key: cord-0697042-4xc8sxcy
authors: Kalicharran, Kishna; Mohandas, Devaki; Wilson, Greame; Dales, Samuel
title: Regulation of the Initiation of Coronavirus JHM Infection in Primary Oligodendrocytes and L-2 Fibroblasts
date: 1996-11-01
journal: Virology
DOI: 10.1006/viro.1996.0572
sha: bfc558a4241a48e116131d26ece914f715b53d10
doc_id: 697042
cord_uid: 4xc8sxcy

Abstract Upon maturation, primary rat oligodendrocytes become resistant to coronavirus JHM (JHMV) infection at an early stage. Involvement of cAMP-dependent protein kinase (PK) in the regulation of oligodendrocyte differentiation has been established (S. Beushausenet al.(1987).J. Virol.61, 3795–3803). An inducer which accelerates maturation, dibutyryl cyclic AMP (dbcAMP) also upregulates the expression of the regulatory subunit, R1 of PK1. Since (i) early block preventing infection of mature oligodendrocytes can be bypassed when transfection with genomic RNA is used and (ii) inhibitors of PKs counteract the dbcAMP effect, so as to alleviate the inhibition of JHMV, enhanced expression of R1 appeared to be connected with virus restriction. This idea was confirmed following upregulation of the R1 gene in fully permissive L-2 cells. There was a connection between an effect due to R1 and dephosphorylation of the nucleocapsid protein N by an endosomal phosphoprotein phosphatase (PPPase) having the properties of types 1 or 2A enzyme which occurs during penetration of inoculum virions. An inhibitionin vitro(cell free) of N dephosphorylation by R1 together with evidence thatin vivo(cell culture) overexpression of R1 inhibited the endosomal PPPase as well as replication of JHMV supports the hypothesis that uncoating of the JHMV inoculum occurs after dephosphorylation, a step obligatory for dissociation of the N protein from the genome. Thus inhibition by R prevents uncoating and thereby interferes with the commencement of replication. These observations intimate the existence of a novel mechanism controlling a virus infection of specific cell target(s) undergoing a process of differentiation and maturation in the central nervous system.

1982; Robbins and Rapp, 1980) , but neural cell infections with rubella virus (Van Alstyne and Paty, 1983 ) and poly-The appearance and type of neurological disease apoma virus (Baru and Manor, 1988) are enhanced. In the parent in the central nervous system of rats inoculated above systems the cAMP effect has not been explained, with the neurotropic coronavirus JHM (JHMV) is related although previous evidence (Beushausen et al., 1987) to the postpartum age at challenge. Onset of resistance has suggested that the regulatory (R) subunit of cAMPto a demyelinating form of white matter disease occurs dependent protein kinase (PK) may somehow be inaround the time that CNS myelination is at the stage of volved. completion and may be due to the paucity of undifferenti-With respect to gene expression as it may be conated progenitor cells of the oligodendrocytic lineage (Sornected to differentiation, elevation of intracellular cAMP ensen et al., 1980) . This presumption is in line with data is believed to increase amounts of the cAMP-dependent from in vitro studies revealing that JHMV replication is PKs of type 1 (PK1) and 2 (PK2) (Beushausen et al., 1985 ; arrested at an early stage when explanted oligodendro- Flockhart and Corbin, 1982; Scott, 1991 ; Rubin and Rocytes are allowed to mature slowly or are driven into sen, 1975) . However, when Wistar Furth (WF) primary rapid differentiation by modulation of the adenylate cyoligodendrocytes, from which the PK1 isoform is absent, clase metabolic pathway (Beushausen and Dales, 1985;  are induced to differentiate by application of dbcAMP, Beushausen et al., 1987; Wilson et al., 1986) . It is known R1 is massively induced without increasing PK2 activity that infection of neural cells by other agents can also be (Beushausen et al., 1987) . While the assumed function influenced, either positively or negatively, by elevation of of R as a component of the holoenzyme is to form a intracellular cAMP. In the case of measles, infection of reversible complex with the catalytic (C) subunit, thereby neuroblastoma cells is suppressed (Miller and Carrigan, controlling phosphorylating activity, the R2 of PK2 was shown to exert a second property, as an inhibitor of some phosphoprotein phosphatase(s) (PPPase) ( al., 1985; Khatra et al., 1985; Shenolikar, 1994 period they are in a highly proliferative O-2A progenitor cubation for 10 hr at 37Њ, the supernatant containing progeny virus was titrated on L-2 cells. phase and the time they become fully mature oligodendrocytes (Pasick and Dales, 1991) . The block in virus

