key: cord-0724516-p8bzkmas
authors: Koolen, Marck J.M.; Osterhaus, Albert D.M.E.; Van Steenis, Gijsbert; Horzinek, Marian C.; van der Zeijst, Bernard A.M.
title: Temperature-sensitive mutants of mouse hepatitis virus strain A59: Isolation, characterization and neuropathogenic properties
date: 1983-03-31
journal: Virology
DOI: 10.1016/0042-6822(83)90211-8
sha: be0f1cd4fef17aac67f5c3d2d95935b582dae9c6
doc_id: 724516
cord_uid: p8bzkmas

Abstract Twenty 5-fluorouracil-induced temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 were isolated from 1284 virus clones. Mutants were preselected on the basis of their inability to induce syncytia in infected cells at the restrictive temperature (40°) vs the permissive temperature (31°). Of these mutants, only those with a relative plating efficiency 40° 31° of 3 × 10−3 or smaller were kept. Virus yields at 40° compared to 37° and 31° (leakiness) were determined. Most mutants (16) were RNA−, i.e., unable to synthesize virus-specific RNA at the restrictive temperature. The other four were RNA+. No qualitative differences were detected in the virus-specific RNAs in cells infected with RNA+ ts-mutants, both at 31° and 40°. Virus-specific proteins present in cells infected with is-171 (RNA−) and the RNA+-mutants (ts-43, is-201, is-209, and is-279) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates. No qualitative differences in the pattern of virus-specific cellular proteins were detected among the mutants except for an additional polypeptide of about 46,000 daltons in ts-209-infected cells. Finally, the neuropathogenic properties of eight of the mutants were investigated. Whereas 102 PFU of wild-type virus injected intracerebrally killed 50 to 100% of 4-week-old Balc/c mice within 1 week, the mutants were highly attenuated. A dose of 105 PFU lead to no or transient disease. However, 4 weeks after infection with ts 342, is-43, or is-201 obvious histological changes were observed in brain and spinal cord of clinically healthy mice.

Twenty 5-fluorouracil-induced temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 were isolated from 1284 virus clones. Mutants were preselected on the basis of their inability to induce syncytia in infected cells at the restrictive temperature (40") vs the permissive temperature (31"). Of these mutants, only those with a relative plating efficiency 40"/31" of 3 X lo-' or smaller were kept. Virus yields at 40' compared to 37" and 31" (leakiness) were determined. Most mutants (16) were RNA-, i.e., unable to synthesize virus-specific RNA at the restrictive temperature.

The other four were RNA+. No qualitative differences were detected in the virus-specific RNAs in cells infected with RNA+ ts-mutants, both at 31" and 40". Virus-specific proteins present in cells infected with ts-171 (RNA-) and the RNA+-mutants (ts-43, ts-201, ts-209, and ts-2'79) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates.

No qualitative differences in the pattern of virus-specific cellular proteins were detected among the mutants except for an additional polypeptide of about 46,000 daltons in ts-209-infected cells. Finally, the neuropathogenic properties of eight of the mutants were investigated.

Whereas 102 PFU of wild-type virus injected intracerebrally killed 50 to 100% of I-week-old Bale/c mice within 1 week, the mutants were highly attenuated. A dose of 16 PFU lead to no or transient disease.

However, 4 weeks after infection with ts-342, ts-43, or ts-201 obvious histological changes were observed in brain and spinal eord of clinically healthy mice.

We have undertaken to isolate temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 (MHV-A59), with the expectation that these mutants will be useful for further studies on the replication and pathogenesis of this virus. MHVis a member of the family Coronaviridae, a group of lipid-enveloped viruses with a single strand of infectious RNA of about 6 X lo6 molecular weight (Tyrrell et al, 1978; Ter Meulen et aZ., 1981) .

