key: cord-271526-14nfqusv
authors: Molenkamp, Richard; Spaan, Willy J.M.
title: Identification of a Specific Interaction between the Coronavirus Mouse Hepatitis Virus A59 Nucleocapsid Protein and Packaging Signal
date: 1997-12-08
journal: Virology
DOI: 10.1006/viro.1997.8867
sha: 
doc_id: 271526
cord_uid: 14nfqusv

Abstract The coronavirus mouse hepatitis virus (MHV) is an enveloped positive stranded RNA virus. In infected cells MHV produces a 3′ coterminal nested set of subgenomic messenger RNAs. Only the genomic RNA, however, is encapsidated by the nucleocapsid protein and incorporated in infectious MHV virions. It is believed that an RNA packaging signal (Ps), present only in the genomic RNA, is responsible for this selectivity. Earlier studies mapped this signal to a 69-nt stem–loop structure positioned in the 3′ end of ORF1b. The selective encapsidation mechanism probably initiates by specific interaction of the packaging signal with the nucleocapsid protein. In this study we demonstrate thein vitrointeraction of the MHV-A59 nucleocapsid protein with the packaging signal of MHV using gel retardation and UV cross-linking assays. This interaction was observed not only with the nucleocapsid protein from infected cells but also with that from purified virions and from cells expressing a recombinant nucleocapsid protein. The specificity of the interaction was demonstrated by competition experiments with nonlabeled Ps containing RNAs, tRNA, and total cytoplasmic RNA. The results indicated that no virus specific modification of the N-protein or the presence of other viral proteins are required for thisin vitrointeraction. The assays described in this report provide us with a powerful tool for studying encapsidation (initiation) in more detail.

gion a domain (from here on called Ps) of 69 nt could be identified that is probably required for the encapsidation The murine coronavirus mouse hepatitis virus (MHV) of defective genomes (Fosmire et al., 1992) . This signal is an enveloped virus containing a positive stranded RNA is present in genomic RNA, but not in sgRNAs and it is genome of about 31 kb (Holmes, 1991; likely that it has also a similar function in the encapsida-1988). The virion envelope is composed of a lipid bilayer tion of genomic RNA. Recently, it was demonstrated that derived from an internal compartment of the host cell the encapsidation of a heterologous RNA by MHV was and three or four virus-encoded structural membrane fully dependent on the presence of this Ps (Woo et al., proteins (Luytjes, 1995) : the spike protein (S), the mem-1997). Furthermore, it was shown by Bos et al. (1997) that brane protein (M), the small membrane protein (E), and transferring the Ps to a sgRNA resulted in the specific the optional hemagglutinin-esterase protein (HE). The viencapsidation of this sgRNA, though with reduced effiral envelope surrounds a nucleocapsid with helical symciency. metry composed of the genomic RNA and multiple copies

