pseudotypevirionsformedbetweenmousehepatitisvirusand...

JOURNAL OF VIROLOGY, July 1995, p. 4237–4244 Vol. 69, No. 70022-538X/95/$04.0010Copyright q 1995, American Society for Microbiology

Pseudotype Virions Formed between Mouse Hepatitis Virus andLactate Dehydrogenase-Elevating Virus (LDV) Mediate LDVReplication in Cells Resistant to Infection by LDV Virions

CHEN EVEN AND PETER G. W. PLAGEMANN*

Department of Microbiology, Medical School, University of Minnesota, Minneapolis, Minnesota 55455

Received 3 January 1995/Accepted 20 March 1995

Infection of cultures of peritoneal macrophages with both lactate dehydrogenase-elevating virus (LDV) andmouse hepatitis virus (MHV) resulted in the formation of pseudotype virions containing LDV RNA whichproductively infected cells that are resistant to infection by intact LDV virions but not to infection by MHV.These cells were mouse L-2 and 3T3-17Cl-1 cells as well as residual peritoneal macrophages from persistentlyLDV-infected mice. Productive LDV infection of these cells via pseudotype virions was inhibited by antibodiesto the MHV spike protein or to the MHV receptor, indicating that LDV RNA entered the cells via particlescontaining the MHV envelope. Simultaneous exposure of L-2 cells to both LDV and MHV resulted in infectionby MHV but not by LDV. The results indicate that an internal block to LDV replication is not the cause of theLDV nonpermissiveness of many cell types, including the majority of the macrophages in an adult mouse.Instead, LDV permissiveness is restricted to a subpopulation of mouse macrophages because only these cellspossess a surface component that acts as an LDV receptor.

Lactate dehydrogenase-elevating virus (LDV) of mice, alongwith equine arteritis virus, simian hemorrhagic fever virus, andporcine reproductive and respiratory syndrome virus, belongsto a new, not-yet-named family of positive-strand RNA viruses(reviewed in references 15 and 17). The organization and rep-lication of the genomes of these viruses resemble those ofcoronaviruses. Their replication involves the formation of a39-coterminal nested set of mRNAs (15). On the other hand, inmorphology and genome size (14 to 15 kb), these viruses differgreatly from coronaviruses. They are enveloped viruses with adiameter of 50 to 60 nm and possess a spherical, rather than ahelical, nucleocapsid 25 to 30 nm in diameter and structuralproteins with relatively low molecular weights (15, 17). Thestructural proteins are a 12- to 13-kDa nucleocapsid protein(VP-1/N), an 18- to 19-kDa nonglycosylated envelope protein(VP-2/M), and two envelope glycoproteins, a primary one(VP-3) of 25 to 42 kDa, which functions as virus attachmentprotein, and a minor one of 25 to 30 kDa with unknownfunction (15).These viruses have other properties in common. For exam-

ple, they replicate primarily in macrophages and can establishpersistent infections in their respective hosts (15). LDV repli-cates only in a subpopulation of mouse macrophages. Up to70% of total peritoneal macrophages of 1- to 2-week-old miceare LDV permissive, but during the next 2 to 3 weeks of life,the proportion of LDV-permissive macrophages in the perito-neum decreases to 5 to 15% (7, 14, 15, 17). The decrease in theproportion of LDV-permissive macrophages coincides with thecessation of the early replication of macrophages in the peri-toneum (12) and probably reflects a decrease in the proportionof macrophages that express a surface component that acts asan LDV receptor. The presence of such an LDV receptorprotein on LDV-permissive macrophages is indicated by the

finding that incubation of macrophages with trypsin rendersthem resistant to LDV infection but that the cells again be-come susceptible after another 24 h of propagation in theabsence of trypsin (8). Electron microscopic studies have sup-ported the view that LDV infection of macrophages is viareceptor-mediated endocytosis (8). Numerous macrophagecell lines, whether generated spontaneously or by transforma-tion with simian virus 40, murine leukemia viruses, or variousoncogenes, fail to support significant LDV replication, and sodo various other cell lines that have been tested (14, 15, 17, 22)or in vivo-activated mouse macrophages (8). It has been re-ported by Inada et al. (6) that various types of cells that areactively infected with ecotropic, xenotropic, or polytropic mu-rine leukemia viruses support a productive LDV infection, butother investigators have not been able to confirm this finding(14, 14a). The LDV receptor is probably a surface componentthat is expressed on only a specific subpopulation of residentmacrophages or during a specific stage of macrophage differ-entiation, but the nature of the receptor has not yet beenelucidated.A corollary to these conclusions is that the failure of most

cells, including the majority of peritoneal macrophages in anadult mouse, to support LDV replication is due to the lack ofthe LDV receptor. This hypothesis seems to be strengthenedby a recent report indicating that up to 40% of cells of variousdiverse lines can be productively infected by transfection ofLDV RNA (6). We have been unable to duplicate this highefficiency of transfection (4a). After transfection of cultures ofRAW264.7 and L929 cells with pure genomic LDV RNA (3)via lipofection, we observed a slight production of infectiousLDV (;104 50% infectious doses [ID50] per ml of culturefluid), but the number of infected cells was too low (,1%) tobe detectable by indirect fluorescent-antibody (IFA) staining(2, 14) with any degree of accuracy. Therefore, we used an-other approach to demonstrate that LDV tropism is primarilydetermined by the presence of an appropriate receptor, whichinvolved the infection of cells with pseudotype virions betweenLDV and mouse hepatitis virus (MHV) by utilizing the MHVreceptor. The surface components of different cells that func-

