key: cord-0004913-q90mkw8r
authors: Leibowitz, J. L.; Bond, C. W.; Anderson, K.; Goss, Susan
title: Biological and macromolecular properties of murine cells persistently infected with MHV-JHM
date: 1984
journal: Arch Virol
DOI: 10.1007/bf01311222
sha: 4f58ba92c5fcce5d97aa1cf882941a959768ab8d
doc_id: 4913
cord_uid: q90mkw8r

A persistently-infected neuroblastoma culture [Neuro-2A (JHMV)] was established with the murine hepatitis virus JHM [MHV-JHM]. After 100 days of passage, the endogenous virus [Neuro-2A (JHMV) end] released by this culture was unable to induce the syncytia typical of MHV-JHM and the endogenous virus was not temperature-sensitive. The Neuro-2A (JHMV) culture was cured of virus production by passage under neutralizing antibody [Neuro-2A (JHMV) Ab]. The Neuro-2A (JHMV) and the Neuro-2A (JHMV) Ab cultures were as susceptible to heterologous infection with mengovirus and vesicular stomatitis virus as the uninfected Neuro-2A culture. However, the Neuro-2A (JHMV) and Neuro-2A (JHMV) Ab cultures were partially resistant to homologous superinfection by MHV-JHM and the closely related MHV-A59. Virus related to MHV-JHM was rescued from the antibody-cured cells by cell fusion. The synthesis of MHV-JHM specific antigens by Neuro-2A (JHMV) cells, Neuro-2A (JHMV) Ab cells and 17 Cl-1 cells infected by Neuro-2A (JHMV) end was studied by SDS-PAGE. The genomic RNAs of MHV-JHM and Neuro-2A (JHMV) end were compared by oligonucleotide mapping. The results of the protein and RNA studies indicated that the genome of Neuro-2A (JHMV) end was substantially modified from the genome of MHV-JHM, but the modifications did not significantly alter the molecular size of the viral-specific proteins.

The 17Cl-1, L2 and DBT cell lines have been previously described (2, 6, 28) . The Neuro-2A clone of the C 1300 mouse neuroblastoma cell line (9) was obtained from Dr. CAROL MILnE~. The BHK-21 cell lines wa obtained from Dr. Jo~ I-IoLLA~D. The cells were grown at 37 ° C as monolayer cultures in prescription bottles using Dulbecco's modified Eagle's medium (DME) supplemented with 10 percent newborn or fetal bovine serum as previously described (2, 21) .

The origin and growth of the MHV-JHM and MHV-A59 have been described (21) . Vesicular stomatitis virus (VSV), Indiana serotype, was supplied by Dr. JOt~N HOI~I~ND. Mengovirus has been previously described (17) . VSV and mengovirus stock were grown in BI4K-21 cells at a multiplicity of infection (NOI) of 0.1. Cells were infected with virus in suspension as previously described (2) or monlayers of cells were infected and incubated at 37 ° C.

The titers of MItV-JttM and MHV-A59 were determined by an endpoint dilution assay (21) or by plaqne assay on 17C1-1 cells. The titcrs of VSV and mengovirus were determined by plaque assay on BtIK-21 cells. The results of the plaque assay were quantitatively equivalent to the end-point dilution assay.

To determine the number of cells releasing infectious virus, an infectious center assay was done. The cells to be tested were suspended at 1 × 106 cells per ml in DME containing 10 percent heat-inaetivated mouse-anti-MttV-Jtt3/i serum and incubated at 37 ° C for 30 minutes. Ten-fold dilutions of these cells were prepared in DME 2 and added to a suspension of indicator cells (17CI-I) and seeded into Falcon Mierotest plates. The number of infectious centers per ml (IC/ml) was eMeulated on the basis of eytopathic effect from the Poisson distribution. An aIternative method of determining infectious eenters was also used. Cells to be tested were suspended at 1 × 106 cells per ml in DME containing 10 percent heat-inactivated mouse-anti-MI-IV-JttM serum and incubated at 37 ° C for 30 minutes. Ten-fold dilutions of these eells were prepared in DNIE2 and 0.5 ml volumes were added to confluent monolayers of 17CL1 cells. The monolayers were incubated at 37 ° C to allow attachement of the test cells, aspirated and overlaid with ])ME 2 containing one percent agarose. The dishes were incubated at, 37 o C for 72 hours and the plaques were counted.

