key: cord-0839019-pd5n5qq1
authors: Astohlman, Stephen; Kyuwaj, Shigeru; Cohen, Michael; Bergmann, Cornelia; Polo, John M.; Yeh, Jason; Anthony, Richard; Keck, James G.
title: Mouse hepatitis virus nucleocapsid protein-specific cytotoxic T lymphocytes are Ld restricted and specific for the carboxy terminus
date: 1992-07-31
journal: Virology
DOI: 10.1016/0042-6822(92)90697-n
sha: 6f353ece91ff4775e66f8d9467ad697dd31c3037
doc_id: 839019
cord_uid: pd5n5qq1

Abstract Infection of mice with the JHM strain of mouse hepatitis virus (MHV) results in an acute encephalomyelitis associated with primary demyelination of the central nervous system. Efforts at understanding the components of the immune response in the development of chronic MHV-induced demyelination have implicated the antibody response and both the CD4+ and CD8+ T cell responses. In this report, we demonstrate that Balb/c (H-2°) mice immunized with the JHM (JHMV) strain of MHV develop a CD8+ cytotoxic T lymphocyte (CTL) response. One population of these virus-specific CTL recognize the nucleocapsid (N) protein. Recombinant vaccinia viruses expressing either the entire N protein or carboxy-terminal deletions were used to determine the number and location of the epitope(s) recognized. The CTLs were found to recognize a peptide contained within the carboxy-terminal 149 amino acids of the N protein. Analysis of infected cell lines expressing transfected major histocompatibility genes demonstrated that the anti-N protein CTLs were restricted exclusively to the L° molecule. These data provide the first definition of a MHV-specific CTL response directed to a viral protein and suggest that the anti-N protein CTL response is one potential mechanism used by the host to clear JHMV from the central nervous system.

Coronaviruses are enveloped, plus-stranded RNA viruses containing a genome of approximately 3 1 kb and three or four structural proteins (Lai, 1990) . The murine coronavirus, mouse hepatitis virus (MHV), produces a variety of acute and chronic infections in its natural host, the mouse. The first isolate, designated JHM virus (JHMV), was derived from a single mouse with hind leg paralysis (Cheever et a/., 1949) . Interest in the pathogenesis of these viruses has centered on their ability to produce both acute and chronic central nervous system (CNS) demyelination in mice (Weiner, 1973; Stohlman and Weiner, 1981; Kyuwa and Stohlman, 1990 ). lmmunosuppression of JHMV-infected mice results in an acute fulminant encephalomyelitis with little or no evidence of demyelination, indicating that the immune response plays a critical role in pathogenesis (Williamson and Stohlman, 1990) . The passive transfer of monoclonal antibodies (mAb) specific for each of the four structural proteins provides protection from the acute phase of the disease; however, they appear to exacerbate the chronic form of the disease (Buchmeier eta/., 1984; Wege et a/., 1984; Fleming et a/., 1988; Yokomori, unpublished data) . Similarly, the adoptive transfer of MHV-specific CD4+ delayed-type ' To whom reprint requests should be addressed. hypersensitivity (DTH)-inducer Th 1 T cells prevents the initial acute form of the disease (Stohlman et al., 1986) . The immune response in the CNS induced by the transfer of these cells appears to resemble a typical DTH response; however, similar to the passive transfer of anti-viral mAb, no reduction in virus titer was found in the recipients. In addition, CD4+ T cell-mediated protection also exacerbated the late form of the disease. Nonviral-specific DTH responses did not similarly effect the course of the disease (Stohlman et al., 1988) , suggesting the requirement for a viral-specific component. A common theme in these studies is that animals protected from the acute disease, in which virus replication is not suppressed, appear to be more susceptible to the chronic form of JHMV-induced CNS disease.

CytotoxicT lymphocytes (CTL) are an important component of the immune response to many viral infections. CTL have been implicated in the pathogenesis of MHV infection by the demonstration that a reduction in JHMV titer during infection of C57Bl/6 mice requires a CD8+ T cell (Sussman et al., 1987 (Sussman et al., , 1989 . Further, the clearance of virus requires compatibility between donor and recipient at the major histocompatibility complex (MHC) Class 1 genes. These data also indicated that a CD4+ helperT cell is required for the activation of the CD8+ effecters (Sussman et al., 1989) . This dependence on CD4+ T cells has been confirmed by the in viva depletion of CD4+ and CD8' T cells (Williamson and Stohlman, 1990) . Depletion of either subset pre-Generation of recombinant vaccinia viruses vents the reduction of infectious virus, consistent with a requirement for CD4+ T cell help in the induction of JHMV-specific CD8+ T cells. These data suggest that the anti-JHMV CTL response may be critical not only in effectively reducing virus in target organs but also may play a pivotal role in preventing the chronic phase of CNS disease.