* Corresponding author. Mailing address: Department of Microbi-ology, University of Minnesota, Box 196 UMHC, 1435 Mayo, 420Delaware St. S.E., Minneapolis, MN 55455. Phone: (612) 624-3187.Fax: (612) 626-0623. Electronic mail address: [email protected].

4237

on July 8, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

http://jvi.asm.org/

tion as MHV receptors are isoforms of biliary glycoprotein(Bgp) of the carcinoembryonic antigen family (13, 24).

MATERIALS AND METHODS

Mice. FVB mice 4 to 6 weeks of age were provided by the transgenic facilityof the University of Minnesota. C58 mice were bred in the Department ofMicrobiology animal facilities. Pathogen-free B10.A mice were kindly suppliedby John Torseth and Dale Gregerson and were used in experiments immediatelyafter receipt, maintained separately from other mice, and monitored for anti-MHV antibodies.Cell cultures. Macrophage cultures were prepared from FVB and pathogen-

free B10.A mice in 60-mm-diameter petri plates or multiwell microscope slides(seeded with 53 104 peritoneal cells per well) as described previously (1, 14, 21).The macrophages were cultured in RPMI supplemented with 10% (vol/vol) fetalbovine serum and 10% (vol/vol) L-cell conditioned medium. L-2 and 3T3-17Cl-1cells were kindly supplied by Kathryn Holmes and propagated in RPMI supple-mented with 10% (vol/vol) fetal bovine serum. For experiments, the cells werecultured in 60-mm-diameter petri plates or in multiwell microscope slides seededwith 5 3 104 cells per well.LDV and MHV. LDV-P is a strain of LDV previously isolated in this labora-

tory (1). To obtain LDV-P free of anti-MHV antibodies, a pool of LDV-P wasprepared in pathogen-free B10.A mice (LDV-P-PF). It consisted of plasmaharvested from 1-day-infected mice and contained about 1010.0 ID50/ml. LDVwas titrated by an endpoint dilution assay (16) in FVB mice.MHV-A59 (23), a goat antiserum to the MHV spike (S/E2) protein (A04aE2),

and monoclonal antibody (MAb) CC1 to the MHV (Bgp) receptor (24) werekindly supplied by Kathryn Holmes. A pool of MHV-A59 was prepared byinfection of L-2 cells and contained about 107 PFU/ml. The virus was titrated byplaque assay in L-2 cells (23).Pseudotype virions. LDV-MHV pseudotype virions (PLMs) were produced by

infecting 1-day cultures of peritoneal macrophages simultaneously with LDV andMHV at multiplicities of about 100 ID50 per cell and 5 to 10 PFU per cell,respectively, and harvesting the culture fluid 24 h postinfection (p.i.). Cultures ofL-2 and 3T3-17Cl-1 cells or peritoneal macrophages were infected with PLMs asdescribed for the appropriate experiments. In antibody inhibition studies, MAbCC1 and anti-S/E2 antiserum were added to cell cultures simultaneously withMHV or PLMs at concentrations that inhibited plaque formation by MHV onL-2 cultures by .3 log10 units. At these antibody concentrations, the degree ofinhibition of plaque formation was about the same whether the antibodies wereadded to L-2 cell cultures together with MHV, the L-2 cells were preincubatedwith MAb CC1 for 30 min, or MHV was incubated with anti-S/E2 antiserum at378C for 30 min prior to use in the infection. Cell cultures were treated withmouse fibroblast alpha/beta interferon (IFN-a/b) and antibody to IFN-a/b asdescribed for the appropriate experiments. IFN-a/b and antibody to IFN-a/bwere purchased from Lee Biomolecular Research, Inc., San Diego, Calif.IFA staining. LDV-infected cells in cell cultures were detected by IFA staining