The effect of calyculin A on the processing of N within the virus inoculum was determined using confluent L-2 replication found with oligodendrocytes at an early stage in the viral cycle, before onset of transcription but after cultures pretreated 30 min prior to JHMV absorption at 4Њ for 60 min using various concentrations of the drug. adsorption, does not occur in fully permissive L-2 fibroblasts, which served as the controls throughout the pres-After 1 hr incubation at 37Њ, following absorption, the cytoplasmic fraction was processed for immunoblotting ent investigation. Previous studies revealed that during virus assembly the nascent N protein of JHMV becomes with anti-N antibodies. phosphorylated on serine residues, which is the only Preparation of purified N protein posttranslational modification to occur (Cheley and Anderson, 1981; Stohlman et al., 1983; Wilbur et al., 1986) .

Isolation of unlabeled and 32 P-labeled N protein from Conversely, during virus disassembly and uncoating, N infected L-2 mouse fibroblasts conformed to procedures undergoes a molecular weight reduction from 56 to 50 previously described by Mohandas and Dales (1991) . kDa, perhaps due to specific dephosphorylation of N by an endosome-associated PPPase (Mohandas and Dales, Photoaffinity labeling of R subunit in cell extracts 1991), evidently an essential step for initiation of the After treatment with H-8 and/or dbcAMP, individual JHMV replication cycle. This study was concerned with cultures of 2 1 10 6 oligodendrocytes per 35-mm plate regulation over virus infections by host cells from the were rinsed twice with ice-cold PBS, the cells were recentral nervous system (CNS), exemplified by interacleased by scraping and collected into pellets, then resustions between JHMV and differentiating oligodendropended at 0Њ in 500 ml buffer containing 10 mM Triscytes.

HCl at pH 7.4, 1 mM ethylene glycol-bis-(a-aminoethyl ether)N,N,N,N-tetra acetic acid (EGTA), and 50 mg MATERIALS AND METHODS PMSF/ml. Thorough cell disruption was effected by squeezing the cell suspension repeatedly (110) through Culture of L cells, preparations of virus, and assays a No. 30 gauge needle of a 1-ml hypodermic syringe. The Details concerning L-2 cell cultures, preparation of homogenates obtained were frozen and thawed twice, JHMV stocks, methods of virus infection, and plaque sedimented at 15,000g for 10 min using an Eppendorf assays were as previously described (Beushausen and microfuge at 4Њ. The cytosol fraction was affinity-labeled .

with 8-azido-[ 32 P]cAMP (61.4 Ci/nmol; ICN Pharmaceutical Inc., Irvine, CA) as described (Budzilowicz et al., Treatment of cells with protein kinase and 1985) . Autoradiograms were prepared after separating phosphatase inhibitors the proteins in 10% SDS-PAGE gels (Laemmli et al., 1970) . Primary WF cultures enriched over 90% with respect to oligodendrocytes were established from cerebral corti-Preparation of catalytic and regulatory subunits from ces of neonatal rats as in Beushausen and Dales (1985) .

the holoenzyme and PK1 assay Multiwell dishes containing 5 1 10 5 cells per cm 2 were maintained for 3 days at 37Њ in modified basal Eagle's Affinity columns were employed to separate the R and C subunits, as previously outlined (Beavo et al., 1974) . medium plus 10% fetal calf serum (BME10) or in BME10 supplemented with dibutyryl cAMP (dbcAMP) (Sigma)

Briefly, dehydrated preparations of rabbit PK1 (Sigma) were reconstituted in distilled water to give a final con-and/or with the protein kinase inhibitors H-8 and HA1004 (Seikagaku Kogyo Co.) (Hidaka et al., 1984) . For the sub-centration of 5 mg/ml. The material was introduced into columns of 5 ml packed volume containing N6-2C-cAMP sequent inoculation, JHMV was used at a multiplicity of infection (m.o.i.) of 1 plaque forming unit (PFU) per cell.