The nucleocapsid protein of MHV is a basic phosphoof the nucleocapsid protein (N). Evidence for the presprotein of 454 amino acids and has an apparent molecuence of a fifth structural envelope protein translated from lar weight of approximately 55 kDa (Armstrong et al. , an internal open reading frame (ORF) within the N gene 1983; Parker and Masters, 1990 ; Laude and Masters, has been published recently (Fischer et al., 1997 (Fischer et al., ). 1995 . It is phosphorylated exclusively on serine residues In infected cells MHV produces a 3 coterminal nested (Stohlman and Lai, 1979) . The N protein contains 41 of set of subgenomic mRNAs (sgRNAs) which possess an these potential phosphorylation sites, but the exact numidentical 5 leader sequence derived from the 5 end of ber and location of phosphoserines have not been identithe genome (Lai et al., 1984; Spaan et al., 1983 Spaan et al., , 1988 . fied yet. The basic amino acids are not clustered in Only genomic length RNA is packaged into virus partistrings, but local densities of positive charge can be cles; however, trace amounts of sg RNAs are sometimes found, particularly in two regions in the middle of the N detected in purified virus (Makino et al., 1988) . Earlier protein (Laude and Masters, 1995) . In contrast, the Cstudies (Fosmire et al., 1992; Most et al., 1991) have terminus is quite acidic. The MHV N protein does not mapped a region in the 3 end of ORF1b that is essential contain known RNA binding motifs, like the arginine-rich for encapsidation of defective genomes. Within this remotif (ARM) or zinc fingers (Draper, 1995; Holmes and Behnke, 1981; Burd and Dreyfuss, 1994) . leader RNA has been reported although there is some analysis. All enzyme incubations and biochemical reactions were performed according to the instructions of the discrepancy about the specificity of this interaction (Stohlman et al., 1988; Bredenbeek, 1990) . A leader-RNA manufacturers. binding domain in the N protein was mapped and com-Construction of plasmids prises the two basic regions mentioned above (Nelson and Stohlman, 1993; Masters, 1992) . Furthermore, spe-(i) pPs290. A 204-nt fragment containing Ps was obcific interaction of the coronavirus infectious bronchitis tained by polymerase chain reaction (PCR) using pMIDIvirus (IBV) nucleocapsid protein with the 3 terminus of C as a template (Most et al., 1991) and oligonucleotide the genome has recently been reported (Zhou et al., primers C060 and C061 (Table 1) . To obtain pPs290 this 1997). fragment was cloned in pCRII using the TA cloning kit The 69-nt Ps is able to form a stable secondary struc-(Invitrogen) according to the instructions of the manufacture and the integrity of this structure is essential for turer. the encapsidation of defective genomes (Fosmire et al., (ii) pEMCV-N. A NcoI restriction site was created at 1992). It has been postulated (Fosmire et al., 1992) that the position of the AUG start codon of the N gene by the Ps functions as an encapsidation initiation site, prob-PCR mutagenesis using oligonucleotide primers C261 ably by interacting specifically with the N protein. Initiaand C224 (Table 1 ). The PCR fragment was digested with tion of encapsidation by packaging signal/(nucleo)capsid