(2, 14). In brief, cultures in multiwell microscope slides were fixed in cold acetoneat various times p.i. and incubated sequentially, first with a MAb to the VP-3envelope glycoprotein of LDV (159-12) (5) and then with fluorescein isothio-cyanate-conjugated (FITC) goat anti-mouse immunoglobulin G (IgG) (SigmaChemical Co., St. Louis, Mo.) or with goat F(ab9)2 anti-mouse IgG (Caltag Labs,San Francisco, Calif.) to avoid any possibility of nonspecific staining of macro-phages. MHV-infected cells in cell cultures were detected by IFA staining asdescribed for the detection of LDV-infected cells, except that MAb 1.16.1,specific for the MHV nucleocapsid protein (4) (kindly supplied by Julian Lei-bowitz), was substituted for anti-LDV MAb 159-12. No nonspecific staining ofLDV-, MHV-, or PLM-infected mouse macrophages or L-2 cells was observed byincubation with an irrelevant MAb (kindly supplied by K. Holmes) or normalplasma from pathogen-free mice and FITC goat anti-mouse IgG or FITC goatF(ab9)2 anti-mouse IgG.Mouse anti-MHV antibodies were titrated as described previously for LDV

(2). Acetone-fixed replicate cultures of 7-h MHV-A59-infected cultures of L-2cells in multiwell microscope slides were incubated with twofold dilutions ofmouse plasma to be titrated for anti-MHV antibodies and then with FITC goatanti-mouse IgG. The antibody titer was expressed as the reciprocal of the highest

FIG. 1. Infection of L-2 cells with LDV-P-PF (A) or PLM-1 (B and C). Cultures of L-2 cells in multiwell microscope slides were infected with about 100 ID50 ofLDV-P-PF per cell (A) or with PLM-1 yielding multiplicities of infection of about 100 ID50 of LDV per cell, of ,1 PFU of MHV per cell, and of an unknown numberof PLMs containing LDV RNA (B and C). At 7 h p.i., the cells were fixed and assayed by IFA for LDV antigen-positive (A and B) or MHV antigen-positive (C) cells.Magnification, 3100.

TABLE 1. Yields of infectious LDV-P and MHV from peritonealmacrophage cultures infected with LDV or MHV alone

or in three mixed infections yielding PLMsa

Infecting virusYield of:

LDV (ID50/ml) MHV (PFU/ml)

LDV-P 1 3 108–1 3 109b

LDV-P-PF 1 3 109

MHV 6 3 105

LDV-P 1 MHV (PLM-1) 33 107 3 3 104

LDV-P-PF 1 MHV (PLM-2) 33 108 1.5 3 105

LDV-P-PF 1 MHV (PLM-3) 13 109 6 3 104

a One-day cultures of mouse peritoneal macrophages were infected with 100ID50 of conventional LDV-P (containing anti-MHV antibodies) or LDV-P-PFper cell and/or with 10 PFU of MHV-A59 per cell. The culture fluid washarvested at 24 h p.i. and titrated.b Range observed in several experiments.

4238 EVEN AND PLAGEMANN J. VIROL.


.org/D

ownloaded from

http://jvi.asm.org/

dilution of plasma that yielded recognizable staining of MHV-infected cellswithout staining any cells in uninfected cultures.

RESULTS

In initial experiments we found that coinfection of culturesof peritoneal macrophages from FVB or C58 mice with MHVand LDV-P, which consisted of plasma from 1-day-LDV-in-fected FVB or C58 mice raised in our facilities, resulted in amarked inhibition of MHV replication, while LDV replicatednormally. Whereas practically all macrophages became in-fected with MHV in the absence of LDV-P (20), only about20% of the cells became infected in the presence of LDV-P,and the 1-day MHV yield was reduced from 63 105 to 33 104

PFU/ml, respectively (Table 1). This finding was surprising,since the proportion of LDV-infected macrophages was only 5to 10% whether the macrophages were infected with LDValone or in combination with MHV. It turned out that all ofour LDV-P preparations contained high levels of anti-MHVantibodies (IFA titer 5 512), which might have been respon-sible for the inhibition of MHV replication. Thus, at least someof the mice used to generate our LDV-P pools had beenexposed to MHV. Subsequent studies demonstrated that by 4to 5 weeks of age practically all mice raised in our facilitiespossessed anti-MHV antibodies without ever showing any clin-ical symptoms.Regardless of the reduction of MHV replication by coinfec-

tion with anti-MHV antibody-contaminated LDV-P, PLMs

were produced in the mixed infection. When the virus pro-duced in the mixed infection (PLM-1; Table 1) was used toinfect L-2 cells and the cultures were fixed 7 h p.i., between 25and 50% of the cells were LDV antigen positive in repeatedexperiments (Fig. 1B), whereas none of the L-2 cells becameinfected when exposed to LDV-P alone (Fig. 1A). About 20%of the L-2 cells contained MHV antigen after infection with thepseudotype virion preparation, and some syncytium formationwas apparent at 7 h p.i. (Fig. 1C). Most of the LDV antigen waspresent in individual cells and not in the syncytia caused byMHV infection (Fig. 1B), indicating that LDV did not enterL-2 cells via formation of syncytia. This was also indicated bythe finding that no L-2 cells became LDV infected when L-2cell cultures were exposed to a mixture of MHV and LDV (seebelow). Furthermore, the infection of L-2 cells by PLMs wasinhibited by the presence of antibodies to the spike protein ofMHV (aE2) as well as by MAbs to the receptor for MHV(CC1). This finding will be documented in the next set ofexperiments.In attempts to improve the production of PLMs, we used

pathogen-free B10.A mice which proved to be devoid of MHVantibodies. We also prepared a pool of LDV-P in pathogen-free mice (LDV-P-PF). First, we found that an unexpectedlyhigh proportion (30 to 50%) of the peritoneal macrophagesfrom pathogen-free adult mice could be infected with LDVwhether or not the LDV pool contained anti-MHV antibodies(Fig. 2A). Second, we found that in a mixed infection with

