Vaccinia virus proteins on the plasma membrane of infected cells III. Infection of peritoneal macrophages

VIROLOGY

147.354-372

(1985)

Vaccinia Virus Proteins on the Plasma Membrane of Infected Cells III. Infection of Peritoneal Macrophages

ROBERT J. NATUK’ AND J. A. HOLOWCZAK2 Department

of Microbiology, University of Medicine and De&L&-g of New Jersey, Rutgers Medic& School, Pkdaway, New Jersey 08854 Received June 19, 1985; accepted August 16, 1985

Primary macrophage cultures were prepared from the peritoneal exudate cell population harvested from mice challenged intraperitoneally with saline, thioglycollate, or vaccinia virus. Vaccinia virus was adsorbed and penetrated into primary macrophages and L-cells with similar kinetics. As evidenced by the expression of some “early” virus-specified proteins, partial uncoating and activation of the virion-associated DNA-dependent RNA polymerase occurred in the infected macrophages. Subsequently, the viral replication cycle in macrophages was aborted, with time after infection, viral DNA and virion proteins initially associated with infected cells could be detected in an acid-soluble form in the medium harvested from infected macrophage cultures. The results suggest that at the time that the final stages of virus uncoating should have occurred, intracellular subviral particles were, instead, degraded in the infected, primary macrophages. Viral DNA synthesis could not be measured in vaccinia virus-infected macrophages, no “late” virus functions were expressed, and progeny virions were not assembled. As measured by the binding of antiviral-antibody-‘261-protein A complexes to the surface of vaccinia virus-infected cells, the expression of virus-specified antigens on the surfaces of infected macrophages was significantly reduced and never exceeded that measured at 2 hr after infection on the surfaces of infected L-cells. The expression of virus-specified polypeptides with mol mass of 48-50, 45-46, 36-3’7, and 25 kDa on the plasma membranes of vaccinia virus-infected, thioglycollate-elicited macrophages, rendered the infected macrophages susceptible to lysis by vaccinia virus-specific cytotoxic T-cells. Q 1985 Academic Press, Inc. INTRODUCTION

The wide distribution of macrophages throughout all of the major body compartments, favors the interaction of macrophages with invading infectious agents such as viruses (Mims, 1964). The result of an encounter of macrophages with viruses can therefore determine or significantly influence the course of the virus infection (Mims, 1964; Silverstein, 1970, 1975; Allison, 1974; Morahan and Morse, 1979; Mogensen, 1979; Virelizier, 1975). The replication or the failure of poxviruses to replicate within cells of the macrophage 1Present address: Department of Pathology, University of Massachusetts Medical Center, Worcester, Mass. 01605. ’ To whom reprint requests should be addressed. 0042-6822135 $3.00 Copyright All righta

@ 1985 by Academic Press, Inc. of reproduction in any form reserved.

lineage has been shown to play an important role in the subsequent course of infection in rabbits (Buchmeier et al, 1979), rats (Jandasek and Votava, 1970), and mice (Mims, 1964; reviewed by Dales and Pogo, 1981). Examination of the replication of vaccinia virus in murine macrophages in vitro has yielded two kinds of results. Investigators have reported that thioglycollateelicited macrophages were permissive for vaccinia virus but that macrophages, harvested from vaccinia virus-infected mice, failed to support the replication of vaccinia virus (Ueda and Nozima, 1973;Koszinowski et a& 1975; Rama-Roa et d, 1977). Other investigators have reported that vaccinia virus replication failed to occur in macrophages, whether derived from uninfected or previously infected mice (Nishmi and 354