NcoI and ApaI and fused to the remaining 3 sequences protein interactions has been observed for several other of the N gene. A consequence of this procedure was RNA viruses, including alphaviruses, retroviruses, and that the second amino acid of the N protein was changed Escherichia coli bacteriophages. (Owen and Kuhn, 1996;  from a Ser to an Ala. The reconstituted N gene was Berkowitz et al., 1995; Zhang and Barklis, 1995; Schle- then exchanged with the NcoI-BamHI fragment of pL1a singer et Dupraz and Spahr, 1992; Witherell et (Snijder et al., 1996) and the sequence of the NcoI-ApaI al., 1991; Aldovini and Young, 1990; Weis et al., 1989) . fragment was confirmed by sequence analysis. The final However, a specific interaction of the MHV N protein expression vector, pEMCV-N, contained a T7 promoter with the Ps has not been demonstrated yet. and EMCV-NTR, followed by the entire N gene and a T7 In this report we have used gel retardation and UV terminator sequence. cross-linking assays to study the in vitro interaction of MHV-A59 nucleocapsid protein and a small RNA con-Preparation of riboprobe taining the Ps domain. We observed specific interaction In order to serve as template DNA, pPs290 was linearbetween in vitro transcripts containing the Ps and N proized with BamHI, extracted with phenol/chloroform, and tein isolated from infected cells, but also with N protein precipitated with ethanol. Alternatively, templates for the extracted from purified viruses. Furthermore, we were production of Ps180 and PsDHP RNA were produced by able to identify a similar interaction with recombinant PCR using a 5 oligonucleotide containing the T7 pronucleocapsid protein expressed in the vaccinia T7 exmoter sequence in addition to MHV specific sequences pression system. These experiments underline the possi- (Table 1) . Radiolabeled RNA was synthesized by T7 tranbility of studying the encapsidation of MHV-A59 RNA at scription for 1.5 h at 37Њ. Reactions contained 1 mg of a molecular basis and allow us to map important dolinearized plasmid DNA, 1 mM (each) ATP, CTP, and mains in both the nucleocapsid protein as well as the GTP, 50 mM UTP, 10 mCi of [a-32 P]UTP (3000 Ci/mmol), RNA packaging signal. and 25 units of T7 polymerase in a final volume of 25 ml transcription buffer (Gibco BRL). The reaction products MATERIALS AND METHODS were extracted with phenol/chloroform, purified on a Sephadex G50 collumn and precipitated by adding 1/10 Cells and viruses volume of 5 M NH 4 Ac (pH 5.2) and 2 vol of ethanol. The Mouse L cells were grown in Dulbecco's modified Eaamount of incorporated label was determined by TCA gle's medium (DMEM; Gibco) supplemented with 8% fetal precipitation. calf serum. MHV-A59 stocks were grown as described (Spaan et al., 1981) phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol 10 ml binding buffer (5 mM HEPES (pH 7.9), 50 mM KAc, 2.4 mM MgAc 2 , 0.1 mM EDTA, 0.01 mM DTT, 1 mM ATP, (DTT)) (Dignam et al., 1983) . Subsequently, the cells were disrupted by freezing (080Њ) and thawing (37Њ) once or 0.4 mM GTP). Where necessary specific or nonspecific competitor RNA was added as indicated in the figure by 15 strokes of a Dounce homogenizer. Cellular debris and nuclei were removed by centrifugation for 30 min at legends. In supershift experiments, 1 ml of the N-specific monoclonal antibody 5B188.2 (Talbot and Buchmeier, 13,000 rpm at 4Њ. The supernatant was aliquoted and stored at 080Њ. The protein concentration was deter-1985) or b-galactosidase monoclonal antibody (Boehringer Mannheim) and 18 units of RNAGuard (Pharmacia) mined using the Bicinchoninic Acid Protein Assay Kit (Sigma).

were added. The mixtures were incubated at room temperature for 20 min, after which 5 ml of 50% glycerol (ii) Protein lysates from purified viruses. Virus was purified as described before (Luytjes et al., 1997) . Briefly, was added to each reaction. The mixtures were then separated by electrophoresis on a 5% polyacrylamide/ viruses harvested from 8 1 10 7 cells were precipitated with polyethylene glycol and loaded on top of a 20 to 50% 10% glycerol gel (mono:bis Å 37.5:1) in 0.51 TBE (45 mM Tris-Cl (pH 7.5)), 45 mM boric acid, 1 mM EDTA) for 6 linear sucrose gradient. The gradient was centrifuged for 16 h at 16,000 rpm at 4Њ in a SW40Ti rotor. Subsequently h at 5 mA (fixed). Subsequently, the gel was dried and exposed to X-ray film with an intensifying screen at 080Њ. the gradient was fractioned into 16 fractions. All fractions were assayed for viral proteins by Western blot using UV cross-linking the rabbit polyclonal MHV-A59 antiserum k134. The two fractions corresponding to the virus peak and two bottom RNA binding reactions were performed as described fractions (control) were combined, and their volumes above. Subsequently the samples were irradiated at a were adjusted to 5 ml with TESV (20 mM Tris (pH 7.4), 2-cm distance with 1.8 J/cm 2 of 254-nm UV light in a 1 mM EDTA, 100 mM NaCl). Virus was pelleted by centrif-Stratalinker 1800 (Stratagene). The complexes were then ugation in a SW55Ti rotor for 3 h at 35,000 rpm at 4Њ.