Antisera were prepared against MttV.JHM in BALB/e mice (21) . The antisera were adsorbed against methanol fixed ] 7 CI-1 cells to remove nonspecifie antibodies except where otherwise indicated. The antisera were polyspeeifie and reacted with all of the MttV-JttM specific proteins detectable by SDS-polyaerylamide gel electrophoresis (SDS-PAGE) (t, 2). Immunofluoreseence was done by the indirect immunofluoreseence technique previously described (2t).

The same antiserum used for immunofluorescenee (unadsorbed) was used in these studies. The antiserum had a 50 percent plaque reduction titer of 1 : 1280 and was added to DME 10 to a final eor~centration of 5 percent (final antibody titer of the medium was 1:64). Cells were resuspended in medium containing antiserum at the time of passage. The medium was replenished after 3 days and the ceils were passaged weekly.

Cells were fused by treatment with polyethylene glycol (18, 24, 25) . Cells to be fused (1.5X l0 s cells of each line in DME10) were coeuttivated in 6 cm dishes for 24 hours at 37 o C. The cells were washed twice with serum-free DME (DME 0) and one ml of 50 percent polyethylene glycol 6000 (Sigma) in DME 0 (pI{ 7.8) was added to each culture and the dishes were incubated at room temperature for 3 minutes. The medium was aspirated and at one minute intervals, the following volumes of DME 0 were added, 0.2 m], 0.5 ml, 0.5 ml, 1.0 ml and 2.0 ml. The DME0 was aspirated one minute later a:nd the monolayers were washed once with DME i0 and fed with 3 ml of ])ME I0. The fused cultures were incubated at 37 ° C for 18 hours. The medium was replaced with 3 ml of ])ME 2, and incubated at 37 ° C for an additional 78 hours. The dishes were monitored for CPE at 12 hour intervals.

tVIonolayers of 17CI-1 cells were infected with virus at an NIOI of 0.3 PFU/cell and incubated at 37 ° C. The infected cells [MttV-JIe_M at 6.5 hours PI, Neuro-2A (JttMV) end at 8.5 hours PI] were washed t~vice with methionine-free DME 2, and radiolabeled with 3~S-methionine (300 BCi/ml) in methionine-free medium for 1 hour at 37 ° C. Persist,entlydnfeeted ceils (2 × i0~ cells in DES110) were seeded into 35 mm dishes and J.L. LEIBOWZTZ, C. W. BOND, K. ANDERSON, and S~-SAN Goss: incubated at 37 ° C for 24 hours. The cell monolayers were washed twice with mcthioninefree DME 2 and radiolabeled with ssS-methionine (300 IxCi/ml) in methionine-h'ee medium for 3 hours at 37 ° C. At the end of the labeling period, the monolaycrs were lysed in situ at 0 ° C with buffer B 10 (1) and the cytoplasmic lysates were immunoprecipitated as described previously (1). SDS-PAGE was done as described by LA~IH and FARCE (10) except the resolving gel was supplemented with 0.5 percent linear polyaerylamide. Volumes representing equal cell numbers were loaded in each lane of the slab gel. Fluorographs were prepared by the method of L~SKE¥ and MILLS (12) .

A stock of Neuro-2A (JHMV) end was prepared from virus isolated from the Neuro-2A (JHMV) cell line at, 178 days PI. Cells (17C1-1) were infected with the Neuro-2A (JHMV) end stock or MHV-JHM at an MOI of 0.1 PFU/celI in phosphatefree medium and radiolabeled with 3~P-orthophosphate (150 ~Ci/ml) throughout the multiplication cycle. Virions were purified, the genomie I~NA was extracted and fingerprinted as previously described (14) .

A persistent infection of Neuro-2A cells was initiated by infecting cells with MHV-JHM at an MOI of 0.1 IU/cell. After the acute phase of the multiplication cycle was completed, characterized by minimal cell fusion and cytolysis, a resistant population of ceils repopulated the flask in about 3 days. The culture was designated Neuro-2A(JHMV) and was passaged at weekly intervals at 37 ° C for over 280 days.

