Replication of lactic dehydrogenase virus and Sindbis virus in mouse peritoneal macrophages. Induction of interferon and phenotypic mixing

V~OLOCY 65,204-214(1975)

Replication in Mouse

of Lactic Dehydrogenase Peritoneal

Macrophages.

Induction

Phenotypic ELIZABETH

LAGWINSKA,’

Virus and Sindbis of Interferon

Virus and

Mixing

CARLETON C. STEWART,* SONDRA SCHLESINGER’

CHERYL

ADLES,*

AND

Department of Microbiology,’ Division of Biology and Biomedical Sciences,’ Section of Cancer Biolog.y,z Division of Radiation Oncology, Washington University School of Medicine, St. Louis, Missouri 63110 Accepted

January

28, 1975

Mouse peritoneal macrophages stimulated by thioglycollate can proliferate in culture for over 2 wk. Sindbis virus replicates in these cells, but not in the initial exudate culture. Lactic dehydrogenase virus (LDV) also produces higher yields in the exponentially growing cells. Proliferating macrophages infected with LDV induce a factor that inhibits the replication of Sindbis virus. The factor was identified as interferon by its species specificity and its acid stability. Macrophages that have been in culture for only 24 hr do not produce interferon. Replication of Sindbis virus in LDV-infected macrophages leads to phenotypic mixing between the two virions. We have detected both pure pseudotypes-virions containing the RNA of Sindbis virus and the envelope of LDV-and particles containing the envelope proteins of both virions. Our results demonstrate that the restricted host range of LDV is not due to the inability of the virus to adsorb to cells other than macrophages. INTRODUCTION

Lactic dehydrogenase virus (LDV) is a small RNA enveloped virus first identified as a passenger in murine tumors (Riley et al., 1960). The virus by itself is not tumorogenie but does establish a persistent infection in the mouse. Although infection by LDV was first considered “benign” (Riley, 1968) more recent studies have demonstrated rather diverse effects of this virus on the immune system (Notkins, 1971). Thus, acute infection with LDV causes destruction of the thymus-dependent areas of the spleen and the paracortical regions of the mesenteric lymph nodes (Snodgrass et al., 1972; Proffitt et al., 1972) and leads to enhanced tumorogenicity of the plasmacytoma MOPC-315 (Michaelides and Schlesinger, 1974). Chronic infection with this virus appears to protect NZB mice from the autoimmune disease characteristic of this strain (Oldstone and Dixon, 1972). Although LDV is similar to members of

the group B togaviruses or flaviviruses with respect to structural proteins (Michaelides and Schlesinger, 1973) and the size of the virion RNA (Darnell and Plagemann, 1972), it is peculiar in its very limited host range. LDV appears to replicate only in the phagocytic cells of the reticulum of the mouse (Snodgrass et al., 1972). The virus has been propagated in vitro in peritoneal macrophages (Evans, 1970) and in primary mouse embryo cells (Yamazaki and Notkins, 1973). Primary mouse embryo cells also contain macrophages (Stewart et al., unpublished results) and it is most likely that LDV is replicating in the latter. Yamazaki and Notkins (1973) reported that LDV is present in less than 2% of the primary mouse embryo cells. The ability of peritoneal macrophages to proliferate in culture is greatly enhanced by the addition of conditioned medium as first introduced by Virolainen and Defendi (1967). Their initial studies have been extended by Stewart (1973) and Stewart et al. (1975) who have demonstrated that 204

Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

RESTRICTED

HOST

peritoneal exudate cells stimulated by thioglycollate will proliferate in culture for 18 days in the presence of L cell conditioned medium providing the initial cell density is low enough so that nutrient depletion does not occur too rapidly. The proliferating cells are capable of forming colonies in liquid medium and have been identified as macrophages by morphology and by tests for phagocytic function using yeast and antibody-coated sheep erythrocytes. The establishment of conditions for obtaining exponentially growing macrophages provided the opportunity to investigate the replication of LDV in a more uniform population of cells. One of our initial goals was to develop an assay for LDV superior to the present one which depends on the ability of this virus to raise the plasma levels of lactic dehydrogenase in mice (Riley, 1968). We considered that LDV might affect the replication of another virus in cultured macrophages and that an interference assay could be developed. In this report we present evidence that LDV is quite effective in inhibiting the replication of Sindbis virus. Our studies on the mechanism of this inhibition establish that it is due to the production of interferon. This finding was unexpected because previous reports indicated that LDV does not induce interferon in vitro (Evans, 1970; Yamazaki and Notkins, 1973). During the course of Sindbis virus replication in LDV-infected macrophages phenotypic mixing between the two virions occurs. Our results indicate that both pure pseudotypes-virions containing the RNA of Sindbis virus and the envelope of LDV-and particles containing the envelope proteins of both virions are formed. MATERIALS