incubated with a mixture of 500 ng RNase A (Pharmacia) The resulting virus pellet was lysed in 100 ml buffer C and 2.5 units RNase T1 (Gibco BRL) for 20 min at 37Њ. The supplemented with 0.5% NP-40. complexes were analyzed by sodium dodecyl sulfate-(iii) Recombinant N protein lysates. Vaccinia virus inpolyacrylamide gel electrophoresis on 15% gels. fections and DNA transfections were performed as de-Alternatively, cross-linked and RNase treated samples scribed previously . Briefly, RK13 cells were immunoprecipitated prior to electrophoresis as dewere grown to subconfluency in 5-cm-diameter petri scribed earlier with monoclonal antidishes (4 1 10 6 cells per dish) and infected with the T7 body 5B188.2 (Talbot and Buchmeier, 1985) or rabbit RNA polymerase expressing vaccinia virus recombinant polyclonal MHV-A59 antiserum k134. (vTF7.3) at a m.o.i. of 5. At 1 h postinfection the cells were transfected with 20 ml lipofectin (Gibco BRL) containing 5 RESULTS mg of pEMCV-N. After an incubation of 16 h protein ly-Ps290 RNA binds specifically to proteins from MHVsates were made as described above.

infected and mock-infected cells Gel mobility shift assays

In the studies described here we have analyzed the interaction between the MHV-A59 N protein and RNAs RNA binding reactions contained 10 ng of radiolabeled riboprobe and 4 mg of protein lysate in a final volume of containing the 69-nt Ps signal. In order to identify this 15). These observations indicate that there is a specific interaction between Ps290 RNA and proteins from MHVinfected cells as well as with proteins from mock-infected cells.

The MHV-A59 nucleocapsid protein interacts specifically with Ps290 RNA To investigate whether the N protein is part of the protein-Ps290 RNA complex, a supershift assay using a N-specific antibody was performed. If the N protein is indeed part of the complex, binding to N of a N-specific antibody should result in the formation of a large complex composed of Ps290 RNA, N protein, and N-specific antibody. This complex is expected to migrate slower in the gel as compared to the protein-Ps290 RNA complex and meier, 1985) was used to analyze the protein-Ps290 RNA complex and the monoclonal b-galactosidase-specific antibody was used as a control. When N-specific antiinteraction we have first used gel mobility shift assays body 5B188.2 was added to the complex formed between using protein lysates from both MHV-infected and mock-Ps290 RNA and proteins from the I-lysate, a second cominfected cells and an in vitro transcript containing the Ps plex, migrating slightly slower than the protein-Ps290 (Fig. 1) . From pilot experiments using 10 ng of labeled RNA complex was readily observed (Fig. 3, lane 5) . In RNA we determined that approximately 4 mg of protein contrast, addition of 5B188.2 to the complex formed belysate was required to observe a distinct retarded band tween Ps290 RNA and proteins from the MI-lysate did (data not shown). We therefore used these amounts as not result in a supershift (Fig. 3, lane 13) . This demonstandard in all binding experiments.

strates the presence of the MHV-A59 N protein in the An interaction between Ps290 RNA and proteins from complex formed between Ps290 RNA and proteins from infected cells (I-lysate), as well as from mock-infected the I-lysate. cells (MI-lysate) was readily observed (Fig. 2, lanes 2 The b-galactosidase-specific antibody was not able to and 9). However, the complex formed between Ps290 shift the complex formed between Ps290 RNA and pro-RNA and proteins from the MI-lysate migrated slightly teins from the I-lysate or from the MI-lysate (Fig. 3 , lanes slower in the gel and appeared to be more diffuse, indi-11 and 19). Furthermore, incubation of Ps290 RNA and cating that there are some differences in binding activity antibody 5B188.2 or b-galactosidase-specific antibody between I-and MI-lysates.