FIG. 2. Infection of macrophage cultures with LDV-P-PF alone (A), MHV alone (B), or LDV-P-PF plus MHV (C and D). One-day cultures of peritonealmacrophages from pathogen-free mice in multiwell microscope slides were infected with about 100 ID50 of LDV-P-PF per cell (A), 10 PFU of MHV per cell (B), orboth (C and D). At 7 h p.i., the cultures were fixed and assayed by IFA for LDV antigen-positive cells (A and C) or MHV antigen-positive cells (B and D).Magnifications, 3128 (A, C, and D) and 380 (B).

VOL. 69, 1995 MHV-LDV PSEUDOTYPE VIRIONS MEDIATE LDV REPLICATION 4239


.org/D

ownloaded from

http://jvi.asm.org/

LDV-P-PF, MHV replication in macrophage cultures was stillreduced (Table 1), even though the LDV-P-PF was free ofanti-MHV antibodies. We attribute this reduction to the pres-ence of IFN-a/b in the LDV-P-PF preparation (see below).Nevertheless, considerable MHV replication was apparent inthese mixed infections, as evidenced by the formation of syn-cytia (Fig. 2C and D) and the presence of MHV antigen-positive cells (Fig. 2D), similar to the case of cultures infectedwith MHV alone (Fig. 2B). However, although the majority ofthe macrophages in the mixed infection contained MHV anti-gen at 7 h p.i., many cells seemed to contain only low levels ofthe nucleocapsid protein (Fig. 2D), probably as a result of asuppression of MHV replication by the IFN-a/b in the mixedinoculum. About 25% of the cells became LDV infected in themixed infection (Fig. 2C). The virus titers in the culture fluid(PLM-3) at 1 day p.i. were 1 3 109 ID50/ml for LDV and 6 3104 PFU/ml for MHV, and similar yields were observed inanother such mixed infection (PLM-2; Table 1).Upon infection of L-2 cells with the virus produced in one

such mixed macrophage infection (PLM-3), about 50% of thecells became infected with LDV (Fig. 3A and B). In thisPLM-3 infection, most of the L-2 cells became infected byMHV, as illustrated by the massive formation of syncytia (Fig.3A and B) and IFA staining (see below). Many LDV-infectedcells were present in syncytia (Fig. 3A). However, single LDV-infected L-2 cells showed a perinuclear accumulation of LDVantigen (Fig. 3B) typically found in LDV-infected macro-

phages (14, 15, 17). The LDV titer of the culture fluid at 24 hp.i. was 109.0 ID50/ml, which is comparable to the yield of LDVfrom macrophage cultures (Table 1) and indicated that theLDV infection of L-2 cells was productive. Infections of theL-2 cultures with either LDV via pseudotype virions or MHVwere almost completely inhibited by addition of antibodies tothe S/E2 protein of MHV (Fig. 3C) or antibodies to the MHVreceptor (Fig. 3D). The inhibition of MHV replication by theaE2 and CC1 antibodies was indicated by the absence of syn-cytium formation (Fig. 3C and D, respectively), the lack of IFAstaining for MHV antigens (data not shown), and a reductionin virus yield of about 4 log10 units. These findings prove thatthe productive LDV infection of L-2 cells via the pseudotypevirions was mediated by an interaction between the MHVspike protein and the MHV receptor. The lack of any LDVreceptor on L-2 cells was indicated by the failure of LDV tobind to L-2 cells. Four successive 30-min adsorptions of asuspension of LDV (106.0 ID50/ml) to L-cell cultures at 378Cdid not reduce the titer significantly, whereas a similar passageof the LDV suspension over macrophage cultures reduced theLDV titer by 1 log10 unit.IFA staining for LDV antigen at 7 h p.i. (Fig. 3) detects only

cells that have initially become infected with pseudotype viri-ons, since little, if any, progeny virus is released from infectedcells before this time. In any case, since LDV does not infectL-2 cells, any progeny LDV virions cannot infect other cells inthe culture. However, since the PLM preparations also con-

FIG. 3. Infection of L-2 cells with PLM-3 and effect of anti-MHV S/E2 protein antibody (aE2) or anti-MHV receptor antibody (CC1) on the infection of the cellsby pseudotype virions containing LDV RNA. Cultures in multiwell microscope slides were infected with PLM-3 to yield multiplicities of infection of about 100 ID50of LDV per cell, of ,1 PFU of MHV per cell, and of an unknown number of pseudotype virions containing LDV RNA. For panels C and D, anti-S/E2 antiserum orMAb CC1, respectively, was added at the same time as PLM-3. At 7 h p.i., the cultures were fixed and all were assayed by IFA staining for LDV antigen-positive cells.Magnifications, 3100 (A, C, and D) and 3160 (B).