The titers of endogenous virus in the form of cell-free virus or cellassociated virus were determined at weekly intervals (Fig. 1) . The amount of endogenous virus produced varied in a cyclical manner between 1.1 × 10 ~ and 2 × 10 v IU/106 cells. The proportion of virus that remained cell-associated varied between 2 and 56 percent. The fluctuations in the amount of virus produced did not correlate well with either the fluctuations in the fraction of ceil-associated virus or the fraction of cells stained for MHV-JItM specific antigen by immunofluorescence. The percentage of cells capable of producing infectious centers was 1.2 to 2.5 fold greater than the percentage of cells containing virus-specific antigen (data not shown).

After establishment, no cytopathic effect (CPE), including syncytia formation, was ever observed in the Neuro-2A(JHMV) persistent culture. It was not possible to distinguish the Neuro-2A(JHMV) cells from the Neuro-2A parent cells by phase-contrast microscopy (Fig. 2) . Both cell lines expressed dendritic processes to a similar extent. An approximate generation time of 30--36 hours was estimated for both lines over the first 21 passages. At 91 and 105 days, generation times of 30 hours were measured for both cell lines.

The Neuro-2A(JHMV) culture was passaged in the presence of MHV-JttM specific antibody for 4 weeks. Before treatment at 190 days postestablishment, the culture released 4.7 × 105 infectious units (IU) of endogenous virus per ml of culture fluid, Viral antigens were detected by immunofluorescenee in 9 percent of the cells of the culture ~nd 15 percent of the cells produced infectious centers. After treatment with neutralizing antiserum, no endogenous virus or viral antigens were detected and infectious centers were not produced. The antibody-cured culture was passaged without neutralizing antiserum for ~n additional 10 weeks. The properties of the antibody-cured cells did not change during this period. The antibody-cured cell line was designated Neuro-2A(JHMV)Ab. 

At approximately 100 days postestablishment, a change was observed in the acute CPE produced in 17C1-1 cells by endogenous virus [Neuro-2A (JHMV)end] from the persistently-infected culture. Endogenous virus was isolated from the Neuro-2A (JHMV) culture at 196 days postestablishment and the cytopathic effect in an acute infection of 17C1-1 cells was determined (Fig. 3) . The CPE characteristic of MHV-JHM in 17C1-1 cells at 37 ° C was syncytia formation followed by lysis with a fraction of the cells surviving. Syncytia were not observed in the cells infected by the endogenous virus at any time PI, but extensive lysis was evident in all cultures by 24 hours PI. An examination of the clones used to test for tempera.turesensitivity [cloned from Neuro-2A (J~IMV)end isolated at 118 days postestablishment] indicated that none of the clones were able to induce syncytia formation [syn (--)]. The plaques obtained in 3 d~ys with MHV-JHM on 17 Cl-1 cells were uniform in size and had a mean plaque diameter (n-= 10) of 2.23 mm with a standard deviation of 0.13 mm. In the same experiment, Neuro-2A(JttMV)end plaques (n----10) had a mean plaque diameter of 1.67 mm with a standard deviation of 0.30 ram.

The persistently-infected and antibody-cured cultures were tested for their susceptibility to heterologous (mengovirus or VSV) infection (Table 1) 

On the basis of the results from the homologous superinfection experiments, it was suspected that the viral genome was maintained in a covert state in the antibody-cured cells. Several attempts were made to rescue virus from the antibody-cured cells by cocult4vation with 17C1-1 cells. At various times after initiation, the cocultures were tested for viral antigen by immunofluorescence and for virus multiplication by plaque assay and end-point dilution assay. No viral antigens or infectious viruses were detected. When the Neuro-2A(JHMV)Ab cells were fused to 17C1-1 cells with polyethylene glycol, focal lysis was observed in 3 to 4 days in 3 trials (fusions were done at 174, 195 and 209 days after the Neuro-2A (JHMV)Ab line was established). Infectious rescued virus was detected by the endpoint dilution assay. The yield of virus obtained from the 3 trials was in the range of i0~ to 104 IU per 106 cells. Several plaque assays were attempted at 33 °, 37 ° and 39°C to detect infectious rescued virus. The assays were observed daily for 7 days. No plaques were observed at any of the temperatures tested. The reason for this is not known. No infectious virus was detected in parallel unfused cultures. Two clones of rescued virus obtained were recloned by the end-point dilution method. The rescued virus was designated Neuro-2A (JHMV)res.