sepharose preequilibrated with buffer containing 5 mM 2-[N-Morpholino] ethane sulfonic acid (MES), 9 mM NaCl, Virus was adsorbed at 37Њ for 1 hr, cells washed 31 with 1 ml phosphate-buffered saline (PBS), pH 7.2, and 15 mM B-ME, pH 6.5, termed MES buffer. The columns were then washed at 20Њ with 2 M NaCl in MES buffer, incubated at 37Њ in BME10, with supplements where appropriate. Extracellular JHMV was quantitated in the me-followed by washes with MES buffer alone. The C subunit was eluted in the void volume and concentrated on Ami-dium, which was changed daily, as previously described (Lucas et al., 1977) .

con YM-10 membranes as outlined below. To elute R1 from the columns, 3 vol of 30 mM cAMP in MES buffer Confluent L-2 cells in 35-mm dishes were either treated for 30 min before JHMV inoculation at 4Њ for 60 were added during incubation for 1 hr at 30Њ. The eluate, containing R1, was concentrated by Amicon YM-10 mem-min or 2 hr after inoculation with various concentrations of calyculin A (Research Biochemical Inc., MA). After in-brane and dialyzed at 4Њ for 2 days with 4 1 1 liter changes of MES buffer. Protein determinations were per-through a syringe needle only 5 rather than 10 cycles and sedimented for only 5 min to remove the larger par-formed using a Bio-Rad kit with BSA as standard. Another preparation of R1, employed as a control, was obtained ticulate. Membranes in the cytosol fraction were disrupted by means of NP-40 added to a final concentration from skeletal muscle of Wistar rats (a gift from B. D. Sanwal, Department of Biochemistry, UWO). of 0.5%, and brief agitation on a vortex mixer followed by 15 min at 4Њ on a rotary mixer. Proteins in the fractions Samples of isolated C and R subunits were analyzed for presence of residual PK activity, using histone IIA were separated by SDS-PAGE then transferred to nitrocellulose filters for Western blotting (Towbin et al., 1979) ; (Sigma) as substrate by the procedure of Corbin et al. (1975) ; briefly, 84 mM KH 2 PO 4 , pH 6.8, 28 mM Mg acetate, N antigen was identified with monoclonal (MAb) as in Beushausen et al. (1987) . 1.75 mM ATP, histone IIA at 50 mg/ml, 10 mM cAMP, and H 2 O was mixed with an equal volume of 50 mCi [ 32 P]ATP (Amersham; sp act 75,000 Ci/mmol). Then, 50-ml aliquots Specificity of binding between N protein and cDNA of the above mixture were added to approximately 3 mg PK protein in glass tubes, incubated for 15 min at 30Њ, Specific binding of N with a cDNA copy of the gene was tested using the overlay method of Robbins et al. then ''spotted'' onto GF/C glass fiber filters (Millipore). The reaction was terminated by immersing the filters in (1986) . After immobilizing N in nitrocellulose, the blots were rinsed for 30 min with standard binding buffer (SBB) 10% ice-cold trichloroacetic acid (TCA) and extraction achieved by agitation for 1 hr on a shaker apparatus.

consisting of 0.05 M NaCl, 1 mM disodium EDTA, 10 mM Tris-HCl at pH 7, 0.02% BSA, 0.02% Ficoll, and 0.02% Following three washes with TCA the filters were rinsed twice with 100% ethanol and dried at 020Њ. The [ 32 P]dpm polyvinyl pyrolidone (PVP-360), incubated for 90 min at room temperature with 32 P-labeled plasmid cDNA probe were determined in a scintillation counter.

plus calf thymus DNA, washed 61 or more with SSB over Isolation of the endosomal fraction and PPPase assay a 60-min period, then air-dried. Autoradiograms were obtained by exposing X-ray film in the presence of an inten-The endosomal/prelysosomal fractions were isolated sifying screen. The cDNA was constructed from plasmid from disrupted L-2 cells by use of a dual percoll gradient G344 recombined with 1800-bp cDNA from the N gene procedure and characterized with respect to the enzyme of CV A59 (Budzilowicz et al., 1985) , kindly provided by activities as previously described (Mohandas and Dales, S. Weiss (University of Pennsylvania, Philadelphia, PA). 1991). Enzyme assays included those for acid phospha-Labeling of plasmid DNA with [ 32 P] as described pretase and a ''neutral'' PPPase with specificity for N as a viously (Feinberg and Vogelstein, 1983 ) and purification substrate. Where appropriate the additions were 1 mM by means of a spin column according to Maniatis et al., 35 cAMP and 3 mg of either PK holoenzyme or regula- (1982) . tory (R) or catalytic (C) subunits. The mixtures were preincubated for 30 min at 30Њ. The 32 P-labeled or unlabeled