AND

METHODS

Mouse peritoneal macrophages. The preparation of peritoneal macrophages from C!,H/Anf mice, the culture conditions and the method of cell counting have been described in detail (Stewart et al., 1975). Mice were injected intraperitoneally with 1.5 ml thioglycollate medium (Difco, Detroit, Michigan). Three days later the animals were killed by cervical dislocation and injected intraperitoneally with 5 ml

RANGE

OF LDV

205

culture medium containing 5 units heparin/ml. After 2-5 min the medium was withdrawn from the flank opposite the injection site. The medium containing exudate cells were kept on ice in the plastic syringe until all were collected and could be inspected with a phase contrast microscope for contamination by erythrocytes or bacteria. Suspensions were pooled, centrifuged at 200g for 10 min, and recentrifuged with fresh culture medium. All procedures were carried out at 4°C to reduce the adherence of cells to the tube. The identification of the adherent cells as macrophages was based on their phagocytic activity. The studies of Stewart et al. (1975) demonstrated that 74% of the initial adherent exudate were phagocytic for yeast. From the fourth day on 99-100% were scored positive for phagocytosis of yeast. Medium. Peritoneal exudate cells were cultured in a-MEM (Flow Laboratories, Rockville, Md.) containing 10% fetal calf serum, 5% horse serum, and 10% L-cell conditioned medium (Stewart et al., 1975). Mouse L cells and chicken embryo fibroblasts were cultured in MEM with 10 and 3% fetal calf serum, respectively. Viruses. The preparation of LDV from infected mice and the infectivity assay have been described previously (Michaelides and Schlesinger, 1973). Sindbis virus used in most of the experiments was obtained from mouse plasmacytoma cells (Symington and Schlesinger, 1975). Vesicular stomatitis virus, a gift from Alice Huang, was propagated on L cells. The virus was passaged at a low m.o.i. to avoid the accumulation of defective-interfering particles. The yield in peritoneal macrophages was essentially the same at a m.o.i. of 20 or 200 indicating that defective-interfering particles were not present. Sindbis virus was titered on chicken embryo fibroblasts and VSV was titered either on these cells or on L cells. Protocol for infection of macrophages with LDV and Sindbis virus. The following procedure was used in all experiments unless noted otherwise in the text. Peritoneal exudate cells were seeded in 30 ml of medium onto 100 mm plates so that on the

206

LAGWINSKA

ET AL.

day of infection (usually 10 days after with antiserum, Sindbis virus from normal culturing) there would be lo6 cells per dish. macrophages and from LDV-infected macThese cells have a doubling time of 68 hr rophages was centrifuged for 2 hr at (Stewart et al., 1975). The medium was 100,OOOg and resuspended in phosphate removed from the plates and LDV (m.o.i. buffered saline containing 1% fetal calf = 200) was added in a total volume of 1.5 serum. This step was included to remove ml of MEM with 3% fetal calf serum. After any soluble proteins in the medium that assay. the plates were incubated for 2 hr, the might interfere in the neutralization inoculum was removed and 10 ml of IX- For all tests Sindbis virus was diluted to MEM with the supplements described lo8 PFU/ml and then mixed with an equal above added. Control cells were treated in volume of rabbit antiserum. Samples were incubated at 37°C for 1 hr. Goat anti-raban identical manner except that no LDV was added. Twelve hours later the medium bit gamma globulin was then added and incubation continued for 2 hr at 37°C. The was removed and stored at -70°C. The goat serum was added either in an amount cells were then infected with Sindbis virus (m.o.i. = 200). Adsorption was carried out fivefold greater than that of the rabbit serum or at a 1:500 dilution, whichever was for 1 hr at 37°C in a final volume of 1.5 ml. All dilutions were The inoculum was removed and 10 ml of more concentrated. fresh a-MEM added. The plates were incumade in phosphate buffered saline containbated for an additional 16 hr. The medium ing 1% fetal calf serum. Samples were was harvested and titered for Sindbis vi- usually stored at -70°C before titering on rus. All incubations were carried out at chicken embryo fibroblasts. Neutralization tests with mouse serum. 37°C in an humidified incubator with a 5-10% CO, atmosphere. The protocol was essentially the same as that described above except that Sindbis Antisera. For immunizing rabbits Sindvirus was diluted to approximately 1500 bis virus was mixed with an equal volume of complete Freund’s adjuvant. Each rab- PFU/ml before being mixed with mouse serum. Only one further dilution was necbit received 50 pg of virus in a total volume of 2.0 ml (0.2 ml/footpad and 1.2 ml essary to titer samples on chicken embryo fibroblast monolayers. In this way possible subcutaneously). Approximately 1 month later, a similar dose was administered and variations due to serial dilutions were minafter 1 wk the rabbits were bled from the imized. ear. The clarified serum was used without RESULTS further treatment. Replication of Sindbis virus and LDV in Mouse serum was obtained from mice mouse peritoneal macrophages. Two facchronically infected with LDV and from tors are crucial for the replication of Sindnormal mice. Both sera were centrifuged bis virus in mouse peritoneal macrophages: for 1 hr at lOO,OOOg-a procedure designed the source of the virus and the state of the to remove the infectious virus-antibody cells. Sindbis virus passaged on chicken complexes present in the serum of infected embryo fibroblasts or BHK cells is unable animals. A 1:lO dilution of the serum from to infect mouse peritoneal macrophages chronically infected animals in conjunction and gives virus yields < 1 PFU/cell. If the with goat antimouse IgG reduced the titer of LDV from lo8 infectious units/ml to less virus is first passaged on mouse cells it will than lo5 units/ml. No reduction of the produce high titers on macrophages. We have passaged Sindbis virus on mouse LDV titer was observed when normal mouse serum was substituted for the in- plasmacytoma cells and on L929 cells. The virus from these sources produced yields on fected mouse serum. Goat anti-rabbit gamma globulin and anti-mouse IgG were macrophages between 103-10” PFU/cell-a purchased from Gateway Immunosera Co. value comparable to that obtained with chicken or BHK cells. An extensive study (Cahokia, Illinois). of Sindbis virus obtained from mouse plasNeutralization tests with rabbit antimacytoma cells demonstrated that this Sindbis virus serum. Before incubation