CTLs specific for JHMV have been previously described; however, neither the viral protein(s) recognized nor the restriction element(s) has been determined (Yamaguchi et al., 1988) . To begin to understand the role of the viral proteins in the anti-JHMV CTL response, recombinant vaccinia viruses were constructed which express the JHMV nucleocapsid (N) protein and a series of deletions from the carboxy terminus. The N protein was chosen because it is the major virus protein synthesized in infected cells (Spaan et a/., 1988; Lai, 1990) , it is a highly conserved protein (Parker and Masters, 1990) , mAb specific for the N protein can protect mice from MHV infection (Nakanaga et al., 1986; Lecomte et al., 1987; Stohlman, unpublished) , and expression of the N protein is used as a marker for chronic CNS infection (Erlich et a/., 1987 (Erlich et a/., , 1989 Perlman and Ries, 1987) . Our data demonstrate that JHMV elicits an anti-N protein CTL response in Balb/c (H-Zd) mice, that the only detectable epitope of the N protein resides within carboxy-terminal amino acids 306 to 455, and that the N-specific CTL response is restricted to the Ld Class 1 molecule.

The derivation of clonal JHMV has been described previously (Stohlman et a/., 1982) . The wild-type WR strain of vaccinia virus was obtained from the American Type Culture Collection (ATCC). Recombinant vaccinia virus, vSC8, created by recombining the pSC-1 l,, plasmid into the WR strain, was supplied by Dr. 6. Moss, Laboratory of Viral Diseases, NIAID.

The JHMV N gene coding sequence was amplified by PCR from a cDNA encoding the JHMV N protein contained in a pT7-3 plasmid (supplied by Dr. M. M. C. Lai, University of Southern California). The oligonucleotides IF-5 @'-GGGGGGTCGACllTAAGGATGGTCTT-TTGTTCCTGGGC-3') and IR-5 (5'-GGGGACCCCTTC-TCGAGTGTAGTCCCGGAGGGGG-3') which contain Sal1 and Stul sites, respectively, were used for PCR amplification. The PCR product was digested with SalI and Stul and ligated into similarly digested vaccinia virus shuttle vector pSC-11 ss (supplied by Dr. 6. Moss, Laboratory of Viral Diseases, NIAID). The resulting construct, pSC-1 1 N, which contains the entire N gene coding sequence, was used for the generation of all subsequent deletion constructs. Deletions were made by digestion with lvrul (located 41 nt 3'-of the N gene stop codon) and Apal (nt 402) Nael (nt 746) or Spel (nt 915) which are contained within the N gene coding sequence. Termini were blunt-ended and the plasmids were religated, generating N gene truncations while also creating new, in-frame stop codons. The plasmid constructs also contain three stop codons immediately 3'-of the N gene in addition to the newly created stop condons. The deletion constructs, including both the initiation and stop codons, were verified by sequence analysis using the Sequenase version 2.0 DNA sequencing kit (U.S. Biochemical) and two flanking pSC 1 1 -specific oligonucleotides: pSC11 L (5'-CCAAACC-CACCCGCTTTlTATAG-3') and pSC 11 R (5'-CGlTGA-AATGTCCCATCGAGTGCGGC-3'). The constructions were then introduced into the WR strain of vaccinia virus by homologous recombination in CV-1 cells (Mackett er al., 1984) . Recombinant vaccinia viruses were selected by three or four plaque purifications on 143 Tk-cells using 25 pg/ml bromodeoxyuridine in the initial agarose overlay. Recombinant plaques werevisualized using 300 pg/ml of 5-bromo-4-chloro-3-indolylb-p-galactopyranoside (X-Gal) in a second agarose overlay as described by Chakrabarti et a/. (1985) . Stocks were prepared by propagation of the plaque isolates in CV-1 cells followed by expansion in HeLa cells (Mackett et a/., 1984) . Virus titers were determined by plaque assay on BSC-1 cells (Mackett et a/., 1984) . The recombinant vaccinia virus expressing the full-length N protein was designed VVJN, while those expressing the carboxy-terminal deletions were designed VVJNs, for the Spel deletion, VVJNn for the hlael deletion, and VVJNa for the Apal deletion. Western blot analysis DBT cells were infected with vSC8 (containing no insert), or recombinant vaccinia viruses containing ei-ther the full-length or truncated N gene sequences. At 24 hr postinfection, the cells were washed IX in PBS and lysed in Laemmli sample buffer (Laemmli, 1970) . The lysates were subjected to electrophoresis in 12% SDS-polyacrylamide gels and transferred to nitrocellulose. The expression of N gene products was detected by Western blot using a mixture of anti-JHMV N protein-specific mAbs , followed by visualization with ECL reagents (Amersham).