Transfections with RNA of JHMV N was then added and the reaction continued at 30Њ for a further 90 min. The reactions were terminated by addi-

The genomic RNA was derived from JHMV propagated tion of 1% BSA and 25% TCA. The released [ 32 P]dpm on confluent L-2 monolayers in 150-cm 2 flasks. After inocwere determined as described previously (Mohandas ulation using an m.o.i. of 1 for 60 min at Ç20Њ, growth and Dales, 1991). medium was added and incubation carried out at 37Њ until the monolayer became about 95% virus-induced Immunoblotting to demonstrate modulation syncytium. Virus released into the supernatant was colof the N antigen lected by centrifugation and used for preparing genomic RNA as described previously (Banner et al., 1990) . For L-2 monolayers were inoculated with JHMV at an m.o.i. of about 50 PFU/cell. After adsorption at 4Њ for 60 min transfection, 5 mg of RNA was combined with 15 ml of Lipofectin (Life Technologies) and 200 ml Opti MEM (Life unattached virus was removed by thorough washing with cold BME10. Penetration was initiated by incubation in Technologies). After gently mixing the reagents, the RNA was added to purified primary oligodendrocytes, pre-fresh BME10 at 37Њ. Cultures were sampled at intervals for 120 min, commencing with a sample taken at the end pared as described by Pasick and Dales (1991) and seeded at from 2.5 to 5.0 1 10 5 cells per cm 2 . Experimen-of adsorption, designated tO. At the time of sampling, monolayers were washed twice with cold PBS, the cells tal cultures were treated with 3 mM dbcAMP for 24 hr.

As an additional control, L-2 cultures were transfected were scraped off and placed into 500 ml of ice cold buffer, containing 10 mM Tris-HCl at pH 7.4, 1 mM EGTA, 50

in the same manner. The cells were exposed for 12 hr to the RNA transfection mixture and then growth medium mg/ml PMSF, and 2 mg/ml leupeptin (TPEL). A cytosol fraction was derived from infected cells as above under was added and supernatants fluid monitored at 48 and 72 hr for the virus progeny released. ''Photoaffinity labeling'' except that cells were squeezed 

previous results demonstrating that the restriction occurs at an early step, prior to onset of transcription, isolated Multiple copies of the DNA of the rat R1 gene, which JHMV genomic RNA was transfected into primary telenhad been cloned into the vector HL-REV (Corell et al., cephalic cultures established as previously described 1989), were used for transfecting semiconfluent cultures (Pasick and Dales, 1991) from neonatal Wistar Furth of L-2 cells, as described above. After incubation with pups. After 8-10 days in culture, the more loosely adherthe plasmid for 72 hr, the cells were released and subculent O-2A lineage cells, which can be released by sharply tured with growth medium containing 500 mg G418 (Life tapping the culture flask, were plated into fresh growth Technologies). Stable individual transfectants (clones) medium and then treated with 1 mM dbcAMP either 24 were selected, grown into cultures for assaying the quanhr before inoculation or 2 days following inoculation with tity of R1 expressed by each clone, employing anti-R1 JHMV. As shown in Table 1 , pretreatment of cultures to antibodies for immunoblotting. As a control the wild-type induce oligodendrocyte maturation completely blocked cells were transfected with the plasmid from which the replication, whereas posttreatment had no effect as com-R1 gene was absent. The high producer clones 1.2 and pared with untreated cells, confirming our previous data 1.1 were amplified and challenged with JHMV to deter- (Beushausen et al., 1987) . To circumvent events related mine their infectability. Efficiency of virus replication was to penetration and uncoating connected with inoculum determined by the PFU present in the supernatant at 10 virions, virus genomes were introduced by transfection. hr PI. Changes in MW of N during the early stages of The genomic RNA was isolated, combined with Lipofecvirus-cell interactions were monitored by precooling (4Њ) tin (Gibco), and added to mature oligodendrocyte culof host cells and then inoculating them for 60 min at 4Њ tures made nonpermissive by pretreatment with dbcAMP. at an m.o.i. of 10 PFU/cell. The unattached inoculum was Such transfected cells were infectable and were able to washed away, warm nutrient medium added, then incureplicate JHMV as compared with controls inoculated bation carried out for 1 hr. Samples were taken and prowith virions (Table 1) , demonstrating as previously sugcessed for immunoblotting.