Since it was possible that a contaminating virus such as a paramyxovirus may have been rescued in the cell fusion experiment, each rescued clone was tested for its ability to synthesize MHV-JHM specific viral antigen, and induce a CPE typical of MHV. Cells were infected in suspension with 10-fold serial dilutions of each rescued clone. Ten replicas of 10 B1 cultures were seeded in 60 well Microtest dishes and incubated at 37 ° C for 48 hours. The cells were stained for MHV-JHM antigens by the immunofluorescent method. Mock cultures were prepared in the same manner using lysates from fused 17C1-1 cells for the infection. Typical results are shown in Fig. 3 . No CPE and no viral antigen were detectable in the mock cultures. A significant amount, of MHV-JHM specific antigen detected by immunofluorescence and CPE was evident in the cultures infected with rescued virus. At higher virus dilutions, no CPE or viral antigens were evident. The MttV-JHM specific fluorescence observed in the rescued cultures was considerably weaker than observed in the MtIV JIIM cultures (Fig. 3) . No syncytia were observed in any of the rescued cultures. The results indicate that the rescued virus clones were related to MI-IV-JH?¢I.

Viral-specific antigens were detected by immunofluoreseence in the persistently-infected culture Neuro-2A (JHMV) (Fig. 3) . However, no MHV-JHM specific antigens were detectable in the antibody-cured culture Neuro-2A(JHMV)Ab. To further these studies, each of the cell lines were radiolabeled with 35S-methionine, cell lysates were prepared and immunoprecipitared with the adsorbed antisera, used in the immunofluoresceace studies. The immunopreeipitates were analyzed by SDS-PAGE. Seven viral-specific proteins have been identified in 17CI-1 cells infected by MHV-JHN (E2-i50K, N-63K, p6t, p56, p35, p23, p22 and p14; references t and 23). 

The oligonucleotide fingerprints of the genomes of M H V -J H M and Neuro-2A ( J H M V ) e n d were compared in an effort to detect specific modifications in the persistent viral genome (Fig. 5) The persistently-infected culture Neuro-2A (JHMV) and the antibodycured culture Neuro-2A(JHMV)Ab were as susceptible to infection by mengovirus or VSV as the parent culture Neuro-2A (Table i) . There was no effect on the yield obtained when the M0I was varied from 0.1 to i0 PFU per cell. In addition, culture supernatant fluid from Neuro-2A (JHMV) did not reduce the titer of VSV assayed on DBT cells. These experiments suggest that interferon did not play a role in the maintenance of the persistent infection. These results are in general agreement with others (7, 16, 26) .

Supernatant cell culture fluids from the persistent culture Neuro-2A (JHMV) did not interfere with the multiplication of MHV-JI-IM or MHV-A59 in i7Cl-1 cells. No interference in the multiplication of MHV-JHM or MHV-A59 was observed during serial undiluted passage (20) . Further, Neuro-2A(JHMV)end was passaged serially in L2 cells,and no virus was produced that interfered with the multiplication of standard MHV-JHM. These results suggest that the presence of defective-interfering particles in our cultures was unlikely.

Of the 25 clones of endogenous virus tested for temperature-sensitivity, none could be considered to be significantly ts. HOLMES and BEHNKE (8) found ts endogenous virus in their persistently-infected cultures. However, others have found the endogenous virus to be non-ts (7, 25, 26) .

There was an evolving relationship between the virus and the host cell during the first 100 days of establishment. After this period of time, the endogenous virus released by the culture was unable to induce syncytia formation during an acute infection (Fig. 3) . This was also true for the 25 clones established from the released virus.

The plaques of the syn (--) endogenous virus released by the persistent cultures were smaller than the plaques typical of MHV-JHM. Others have reported that the mean plaque size of endogenous virus released by cultures persistently infected with MHV-JHM or MttV-A59 was reduced (7, 8, 26, 33) . However, only YOSttlKURA and TEJIMA (33) reported the loss of ability to induce syncytia during an acute infection.