induction of bulk effector CTL Spleen cell suspensions were prepared from mice primed 3-8 weeks earlier by intraperitoneal (ip) injection of either 4 X 1 O6 PFU of the DL isolate of JHMV (Stohlman et al., 1982) or 5 X 1 O7 PFU of the recombinant vaccinia virus VVJN. Spleen cells (I X 1 O*) from immune mice were cultured for 6 days at 37" with 5 x lo7 irradiated (2500R) syngeneic spleen cells from naive mice infected with either JHMV [multiplicity of infection (m.o.i.) of 0.051 or VVJN (m.o.i. of 5.0) in 40 ml of RPMI 1640 medium supplemented with 10% fetal calf serum (Gemini Bioproducts), 2 mM glutamine, 25 pg/ml gentamicin, 1 mM sodium pyruvate, 5 X 1 O-5 M P-2-mercaptoethanol, and nonessential amino acids (RPMI-10).

In vitro stimulated spleen cells in 100 ~1 of RPMI-10 were added at various ratios to round bottom 96-well plates (Falcon Plastics) containing 1 X 1 O4 target cells in 100 ~1 of RPMI-10. J774.1 cells or the various L929 cell transfectants expressing the individual MHC H-Zd genes were used as targets. J774.1 cells were infected with JHMV or recombinant vaccinia viruses at an m.o.i. of 5 to IO. The L929-derived cell lines were infected with recombinant vaccinia viruses at an m.o.i. of 50. Following incubation for 6 hr at 37" the targets were washed 2x in RPMI and 1 x 10" cells were labeled with 100 &i Na"Cr0, (New England Nuclear) in a volume of 100-200 ~1, washed 4X with RPMI, resuspended in RPMI-10, and added to the effector cells. After 4 hr incubation at 37", 100 ~1 of the supernatant was removed and the radioactivity determined in a gamma counter. 1 . Schematic representation of the pSC1 l,, constructs containing the JHMV N gene and 3'.terminal deletions. The N protein coding sequence was cloned by PCR and ligated into the SalllSrul sites on the pSC-1 1 ss vector (designated pSC-1 1 N). Deletions were constructed by digestion at the Spel, Nael, or Apal sites within the coding sequence and the unique Nrul sate in the 3'kntranslated region and followed by ligation (Materials and Methods). These plasmrds were designated pSC-1 1 Ns, pSC-1 1 Nn, and pSC-1 1 Na, respectrvely. Each plasmid was used to construct a recombinant vaccrnia vrrus by homologous recombination and the vrruses derived from each were designated VVJN, VVJNs, VVJNn, and VVJNa. an m.o.i. of 5 to 10. The cells were washed twice by centrifugation in RPMI, resuspended in 2.0 ml RPMI at 2 X 1 O6 viable cells per milliliter, and incubated for 1 hr at 4" either with mAb specific for CD8 (Sarimento eta/., 1980) , CD4 (Ceredig et al., 1985) , or without antibody. The cells were washed twice in RPMI, resuspended in 1.8 ml RPMI containing 25 mM HEPES, pH 7.2, and 0.3% bovine serum albumin. Low-Tox Rabbit complement (Cedarlane) was added at 0.2 ml and the mixture incubated 45 min at 37". Following two additional washes, the cells were resuspended and tested for residual CTL activity.