gested (Beushausen et al., 1987) that restriction does, indeed, pertain to an early event. Inevitably, the efficiency RESULTS of infecting with isolated RNA was much lower than with virions, less than 1%, as evident with both L-2 cells and Do glial cells exert control over the virus infection at primary oligodendrocytes (Table 1) . The relatively small an early stage? but consistent disparity between yields from untreated JHMV has a tropism for explanted rat oligodendrooligodendrocytes and those treated postinoculation with cytes, although infectability is confined to a discrete dbcAMP, reported previously (Pasick and Dales, 1991), stage in the differentiation process, sometime between remains unexplained. the mitotically active progenitors (O-2A cells) and termi-Effect of PK inhibitors on JHMV replication nally differentiated oligodendrocytes (Pasick and Dales, in oligodendrocytes treated with inducers 1991). Permissiveness is apparently determined by intraof differentiation cellular factor(s) acting after adsorption, which itself is not affected when the nonpermissive cells are chal-

The previously made connection relating modulation of the cAMP dependent-PK(s) caused by dbcAMP with lenged (Beushausen et al., 1987) . To elaborate on our oligodendrocyte differentiation and inhibition of coronavirus expression (Beushausen and Dales, 1985; Beushausen et al., 1987; Wilson et al., 1986) led us to question whether PK inhibitors can counteract the dbcAMP effect and alleviate the restriction on virus replication. The answer was sought by pretreating WF oligodendrocytes with two isoquinoline sulfonamide derivatives, H-8 and HA1004 (Hidaka et al., 1984) , which are able to inactivate cAMP-dependent PKs by binding to the C subunits.

As a confirmation of our previous findings and a control for the inhibition of infection due to differentiation, cultures of oligodendrocytes were pretreated with 1 mM dbcAMP before inoculation. It is evident in Fig. 1A that due to pretreatment, JHMV titer was reduced by 80% within 24 hr and over 90% in 48 hr. Application of 3 mM the untreated controls (Fig. 1A) . After similar treatment with 3 mM dbcAMP the addition of 25 mM H-8 was able to relieve the inhibition only partially, as shown in Fig.  1B . The other inhibitor of PK, HA1004, when added at 50 mM together with 1 mM dbcAMP reversed the dbcAMP effect during the initial 24 hr of the experiment (Fig. 1A) . However, simultaneous treatment with 3 mM dbcAMP and HA1004 affected the inhibition of JHMV replication very little (Fig. 1B) .

To ascertain independently whether reversal of the inhibitory effect due to dbcAMP on JHMV replication by H-8 involves the PK's, oligodendrocytes were treated with either the inducer or the inhibitor or with both compounds. It is evident from lanes 2 and 4 of the autoradiogram in Fig. 2 that, as predicted from Beushausen et al. (1987) , dbcAMP induced the synthesis of large amounts of R1. Treatment with H-8 alone did not appreciably decrease the quantity of R1 present in controls (lane 2 vs 3). When both H-8 and dbcAMP were added R1 induction was abrogated (lane 5), demonstrating that in oligodendrocytes presence of low amounts of R1 appears to be The above results led us to extend our enquiry into changes in phosphorylation of N during virus penetration, specifically whether dephosphorylation is required for initiating JHMV replication. For this purpose, we employed calyculin A, a reversible inhibitor of PPPases (Ishihara et al., 1989) . L-2 cells were treated with the drug for 30 min either before inoculation or 2 hr after initiating penetration. As a measure of JHMV replication, titers of virus released by 10 hr postinfection into the medium were determined. As evident from Fig. 4 , when 100 nM calyculin A was applied in advance of inoculation, the JHMV titer was reduced about 1.5 log 10 PFU/ml as compared with virus generated by cells treated with calyculin A commencing 2 hr postinfection. These data are consistent with the idea that dephosphorylation by a PPPase activity may be re- of N, L-2 cells were treated with various concentrations of the inhibitor prior to inoculation as described for Fig. 4 . Samples taken 1 hr postinoculation were subjected to Evidence that MW modulation of N is related to the immunoblotting employing anti-N antibodies. As evident state of phosphorylation from Fig. 5 , conversion of N from the 56-to 50-kDa MW form was reduced by calyculin A in a concentration-de-A relationship between the 56-kDa N present within virions and a related 50-kDa protein was previously es-pendent manner, as was to be anticipated if dephosphorylation of N was being affected. From these combined re-tablished using 2-D tryptic peptide mapping (Cheley and Anderson, 1981) and anti-N monoclonal antibodies (MAb) sults, it was concluded that dephosphorylation of N is an early event during virus-cell interactions which may be (Coulter-Mackie et al., 1985) . Upon its synthesis, N becomes phosphorylated at serine residues (Stohlman et essential for commencement of replication.