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AND

Bernkopf, 1958; Nishmi and Niecikowski, 1963; Glasgow and Habel, 1963; Silverstein, 1970,1975). Even when complete vaccinia virus replication in macrophages could not be measured, there was, nevertheless, experimental evidence which indicated that limited expression of virus-specified gene products had occurred in the infected macrophages (Silverstein, 1975). In addition, vaccinia virus-infected macrophages have been employed as target cells in studies where vaccinia virus specific cytotoxic Tcell (VV-CTL) activity was measured by employing in vitro 51Cr release assays (Zinkernagel and Doherty, 197’7; Zinkernagel, 1976; Koszinowski et or, 1976) suggesting that virus-specified antigens were expressed on the plasma membrane of virus-infected macrophages. In the experiments to be reported here, we have reinvestigated the infection of primary murine peritoneal macrophages in vitro with vaccinia virus. Our results confirm and extend the observations of previous investigators (Nishmi and Bernkopf, 1958;Glasgow and Habel, 1963;Nishmi and Niecikowski, 1963; Silverstein, 1970, 1975) in that we could show that vaccinia virus replication in primary peritoneal murine macrophages was abortive. After infection of macrophages the expression of the vaccinia virus genome was limited to a portion of the early viral gene products detected after infection of permissive host cells. The expression of virus-specified antigens on the plasma membrane of infected macrophages was demonstrated and the viral antigens expressed on the plasma membrane of macrophages, at the time that such cells were susceptible to lysis by VVCTLs, were indentified. MATERIALS

AND

METHODS

Experirnentul animals. Six- to eightweek-old DBA/2J and C3H/HeJ mice were obtained from Jackson Labs, Maine. Ageand sex-matched animals were employed in all experiments. In the majority of experiments, peritoneal exudate cells were harvested and macrophages prepared from C3H/HeJ mice and all experiments were

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repeated, at least once, with macrophages prepared from this strain of mice. Macrophages from the C3H/HeJ strain of mice do not respond to endotoxin (reviewed by Adams and Hamilton, 1984) which, if introduced accidentally in medium or serum could have “activated” the macrophages and perhaps altered their response to virus infection. CeuSand viru.se.s.Mouse L-929 cells and HeLa cells were maintained and propagated as described previously (Holowczak, 1976; Esteban and Holowczak, 1977). The following cell lines were obtained from the American Type Culture Collection, Rockville, Maryland, and the cells grown as recommended by the suppliers: PU5-1.8, mouse monocyte-macrophage [TIB-611; 5774-1, mouse, monocyte-macrophage [TIB671; WR 19M.1, mouse monocyte-macrophage [TIB-‘IO]. THe P388Dl monocytemacrophage cell-line was generously provided by Dr. Koren, Duke University, North Carolina, and P-815 mouse mastocytoma cells by Dr. Smith, Fox-Chase Cancer Institute, Pennsylvania. The WR and IHDJ strains of vaccinia virus were propagated and cell-associated virus was purified, as described previously (Joklik, 1962; Holowczak, 1976). Vaccinia virus preparations were titrated on monolayers of Vero, L, or primary chick embryo fibroblasts and the number of virus particles or elementary bodies (EBs) present in each preparation was estimated spectrophotometrically (Sarov and Becker, 1967). Vaccinia virions labeled with rH]thymidine in their DNA and (or) [%]methionine or [3H]leucine in their protein components were prepared and purified as described previously (Joklik, 1962). Preparation of peritoneal exudate cells. Mice were challenged ip with purified vaccinia virus, strain IHDJ (0.7-l X lo6 PFU or 0.25 pg of protein as virus), phosphatebuffered saline (PBS), or thioglycollate (1.0 ml of a sterile 3% solution of Thioglycollate Medium; BBL Microbiology Systems, Md.). Five to six days after challenge, animals were sacrificed by decapitation and dissected to reveal the peritoneum. Cells within the cavity were harvested by flush-

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ing the peritoneal cavity with ice-cold RPMI-medium, manipulating the animals and then withdrawing medium and cells from the cavity with a syringe. Pertioneal exudate cells (PEC) were separated from the “wash-out” medium by centrifugation, the cells were washed 2X with RPMI-complete medium [RPM1 medium (GIBCO, N. Y.), supplemented with 510% heat-inactivated fetal calf serum (AFCS) and 10 mM HEPES], and the cell pellet was resuspended in RPMI-complete medium. A portion of the cell suspension was removed for counting and estimation of cell-viability by trypan blue exclusion. Approximately 4-7 X lo6 peritoneal exudate cells were recovered from unmanipulated or PBS-injected animals (resident PECs), 12-20 X lo6 PECs were recovered from animals injected with thioglycollate and 12-35 X lo6 PECs were recovered from animals challenged ip with vaccinia virus, 5-6 days after the indicated injections were administered. Preparation of mucrophage mcmoluyers. Peritoneal exudate cells were plated in tissue culture plates and adherent cells (macrophages) were separated from the nonadherent cell population (nPECs) (Edelson and Cohn, 1976). Preliminary experiments were performed to determine the numbers of PECs to be plated such that a near-confluent monolayer of adherent cells would result. For this purpose, PEC preparations were adjusted to contain 5 X lo6 cells/ml and 1.0 ml of such a cell suspension was plated in 35-mm tissue culture plates or 0.5 ml was plated in 16-mm tissue culture plates. The cells were allowed to adhere for 3 hr at 37“ (5% COzenvironment) and nonadherent peritoneal cells (nPECs) were removed. The resulting adherent-cell monolayers were washed 5X to remove any remaining nonadherent cells. Adherent cell monolayers were incubated for no more than 24-48 hr before being employed for the experiments to be described. In some cases, the cultures were treated briefly with trypsin (Marcelletti and Furmanski, 1978) to ensure that all nPECs had been removed. The adherent cell population was characterized (a) morphologically after acridine orange staining (Jackson, 1961; Allison, 1976) or Wright’s