In the last few years, considerable progress has been made in elucidating the replication strategy and structure of coronaviruses (for reviews see Ter Meulen et UC, 1981; Siddell et al., 1982) . A characteristic 1 To whom reprint requests should be addressed. feature of the coronavirus r~~li~at~~~ strategy is that seven, or six in the avian system, virus-specific RNAs are synthesized in the infected cell. The largest ot?e is the intracellular form of ge~~rne~ the others are a nested set of sub~e~~~i~ mRNAs with common ~-proximal sequences (Spaan et al, 198 al., 1981; Lai et al., 1981; Cheley et al., 1981; bowitz, 1981; Stern and Kenne RNAs 3, 6 , and '7 have be code the three major virion proteins (Slddell et al+, 1980; Rottier et aZ., 19818. et al, 1981; Leibowitz et al., 19 and 2 of MHV encode nonstruc teins Si 1981) . ts mutants will be useful to identify the functions of these and other ~~~~tr~~ 393 0042~6822/X3 $3.00 tural proteins and for the study of the transcription mechanism. In addition they might be helpful to elucidate the process of virus maturation.

Moreover, we were interested to know whether the pathogenic properties of MHV-A59 would change after the induction of mutations into the genome. A59 virus is a hepatotropic strain of MHV, causing extensive hepatic necrosis and leading to death within a week (Manaker et al., 1961) . When injected intracerabrally, the virus leads to encephalitis (Robb et al., 1979) . For the neurotropic strain of mouse hepatitis virus, MHV-4(JHM), a clear difference was observed between wild-type virus and mutant ts-8 (Haspel et al., 1978) . Whereas wild-type virus produced a rapid and fatal encephalomyelitis, the mutant induced a prolonged infection of the central nervous system accompanied by demyelination and mice survived. Immunocytochemical studies in viva have shown that ts-8 has a restricted tropism for neurons and replicates mostly in oligodendrocytes (Knobler et al, 1981) .

cells were grown in Dulbecco's modified Eagle medium containing 10% fetal calf serum (DMEM-10% FCS) supplemented with 100 IU/ml penicillin and 100 pg/ml streptomycin.

The preparation of virus stocks of MHV-A59 has been described previously .

Virus was plaque-titrated on L cells using an overlay with 1.5% Bacto-agar (Difco, Detroit, Mich.) in DMEM-1% FCS unless otherwise indicated. Plaques were read after one (3'7", 40") or three (31") days. A subline of L cells obtained from F. Lehmann-Grube (Hamburg, West Germany) was used. With these cells plaques could be read sooner than reported previously ; also plaque titers were five-to sevenfold higher.

For most experiments Sac( -). cells were grown in 35-mm tissue culture dishes and infected at densities of 1 to 2 X lo6 using 50 PFU/cell in 0.3 ml DMEM-3% FCS. Af-ter 1 hr this inoculum was replaced by 1 ml DMEM-10% FCS.

with doubly cloned MHV-A59 wild-type virus at a multiplicity of infection (m.0.i.) of 10. One hour postinfection (p.i.) inocula were removed and replaced by 10 ml of DMEM-10% FCS supplemented with 150 Kg/ml 5-fluorouracil.

Virus was harvested at 16 hr pi. (37") and stored at -70". Mutagenized virus stocks had an infectivity titer of 1.2 X lo4 PFU/ml(37") a reduction of about 1000 times compared to the untreated control.

Selection of mutants. Mutagenized virus stocks diluted as to give 15 plaques in a 35-mm tissue culture dish were plaqued on L cells at 31". In order to facilitate subsequent elution of virus an overlay of 0.4% agarose (Merck-Schuchard, Darmstadt, Germany) was used. After 3 days, wellisolated plaques were picked using the back of a Pasteur pipet. Plaques were then disrupted by vortexing in 0.5 ml DMEM-3% FCS resulting in a virus suspension containing about lo5 PFU/ml. With this suspension parallel cultures, at 31" and 40", of Sac(-) cells were infected. About lo4 PFU (0.1 ml) was added to 2 X lo5 cells grown in 0.5 ml DMEM-10% FCS in the 16-mm wells of tissue eultures clusters (Costar, Cambridge, Mass.). The cells at both temperatures were observed daily for the appearance of syncytia. Those virus clones that caused syncytium formation 3 days p.i. at 31" but not at 40" 2 days p.i. were considered as potential ts mutants. Each potential mutant was tested to confirm its ts nature by plaquing the medium of the 31" culture on L cells at the restrictive and permissive temperature. Only those mutants having a plating efficiency 40"/31" of 3 X 10e3 or lower were used.