RESTRICTED

HOST

passaging of the virus selects for a variant of the virus that is better able to adsorb to mouse cells (Symington and Schlesinger, 1975). We have used this variant in the studies described here. Even mouse-passaged Sindbis virus is incapable of infecting mouse peritoneal macrophages in the first few days after the cells are placed in culture (Table 1). Almost no replication of Sindbis virus occurs when macrophages are infected 24 hr after they are harvested from animals. A significant increase in yield is noted by 4 days and maximal yields per cell are attained after 7-10 days in culture. The growth rate of Sindbis virus in peritoneal exudate cells after 10 days in culture shows a maximum yield occurring about 12 hr after infection (Fig. 1). An increase in the titer of LDV is also observed from the first to the fourth day of culturing these cells. The difference in the yield of LDV, however, is not as dramatic as that obtained with Sindbis virus. Inhibition of the replication of Sindbis virus by LDV. Mouse peritoneal macrophages infected with LDV remain viable and continue to divide (our unpublished results), but they are altered in their abilTABLE

1

INFECTION OF MOUSE PERITONEAL MACROPHACESWITH SINDBIS VIRUS OR LDV AT DIFFERENT TIMES AFTER CELLS WERE PLACED IN CLJLTURE~ Day of infection

Yield of virus Sindbis PFU/cell

1 4 7 10

2.2 7.0 360 130 1200 2000 4800 2500

LDV infectious units/ceil 103 103 10’ 10’ 10" 10" 10' 10"

a Cells were seeded onto 100 mm plates so that on the day of infection there were approximately 10’ cells per plate assuming a 68-hr doubling time (Materials and Methods). The cell number was determined directly by cell counts. The two values presented fox each day represent two independent experiments. The yield of virus was assayed 16 and 12 hr after infection of Sindbis virus and LDV, respectively.

RANGE

207

OF LDV

TIME AFTER INFECTION

FIG. 1. Growth czm~e of Sindbis virus in peritoneal macrophages. Peritoneal exudate cells were placed in culture so that by day 10 there would be lo6 cells/ plate. These cells were infected with Sindbis virus (m.o.i. = 200) and samples were removed at the indicated times for titering (04). Some plates were infected with LDV (m.o.i. = 20, 60; m.o.i. = 200, 0-O) 12 hr prior to infection with Sindbis virus.

ity to produce Sindbis virus. The yield of Sindbis virus is decreased by about lo- to 50-fold if cells are infected with LDV for 12 hr before being exposed to Sindbis virus (Fig. 1 and Table 2). Mouse serum from uninfected animals has no effect on the replication of Sindbis virus. We varied the time between LDV and Sindbis virus infection to determine what effect this would have on the yield of Sindbis virus. Macrophages infected with Sindbis virus within 2 hr after they are infected with LDV produce a normal yield of Sindbis virus (Table 3). A maximum degree of inhibition is observed, however, by the time 5 hr has elapsed between the two infections. Inhibition is due to interferon. There have been several reports that LDV does not induce interferon when macrophages or primary mouse embryo cells are infected in culture (Evans, 1970; Yamazaki and Notkins, 1973). We felt, however, that it was necessary to determine if the interference phenomenon we were observing might be caused by interferon. The first hint that this was the case was the finding that LDV

LAGWINSKA

208

TABLE 2 EFFECT OF LDV ON THE REPLICATION OF SINDBIS VIRUS IN MOUSE PERITONEAL MACROPHAGES LDV m.o.i.