The Neuro-2A(JHMV) culture was cured of endogenous virus production by passage under neutralizing antibody. MHV-JHM specific antigen was not detectable in the antibody-cured culture by immunofluorescence or by immunoprecipitation of radiolabeled proteins (Figs. 3, 4) . Virus was rescued from these cultures and was found to be antigenically related to MHV-JHM (Fig. 3) . STOYILMAN and ~rEINER (26) antibody cured ~ culture similar to our Neuro-2A(JHMV) by passage under antibody. They were unable to detect endogenous virus in their antibody-cured culture. How-ever, they were able to detect virus-specific antigen by immunofluorescence. Cold-sensitive mutants were isolated from antigen positive and antigen negative clones of their antibody-cured cells (25) . Since the persistent infections could be cured by passage under neutralizing antibody, it seems reasonable that the persistent infections were maintained by cell to cell transfer of virus. However, MHV-JHM related virus was rescued from the antibody-cured cells by cell fusion. The results of these workers and our results indicate that the viral genome was capable of persisting in the absence of infectious virus. The mechanism of genome persistence is under investigation.

The persistently-infected culture Neuro-2A(JHMV) and the antibodycured culture Neuro-2A(JH~{V)Ab were resistant to superinfection by either MHV-JHM or MHV-A59 on the basis that no typical CPE was observed during the infection. These results are similar to those obtained by others (8, 26) . However, the Neuro-2A(JHMV) and Neuro-2A(JHMV) Ab cultures yielded about the same amount of virus as the acutely infected Neuro-2A control culture (Table 1) . Clones of the virus released by the superinfected Neuro-2A(JHMV) were syn(--) suggesting that the progeny virus was endogenous virus. An alternative explanation is that superinfecting virus multiplied, but the CPE typical of MHV was altered by a mechanism such as phenotypic masking (34) . However, this is unlikely because the clones of released virus did not produce syncytia during an acute infection of 17 CI-1 cells.

The MHV-JHM specific proteins synthesized by cells acutely infected by MHV-JHM or by Neuro-2A (JHMV)end were compared on the basis of molecular size (Fig. 4) . The gel profiles were almost identical suggesting that there were no modifications in the genome of Neuro-2A(JHMV)end resulting in changes in the molecular size of the proteins. These data are in general agreement with the data reported by YOSHIKURA and TEJIXgA (33) for MHV-S. However, the profile of viral-specific proteins synthesized by the Neuro-2A(JHMV) cells was somewhat altered from the acute infections. There appeared to be minor modifications in the E 1-23 K protein and the 61K and 56K proteins were not detectable in the Neuro-2A (JH~IV) ]ysate. The change in migration of the E 1-23K protein was probably due to the differences in cell-specific glycosylation of the E 1-22K protein between the 2 cell lines, No viral-specific proteins were detected in the Neuro-2A(JHMV)Ab culture. An additional protein band with an apparent, molecular weight of 120 kilodMtons was observed in the immunoprecipitate of the lysate of t7 C1-1 cells infected by Neuro-2A (JHMV)end. A protein with a similar size was shown to be the precursor to E2-150 in in vitro translation studies (13) .

We previously identified 7 virus specific I~NAs in cells infected by MHV-JHM (14) . These RNAs formed a nested set of 6 subgenomic sized RNAs in addition to the genomic sized RNA as determined by oligonucleotide mapping. The genomic sized RNA was designated RNA-1. The subgenomie species were designated RNA-2 to RNA-6 in order of decreasing size. At least. 6 differences were observed in the oligonucleotide patterns of MHV-JHM and Neuro-2A(JHMV)end (Fig. 5) . One spot (arrowhead 1) was present in the Nenro-2A(JHMV)end genome, but not in the MHV-JHM genome. This probably represents an alteration in the oligonucleotide indicated by arrowhead 2 in the MHV-JHM genome. Arrowheads 2---6 indicate oligonucleotides detected in the MHV-JHM genome, but not in the Neuro-2A (JHMV)end genome. We have determined that oligonucleotide 2 was from the 5' region of RNA-3, oligonucleotides 3 and 4 were from the 5' region of RNA-1, and oligonucleotides 5 and 6 were from the 5' region of RNA-2. SIDD~:LL et al. (22) demonstrated that I~NA-3 codes for the precursor to the glycoprotein E 2. Others have implicated the E2 protein in MHV-induced cell fusion (3, 8) . Thus, the modification in the t~NA-3 portion of the genome which codes for E 2 may result in the syn(--) phenotype.

The results described in this paper suggest that it is unlikely that the induction of interferon, the development of defective-interfering particles or the development of ts mutants play a role in the persistent infection of Neuro-2 A cells with MHV-JHM. However, a particular phenotype, syn(--), developed during the course of the infection. The syn(--) phenotype was presumably selected by the host cell on the basis of its decreased cytopathogenicity. 

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