To connect more directly dephosphorylation of the 56-al., 1983; Wilbur et al., 1986) . To determine whether MW modulation of N following its synthesis is due to phos-kDa form of N with an endosomal PPPase activity, in vitro assays were conducted. As shown in Fig. 6 , after phorylation, the nutrient medium of infected cultures was varied in the phosphate concentration. The 50-kDa species of N was reduced in proportion to increasing phosphate concentration, being absent when phosphate was at 5 mM (data not shown), demonstrating a regulation of the relative amounts of 50-and 56-kDa N protein in infected cells by phosphate concentration in the growth medium. The 56-kDa MW of N is the predominant form within mature JHMV particles.

From previous evidence suggesting that N is modified during JHMV penetration, MW of the inoculum N protein was ascertained by immunoblotting at intervals for the first 120 min. The results shown in Fig. 3 indicated that between t0 and 30 min after warming to 37Њ, N existed predominantly as the 56-kDa species. At 45 min, there was some processing of the 56-kDa antigen evident as several lower MW bands, including one at 50 kDa and some below 40 kDa. At 60 min, the 56-kDa material was completely absent and only a faint 50-kDa band remained while the remainder of N was at lower MW. By 120 min, the N antigen had disappeared altogether. increased according to the quantity of endosomes used. When 32 P-labeled N was reacted with endosomes in the same manner, about one-half of the protein bound [ 32 P] culin A, acting as a specific inhibitor of neutral PPPases, was solubilized (Mohandas and Dales, 1991) . Evidently, depressed the conversion of 56 to 50 kDa (lane 5 in Figs. conditions used for the in vitro assays were appropriate 7A and 7B). Since the MW shift was unaffected in the for demonstrating that dephosphorylation of N brings presence of protease inhibitors in the reaction mixture, about a shift down of the MW. the observed change in MW of N must have been due As a confirmatory approach for detecting the MW modto dephosphorylation, not proteolysis. From the above ulation of N due to the endosomal PPPase activity, we observations, it was concluded that, during the interaccorrelated the binding of a radioactive cDNA probe with tion of JHMV with host cells, the dephosphorylation of N the N antigen, bound to nitrocellulose as detected by by an endosomal PPPase most probably causes the 56immunoblotting. Samples were monitored following incuto 50-kDa MW conversion, as a step in the uncoating of bation with the endosomal fraction, as described above.

inoculum genomes. The results presented in Fig. 7A indicated that the cDNA copied from N RNA could recognize both the 56-and 50-Inhibition of endosomal PPPase activity by the R kDa forms of N in an nondiscriminating manner. Howsubunit of cAMP-dependent PKs ever, a specific binding of the 56-kDa form of N to viral