HOLOWCZAK

staining (as per manufacturer’s suggested procedure for blood smears, Cambridge Chem., Fla.); (b) staining for nonspecific esterases (Koski et c& 1976; Tucker et al, 1977); and (c) ability to phagocytize latex particles (Roberts and Quastel, 1963). By all these criteria, the adherent cell population was determined to consist of mononuclear phagocytes or macrophages (9092%)and we will refer to them as primary macrophage cultures. The remaining 810% of the cells in the cultures, while sharing some of the characteristics expected for macrophages (Dvorak and Dvorak, 1982) were found deficient in one or another of the criteria described above, which were rigorously applied in characterizing the cultures. Ir4fecticrus center assays, indirect-immunoJluorescence assays for viral antigen expression and Hoed& staining to reveal cytophsmic viral factmees. After infecting L-cells or macrophages with vaccinia virus (500 EB or 12.5 PFU/cell), the infected cultures were divided and half incubated with anit-IHDJ or anti-WR antisera, the other half with PBS (60 min, room temperature, gentle mixing). Infected macrophages were released from their substratum by gentle scraping with a rubber policeman. Infected L-cells and macrophages were washed (2X, ice-cold PBS), serially diluted, appropriate dilutions were plated onto near-confluent monolayers of L-cells or Vero cells (Plaeger-Marshall et aZ.,1982), and the number of infected cells which gave rise to a plaque were determined. The expression of viral antigens in cells infected with vaccinia virus was detected by indirect-immunofluorescence staining. Cells propagated on coverslips were infected (500 EB/cell) and at various times after infection mock-infected and infected cultures were washed and fixed in acetone (-20”). The cells were incubated with rabbit pre-immune or rabbit anti-IHDJ antisera, washed, and then incubated with FITC-conjugated goat-anti-rabbit (FAB’)z (Cappel Labs, Pa.). Selected cultures were incubated with buffer followed by FITCgoat anti-rabbit (FAB’)a. For quantitation, 106 cells in duplicate cultures were evaluated.

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Infected and uninfected cells were also stained with Hoechst compound (Flow Labs, Va.) to reveal viral factories which developed in the cytoplasm of vaccinia virus-infected cells indicating that viral DNA synthesis had progressed in a normal fashion (Esteban, 1977). As above, 100 cells in duplicate infected or mock-infected cultures were evaluated as described previously (Esteban, 1977). Vaccinia virus replication in macrcb phages as compared to L.-cells. Various stages in the replication cycle of vaccinia virus after infection of primary macrophages or L-cells were studied. Unless otherwise noted, infected cells were lysed with Nonidet-P40 (NP-40) and a cytoplasmic and nuclear fraction prepared as described by Penman (1969). In all experiments, mock-infected cells were analyzed in parallel with virus-infected cells. The macrophage-like cell lines described above and in particular, the P388Dl cell line were employed initially to test the experimental protocols which were then applied to the analysis of the virus-infected, primary macrophages. The following stages in the interaction of virus with cells or virus replication were analyzed: (a) Adsorption (Dales, 1965; Payne and Norrby, 1978). The adsorption of rH]thymidine- or [%Imethionine-labeled virions was measured. Studies were conducted at 4 and 37” in order to separate virus uptake by phagocytosis as compared to adsorption (Svennerholm et al, 1982). (b) Unwating (Joklik, 1964a, 1964b). Cells were infected with [3H]thymidine-labeled virus and the hydrolysis of cell-associated parental genomes, present in the cytoplasmic fraction prepared from cells at various times after infection, by exogenous DNases was measured. When nonionic detergent extracts of virus-infected or mock-infected macrophages were incubated with purified E3H]thymidine-labeled vaccinia DNA, less than 15% of the viral DNA was hydrolyzed to acid soluble products. When exogenous DNase was added, greater than 95% of the purified labeled viral DNA was hydrolyzed to acid soluble products. (c) Viral DNA repkaticm (Esteban and Holowczak, 1977). The incorporation of rH]thymidine into