.org/D

ownloaded from

http://jvi.asm.org/

tained low concentrations of MHV, it is possible that mixedinfections of some single cells with both pseudotype virions andMHV could have resulted in the production of new pseudotypevirions, which later could have spread the LDV infection toadditional cells in culture.LDV infection was also mediated by PLMs in 3T3-17Cl-1

cells (Fig. 4A). Between 2 and 17% of the 3T3-17C1 cellsbecame infected with LDV after exposure to PLM-2 or PLM-3in repeated experiments (Fig. 4A), and most cells were in-fected with MHV (Fig. 4B). No LDV infection was observed in3T3-17Cl-1 cells that were exposed to LDV-P-PF alone (Fig.4C). LDV infection of 3T3-17Cl-1 cells via PLMs or MHV wasalso inhibited by antibodies to the MHV S/E2 protein or to theMHV receptor (data not shown).In another experiment, we demonstrated that residual mac-

rophages from persistently LDV-infected mice can be infectedby PLMs. Upon an initial infection of a mouse by LDV, allavailable LDV-permissive macrophages are infected and de-stroyed within the first 2 days p.i. (15, 17, 19, 21). Thereafter,a lifelong persistent infection is established by replication ofLDV in new permissive macrophages that are slowly but con-tinuously regenerated in lymphoidal tissues of the mouse (15,17–18). None of the macrophages in the peritoneum in thesemice was found to support LDV replication in vitro (15, 21,22), but whether this nonpermissiveness was due to a lack of anLDV receptor or an internal block to LDV replication was notknown. In order to resolve this uncertainty, peritoneal macro-phages were harvested from 5-day-LDV-infected mice and cul-tured for 1 day, and replicate cultures were then superinfectedwith LDV or PLMs or were not treated. Seven hours later, thecultures were fixed and assayed by IFA staining for LDV-infected cells. The results in Fig. 5A and B show that none ofthe peritoneal macrophages from a 5-day-LDV-infectedmouse contained LDV antigen or could be productively in-fected by LDV, respectively, in agreement with previous re-sults (21, 22). On the other hand, exposure to PLM-2 orPLM-3 resulted in infection of between 20 and 40% of the

macrophages in repeated experiments (Fig. 5C to E). SomeLDV-infected cells had rounded up by 6.5 to 7 h p.i. andformed clumps (Fig. 5D), but some single infected macro-phages had retained their morphology and exhibited typicalstaining for LDV antigens (Fig. 5C and E). There was alsoalready considerable syncytium formation at 7 h p.i. withPLM-3 (Fig. 5D). The results demonstrate that the residualmacrophages in LDV-infected mice are not intrinsically resis-tant to LDV infection but cannot be infected with intact viri-ons, presumably because they lack a surface component thatacts as an LDV receptor.In control experiments, we observed that LDV-P-PF in a

mixed infection of L-2 cells with MHV also reduced syncytiumformation by MHV as observed in macrophage cultures (Fig.6A and B). Whereas infection of L-2 cells by MHV aloneresulted in fusion of practically all cells in the culture by 24 hp.i. and the syncytia contained large amounts of MHV antigen(Fig. 6A), no syncytia were apparent in the L-2 cell culturesinfected simultaneously with MHV and LDV-P-PF, even at 24h p.i. (Fig. 6B). The data in Fig. 6B also show that none of theL-2 cells became infected with LDV in a mixed infection withMHV, which contrasts sharply with the results of infection bythe pseudotype virions prepared in macrophage cultures (Fig.6C). Since the LDV-P-PF preparation was free of anti-MHVantibodies and LDV did not infect the L-2 cells under theseconditions (Fig. 6B), a factor(s) other than LDV must beresponsible for the inhibitory effect. A soluble factor seemedresponsible, since LDV-P-PF partially purified by centrifuga-tion through a 0.5 M sucrose layer (2, 3) or pseudotype virionsharvested from macrophage cultures had no significant effecton MHV infection of L-2 cells at comparable multiplicities ofinfection (Fig. 6C and data not shown). It seems most likelythat IFN-a/b is the responsible factor, since (i) IFN-a/b isknown to be present in high concentrations (2,000 IU/ml) inthe plasma of 1-day-LDV-infected mice (11, 15, 19), probablybeing generated by the large number of macrophages thatbecome infected in a mouse during the first day p.i. (15, 17),

FIG. 4. Infection of 3T3-17Cl-1 cells with PLM-2 (A and B) or LDV-P-PF alone (C). Multiwell microscope cultures were infected with PLM-2 to yield multiplicitiesof infection of about 100 ID50 of LDV per cell and ,1 PFU of MHV per cell plus an unknown number of PLMs or with 100 ID50 of LDV-P-PF per cell alone. At7 h p.i., the cultures were fixed and assayed by IFA for LDV antigen-positive cells (A and C) or MHV antigen-positive cells (B). Magnification, 3100.



