. & Makino, S. (2000).Characterization of the coronavirus M protein and nucleocapsid

interaction in infected cells. J Virol 74, 8127–8134.

Nichol, S. T., Beaty, B. J., Elliott, R. M., Goldbach, R., Plyusnin, A.,Schmaljohn, C. S. & Tesh, R. B. (2005). Family Bunyaviridae. In Virus

Taxonomy: Eighth Report of the International Committee on Taxonomyof Viruses, pp. 695–716. Edited by C. M. Fauquet, M. A. Mayo,

J. Maniloff, U. Desselberger & L. A. Ball. San Diego, CA: Elsevier

Academic Press.

Overby, A. K., Pettersson, R. F. & Neve, E. P. (2007a). The

glycoprotein cytoplasmic tail of Uukuniemi virus (Bunyaviridae)

interacts with ribonucleoproteins and is critical for genomepackaging. J Virol 81, 3198–3205.

Overby, A. K., Popov, V. L., Pettersson, R. F. & Neve, E. P. (2007b).The cytoplasmic tails of Uukuniemi virus (Bunyaviridae) GN and GC

glycoproteins are important for intracellular targeting and the

budding of virus-like particles. J Virol 81, 11381–11391.

Pensiero, M. N. & Hay, J. (1992). The Hantaan virus M-segment

glycoproteins G1 and G2 can be expressed independently. J Virol 66,

1907–1914.

Persson, R. & Pettersson, R. F. (1991). Formation and intracellular

transport of a heterodimeric viral spike protein complex. J Cell Biol112, 257–266.

Pini, N. (2004). Hantavirus pulmonary syndrome in Latin America.

Curr Opin Infect Dis 17, 427–431.

Plyusnin, A. & Morzunov, S. P. (2001). Virus evolution and genetic

diversity of hantaviruses and their rodent hosts. Curr Top MicrobiolImmunol 256, 47–75.

Plyusnin, A., Vapalahti, O. & Vaheri, A. (1996). Hantaviruses: genome

structure, expression and evolution. J Gen Virol 77, 2677–2687.

Plyusnin, A., Kukkonen, S. K., Plyusnina, A., Vapalahti, O. & Vaheri,A. (2002). Transfection-mediated generation of functionally compet-ent Tula hantavirus with recombinant S RNA segment. EMBO J 21,

1497–1503.

Razzauti, M., Plyusnina, A., Sironen, T., Henttonen, H. & Plyusnin, A.(2009). Analysis of Puumala hantavirus in a bank vole population in

northern Finland: evidence for co-circulation of two genetic lineages

and frequent reassortment between strains. J Gen Virol 90, 1923–1931.

Ribeiro, D., Borst, J. W., Goldbach, R. & Kormelink, R. (2009).Tomato spotted wilt virus nucleocapsid protein interacts with bothviral glycoproteins Gn and Gc in planta. Virology 383, 121–130.

Rodriguez, L. L., Owens, J. H., Peters, C. J. & Nichol, S. T. (1998).Genetic reassortment among viruses causing hantavirus pulmonarysyndrome. Virology 242, 99–106.

Hantavirus glycoprotein–RNP interaction

http://vir.sgmjournals.org 2349

Downloaded from www.microbiologyresearch.org by

IP: 54.162.133.179

On: Wed, 24 Feb 2016 13:30:39

Schmaljohn, C. & Hjelle, B. (1997). Hantaviruses: a global disease

problem. Emerg Infect Dis 3, 95–104.

Schonrich, G., Rang, A., Lutteke, N., Raftery, M. J., Charbonnel, N. &Ulrich, R. G. (2008). Hantavirus-induced immunity in rodent

reservoirs and humans. Immunol Rev 225, 163–189.

Shi, X. & Elliott, R. M. (2002). Golgi localization of Hantaan virus

glycoproteins requires coexpression of G1 and G2. Virology 300, 31–38.

Snippe, M., Willem Borst, J., Goldbach, R. & Kormelink, R. (2007).Tomato spotted wilt virus Gc and N proteins interact in vivo. Virology

357, 115–123.

Spiropoulou, C. F. (2001). Hantavirus maturation. Curr Top

Microbiol Immunol 256, 33–46.

Sturman, L. S., Holmes, K. V. & Behnke, J. (1980). Isolation of

coronavirus envelope glycoproteins and interaction with the viral

nucleocapsid. J Virol 33, 449–462.

Vaheri, A., Vapalahti, O. & Plyusnin, A. (2008). How to diagnose

hantavirus infections and detect them in rodents and insectivores. Rev

Med Virol 18, 277–288.

Vapalahti, O., Kallio-Kokko, H., Narvanen, A., Julkunen, I., Lundkvist, A.,

Plyusnin, A., Lehvaslaiho, H., Brummer-Korvenkontio, M., Vaheri, A. &Lankinen, H. (1995). Human B-cell epitopes of Puumala virus

nucleocapsid protein, the major antigen in early serological response.

J Med Virol 46, 293–303.

Vapalahti, O., Lundkvist, A., Kallio-Kokko, H., Paukku, K., Julkunen, I.,Lankinen, H. & Vaheri, A. (1996). Antigenic properties and diagnostic

potential of Puumala virus nucleocapsid protein expressed in insectcells. J Clin Microbiol 34, 119–125.

Vapalahti, O., Mustonen, J., Lundkvist, A., Henttonen, H., Plyusnin, A.& Vaheri, A. (2003). Hantavirus infections in Europe. Lancet Infect Dis3, 653–661.

Vaux, D. J., Helenius, A. & Mellman, I. (1988). Spike–nucleocapsidinteraction in Semliki Forest virus reconstructed using networkantibodies. Nature 336, 36–42.

Venien-Bryan, C. & Fuller, S. D. (1994). The organization of the spikecomplex of Semliki Forest virus. J Mol Biol 236, 572–583.

Vogel, R. H., Provencher, S. W., von Bonsdorff, C. H., Adrian, M. &Dubochet, J. (1986). Envelope structure of Semliki Forest virusreconstructed from cryo-electron micrographs. Nature 320, 533–535.

Wang, Y., Boudreaux, D. M., Estrada, D. F., Egan, C. W., St Jeor, S. C.& De Guzman, R. N. (2008). NMR structure of the N-terminal coiledcoil domain of the Andes hantavirus nucleocapsid protein. J BiolChem 283, 28297–28304.

Wang, H., Alminaite, A., Vaheri, A. & Plyusnin, A. (2010). Interactionbetween hantaviral nucleocapsid protein and the cytoplasmic tail ofsurface glycoprotein Gn. Virus Res 151, 205–212.

Weaver, S. C., Frey, T. K., Huang, H. V., Kinney, R. M., Rice, C. M.,Roehrig, J. T., Shope, R. E. & Strauss, E. G. (2005). FamilyTogaviridae. In Virus Taxonomy: Eighth Report of the InternationalCommittee on Taxonomy of Viruses, pp. 999–1008. Edited by C. M.Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. SanDiego, CA: Elsevier Academic Press.

J. Hepojoki and others

2350 Journal of General Virology 91