We obtained evidence in this study that N is dephos-RNA has been noted ( Baric et al., 1988) . The specificity phorylated during JHMV penetration into L-2 cells. Both of the endosomal PPPase activity for N as a substrate previously and in the present study, it was demonstrated was checked by means of okadaic acid which, like calythat an endosome-associated PPPase of L-2 and glial cells can specifically dephosphorylate N in vitro (Mohandas and Dales, 1991), indicating that this enzyme activity has a function during the interaction between JHMV and explanted oligodendrocytes. Since inducers of differentiation, among them cAMP, can regulate PK expression and suppress JHMV replication in oligodendrocytes at an early stage (Beushausen and Dales, 1985; Beushausen et al., 1987) , it was hypothesized that PK, or one of the holoenzyme components, can influence the endosomal PPPase. This idea is consistent with the wellrecognized interplay between PKs and PPPases in metabolic pathways involving these enzymes (for reviews, see inhibition of N dephosphorylation (data not shown). (4) was inhibited at an early stage of replication in the transfectant clones, with a greater restriction evident in Effects of adding PK1, PK2, R1, or C subunits to the the higher R1 expressing clone 1.2 (Fig. 9C ). These findreaction mixture containing endosomal PPPase and 32 Pings are entirely consistent with above data from celllabeled N protein are summarized in Table 2 The multifactorial control over replication of coronavimum [ 32 P] released from N was not above 50%, but usuruses in the CNS has been of continuing interest in this ally 30-40% of the [ 32 P] residues were solubilized. It is laboratory. One of the most challenging aspects in virusevident that presence of either holoenzyme PK1 or PK2 cell interactions concerns the influence of differentiation had no inhibitory effect on the dephosphorylation reacon the infectability of oligodendrocytes by JHMV. When tion. Inclusion of cAMP along with the holoenzymes, this cell type is driven toward differentiation by modulatwhich brings about PK dissociation into R and C subunits, ing the adenylate cyclase pathway, resistance becomes resulted in a minor inhibition of [ 32 P] release. However, manifested at a stage subsequent to adsorption and inaddition of the purified R1 subunit, whether in the abternalization but antecedent to expression of the viral sence or presence of cAMP, reduced PPPase more progenome. Since the restriction in mature oligodendrocytes foundly, but addition of the C subunit had no effect. These of JHMV could be circumvented by transfecting into them results imply that the R subunit can inhibit directly the isolated genomic RNA, the arrest involves an early step endosomal PPPase. A graphic confirmation of the R1 of the replication cycle. Such resistance to infection apeffect was obtained by subjecting to autoradiography isopears to be connected with regulation of the cAMP-detopically labeled N following SDS-PAGE in 10% gels. It pendent PK, as evident following dbcAMP treatment is evident from Fig. 8 that N dephosphorylation, demonwhich induces abundant synthesis of R1, despite abstrated in lane 2 was inhibited by the presence of R1, as sence of PK1 from these cells (Beushausen et al., 1987) . evident from lane 3.

Involvement of PKs was extended by our current data The species specificity of R1 was tested by comparing R1 from rabbit skeletal muscle with R1 isolated from skeletal muscle of Wistar Furth rats. At the standard concentration of 3 mg per reaction mixture, the rabbit and rat subunits inhibited endosomal PPPase to the same extent (data not shown).

The consequence of overexpressing R1 on the MW of PPPase activity in vitro and may affect virus replication and Flintoff, 1987; Yokomori et al., 1993) and facilitation of the uncoating process (Asanaka and Lai, 1993; Beushausen et al., 1987; Flintoff and van Dinter, 1989; Kooi et al., 1988) . Other evidence from several laboratories supports the view that MHVs, including JHMV, can penetrate by receptor-mediated endocytosis into vesicles possessing the structure of ''coated pits'' (David-Ferreira and Manakar, 1965; Flintoff and van Dinter, 1989; Krzystyniak and Dupuy, 1984; Mizzen et al., 1985) . The current study reveals that dephosphorylation of N from inoculum virions is most probably catalyzed by a PPPase associated with endosomes. Combined evidence from this study and a previous investigation (Mohandas and Dales, 1991) characterized the relevant PPPase as one which functions optimally at neutral pH and is sensitive to okadaic acid and calyculin A, two inhibitors of phosphatases type 1 and 2A. These observations led us to the hypothesis that JHMV uncoating may be initiated within early endosomes. The uncoating process whereby the RNA genome dissociates from the capsid N protein, follows dephosphorylation and subsequent breakdown of N. Presumably, the relevant endosomal PPPase is maximally active at neutrality, the pH milieu likely to prevail on the cytosolic face of the membrane, regardless of the pH optimum for virus-cell membrane fusion (Gallagher et al., 1991; Kooi et al., 1988) . Latency of this PPPase, i.e., it becomes more active after solubilization of membranes, as demonstrated in Mohandas and Dales (1991) , implies that in host cells the enzyme occurs in membranes at a site where it can function to dephosphorylate N as the N-RNA complex is released from inoculum viri- PPPase becomes directly relevant to interference with the uncoating process of JHMV.