Yield of Sindbis virus PFU/ml x 109

A 0 2 20 200

3.0 2.7 0.7 0.25

0 200 Control mouse serumb

8.5 0.16 9.5

B

n Mouse peritoneal macrophages were cultured for 10 days before infection. The cell concentration in this experiment was 2 x 10B/dish. The cells were infected with LDV for 12 hr before being exposed to Sindbis virus. bMouse serum obtained from uninfected animals was added in a volume equivalent to 200 infectious units of LDV per cell. TABLE

3

THE EFFECT OF VARYING THE TIME BETWEEN LDV AND SINDBIS VIRUS INFECTION ON THE YIELD OF SINDBIS VIRUSa Time between LDV and Sindbis virus infection

Yield of Sindbis virus PFU/ml x lOa

hr 0 2 5 7 10 No LDV infection

3.0 3.7 0.12 0.10 0.17 4.0

“The procedure for infecting macrophages with these viruses is described in Materials and Methods.

also inhibits the replication of vesicular stomatitis virus (VSV) in these cells-a result different from that reported previously (Yamazaki and Notkins, 1973). Our original observations were made with LDV obtained from the serum of acutely infected animals, but LDV produced by macrophages in culture produces the same degree of interference (Table 4, line 2). If macrophages were producing an inhibitory factor such as interferon in response to LDV infection, it should remain in the medium under conditions in which the virus is removed by sedimentation.

ET AL.

When the LDV-containing medium was subjected to high speed centrifugation the virus titer in the supernatant fraction dropped from lo9 to lo7 infectious units/ ml, but all of the inhibitory activity remained in this fraction (Table 4, line 3). The residual titer of LDV would result in an m.o.i. of 2 which would be too low to affect the yield of Sindbis virus (Table 2). Two specific characteristics of interferon are its acid stability and its species specificity. The inhibitory factor produced by LDV-infected cells could be identified as interferon more definitely by these two properties. Thus, exposure of the supernatant fraction to pH 2 for 3 days at 4°C had no effect on the inhibitory activity (Table 4, line 4). The species specificity of this factor was examined by comparing its effect on the replication of VSV in mouse L929 cells and in chicken embryo fibroblasts. VSV was used in this experiment so that we could plaque directly on the two different cell lines as Sindbis virus cannot be titered on L cells. The number of VSV plaques decreased on mouse cells, but not on avian cells, with increasing amounts of the acid stable factor (Table 5). TABLE

4

PRESENCE OF AN ACID-STABLE INHIBITORY FACTOR IN MEDIUM OF MACROPHAGESINFECTED WITH LDV Addition to macrophages before infection with Sindbis virus” 1. None 2. LDV obtained from macrophages (m.0.i. = 200) 3. Supernatant from aboveb 4. Acid-treated supernatant’ 5. Medium from uninfected macrophages

Yield of Sindbis virus PFU/ml x lo9 3.0 0.2 0.08 0.08 1.9

a Cells were incubated with the various additions for 12 hr before infection with Sindbis virus. The samples described in lines 3-5 were added in a volume equivalent to a m.o.i. of 200 for the original LDV-containing medium (line 2). bThe medium containing LDV (lo9 infectious units/ml) was centrifuged for 4 hr at 100,OOOg. The titer of LDV in the supernatant fraction was 10’ infectious units/ml. c The supematant fraction was acidified to pH 2.0 and kept at 4°C for 3 days. The pH was adjusted to 7.4 before use.

RESTRICTED TABLE

HOST

5

THE EFFECT OF THE ACID STABLE FACTOR ON THE REPLICATION OF VESICULAR STOMATITIS VIRUS IN MOUSE L CELLS AND CHICKEN EMBRYO FIBROBLASTS Addition to cells prior to infection”

None Acid-treated supernatant fraction from LDV-infected macrophapes

Acid-treated supernatant fraction from uninfected macrophages LDVb m.0.i. = 20 m.0.i. = 200

ml

0.1 0.3 0.6 0.9 2.7 2.7

PFU/plate L cells

CEF

38 29 15 9 1 0 35

31 33 33 31 34 32

33 37

“The cells were treated for 12 hr with the various additions before being infected with VSV. 0 LDV was obtained from mouse serum. L cells were exposed to LDV for 12 hr before being infected with VSV.