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acid-soluble material in the cytoplasmic fraction prepared from vaccinia virus-infected cells was measured. (d) Assembly of progeny virions (Moss et al., 1969). The cytoplasmic fraction prepared from [3H]thymidine-labeled, virus-infected or mockinfected cells was analyzed by sedimentation in preformed, linear 20-40s (W:W) sucrose gradients (Holowczak and Joklik, 1967). Purified virions, labeled with [35S]methionine or [14C]thymidine were added to samples to provide an internal sedimentation marker. (e) Protein synthesis (Holowczak and Joklik, 1967;Moss, 1968). Total protein synthesis was determined by measuring the incorporation of rH]leucine or [35S]methionine into acid insoluble material in the cytoplasmic fraction prepared from virus-infected cells as compared to mock-infected cells. In some experiments rH]leucine- or [?S]methionine-labeled infected and mock-infected cells were solubilized with ionic detergent and after trichloroacetic acid (TCA) precipitation, acid insoluble, labeled proteins were washed with ice-cold acetone, lyophilized, and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (Soloski et al., 1978). (f) Immunoprecipitation studies (Mallon et al, 1985). For this purpose [35S]methionine- or [3H]leucine-labeled, infected and mock-infected cells were solubilized using the double-detergent procedure described by Gomer and Lazarides (1983). We were able to determine that this procedure was superior to extraction with Nonidet-P40 alone in that the majority of viral proteins, detected after SDS-PAGE of whole cell preparations as described above, were represented in the double-detergent extracts. Extracts were clarified by centrifugation, portions of the supernatant fraction were removed, incubated with preimmune or antiviral serum, and antibody-antigen complexes were collected using the Protein A adsorbent procedure (Kessler, 1975) as modified by Soloski et aL, 1981. Immune complexes were then processed and analyzed by SDS-PAGE. (g) Expression ofvirus-speci$ed antigens on iqfected cell surfaces (Goding, 1978). To detect the expression of viral antigens on the surfaces of

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infected L-cells or macrophages, infected cell cultures were incubated at various times after infection with antiviral antisera, washed, and then incubated with ‘%IProtein A (Mallon and Holowczak, 1985) (sp act was 4.2 X lo4 cpm/pg protein A). Preparation and purification of plasma membranes. Plasma membranes from virus-infected and mock-infected cells were isolated and purified by the two-phase fractionation technique of Brunette and Till (1971) as modified by Stone et al. (1974). A similar technique was previously employed to isolate plasma membranes from vaccinia virus-infected cells (Weintraub and Dales, 1974; Mallon and Holowczak, 1985). Because the number of macrophages available for this purpose was limited, we mixed 1.5 X lo7 mock-infected or virus-infected macrophages with l-2 X lo* unlabeled and uninfected carrier L-cells to maximize the conditions for membrane isolation (Lasfargues, 1976; Mallon et al, 1985). In vitro assay for vaccinia virus spe&&c cytotuxic T-cell (W-CTL) activity. Splenic effector cell populations were prepared 56 days after infection of C3H/HeJ or DBA/ 2 mice with vaccinia virus and the activity of VV-CTLs was assayed by measuring the lysis of 51Cr-labeled vaccinia virus-infected syngeneic target cells in vitro. The percentage specific lysis was calculated as described previously (Byrne et u& 1983; Mallon et al, 1985). SDS-polyacrylumide gel electrophoresis. A modification of the procedure described by Laemmli (1970) was employed (Soloski et al, 1978). Stained gels were treated with “EN3HANCE” (New England Nuclear, Mass.), dried, and Kodak X-Omat-R film was exposed to the dried gels at -70”. The resulting radiofluorograms were analyzed with a Model 800 Fiber Optic Scanner (Kontes Scientific, N. J.) and a 3390A Reporting Integrator (Hewlett-Packard, Pa.). purchased Reagents. Radioisotopes from the New England Nuclear Corporation, ICN-Pharmaceuticals, California, and Amersham Searle Company, Illinois, included: rH]thymidine (20.0 CYmmol), rH]leucine (146.5 Ci/mmol), sterile sodium[51Cr] (200-900 Ci/g), [%!l]methionine