Based on the assumption that dephosphorylation of N showing that application of specific PK inhibitors can counteract the dbcAMP effect so as to maintain the oligo-and release of the viral genome are connected, it is quite possible that JHMV uncoating is depressed in mature dendrocytes in a permissive state, depending on both the concentration of dbcAMP inducer used and inhibitor oligodendrocytes because R1 synthesis is upregulated, as demonstrated by Beushausen et al. (1987) . This con-affinity for the PKs. While data from studies employing inhibitors do not provide unambiguous identification as cept has support from our in vitro experiments and those of Jurgensen et al. (1985) , Khatra et al. (1985) , and Sriva-to which among the various cellular PKs is pertinent to JHMV, our previous investigation (Beushausen et al., stava et al. (1988) , who demonstrated that R can inhibit PPPase, including the endosomal enzyme. One should 1987) indicated that neither the type C or cGMP-dependent PK's are involved in controlling the infection.

not, however, overlook other metabolic derangements effected by R, including a reduction in the basal level Data obtained from other studies, among them cell fusion analyses, demonstrated that other cellular factors of cAMP-responsive transcription (Mellon et al., 1989; Shenolikar, 1994) ; however, in the present context, an apart from the presence of specific viral receptors are required for JHMV penetration (Asanaka and Lai, 1993; effect by R on transcription does not explain the resistance manifested by mature oligodendrocytes because Flintoff and Van Dinter, 1989; Kooi et al., 1988; van Dinter Flockhart, D. A., and Corbin, J. D. (1982) . Regulatory mechanisms in early block to infection can be circumvented by transfecthe control of protein kinases. Crit. Rev. Biochem. 12, tion with free JHMV RNA. Gallagher, T. M., Escaramis, and Buchmeier, M. J. (1991) . Alteration of the pH dependence of coronavirus-induced cell fusion: Effect of mutations in the spike glycoprotein. J. Virol. 65, 1916 Virol. 65, -1928 

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The structure and regulation of protein phosphatases

Attenuation of murine coronavirus infection by ammonium chloride

Endosomal association of a protein kinases in adipose, cardiac and other tissues

Infection by Coronavirus JHM of rat neurons and oligodendrocyte-type-2 astrocyte lineage cells durof type 1 protein kinase

In vivo ing distinct developmental stages

and Holmes, and in vitro models of demyelinating disease. XII. Persistence and expression of corona JHM virus functions in RN2-2 Schwannoma K

A technique for radiolabelling assay

Inhibition of measles virus replication DNA restriction endonuclease fragments to high specific activity

Protein phosphorylation

An electron microscope study of the development of a mouse hepatitis virus in tissue culture Biochem

Cyclic nucleotide-dependent protein kinase

Several rat cell lines share a Ther

Protein serine/threonine phosphatases: new avecommon defect in their inability to internalize murine coronaviruses efficiently

and Veron, dure and some applications

The effect of dibutyryl cyclic dependent protein kinase blocks the differentiation of Dictostelium AMP on restricted replication of rubella virus in rat glial cells in discoideum

Rat glial C6 cells are defective models of demyelinating disease III. JHM virus infection of rats. Arch. in murine coronavirus internalization

the regulation of coronavirus infection in primary explants of mouse Synthesis and subcellular localization of the murine coronavirus nu-CNS

Electrophoretic transfer A spike protein dependent cellular factor other than the virus receptor is required for mouse hepatitis virus entry

ACKNOWLEDGMENTS Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984). Isoquinoline sulfonamides, novel and potent inhibitors of cyclic nucleotide We thank A. Hanington for expertise in maintenance of the animal dependent protein kinase and protein kinase C. Biochemistry 23, colony. This work was supported by the Medical Research Council of 5036-5041. Canada. K.K. was the recipient of a studentship from the Multiple Scle-Ingebritsen, J. S., and Cohen, P. (1983). Protein phosphatases: Properrosis Society of Canada.ties and role in cellular regulation. Science 221, 331-338. Ishihara, H., Martin, B. L., Brautigan, D. L., Karaki, H., Ozaki, H., Kato, Y., Fusetani, N., Watabe, S., Hashimoto, K., Uemura, D., and Hart-