without protein lysate, did not result in the formation of In order to determine the specificity of the interaction, competition experiments were performed. Competition of the interaction between Ps290 RNA and proteins from the I-lysate with a 10-fold molar excess of nonlabeled Ps290 RNA resulted already in a decrease of intensity of the retarded band (Fig. 2, lane 5) . At a level of 30fold molar excess of nonlabeled Ps290 RNA the retarded band was no longer visible and all of the labeled RNA migrated at the position of the unbound RNA probe (Fig.  2, lane 7) . Competition with a 100-fold molar excess of yeast tRNA did not affect the intensity or the mobility of the retarded band, indicating that the observed interaction between Ps290 RNA and proteins from the I-lysate is specific for the Ps290 RNA (Fig. 2, lane 8) . Competition of the interaction between Ps290 RNA and proteins from the MI-lysate with non-labeled Ps290 RNA or yeast tRNA beled Ps290 RNA competed entirely for protein binding formed using a 1-to 30-fold molar excess of unlabeled Ps290 RNA or (Fig. 2, lane 14) , whereas a 100-fold molar excess of a 1-to 100-fold molar excess of yeast tRNA. The positions of the unbound RNA (probe) and complexed RNA (complex) are indicated.

yeast tRNA had no effect on the interaction (Fig. 2, lane tor RNA. Cross-linked and RNase treated samples were immunoprecipitated with the monoclonal N-specific antibody 5B188.2 or the rabbit polyclonal MHV-A59 antiserum k134 as indicated in the figure legends.

An interaction between the N protein and Ps180 RNA was readily observed (Fig. 4, lane 1) , but no interaction was observed between the N-protein and PsDHP RNA (Fig. 4, lane 2) . Complexes formed between proteins from the MI-lysate and Ps180 RNA or PsDHP RNA could not be immunoprecipitated with the N-specific antibody (Fig.  4, lanes 3 and 4) . When the Ps180 and PsDHP RNAs were used in a competitive gelretardation experiment with radiolabeled Ps290 and the I-lysate, complete competition was observed with the Ps180 RNA, whereas no immunoprecipitation and that this interaction is dependent on the presence of the 69-nt Ps hairpin.

To determine the specificity of the nucleocapsid pro-RNA-protein complexes (Fig. 3, lanes 8-10 and 12) , tein-Ps180 RNA interaction in UV cross-linking assays, clearly indicating that the observed supershift was not competition experiments were performed. Addition of a the result of an interaction between the RNA probe and 100-fold molar excess of nonlabeled Ps180 RNA resulted the N-specific antibody.

in the complete inhibition of N protein binding (Fig. 5 , Addition of a 100-fold molar excess of nonlabeled lane 2), whereas, a 100-fold molar excess of tRNA had Ps290 competitor RNA resulted in the complete inhibition no effect on the interaction between the N protein and of protein binding to the RNA probe (Fig. 3, lane 6) . In Ps180 RNA (Fig. 5, lane 3) . This clearly demonstrates the contrast, a 100-fold molar excess of tRNA did not affect specificity of the interaction between the N protein and the formation of RNA-protein and RNA-protein-anti-Ps180 RNA. body complexes (Fig. 3, lane 7) , which demonstrates the specificity of the interaction between the N protein and Ps290 RNA.

Nucleocapsid protein from purified viruses and recombinant expressed nucleocapsid protein interact specifically with Ps180 RNA UV cross-linking demonstrates specific nucleocapsid protein Ps180-RNA interaction During RNA encapsidation or particle formation, the N protein might undergo structural changes or other modifi-Another assay for studying the interaction between the cations. It would therefore be interesting to see if the N N protein and Ps containing RNAs is UV cross-linking. protein incorporated in virus particles is still able to inter-To exclude the possibility that the non-MHV-specific sequences present in Ps290 RNA are involved in the interaction between the nucleocapsid-protein and Ps290 RNA, an RNA probe (Ps180) containing only MHV-specific sequences was constructed. Furthermore, in order to investigate whether the 69-nt hairpin of Ps180 (and Ps290) RNA is responsible for specific nucleocapsid protein binding, PsDHP RNA was constructed. This RNA is identical to Ps180 RNA except that the largest part of the 69-nt Ps hairpin has been removed. The U contents of Ps180 and PsDHP are similar (approx. 25%).