.org/D

ownloaded from

http://jvi.asm.org/

(ii) the effect was mimicked by addition of IFN-a/b. (MHVreplication is relatively susceptible to inhibition by IFN-a/b,whereas LDV replication is relatively resistant [20, 22]), and(iii) the inhibition by LDV-P-PF was partially prevented by thepresence of anti-IFN-a/b antibodies.These results are documented in the following experiment.

To allow direct comparison of the data, replicate cultures ofmacrophages and L-2 cells were infected with MHV in theabsence and presence of LDV-P, LDV-P-PF, and IFN-a/b. At1 day p.i., the culture fluid was assayed for infectious MHV(Table 2), and the cells were analyzed for MHV antigen andsyncytium formation. LDV-P, which contains anti-MHV anti-bodies (see above), strongly (99 to 99.9%) inhibited MHVreplication in both types of cells. LDV-P-PF, which lacks theseantibodies, was also inhibitory but less so (about 90%). Theinhibition was largely prevented by the presence of anti-IFNa/b antibodies. Addition of normal mouse plasma at thetime of MHV infection had no detectable effect, but additionof IFN-a/b at concentrations comparable to those found in theplasma of 1-day-LDV-infected mice mimicked the effect ofLDV-P-PF. This treatment reduced the production of infec-tious MHV, and, just as observed for LDV-P-PF, syncytiumformation was completely blocked and little new MHV antigenwas detectable in cells at 24 h p.i. (data not shown; see Fig. 6B).The inhibitory effect of IFN-a/b was surprising, since it wasadded simultaneously with MHV, but it might have resultedfrom the use of relatively high concentrations of IFN-a/b. We

thought that preincubation of the L-2 cells with IFN-a/b orLDV-P-PF might enhance the degree of inhibition of virusproduction, but this was not found to be the case, except forthe treatment of macrophage cultures by IFN-a/b (Table 2).

DISCUSSION

It was found in earlier studies that pseudotypes containingthe Sindbis virus genome and an LDV envelope can infectchicken cells which are resistant to productive LDV infection(9). The results were interpreted to indicate that, contrary toour results, resistance by many cell types to LDV is not due toa lack of a receptor but rather to an internal block to LDVreplication. However, it was not ruled out in those experimentsthat infection of the chicken cells was via pseudotype virionsthat contained both LDV and Sindbis virus envelope glyco-proteins (17). Such pseudotype virions are not a problem inour present study, which demonstrates that LDV replication isinitiated by PLMs in various types of cells that are normallyresistant to LDV infection but susceptible to MHV infection.These pseudotype virions must possess the LDV genome andan envelope containing the MHV spike (S/E2) protein, whichallows interaction with the MHV receptor on the host cells.Our results clearly show that such pseudotype virions con-

taining the LDV genome and MHV envelope glycoproteinsare readily formed during mixed infection of macrophages withthe two viruses. The structure of the pseudotype virions is

FIG. 5. Infection of peritoneal macrophages from 5-day-LDV-infected mice with PLMs. Pathogen-free B10.A mice were injected intraperitoneally with 106 ID50of LDV-P-PF. At 5 days p.i., their peritoneal macrophages were harvested and cultured for 1 day in multiwell microscope slides. Duplicate wells were infected withabout 100 ID50 of LDV-P-PF per cell (B), with PLM-2 (C and E), or with PLM-3 (D) to yield multiplicities of about 100 ID50 of LDV per cell and ,1 PFU of MHVper cell, or they were untreated (A). At 6.5 h (C and D) or 7 h (A, B, and D) later, the cultures were fixed and all were assayed by IFA for LDV antigen-positive cells.Magnifications, 380 (A and C), 3100 (D), and 3128 (B and E).



.org/D

ownloaded from

http://jvi.asm.org/

unclear, but they might be MHV-like particles that contain oneor more LDV spherical nucleocapsids instead of the MHVhelical nucleocapsid. These pseudotype virions efficiently in-fect L-2 and 3T3-17Cl-1 cells, as well as residual macrophagesin persistently LDV-infected mice, all of which are resistant toinfection by LDV virions. The infection is productive, resultingin the release of between 103 and 104 ID50 of LDV per cell, ayield comparable to that from LDV-permissive macrophages(17, 21). The results support the view expressed previously (8,

15, 17) that LDV infection is restricted to a subpopulation ofmacrophages possessing a specific surface component that actsas an LDV receptor and is not present on all other types ofcells that have been examined, including the majority of mac-rophages in mice.LDV-infected mice do not develop clinical symptoms, in

spite of the continuous destruction of the LDV-permissivemacrophages, because the subpopulation of LDV-permissivemacrophages is relatively small and does not possess an essen-tial irreplaceable function (15, 17). However, these macro-phages play a role in the clearance of muscle-type lactatedehydrogenase and some other blood enzymes, and continu-ous destruction of this subpopulation leads to impaired clear-ance of lactate dehydrogenase and the other enzymes andtherefore to their elevation in the blood (15, 17, 19).Our results also reemphasize potential pitfalls in studying

mixed infections involving MHV and another agent propa-gated in mice. Since inapparent MHV infections are wide-spread in mouse colonies (10), material prepared in mice maycontain high concentrations of anti-MHV antibodies that blockMHV infections of cells in culture. In addition, in the case ofLDV, 1-day LDV pools prepared in mice contain high concen-trations of IFN-a/b, which, as shown in the present study, canalso strongly inhibit MHV replication in cell culture even whenadded simultaneously with MHV.