The leakiness of the mutants was determined by infecting 5 X lo5 Sac(-) cells grown in 16-mm wells of tissue culture clusters with wild-type virus or ts mutants in 0.1 ml of DMEM-3% FCS at an m.o.i. of 50 at 31". After 1 hr the inocula were removed and replaced by 0.5 ml DMEM-10% FCS, and incubation was continued at 31", 37", or 40" until respectively 16,12, and 9 hr p.i. Virus titers in the culture

OF MHV-A59 3%

HnC?diU were determined by plaque titration on L cells at 31".

Stocks of the mutant viruses were preared by infecting 75-cm2 tissue culture flasks of Sac(-) cells with about lo3 PFU of the original virus clones eluted from plaques. Virus was adsorbed for 1 hr in 1.5 ml DMEM-3% FCS at 31' before 10 ml -10% FCS was added. Stocks were harvested after an additional 48-72 hr at the permissive temperature and stored at -70".

High-titered virus stocks needed for infection of cells at high m.o.i. were prepared by infecting roller flasks (1200 cm') containing about 2 X lo* Sac(-) cells with the ts mutants at 0.2 PFU/cell in 25 ml EM-3% FCS for 1 hr at 31". Inocula were removed and 100 ml DMEM-10% FCS was added and incubation was continued at 31". The virus was harvested at 48 p.i. and precipitated from the medium with lyethylene glycol . The ulting virus pellet was resuspended in 1 to 2 ml DMEM-3% FCS per roller flask. Determination of the kinetics of virus formation and the synthesis of virus-speci$c RN& at 31" and 40" in wild-type virus-infected cells. Sac(-) cells in 35-mm ulture dishes were infected as deabove. After adsorption, the cells were washed once with PBS and medium was added. Samples from the culture fluid were taken at various times p.i. and plaquetitrated. The kinetics of virus-specific RNA synthesis were determined by following the incorporation of rH]uridine into actinomycin D-treated Sac(-) cells as described previously .

Analysis of virus-speci& proteins in cells infected with ts mutants. Sac( -) cells in 35mm dishes were infected at 31" and replicate cultures were incubated at 31" and 40". Seven hours p.i. (40") or 13 hr p.i. (31") the cells were washed twice with PBS and labeled with 1 ml methionine-deficient MEM, supplemented with 5% FCS and 10 &i r5S]methionine (-1000 Ci/mmol, The Radiochemical

Centre, Amersham, England) until 9 hr pi. (40") or 16 hr p.i. (31"). After the labeling period, cells were washed with PBS and lysed in 0.15 ml lysis riton X-100, 0.5 thalene-disul~o~ate-disodium mM phenylmethylsulfonyl fluo buffer (20 mM Tris, pH 7.4, 1 m 100 mlM NaCl). The lysates were centril fuged for 5 min at 000 .q and prQ~~ss~~ directly or kept at Virus-specific proteins were im precipitated by incubating 30 ~1 clarified samples with PO ~1 mouse anti-MHV-A59 serum. The serum was obtainer from Balb/c mice surviving an exper' tal MHV-A59 infection (Spaan et aL, After overnight incubation at 4", 0.2 vsi Finally, the adsorbed proteins were dissolved in 50 ~1 ~lectro~boresis s buffer (10 m2M Tris-HCl, pH 8.0, EDTA, 10% glycerol, 2% SDS, 5% captoethanol, 0.001% brorn~~be~o To avoid aggregation of the species (Sturman, 1977) , sampl boiled prior to electrophoresis in I%% a,erylamide-0.085% bisacrylamide ge tier et al., 1981b). Gels were loade 10,000 cpm of radioactivity of the noprecipitated virus-specific prote thesized at 31", which were compared with material from an equal number of cells incubated at 40". Proteins were visuali hy on preglashed Kodak washed and RNA was isolated by phenol extraction as described previously (Jacobs et ab, 1981) . RNAs were analyzed by electrophoresis in 6 M urea-containing agarose gels (Rottier et aZ., 1981a) .

Pathology in mice. The neuropathogenic properties of the ts mutants were determined by intracerebral injection of groups of four 4-week-old Balb/c mice (Centraal Proefdierenbedrijf TNO, Zeist, The Netherlands) with lo5 PFU in 0.1 ml PBS. Before injection, animals were bled and sera were checked for the absence of anti-MHV antibodies in a plaque 'reduction assay. Control animals received 0.1 ml PBS without virus. The mice were kept in isolators and were observed daily for clinical disfunctions. Four weeks after injection, they were bled and fixed by total perfusion through the heart with about '7 ml of 4% formaldehyde in phosphate-buffered saline. Brains and spinal cords were embedded in paraffine, and sections were stained with haematoxylin-eosin.