Pilot experiments were performed to estimate the optimal UV dose. From these experiments it was determined that an UV dose of 1.8 J/cm 2 gave the most distinct bands with the least background (data not shown). from uninfected cells was added as nonspecific competi-observed using the vTF7-lysate (Fig. 6C, lane 2) . These experiments clearly demonstrate that no virus specific modifications of the N protein or any other viral proteins are required for the specific interaction with Ps180.

Initiation of encapsidation by a specific interaction between the (nucleo-) capsid protein and a RNA packaging signal is a common mechanism used by positive stranded RNA viruses. Such an interaction has already Weis et al., 1989) , retroviruses (Zhang were immunoprecipitated with N-specific monoclonal antibody and Barklis, 1995; Berkowitz et al., 1995; Dupraz and 5B188.2 . Competition was performed with unlabeled Ps180 RNA or yeast tRNA as indicated above the lanes. The position of the N-protein Spahr, 1992; Aldovini and Young, 1990) but also in E. coli specific band is indicated.

bacteriophages like R17 and MS2 (Witherell et al., 1991) . In this report we describe the specific in vitro interaction of the mouse hepatitis virus A59 N protein with tranact specifically with Ps180. In order to address this quesscripts containing the RNA packaging signal of MHV. tion, virus particles were purified on a sucrose gradient.

This interaction was studied by gel retardation and UV Lysates were prepared from the virus peak fractions (pcross-linking assays using an RNA probe containing the lysate) and from the gradient bottom fractions (b-lysate; 69-nt Ps and the N protein from MHV-A59 infected cell negative control). UV cross-linking was performed using lysates. A similar interaction was identified between the both lysates, radiolabeled Ps180 RNA, and competitor N protein from purified virus and recombinant N-protein RNA (5 mg of total cytoplasmic RNA extracted from uninexpressed by the vTF7 expression system. fected cells; Fig. 6B ). A specific interaction was observed In addition to the N-Ps RNA complex we also observed between the N-protein from the p-lysate and Ps180 RNA that cellular proteins bind to Ps containing RNAs. The (Fig. 6B, lanes 2 and 4) . In contrast, no complex could nature of these cellular proteins and the significance of be immunoprecipitated with N-specific antibodies when this interaction, however, remains unclear. the b-lysate was used (Fig. 6B, lanes 1 and 3) . This indicates that any structural change of the N protein which might occur during virus assembly does not affect the ability of the N protein to interact with Ps180 RNA.

In MHV-infected cells, the N protein might also undergo virus-specific or virus-dependent posttranslational modifications. These modifications might play a role in the specific interaction with the Ps. Furthermore, it is not yet known if the interaction between the N protein and the Ps involves other viral proteins as well. In order to investigate this, recombinant N protein was expressed by the vTF7.3 expression system. A N protein expression vector (pEMCV-N) was transfected in vTF7.3-infected RK13 cells and protein lysates were made 16 h posttransfection (vTF7pN lysate). As a control protein lysates from mock-transfected but vTF7.3-infected RK13 cells were made (vTF7-lysate). UV cross-linking experiments, followed by immunoprecipitation with N-specific anti- Fig. 6C ). Using the vTF7pN lysate, a clear band was fraction lysate and p denotes the peak-fraction lysate. Immunoprecipitaobserved migrating at the expected position of the nution with 5B188.2 and k134 were performed as indicated above the lanes. The position of the N-protein-specific band is indicated. cleocapsid protein (Fig. 6C, lane 1) . This band was not

The nature of the nucleocapsid protein-Ps RNA cells (Woo et al., 1997) . Although it is not known whether this encapsidation was efficient, it might be possible that interaction the heterologous flanking sequences have increased the The N-protein of MHV does not posess any well known flexibility of the RNA molecule, allowing it to adapt the RNA binding domains, such as the ARM (Lazinski et al., proper structure for encapsidation. 1989; Talbot and Buchmeier, 1985) or zinc fingers (Draper, 1995) . It interacts, although, specifically with