HOLOWCZAK

(1107.4 Ci/mmol), and sodium[‘251] (17 Ci/ mg/mmol). RESULTS

Preparation and characterixation of primarl/ macrophage cultures. Sex- and agematched (8-9 weeks) groups of DBA/Z and C3H/HeJ mice were injected, intraperitoneally with thioglycollate, vaccinia virus, or PBS as described under Materials and Methods. Six days later, the animals were sacrificed, peritoneal exudate cells were harvested, and primary macrophage cultures were prepared and charaterized as described under Materials and Methods. Primary macrophage cultures prepared from virus-infected animals (which will be referred to as virus-elicited macrophages) were analyzed for the presence of infectious virus particles. No virus was detected in such cultures and no cytopathic effect (CPE) developed in the cultures upon incubation for 3-5 days (data not shown). It could be demonstrated that virus-infected macrophages failed to adhere to tissue culture plates. Therefore, the “adherence” method used for preparing the primary macrophage cultures effectively selected against any infected macrophages which may have been present in the peritoneal exudate cell population harvested from virus-infected animals. Ir&ction of primary mucrophuge cultures with vaccinia virus: I~ectiws center assays and yield of progeny wi&ns. Near-confluent monolayer cultures of primary macrophages and a variety of established cell lines were infected with vaccinia virus strain, IHDJ, or strain WR (500 EB/cell). The results of a number of experiments are summarized in Table 1 including those in which (a) the yield of progeny virions was determined; (b) the number of infected cells which gave rise to infectious centers was measured, (c) indirect immunofluorescence was employed to determine the number of virus-infected cells in which viral antigens were expressed; and (d) Hoechst staining was employed to determine whether viral DNA synthesis had occurred in the cytoplasm of virus-infected cells. As compared to L-cells and some of the

1

6 24

6 24

6 24

6 24

6 24

Thioglycollate-elicited macrophages

Virus-elicited macrophages

Resident macrophages

P388Dl cells

WR19M.l

6 24

5774.1 cells

N.D. 21

N.D. 43

N.D. 76

N.D. 95

N.D. 1.6

1.0 0.6

0.7 1.8

12.3 132.5

-Trypsin

N.D. N.D.

N.D. N.D.

N.D. N.D.

N.D. N.D.

N.D. 1.6

1.2 0.6

0.3 0.5

N.D.O N.D.

+Trypsin

N.D.

N.D.

N.D.

N.D.

N.D.

50

72

63

-Ah

83% >83%

+ + + + + +++ N.D. N.D.

2 8 2 8 2 8 N.D. N.D. N.D.

5 3.2

N.D. N.D.

53

56

N.D.

45%

+ +

2 8

13

<50%

>50%

>90%

+ +++

55

2 8

Hoeehst stainr % Infected cell with cytoplasmic inclusions

Intensity

Indirect immunofluorescencee Time p.i.

+Ab

% Infectious centersd

a Cell cultures were washed and infected with vaccinia virus strain IHDJ or WR (500 EB or 10 PFU/cell). Identical results when obtained with these two strains were employed in experiments. Data obtained with the IHDJ strain of vaccinia virus is presented. b At the times indicated, cultures were harvested, sonicated, cell extracts prepared, serially diluted and aliquots of each dilution were titrated on Vero cell monolayers. ’ Half of the primary macrophage cultures employed were treated briefly with trypsin [trypsin (+)I, prior to infection, as an additional step for removing nPECs (Marcelletti and Furmanski, 1978). d Infectious center assays were performed in the presence (+) or absence (-) of heat-inactivated (50”, 30 min) rabbit polyclonal, anti-IHDJ antiserum, as described under Materials and Methods. Vaccinia virus has been reported to persist on macrophage surfaces for 12-16 hr postinfection (Nishmi and Niecikowski, 1963). e Greater than 80% of virus-infected L-cells and greater than 50% of the virus-infected primary macrophages could be demonstrated to contain viral antigens in their cytoplasm by indirect immunofluorescence staining as described under Materials and Methods. When the pattern of immunofluorescence resembled that observed for virus-infected L-cells harvested at 2 hr p.i., the cultures were scored as (+) while an immunofluorescent pattern like that observed for virusinfected L-cells, harvested at 8 hr p.i. was scored (+++) (see under Materials and Methods). f Cells were stained with Hoechst reagent and evaluated as described previously (Esteban, 1977). @N.D.-Not done.