LDV obtained from mouse serum was also added to L cells 12 hr before the addition of VSV but had no effect on the number of plaques observed (Table 5). This latter result is consistent with previous findings that LDV is unable to replicate in most cultured cells. Production of interferon by peritoneal macrophages. It is apparent that with proliferation in culture, peritoneal exudate cells develop the capacity to replicate Sindbis virus and enhance their ability to produce LDV (Table 1). We have demonstrated that after 10 days in culture these cells produce interferon in response to LDV infection. An examination of the ability of macrophages to induce interferon as a function of time after culturing reveals a striking increase in this property as well. Cells were infected with LDV at several time intervals after being placed in culture. The medium from infected cells was centrifuged and the supernatant fraction acidified as described in Table 4. L cells were treated with varying amounts of the acidtreated medium and subsequently challenged with VSV (Fig. 2). Essentially no interferon is produced by cells 24 hr after

RANGE

209

OF LDV

they are placed in culture even though they are capable of replicating LDV at that time. The cells do synthesize interferon in response to LDV infection after 4 days in culture and the response appears to be maximal by 7 days. The difference between the amount of interferon found at 4 and 7 days may reflect a difference in the number of cells capable of inducing interferon or a difference in the amount of interferon synthesized per cell. We have not yet distinguished between these possibilities. Phenotypic mixing between Sindbis virus and LD V. There are several examples in which unrelated RNA enveloped viruses replicating in the same cell will form phenotypically mixed particles (Choppin and Compans, 1970; Zavada, 1972). Both pure pseudotypes containing the nucleic acid of one virus and the glycoproteins of the other and mosaics containing glycoproteins of both viruses have been detected (Choppin and Compans, 1970). We undertook a search for the presence of pseudotypes containing the RNA of Sindbis virus and the envelope protein of LDV. The existence of this particular pseudotype would be valuable in determining if the restricted host range of LDV is due to

2

4

6

IO

ml OF ACID-TREATED

20

30

MEDIUM

FIG. 2. Production of interferon by macrophages as a junction of time after being placed in culture. Peritoneal exudate cells were seeded in 100 mm dishes to obtain lo6 cells/plate on the appropriate day. On the days indicated cells were infected with LDV (m.o.i. = 200) for 12 hr. The medium was removed, subjected to centrifugation for 4 hr at 100,OOOg and then acidified to pH 2 for 3 days (Table 5). Samples of the neutralized medium were then added to L cells for 12 hr prior to infection with VSV (see Table 5). Time between culturing and infection: 1 day (0-O); 4 days (O----O); 7 days (A-A); 10 days (A-A).

210

LAGWINSKA

an inability of the virus to adsorb to cells other than macrophages. As a first step before beginning this search we examined the neutralization of Sindbis virus by rabbit anti-Sindbis serum (Fig. 3). This serum decreased the virus titer by 30-fold but the titer can be reduced several orders of magnitude by the addition of goat serum directed against rabbit gamma globulin. Rabbit serum containing antibodies to an unrelated antigen had no effect on the titer of Sindbis virus. It appears that Sindbis virus-like LDV (Notkins et al. 1966) -forms infectious antigen-antibody complexes (Symington and Schlesinger, unpublished results). These complexes are inactivated by the addition of serum containing antibodies to the rabbit gamma globulin. Evidence for the presence of LDV pseudotypes of Sindbis virus can be seen by comparing the neutralization of Sindbis

00001

:yoIO ANTISERUM

DILUTION

x lO-3

FIG. 3. The ability of rabbit serum and rabbit serum plus goat-anti-rabbit gamma globulin to neutralize Sindbis virus. The details of the conditions of incubation are described in Materials and Methods. Rabbit serum containing antibodies to trinitrophenyl bovine gamma globulin (anti-TNP) (04); antiTNP plus goat anti-rabbit gamma globulin (A -A); rabbit serum containing antibodies to Sindbis virus, (anti-Sindbis) (O----O); anti-Sindbis plus goat anti-rabbit gamma globulin (A-A).

ET AL.