Significance of the nucleocapsid protein-Ps RNA leader-RNA (Stohlman et al., 1988; Baric et al., 1988) and interaction an RNA binding domain has been identified (Nelson and Stohlman, 1993) . This domain comprises the two major Since MHV virions contain only MHV genomic RNA, it hydrophobic basic regions in the middle of the protein.

is evident that RNA encapsidation is a highly specific Since this central part of the N protein contains a high process. The location of the encapsidation signal in degree of basic amino acids, it is possible that the Ps ORF1b ensures the specific encapsidation of only genorecognition domain might also be positioned somewhere mic RNA. It is unclear if the MHV Ps is by itself sufficient within this part of the N protein.

for efficient encapsidation. It is known that Rous sarcoma In infected cells the N protein of MHV is very rapidly virus has multiple encapsidation sites, which are rephosphorylated on serine residues (Stohlman and Lai, quired to interact for efficient packaging of the genome 1979) and part of it concomitantly becomes associated (Sorge et al., 1983; Pugatsch and Stacey, 1983) . Recently, with a cell membrane fraction (Stohlman et al., 1983;  it was shown that a subgenomic RNA of MHV containing Anderson and Wong, 1993) . It is still unknown if phosthe Ps can be encapsidated specifically but the efficiency phorylation is carried out by a host cell or viral encoded was much lower as compared to the encapsidation effiprotein kinase and its (biological) role remains unclear.

ciency of a defective interfering RNA (Bos et al., 1997) . It has been suggested that it might govern the tightness Preassembled nucleocapsids have never been obof the interaction between N and RNA (Laude and Masserved in coronavirus infected cells, but an electronters, 1995). It will be interesting to compare the phosphordense structure, which may correspond to the nucleoylation of the N protein from infected cells and the recomcapsid, can be found at the cytoplasmic face of the binant N protein and to investigate the possible role of budding site (Dubois-Dalcq et al., 1982; Holmes, 1991) . phosphorylation in RNA binding. Phosphorylation and Nucleocapsid incorporation in budding virions is exlikewise dephosphorylation could also induce a major pected to be mediated by M-N protein interactions. Asconformational change of the N protein. It has been sugsociation of the M protein to the nucleocapsid in NP-40gested that dephosphorylation of the nucleocapsid after disrupted virions has been reported (Sturman et al., virus entry is involved in uncoating of the viral RNA (Ma-1980) ; however, the same study demonstrated that the hondas and Dales, 1991) .