Choice of the Restrictive (40°) and Permissive @lo) Temperature, Growth Kinetics of Wild-Type MHV-A59 at These Temperatures Wild-type virus plaqued equally well on mouse L cells at 31", 37", and 40". Also in Sac(-) cells similar virus titers were reached at the three temperatures ( Fig.  1A ; Spaan et al., 1981) but whereas maximum virus yields in the medium were obtained at 8 hr at 40", about 16 hr was needed at 31". As described before (Spaan et aZ., 1981) , virus yield at 37" is maximal at 10 hr p.i. The accumulation of virusspecific RNA was determined by measuring the incorporation of rH]uridine into actinomycin D-treated Sac(-) cells. Figure 1B shows that it closely followed the curve for virus release. On the basis of this experiment virus-specific RNA synthesis in mutants was studied from 8 to 16 hr at 31" and from 4 to 9 hr at 40'.

EGFluorouracil was chosen as a mutagen under conditions where it induced l-2% ts mutants (Van Berlo et cd, 1980), a pro- portion at which multiple ts lesions are not likely to occur. A prescreening of ts mutants was made on the basis of their inability to induce syncytia at 40'. This resulted in 148 potential mutants out of a total of 1284 isolated plaques. The final selection was made by plaquing potential mutants at the permissive and restricted temperature.

Only those mutant having a relative plating efficiency 40"/31" of 3 X 1O-3 or less were kept. This resulted in 20 mutants, i.e., 1.6% of the initial number of virus clones. A number of biological characteristics of the mutants are summarized in Table  . Among them are the yields after one step growth at 40", 37", and 31" which allow calculations of the leakiness of a mutant. Knowledge of virus growth at 37", the approximate body temperature of mice (Weir, 1947) is relevant for the interpretation of in vivo infection experiments.

The ts mutants were tested for their ability to synthesize RNA at the permissive and restrictive temperature.

Infected Sac(-) cells were labeled with rH]uridine in the presence of actinomycin D. Cells infected with wild-type virus incorporated comparable amounts of permissive and r sulting in a 30 synthesis over infected cells. Four of the ts mutants were able to synthesize virus-specific both the restrictive and the temperature, giving a 10-to 25 ulation compared to aetino treated, mock-infected cells. The other 16 ts mutants were RNA-, i.e., vir~s-s~e~i~~ RNA-synthesis at 40" was twofold or less above the background in mock-i~fe~t~~ cells.

Cells infected with the RNA+ rn~t~~~t~ (ts-43, ts-201, ts-209, and ts-379) w no1 extracted and virus-specific RN these cells were analyzed. As show 2, no qualitative differences were between the set of virus-specific fected in these cells compared to th present in cells infected with wild with wild type MHV-A59 (ts') or RNA+ ts mutants grown at 31" and 40'. Gels were loaded with equal amounts (50,000 cpm) of radioactivity of the virus-specific RNAs synthesized at 31", which were compared with RNA extracted from an equal number of cells incubated at 40". Fluorographs were exposed for '7 days.

rus. The RNA-mutants were also analyzed to see whether there was still some preferential synthesis of one or more of the RNAs; none was detected.

Proteins in Cells Infected with ts Mutants RNA+ mutants were tested for their ability to synthesize proteins at the permissive and restrictive temperature.

Virus-specific intracellular proteins were labeled with

[35S]methionine and immunoprecipitated.

As described previously (Rottier et ah, 1981b) , Sac(-) cells infected with wild-type MHV-A59 contain gp150, the precursor of the virion proteins gp90/180/EZ, the nucleocapsid protein pp54/N, from which during the immunoprecipitation procedure usually two smaller polypeptide species arise (Rottier et ak, 1981a) , and the three forms of the matrix protein El, ~24, gp25.5, gp26.5 (Fig.  3) . These same proteins were observed in cells infected with mutants ts-201, ts-209, and ts-379 both at the permissive and restrictive temperature.

However, ts-209-infected cells contained an extra 46K protein band, which could be a degradation product of the nucleocapsid protein generated during immunoprecipitation.