DNA encoding the N protein of JHMV was amplified by PCR using the primers IF-5 and IR-5 and cloned into the Sa/l and Stul sites of pSC 1 l,, as described under Materials and Methods. The resulting construct was designated pSC-11 N and was used for all subsequent N gene deletions. The deletions ranged from the Apal, Spel, or /Gael sites through the lvrul site, yielding serial 3'-terminal truncations within the N gene coding sequence (Fig. 1) . Each deletion construct uses the same plasmid initiation codon and relied on in-frame termination codons generated during the constructions (Fig.  I) . The pSC-1 1 N and N-deletion plasmids were trans- fected into CV-1 cells infected with the WR strain of VV (Mackett er a/., 1984). Recombinant vaccinia viruses were isolated by at least three plaque purifications in the presence of BudR while using X-Gal to visualize recombinant plaques. Expression of the appropriate N gene truncations was demonstrated by Western blot. Figure 2 shows that the complete N protein and two of the carboxy-truncated proteins are recognized by an anti-N mAb pool. The smallest peptide, comprising the amino terminal 134 amino acids (VVNa), was not detected by the mAb pool nor any of the mAbs tested individually. In addition, no reactivity was observed using sera from Balb/c or C57Bl/6 mice immunized with JHMV, suggesting the absence of an immunoglobulin epitope(s) in this region. N protein-specific CTL activity Initially, mice were immunized with either JHMV or VVJN. Spleen cells were prepared from the immunized mice after 3-8 weeks and were cultured with syngeneic irradiated feeder cells infected with either JHMV or VVJN. Figure 3A shows that spleen cells from mice immunized with JHMV and stimulated for 6 days in vitro with JHMV-infected feeder cells specifically lysed J774.1 targets infected with JHMV. Little or no lysis was observed on uninfected cells. In addition, these cells were able to specifically lyse targets infected with VVJN, but not those infected with the vSC8 control. Spleen cells from mice immunized with JHMV and cultured on irradiated feeder cells infected with VVJN also showed specific killing of both JHMV-and VVJN-infected targets, but not uninfected targets (Fig. 3B ). Spleen cells from mice immunized with VVJN and stim-ET AL. ulated with irradiated feeder cells infected with VVJN also exhibited specific lysis of JHMV-infected targets compared to uninfected targets; however, the targets infected with both VVNJ and vSC8 were lysed with nearly equal efficiency (Fig. 3C) . Cytotoxic activity was removed following treatment with anti-CD8 mAb plus complement, but not by treatment with anti-CD4+ or complement only (Fig. 3D) . These data confirm that the killing observed was due to CD8+ anti-viral CTLs.

To initially gauge the heterogeneity of the responding population, mice were immunized with JHMV and cultured with feeder cells infected with VVJN. CTLactivity was tested 6 days later using L929 targets which express the Kd, Dd, or Ld MHC Class 1 molecules infected with VVJN at an m.o.i. of 5. Control J774.1 targets were infected with either VVJN or vSC8 at an m.o.i. of 5. No CTL activity was detected using VVJNinfected K830 (Dd) or H40.5 (Kd) targets. Specific CTL activity was detected using only the K2A7 targets which express the Ld Class I molecule indicating that the anti-N specific CTL response in Balb/c mice is restricted to the Ld molecule (Fig. 4A) . The data shown in Fig. 4B indicate that the J774.1 cells infected with VVJN were also lysed by these CTLs while no lysis was observed on the uninfected targets.

To determine the location of the epitope(s) recognized by the N-specific, Ld-restricted CTL, Balb/c mice were immunized with JHMV and the spleen cells cultured for 6 days with irradiated syngeneic VVJN-infected feeder cells. The Ld-expressing K2A7 cell line was infected with vSC8, VVJN, VVJNs, VVJNn, and VVJNa at an m.o.i. of 50. Fig. 5A shows that CTL activity was observed only when targets were infected with VVJN. No lysis over background (vSC8) was observed for targets infected with recombinant vaccinia viruses expressing any of the carboxy-terminal deletions, (VVNs, VVNn, and VVNa), indicating that the epitope(s) is contained within the carboxy-terminal 149 amino acids. Sequence analysis of the gene encoding the N protein derived from a number of MHV strains demonstrated a region of heterogeneity within the carboxy terminus (Parker and Masters, 1990) . To determine if the epitope(s) was contained within this variable region, the ability of CTLs induced by immunization with JHMV to recognize J774.1 targets infected with the A59 strain of MHV was tested. The data in Fig. 5B shows that the targets infected with the A59 strain of MHV are effectively recognized by these JHMV-in- duced CTLs, suggesting that the epitope is not within this variable region.