M protein was able to bind RNA in the absence of N. It In general the recognition of an RNA signal by a protein is still unclear if the interaction between M and N is a involves secondary structure elements in addition to the prerequisite for RNA encapsidation in vivo. This interacprimary sequence of the RNA signal (Draper, 1995) . tion could position the N protein in a favorable way to There is accumulating evidence for an induced-fit mechinteract with the genomic RNA. This must then be folanism in RNA-protein interactions affecting both the lowed by interaction with additional N proteins, forming RNA and the protein (Beck and Nassal, 1997 ; Allain et the helical nucleocapsid. Involvement of other viral proal., 1996) . Sufficient flexibility in the RNA molecule could teins in Ps RNA binding, however, was not observed in be a major determinant in protein binding. From that our in vitro studies. Recombinant nucleocapsid protein, perspective, the recognition of the Ps by the N protein expressed in the vTF7 expression system, interacts also could be highly dependent on the secondary structure specifically with the Ps RNA (Fig. 6C) , although the effiof the Ps domain. When a short RNA probe consisting ciency of this interaction was not determined. A striking of only the 69-nt Ps was used in our studies, no gel feature of coronaviruses is that the nucleocapsid has a retardation or UV cross-linking to the N protein was obhelical symmetry (MacNaughton et al., 1978;  Holmes and served (unpublished observations). Computer prediction Behnke, 1981) , in contrast to the nucleocapsids of all of the secondary structure of this small 69-nt RNA moleother positive stranded RNA viruses, which are icosaecule showed that the secondary structure was entirely dral or spherical (Murphy et al., 1995) . However, electron different from that of the Ps in the ORF1b context. This microscopy studies on the transmissible gastroenteritis suggests that the flanking ORF1b sequences are necescoronavirus and MHV (Risco et al., 1996) have revealed sary to force the Ps in its specific structure or that the recently a spherical core structure inside the virion (intersmall RNA probe lacks the flexibility to form the specific nal core) and this structure reacted with M-and N-spestructure. Recently, it has been shown that the 69-nt Ps, cific antibodies. A structural model for coronaviruses was flanked by non-MHV sequences could confer specific proposed, in which a spherical core, composed of a combination of N and M proteins, was present in addition to encapsidation to a heterologous RNA in MHV infected INTERACTION BETWEEN NUCLEOCAPSID PROTEIN AND PACKAGING SIGNAL of a murine coronavirus in the absence of helper virus. Virology 218, a helical nucleocapsid. It will be of interest to study the 52-60. possible relationship between the morphology of the nu-Bos, E. C. W., Dobbe, J., Luytjes, W., and Spaan, W. J. M. (1997) . A subcleocapsid or internal core and the mechanism of encapgenomic mRNA transcript of the coronavirus mouse hepatitis virus sidation initiation. strain A59 defective interfering (DI) RNA is packaged when it contains It has been shown that, as opposed to alphavirus asthe DI packaging signal. J. Virol. 71, 5684-5687. Bredenbeek, P. J. (1990) . ''Nucleic Acid Domains and Proteins Involved sembly, nucleocapsid formation and RNA encapsidation in the Replication of Coronaviruses.'' Ph.D. Thesis, University of are not strictly required for virion formation (Vennema et Utrecht, Utrecht, The Netherlands. al., 1996; Strauss and Strauss, 1994) . Burd, C. G., and Dreyfuss, G. (1994) . Conserved structures and diversity Coexpression of M and E protein was sufficient for partiof functions of RNA-binding proteins. Science 265, 615-621. cle formation. However, incorporation of nucleocapsids Dignam, J. D., Lebowitz, R. M., and Roeder, R. G. (1983) . Acurate transcription initiation by RNA polymerase II in a soluble extract from during the budding process could greatly increase the isolated mammalian nuclei. Nucleic Acids Res. 11, 1475 Res. 11, -1489 efficiency of virion formation. Draper, D. E. (1995) . Protein-RNA recognition. Annu. Rev. Biochem. 64,

The study of the encapsidation of coronaviruses is 593-620. greatly hampered by the absence of an infectious clone.

Dubois-Dalcq, M. E., Doller, E. W., Haspel, M. V., and Holmes, K. V.

The in vitro binding assay, described in this report, can (1982) . Cell tropism and expression of mouse hepatitis viruses (MHV) greatly enhance our understanding of the encapsidation in mouse spinal cord cultures. Virology 119, 317-331. Dupraz, P., and Spahr, P. F. (1992) . Specificity of Rous sarcoma virus mechanism. We have shown that recombinant nucleonucleocapsid protein in genomic RNA packaging. J. Virol. 66, capsid protein expressed in the vTF7 expression system 4670.

is able to interact with the Ps in a similar fashion as the Fischer, F., Peng, D., Hingley, S. T., Weiss, S. R., and Masters, P. S. nucleocapsid protein produced in infected cells. Muta- (1997) . The internal open reading frame within the nucleocapsid gene tional analysis of both the nucleocapsid protein and the of mouse hepatitis virus encodes a structural protein that is not essential for viral replication. J. Virol. 71, 996-1003. Ps should provide us with information that allows us to Fosmire, J. A., Hwang, K., and Makino, S. (1992) . Identification and understand the encapsidation mechanism in more detail. characterization of a coronavirus packaging signal. J. Virol. 66, 3522-3530. Holmes, K. V. (1991) . In ''Fundamental Virology'' (B. N. Fields and D. M.

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