6 24

PU5-1.8 cells

cells

6 24

L-Cells

Cells infected”

Time postinfectionb

Yield PFU/eell”

YIELD OF PROGENY, INFECTIOUS CENTERS, VIRAL ANTIGEN EXPRESSION, AND FORMATION OF CYTOPLASMIC FACTORIES AVER INFECTION OF L-CELLS, PRIMARY MACROPHAGES, AND MACROPHAGE-LIKE CELL LINES WITH VACCINIA VIRUS

TABLE

w t2

i 2

3

31 g T1

3

% 02 *

s ?d

2 3 *

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macrophage-like cell lines tested, resident, thioglycollate, or virus-elicited primary macrophages failed to support the complete replication of vaccinia virus (Table 1). Investigators have previously presented data which indicated that infection of resident or thioglycollate-elicited murine macrophages with vaccinia virus resulted in a productive infection (Ueda and Nozima, 1973; Koszinowski et &, 1975; Rama Roa et CL&1977). Where sufficient data was presented (Ueda and Nozima, 1973; Koszinowski et al, 1975) we were able to calculate that these reported productive infections resulted in yields of virus of less than 1 PFU/cell and, it was not clear if this represented progeny virus or virus, derived from the inoculum, which persisted in the infected macrophage cultures (Nishmi and Niecikowski, 1963). The results of the infectious center assays reported here (Table 1) and previously (Koszinowski et al, 1975) indicated that while the majority of the cells in primary macrophage cultures were nonpermissive hosts for vaccinia virus there appear to be a small subpopulation of cells, most evident in the thioglycollateelicited macrophage population, which were productively infected. Whether these productively infected cells were macrophages or some other cell type, with adherence properties similar to macrophages, was not determined. The yield of progeny virions recovered after infection of macrophage-like cell lines was variable; the P388Dl and WR19M.l cell lines were as permissive as L-cells while significantly less progeny virus was measured after infection of the PU5-1.8 and 5774.1 cell lines (Table 1). The reasons for these differences in virus yield remain to be explored. While little or no progeny virus was produced in the virus-infected primary macrophage cultures, indirect-immunofluorescence studies indicated that greater than 50% of the virus-infected macrophages in the cultures contained virus-specified antigens (Table 1). Immunofluorescence techniques have been previously employed to demonstrate that vaccinia virus can replicate in murine macrophages in wivo (Mims, 1964; reviewed by Dales and Pogo,

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1981). Our results (Table 1) confirm that vaccinia virus antigens, either derived from virions or synthesized after infection, can be detected in the cytoplasm of infected macrophages but that the presence or expression of such viral antigens is not necessarily an indication that the infection will be productive (McFadden et aL, 1979). The results of experiments in which vaccinia virus-infected primary macrophages were stained with Hoechst reagent, showed that less than 10% of the virus-infected macrophages developed “factories” in their cytoplasm (Table 1) indicative of viral DNA replication (Cairns, 1960; Esteban, 1977), suggesting that viral replication in primary macrophages was blocked at or prior to the stage of viral DNA replication. While the majority of the cells in primary macrophage cultures were nonpermissive hosts for vaccinia virus, with time after infection, a marked, generalized, cytopathic effect was observed in the infected cultures. Uniformly, the infected macrophages became highly vacuolized, granular, rounded-up, and by 36 hr postinfection, the majority of the virus-infected cells had detached from their substratum and upon extended incubation no surviving cells were observed in virus-infected primary macrophage cultures. This “toxic” effect of vaccinia virus on macrophages has been previously reported (Nishmi and Bernkopf, 1958; Silverstein, 1970) and it was suggested that the “toxic effect” was mediated, in part, by virion components (Silverstein, 1970). We concluded from the results of the experiments summarized in Table 1, that infection of primary macrophages with vaccinia virus resulted in an abortive infection. To further define the stage at which virus replication was aborted we undertook a series of experiments in which we analyzed the replication of vaccinia virus in primary macrophages. Adsorption, uncoating, and viral DNA replication in vaccinia virus-iMected primaw macrcrphages. The adsorption of [3H]thymidine- or [35S]methionine-labeled vaccinia virus was studied as described under Materials and Methods. In agreement with the observations of Silverstein (1970)