00001’

.03 ANTISERUM

06 DILUTIONx10-3

FIG. 4. The effect of oarying the time between LDV and Sindbis virus infection on the formation of pseudotypes. The details of the neutralization assay are described in Materials and Methods. Note that the amount of rabbit antiserum is much less than that in Fig. 3. Goat anti-rabbit gamma globulin was used at a dilution of 1:X0. The four samples of Sindbis virus were obtained from macrophages that (a) had not been infected with LDV (O---O), (b) had been infected with LDV 5 hr prior to addition of Sindbis virus (O-O), (c) had been infected with LDV 7 hr prior to addition of Sindbis virus (O-O), and (d) had been infected with LDV 10 hrs prior to addition of Sindbis virus (A-A) (see Table 3). The sample from (d) that had been incubated with the lowest dilution of anti-Sindbis serum (0.06 x 10-Y was incubated at 37°C with normal mouse serum (A) or with anti-LDV mouse serum (m) (both diluted 1:500) for 1 hr and then for an additional 2 hr with goat anti-mouse -rG (1: 100).

virus obtained from normal and LDVinfected macrophages. The fraction of Sindbis virus from LDV-infected cells that is protected from inactivation by the combined rabbit and goat antisera is considerably higher than that from normal cells (Table 6, column 1). The extent of inactivation of Sindbis virus is unaffected if LDV is added to Sindbis virus at the time of the neutralization assay. We have also examined the degree of phenotypic mixing obtained when the time between infection by LDV and Sindbis

RESTRICTED TABLE

HOST

6

A COMPARISONOF THE NEUTRALIZATION BY ANTI-SINDBIS SPECIFIC ANTISERUM OF SINDBIS VIRUS REPLICATED IN THE PRESENCE AND ABSENCE OF LDV” Antiserum dilution x 10-a

No antiserum 0.031 0.062

RANGE

contain the RNA of LDV. As Sindbis virus is cytopathic, its presence in the mixed population of virions would obfuscate our assays to detect LDV replication.

Residual titer log,, PFU/ml Sindbis + LDV

Sindbis + Added LDVb

Sindbis

7.8 5.3 5.2

7.7 3.9 3.5

7.7 3.8 3.2

a Samples were incubated with rabbit serum containing antibodies to Sindbis virus for 1 hr at 37°C and then for an additional 2 hr at the same temperature with goat anti-rabbit gamma globulin (1:500) (Materials and Methods). b LDV was added to Sindbis virus obtained from normal macrophages immediately before the addition of antiserum.

virus is altered (Fig. 4). The fraction of particles insensitive to anti-Sindbis antiserum increased tenfold as the time between LDV and Sindbis virus infection increased from 5 to 7 hr. No further increase is obtained by extending the time to 10 hr. The fraction of Sindbis virus that is protected from anti-Sindbis antiserum could be more definitely characterized as having the envelope proteins of LDV by testing its sensitivity to mouse serum containing antibodies to LDV. The addition of anti-LDV mouse serum plus goatantimouse rG reduced the surviving fraction of Sindbis virus from 0.0025 to 0.0001 (Fig. 4). Normal mouse serum had no effect on the titer of Sindbis virus. We have also employed mouse serum containing antibodies to LDV to detect the presence of particles that contain both LDV and Sindbis virus antigens. This mouse serum is capable of neutralizing about 40% of the PFU of Sindbis virus obtained from LDV-infected macrophages (Table 7). It did not inhibit Sindbis virus obtained from uninfected macrophages. Furthermore normal mouse serum had no effect on the titer of Sindbis virus. We have not yet made any attempts to detect phenotypically mixed virions that

211

OF LDV

DISCUSSION

In their studies on the colony forming ability of peritoneal exudate cells, Stewart et al. (1975) concluded that only a small fraction of the original adherent cells are capable of extensive proliferation. Two hours after thioglycollate-stimulated periTABLE

7

NEUTRALIZATION OF SINDBIS VIRUS BY ANTISERUM DIRECTED AGAINST LDV” Incubation

Virus virus Sindbis from macrophages

Sindbis virus from LDVinfected macrophages

PFU/ mlb

None

j97 * 29

Normal mouse serum plus goat anti-mouse serum

120 zt 46

Serum from LDVinfected mice plus goat anti-mouse serum

01 + 28

None

‘76 + 21

Normal mouse serum plus goat anti-mouse serum

‘65 zt 40

from LDVSerum infected mice plus goat anti-mouse serum

149 f 13

“The two preparations of Sindbis virus were diluted to 1500 PFU/ml. A sample of the virus (0.15 ml) was diluted with an equal volume of mouse serum (final dilution of mouse serum was 1:500) and was incubated at 37°C for 1 hr. Goat anti-mouse rG was then added (final dilution 1:lOO) and the samples were incubated for an additional 2 hr at 37°C. b Three different aliquots from each sample were titered. The PFU/plate ranged from 17 to 90. The data are normalized back to the original incubation with antiserum and the standard error calculated. ‘This difference is significant by T test, P < 0.0001.