To begin to understand the repertoire of the JHMVspecific CTL response and the contribution of this population of effecters to acute and chronic demyelinating disease, we have constructed recombinant vaccinia viruses that express the JHMV N protein and a series of deletions from the carboxy terminus. CTLs specific for JHMV have been described previously (Yamaguchi et a/., 1988) and recent experiments using these clones have shown that they can protect infected recipients, presumably via the reduction of infectious virus (Yamaguchi eta/., 1991). However, neitherthe restricting element nor the viral protein recognized by these clones has been reported. The N protein plays multiple roles in the structure and replication of coronaviruses, including the encapsidation of genomic RNA to form a helical nucleocapsid structure (Lai, 1990) . It is also the most abundant protein in MHV-infected cells, and its corresponding mRNA is the most abundant species detected in infected cells (Spaan et a/., 1988; Lai, 1990 ). Mabs specific for the N protein protect mice from MHV-induced disease, although the epitope(s) recognized have not been determined (Nakanaga et al., 1986 (Nakanaga et al., , 1987 Lecomte et al., 1987) . In addition, the sequence of the N protein coding region is highly conserved among a number of MHV strains (Parker and Masters, 1990) pressing the S protein (Wysocka et al., 1989) . CTL activity was detected when JHMV-infected targets were tested, confirming that CTLs can be induced in Balb/c mice and indicating that analysis of these cultures would help determine the viral proteins recognized. Although clonal populations of anti-JHMV CTL have been reported previously (Yamaguchi eta/., 1988) , the 31 -kb coding capacity of the MHV genome has hindered a determination of the proteins recognized by these clones. The JHMV N protein-specific CTLs demonstrated in this report were examined for their restriction element(s) using L929 (H-2k) cells transfected with the three Class 1 genes derived from the H-2d haplotype. Analysis of the ability of the anti-N protein-specific CTL to recognize these three cell lines clearly showed that the response was restricted to the Ld molecule. Recombinant vaccinia viruses expressing the carboxy-terminal deletions of the N protein were used to localize the domain(s) of the N protein recognized in the context of the Ld molecule. This analysis demonstrated that only recombinant vaccinia virus expressing the entire N protein was recognized. No CTL activity was detected with recombinant vaccinia virus expressing the N protein from which the carboxy-terminal 149 amino acids had been deleted nor any of the recombinant vaccinia viruses expressing additional carboxy-terminal deletions. These data suggest the epitope(s) of the JHMV N protein recognized is contained within the carboxy-terminal portion of the N protein. The precise epitope has not been defined nor can our present data rule out the presence of additional epitopes at the sites of truncations. It is likely, however, by analogy with other viralspecific CTL responses (Bastin et al., 1987; Whitton et a/., 1988) , that only one or at most a few epitopes are contained within the domain comprising amino acids 307 to 455. A hypetvariable domain (amino acids 38 l-405) has been found within this region (Parker and Masters, 1990) ; however, our data demonstrate that JHMV-specific CTL can recognize J774.1 targets infected with either MHV-A59 (Fig. 5) or MHV-S (data not shown), each of which differ from JHMV in this region. These data suggest that the epitope recognized by the JHMV-specific CTLs is not contained within this hypervariable region.

Immunization with either JHMV or VVJN protected mice from a lethal JHMV infection (unpublished); however, both groups of mice also produced anti-N antibody. Thus, the direct role of N-specific CTL activity in protection is not yet clear. Preliminary data also indicate that mAb specific for the N protein can provide protection from JHMV; however, similar to the other models of mAb-mediated protection, there is no reduction of virus within the CNS (unpublished). Therefore, the possibility that antibody epitopes contained within the carboxy terminus are able to confer protection cannot be ruled out. The isolation and characterization of clonal populations of anti-N-specific CTLs are currently in progress in order to precisely map the epitope and to examine the role of these cells in JHMV-induced CNS disease. Comparison of the epitope(s) within the carboxy terminus recognized by antibody and CTLs should allow a clear distinction to be made between the roles of the anti-N antibodies and the CTL activity in both the acute and chronic forms of JHMV-induced demyelination in mice. It will also be of interest to determine the number of mouse haplotypes capable of recognizing this epitope, since preliminary experiments indicate that JHMV is not able to induce an N protein-specific CTL response in C57BU6 mice (data not shown; Perlman, personal communication).

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Munne hepatitis virus-4 (Strain JHM)-induced neurologic disease is modulated in viva by monoclonal antibody

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Antlgenic relationships of munne coronavlruses: Analysis using monoclonal antibodies to JHM (MHV-4) virus. virology 131

Identification of a Balb/c 1 +2Ld gene by DNA-mediated gene transfer

Antigenic relationship of murine coronaviruses: Analysis using monoclonal antibodies to JHM (MHV-4) virus. virology 131

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Coronavirus: Organization, replication and expression of genome

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General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. 1. Viral

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Delayed-type hypersensitivity response in the central nervous system during JHM virus infection requires viral specificity for protection

Interaction of (~2 region in class I MHC proteins contributes determinants recognized by antibodies and cytotoxic T cells. 1

Immune-mediated clearance of JHM virus from the central nervous system

T-cell-mediated clearance of mouse hepatitis virus strain JHM from the central nervous system

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The technical assistance of Manny Dlmacall, the advlce of Dr. Stanley M. Tahara, and the editorial assistance of Sonia Q. Garcia are gratefully acknowledged.This work was supported by U.S. Public Health Servtce Research Grant NS18146.