The fact that it is also found in straight cell lysates (not shown) argues against this, however.

A protein of about 120K was present in cells infected with ts mutants at 31". Its nature is still unclear. It does not comigrate with ~110, the unglycosylated precursor of gp150, found in tunicamycinetreated cells (data not shown).

Surprisingly, ts-43 although RNA+ was unable to synthesize viral proteins at 40°, although some nucleocapsid protein might be present (Fig. 3 ).

Wild-type MHV-A59 is highly pathogenic for mice as was described before (Manaker et al., 1961) . We have found that 50 to 100% of 4-week-old Balb/c mice infected intraperitoneally or intracerebrally with this virus die within 1 week from hepatitis or encephalitis, respectively. Ts mutants are highly attenuated in their neuropathogenic

properties. Of the mutants tested so far (ts-43, ts-169, ts-201, ts-209, ts-276, ts-299, ts-342, and ts-379) , lo5 PFU injected intracerebrally did not result in any clinical illness, with the exception of ts-169 which caused transient clinical symptoms 6 to 7 days p.i. Full details will be published elsewhere, but relevant at this moment is that histological sections of the central nervous system of mice infected with ts-342 sacrificed 4 weeks after infection showed, compared with control animals, obvious histological changes (Fig. 4) Rottier et al. (1981b) .

although the mice were clinically healthy. areas of spongy degeneration were d in the white matter of the spinal cord. These areas were not restricted to any particular tract. Similar areas were seen scattered throughout the brain stem, where also mainly the white matter appeared to be involved. Occasionally structures resembling swollen degenerating ax-0 and macrophages were noted in the a eted areas. Mild microglial reaction, and especially in the brain stem, astroglial reaction accompanied the lesions. There was otherwise little or no inflammatory reaction, except for a rare perivascular cuff.

In some of the mice, dilation of the ventricular system, especially of the lateral ventricles, occurred, with partial disappearance of the ventricular ependyma and rarefaction of the periventricular cortical aera. Similar, although less extensive, pathological changes were found in sections of the central nervous system obtained from mice infected with ts-43 and ts-201. DISCUSSION 'Ibis paper reports the isolation and initial characterization of a set of MHV-A59 ts mutants. Only 4 of tants were able to i RNA in infected cell temperature. Assum bution of mutations estimating about 55% of the coding capacity specifying replicative functions , this is a su~~~i~in~~~ small proportion. It might reflect a greater sensitivity of the viral polymerize to mutations compared to the viral struetural proteins. A similar large ~ro~~~ti~~ of RNA-mutants was found in a set of ts mutants of ~~V-J~~ (Leibowitz et o2+ 1982) .

The RNA-mutants will be u 1 for study of the viral genes invol in v RNA synthesis. Our preliminary unpublished experiments have demonstrated t in MHV-A59 at least five corn groups are involved in viral sis. This unexpectedly high agreement with data from Leibowitz et cx,l.

Since protein synthesis is unlike1 cur without RNA synthesis, snly mutants were tested for their a~~Iity to There is some confusion about the pathogenic properties of MHV-A59. Some investigators have reported that the virus has a very low virulence and a weak pathogenicity (Lai and Stohlman, 1981; Robb et al, 1979) . This might be explained by assuming that these investigators were working with a nonpathogenic variant of the original virus isolated by Manaker et al. (1961) . A more likely explanation, however, is that the mice used for the pathogenic studies had antibodies against mouse hepatitis virus (Robb et al., 1979) . We have always checked our mice for the absence of anti-MHV antibodies by a plaque reduction assay, before they were used for pathogenic studies. Doing so we were able to confirm the observation of Manaker et al (1961) that the virus is indeed highly pathogenic and virulent, when injected intracerebrally or intraperitoneally. We show here that attenuated virus has drastically attenuated pathogenic properties and is able to cause more prolonged infection of the central nervous system. In this respect there seems to be little differenee between so-called hepatotropic MHV-A59 and ~e~rotro~i~ detailed study on the time course of the development of the lesions in the nervous system and the cells inv in progress.

We assume that the ts defects are due to a change in one single nuele though during mutagenesis i other non-ts lesions will have duced. Therefore, it is not sure the ts lesion itself or additional rn~t~ti~~s are responsible for the altered patho properties of the virus. The high leakiness of ts-342 at 37" suggests that in thi. 

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