212

LAGWINSKA

toneal exudate cells are placed in culture only 10% of the adherent cells are in cell cycle as measured by a 2 hr pulse of 3HTdr. At this time between 74 and 45% of the cells are phagocytic based on their ability to engulf yeast or sheep red blood cells, respectively. After 4 days in culture essentially all of the cells are scored positive for phagocytic activity (> 95%) and about 45% of them are proliferating. By the eighth day about 75% of the cells are calculated to be in cell cycle. Between 4 and 12% of the original cells plated are able to give rise to colonies in agar (Lin and Stewart, 1973) and in liquid (Stewart et al., 1975). In many respects our data parallel these previous findings. Initially peritoneal exudate cells are unable to replicate Sindbis virus. Furthermore, although cells do produce a significant yield of LDV 24 hr after being placed in culture, they apparently are incapable of inducing interferon in response to this infection. Both the ability to replicate Sindbis virus and to induce interferon increase substantially during the first week after the cells are cultured and therefore may be correlated directly with the ability of cells to enter cell cycle. The difference between our results on interferon induction and those reported previously can be explained by differences in the state of the cells. Our data strongly suggest that only proliferating macrophages are capable of inducing interferon. The same conclusion seems to be true for the ability to replicate Sindbis virus. There may be some analogy between the latter observation and the results obtained with sensitized lymphocytes. These cells, but not resting lymphocytes, are capable of replicating certain RNA viruses (Bloom et al., 1970). One of the hallmarks of LDV infection in mice is the persistence of the virus in the animal. There has been some discrepancy however as to the length of time an LDV infection can be maintained in vitro (DuBuy and Johnson, 1966, 1968; Evans, 1970). Realizing that LDV is capable of inducing interferon in vitro, as well as in vivo (DuBuy and Johnson, 1965; Evans and Riley, 1968), it seems most probable

ET AL.

that the persistence of LDV in culture would be correlated inversely with the ability of the cells to induce interferon. One of the most intriguing properties of LDV is its narrow host range which seems to be limited to the phagocytic cells of the reticulum (Snodgrass et al., 1972). One obvious explanation for this property is that only mouse macrophages possess the proper receptors for LDV and the virus is unable to adsorb to other cells. This explanation now seems untenable based on our ability to detect pseudotypes of Sindbis virus-insensitive to antibodies directed against Sindbis virus but neutralized by antibodies to LDV. Our neutralization scheme for Sindbis virus does not depend on the ability of the rabbit serum to inactivate Sindbis virus, but requires only that the rabbit antibodies react with the Sindbis viral proteins. Sindbis virus has two glycoproteins (Schlesinger et al., 1971) and the rabbit anti-Sindbis serum recognizes both of them. The addition of goat anti-rabbit gamma globulin insures that any particle containing Sindbis viral proteins on the surface will be neutralized. The fraction of virions that is insensitive to the anti-Sindbis serum is inactivated by antibodies to LDV. The ability of the anti-LDV mouse serum to neutralize this fraction of Sindbis virus permits us to conclude that the glycoprotein in their envelope is derived from LDV. These pseudotypes are capable of initiating the formation of plaques on chicken embryo fibroblasts. Thus, the steps of adsorption and penetration can not be the ones responsible for the restricted host range of LDV. It would be extremely difficult to analyze the pseudotypes biochemically as they represent less than 1% of the population of Sindbis virions. It appears that a much larger percentage of the population (about 40%) can be neutralized both by anti-Sindbis specific antiserum and by anti-LDV serum. These particles must represent mosaic particles and it should be possible to analyze their protein content. LDV contains three polypeptides (Michaelides and Schlesinger, 1973). One of the proteins is

RESTRICTED

HOST

associated with the nucleocapsid, one is not glycosylated but is found with the envelope fraction of the virion and may be equivalent to the internal viral membrane protein (Michaelides and Schlesinger, 1973), the third protein is the glycoprotein. In a study of phenotypic mixing between VSV and simian virus 5 McSharry et al. (1971) found that the glycoproteins of the simian virus, but not the internal proteins, are present in particles containing VSV nucleocapsids. They concluded that recognition between the capsid and the internal membrane protein of a specific virus is much greater than the recognition of the viral glycoproteins. If their conclusion is correct the only LDV-specific protein in the Sindbis-LDV mixed particles should be the LDV glycoprotein.

RANGE

8.

9.

10.

11.

12.

ACKNOWLEDGMENTS This work was supported Service Grant AI-12242 and S.S.; and U.S. Public Health to C.C.S. The NSF Grant the preparation of cells and

by U.S. Public Health NSF Grant GB-37853 to Service Grant CA-13053 GB-38657 contributed to media.