ACKNOWLEDGMENTS

We thank Anjeli Bandyopadhyay for technical assistance and Col-leen O’Neill for typing the manuscript.

REFERENCES

1. Brinton-Darnell, M., and P. G. W. Plagemann. 1975. Structural and chem-ical-physical characteristics of lactate dehydrogenase-elevating virus and itsRNA. J. Virol. 16:420–433.

2. Cafruny, W. A., S. P. K. Chan, J. T. Harty, S. Yousefi, K. Kowalchyk, D.McDonald, B. Foreman, G. Budweg, and P. G. W. Plagemann. 1986. Anti-

FIG. 6. Infection of L-2 cells with MHV alone (A), with MHV plus LDV-P-PF (B), or with PLM-3 (C). Cultures of L-2 cells in multiwell microscope slides wereinfected with 10 PFU of MHV per cell (A), with MHV plus 100 ID50 of LDV-P-PF per cell (B), or with PLM-3 (C) as described in the legend to Fig. 3. At 24 h p.i.(A and B) or 7 h p.i. (C), the cultures were fixed and assayed by IFA staining for MHV antigen-positive cells (A) or LDV antigen-positive cells (B and C). Magnification,361.

TABLE 2. Effects of LDV and IFN-a/b on MHV replicationin macrophage and L-2 cell culturesa

Preincubation Addition with MHV

MHV yield(PFU/ml) from:

Macrophages L-2 cells

None None 6.4 3 105 4.4 3 106

LDV-P 2.7 3 103 3.2 3 102

LDV-P-PF 3.0 3 104 7.4 3 105

LDV-P-PF 1 anti-IFN-a/b NDb 3.3 3 106

LDV-P-PF None 1.6 3 104 9.0 3 105

NMP-PF 6.0 3 105 5.0 3 106

NMP-PF None 6.4 3 105 4.6 3 106

IFN-a/b 1.2 3 104 2.1 3 105

IFN-a/b None 3.0 3 102 4.5 3 105

a Replicate 1-day cultures of peritoneal macrophages or L-2 cells in multiwellmicroscope slides were infected with about 5 PFU of MHV per cell. Whenindicated, the MHV inoculum was supplemented with about 100 ID50 of LDV-Por LDV-P-PF per cell, 2,500 U of IFN-a/b per well, 500 neutralizing units ofanti-IFN-a/b antibody per well, or 10 ml of normal mouse plasma from pathogen-free mice (NMP-PF) per well, or the cultures were preincubated with LDV-P-PFor IFN-a/b for 1 day, the culture fluid was removed, and the cells were infectedwith MHV. At 22 h p.i. with MHV, the culture fluid was removed and assayed forinfectious MHV by plaque assay. The values are means for triplicate plates. Theremaining adhering cells were fixed and analyzed by IFA for MHV antigens (notshown).b ND, not determined.



.org/D

ownloaded from

http://jvi.asm.org/

body response of mice to lactate dehydrogenase-elevating virus during in-fection and immunization with inactivated virus. Virus Res. 5:357–375.

3. Chen, Z., K. S. Faaberg, and P. G. W. Plagemann. 1994. Detection ofnegative-stranded subgenomic RNAs but not of free leader in LDV-infectedmacrophages. Virus Res. 34:167–177.

4. Denison, M. R., P. W. Zoltick, J. L. Leibowitz, C. J. Pachuk, and S. R. Weiss.1991. Identification of polypeptides encoded in open reading frame 1b of theputative polymerase gene of the murine coronavirus mouse hepatitis virusA59. J. Virol. 65:3076–3082.

4a.Faaberg, K. S., C. Even, and P. G. W. Plagemann. Unpublished data.5. Harty, J. T., and P. G. W. Plagemann. 1990. Monoclonal antibody protectionfrom age-dependent poliomyelitis: implications regarding the pathogenesisof lactate dehydrogenase-elevating virus. J. Virol. 64:6257–6262.

6. Inada, T., H. Kikuchi, and S. Yamazaki. 1993. Comparison of the ability oflactate dehydrogenase-elevating virus and its virion RNA to infect murineleukemia virus-infected or -uninfected cell lines. J. Virol. 67:5698–5703.