REFERENCES 1. BLOOM, B. R., JIMENEZ, LUIS, and MARCUS, P. I. (1970) A plaque assay for enumerating antigensensitive cells in delayed-type hypersensitivity. J. Exp. Med. 132, 16-30. 2. CHOPPIN, P. W. and COMPANS, R. W. (1970) Phenotypic mixing of envelope proteins of parainfluenza virus SV5 and vesicular stomatitis virus. J. Viral. 5,609-616. 3. DARNELL, M. B. and PLAGEMANN, P. G. W. (1972) Physical properties of lactic dehydrogenaseelevating virus and its ribonucleic acid. J. Viral. 10, 1082-1085. 4. EVANS, R. (1970) Further studies on the replication of the lactate dehydrogenase-elevating virus (LDH virus) in mouse peritoneal macrophage cultures. Proc. Sot. Exp. Sol. Med. 133, 831-836. 5. LIN, H. and STEWART, C. C. (1973) Colony formation by mouse peritoneal exudate cells, in uitro. Nature (New Biol.) 243, 176-177. 6. MCSHARRY, J. J., COMPANS, R. W., and CHOPPIN, P. W. (1971) Proteins of vesicular stomatitis virus and of phenotypically mixed vesicular stomatitis virus-simian virus 5 virions. J. Viral. 8, 722-729. 7. MICHAELIDES, M. C. and SCHLESINGER, S. (1973)

13.

14.

15.

16.

17.

18.

19.

20.

OF LDV

213

Structural proteins of lactic dehydrogenase virus. Virology 55, 211-217. MICHAELIDES, M. C. and SCHLESINGER, S. (1974) Effect of acute or chronic infection with lactic dehydrogenase virus (LDV) on the susceptibility of mice to plasmacytoma MOPC-315. J. Immunol. 112, 1560-1564. NOTKINS, A. L. (1971) Enzymatic and immunologic alterations in mice infected with lactic dehydrogenase virus. Amer. J. Pathol. 64, 733-745. NOTKINS, A. L., MAHAR, S., SCHEELE, C., and GOFFMAN, J. (1966) Infectious virus-antibody complex in the blood of chronically infected mice. J. Exp. Med. 124, 81-97. OLDSTONE, M. B. A. and DIXON, F. J. (1972) Inhibition of antibodies to nuclear antigen and to DNA in New Zealand mice infected with lactate dehydrogenase virus. Science 175, 784-786. PROFFITT, M. R., CONGDON, C. C., ~~~TYRDALL, R. L. (1972) The combined action of Rauscher leukemia virus and lactic dehydrogenase virus on mouse lymphatic tissue. Int. J. Cancer 9, 193-211. RILEY, V. (1968) Lactate dehydrogenase in the normal and malignant state in mice and the influence of a benign enzyme-elevating virus. In “Methods in Cancer Research” (H. Busch, ed.), vol. 14, pp. 493-617. RILEY, V., LILLY, F., JUERTO, E., and BARDELL, D. (1960) Transmissible agent associated with 26 types of experimental mouse neoplasms. Science 132, 545-547. SCHLESINGER,M. J., SCHLESINGER,S., and BURGE, B. W. (1971) Identification of a second glycoprotein in Sindbis virus. Virology 47, 539-541. SNODGRASS,M. J., LOWREY, D. S., and HANNA, M. G. (1972) Changes induced by lactic dehydrogenase virus in thymus and thymus-dependent areas of lymphatic tissue. J. Immunol. 108, 877-892. STEWART, C. C. (1973) Nutrient utilization by peritoneal exudate cells. J. Reticuloendothel. sot. 14, 332-349. STEWART, C. C., LIN, H., and ADLES, C. (1975) Proliferation and colony forming ability of peritoneal exudate cells in liquid culture. J. Exp. Med. (In Press). SYMINGTON, J. and SCHLESINGER, M. J. (1975) Properties of a Sindbis virus variant isolated by passage on mouse plasmacytoma cells. J. Viral. (In Press). VIROLAINEN, M. and DEFENDI, C. (1967) Dependence of macrophage growth in vitro upon interaction with other cell types. In “Growth Regulating Substances for Animal Cells in

214

LAGWINSKA

Culture” (V. Defendi and M. Stoker, eds.), Wistar Inst. Symp. Monogr. No. 7, pp. 67-85. Wistar Institute, Philadelphia. 21. YAMAZAKI, S. and NOTKINS, A. L. (1973) Inhibition of replication of lactic dehydrogenase virus by

ET AL actinomycin.

J. Viral. 11, 473-478. of vesicular stomatitis virus with the coat of murine leukaemia and of avian myeloblastosis viruses. J. Gen. Vz’rol. 15, 183-191.

22. Z~VADA, J. (1972) Pseudotypes