7. Inada, T., and C. A. Mims. 1985. Pattern of infection and selective loss of Iapositive cells in suckling and adult mice inoculated with lactic dehydrogenasevirus. Arch. Virol. 86:151–165.

8. Kowalchyk, K., and P. G. W. Plagemann. 1985. Cell surface receptors forlactate dehydrogenase-elevating virus on subpopulation of macrophages.Virus Res. 2:211–229.

9. Lagwinska, E., C. C. Stewart, C. Adles, and S. Schlesinger. 1975. Replicationof lactic dehydrogenase virus and Sindbis virus in mouse peritoneal macro-phages. Induction of interferon and phenotypic mixing. Virology 65:204–214.

10. Lindsey, J. R., D. B. Casebolt, and G. H. Cassell. 1986. Managing conductand data quality of toxicology studies, p. 155–171. Princeton Scientific,Princeton, N.J.

11. Lussenhop, N., B. Holmes, W. A. Cafruny, and P. G. W. Plagemann. 1982.Acute infection of mice with lactate dehydrogenase-elevating virus enhancesFc and complement receptor activity of peritoneal macrophages. J. Gen.Virol. 61:25–32.

12. Metcalf, D., M. J. Elliott, and N. A. Nicola. 1992. The excess numbers ofperitoneal macrophages in granulocyte-macrophage colony-stimulating fac-tor transgenic mice are generated by local proliferation. J. Exp. Med. 175:877–884.

13. Nedellec, P., G. S. Dveksler, E. Daniels, C. Turbide, B. Chow, A. A. Basile,K. V. Holmes, and N. Beauchemin. 1994. Bgp2, a new member of thecarcinoembryonic antigen-related gene family, encodes an alternative recep-

tor for mouse hepatitis viruses. J. Virol. 68:4525–4537.14. Onyekaba, C. O., J. T. Harty, and P. G. W. Plagemann. 1989. Extensive

cytocidal replication of lactate dehydrogenase-elevating virus in culturedperitoneal macrophages from 1–2-week-old mice. Virus Res. 14:327–338.

14a.Palmer, G. A., and P. G. W. Plagemann. Unpublished data.15. Plagemann, P. G. W. 1995. Lactate dehydrogenase-elevating virus and re-

lated viruses, p. 1105–1120. In B. M. Fields, D. M. Knipe, and P. M. Howley(ed.), Virology. Raven Press, New York.

16. Plagemann, P. G. W., K. F. Gregory, H. E. Swim, and K. K. W. Chan. 1963.Plasma lactic dehydrogenase-elevating agent of mice: distribution in tissuesand effect on lactic dehydrogenase isozyme patterns. Can. J. Microbiol.9:75–86.

17. Plagemann, P. G. W., and V. Moennig. 1992. Lactate dehydrogenase-elevat-ing virus, equine arteritis virus, and simian hemorrhagic fever virus: a newgroup of positive-strand RNA viruses. Adv. Virus Res. 41:99–192.

17a.Rowland, R. R. R., et al. Unpublished data.18. Rowland, R. R. R., C. Even, G. W. Anderson, Z. Chen, B. Hu, and P. G. W.

Plagemann. 1994. Neonatal infection of mice with lactate dehydrogenase-elevating virus results in suppression of humoral antiviral immune responsebut does not alter the course of viraemia or the polyclonal activation of Bcells and immune complex formation. J. Gen. Virol. 75:1071–1081.

19. Rowson, K. E. K., and B. W. J. Mahy. 1985. Lactate dehydrogenase-elevatingvirus. J. Gen. Virol. 66:2297–2312.

20. Smith, M. S., R. E. Click, and P. G. W. Plagemann. 1984. Control of mousehepatitis virus replication in macrophages by a recessive gene on chromo-some 7. J. Immunol. 133:428–432.

21. Stueckemann, J. A., M. Holth, W. J. Swart, K. Kowalchyk, M. S. Smith, A. J.Wolstenholme, W. A. Cafruny, and P. G. W. Plagemann. 1982. Replication oflactate dehydrogenase-elevating virus in macrophages. 2. Mechanism of per-sistent infection in mice and cell culture. J. Gen. Virol. 59:263–272.

22. Stueckemann, J. A., D. M. Ritzi, M. Holth, M. S. Smith, W. J. Swart, W. A.Cafruny, and P. G. W. Plagemann. 1982. Replication of lactate dehydroge-nase-elevating virus in macrophages. 1. Evidence for cytocidal replication. J.Gen. Virol. 59:245–262.

23. Sturman, L. S., and K. V. Holmes. 1983. The molecular biology of corona-viruses. Adv. Virus Res. 28:35–112.

24. Williams, R. K., G.-S. Jang, and K. V. Holmes. 1991. Receptor for MHV isa member of the carcinoembryonic antigen family of glycoproteins. Proc.Natl. Acad. Sci. USA 88:5533–5536.



.org/D

ownloaded from

http://jvi.asm.org/

pseudotypevirionsformedbetweenmousehepatitisvirusand...

Documents