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we could show that vaccinia virus was adsorbed to primary macrophages and L-cells with about equal efficiency at both 4 and 37”. This suggests that the adsorption of virus particles to macrophages rather than phagocytosis of virus particles by macrophages was measured in these assays (Svennerholm et a& 1982). When uncoating of vaccinia virus particles containing rH]thymidine-labeled DNA was measured after infection of Lcells (Joklik, 1964a, 1964b) 6575% of cellassociated viral genomes were determined to be uncoated by 3 hr postadsorption (data not shown). When we attempted to perform HOURS POST-ADSORPTION similar uncoating assays after infecting FIG. 1. Release and degradation of [‘HJthymidinethioglycollate-elicited macrophages with labeled vaccinia virus DNA after infection of thioglyvaccinia virus, we found that a significant collate-elicited macrophages. L-cells and thioglycolportion of the [3H]thymidine-labeled viral late-elicited macrophage monolayers in 35-mm culture genomes, associated with the infected cells dishes were infected with [Qlthymidine-labeled vacat 1 hr postinfection were subsequently lost cinia virus, strain IHDJ (500 EB/cell). After adsorpfrom the cells. With time after infection a tion (60 min, 37”) the cell monolayers were washed significant portion of the labeled viral DNA three times with PBS and refed with medium. Imcould be detected in the culture medium mediately after the absorption period and at the inharvested from infected macrophage cul- dicated times postadsorption, duplicate cell cultures tures, in the form of acid-soluble products were harvested. The medium was removed, centri(SOOg,3 min, 4”) and a portion of the clarified (Fig. 1). Similar results were obtained after fuged medium was removed and precipitated with TCA. An infection of virus-elicited macrophages equal portion of the medium was spotted directly onto (data not shown). This loss and conversion GFK glass filters, dried, and counted under a toluene of cell-associated viral DNA to acid-soluble based scintillator to provide a measure of the total products prevented an accurate determi- (acid soluble and insoluble radioactivity) labeled manation of the percentage of genomes which terial present in the medium. The difference between actually became uncoated in the macro- the total radioactivity and acid insoluble radioactivity phages. When [35S]methionine-labeled vi- was used as a measure of acid soluble material in the medium. The cell monolayers were washed, cell lysates rions were employed to infect primary macrophages a significant portion of the were prepared, aliquots of the lysates were removed and precipitated with TCA. The precipitates were col[35S]methionine-labeled, cell-associated vi- lected on GF/C glass filters, washed, dried, and rion proteins were also subsequently de- counted under a toluene based scintillator. The tected in the medium in the form of acid- amount of cell-associated [aI-I]thymidine-labeled viral soluble products. While viral proteins were DNA was then determined with respect to time postalso released from infected L-cells (Joklik, adsorption. Macrophages (O), L-cells (0). At 2 hr pos1964a), the amounts of acid-soluble r”S]- tadsorption 20% of the viral DNA released from macmethionine-labeled material released from rophages into the medium was in the form of acid infected, primary macrophages signifi- soluble products; at 4 hr postadsorption, 40% of the cantly exceeded the amounts released from viral DNA released from macrophages was in the form infected L-cells. These results confirmed of acid soluble products. Less than 10% of the viral DNA lost from infected the observations of Silverstein (1975) in [3H]thymidine-labeled L-cells detected in the culture medium at 4 hr postthat, uncoating of vaccinia virus particles adsorption, was in the form of acid soluble products. in primary macrophages was clearly aberrant and that intracellular subviral particles were degraded and the degradation No viral DNA synthesis could be meaproducts released from virus-infected sured in the cytoplasm of vaccinia virusmacrophages. infected primary macrophages (Fig. 2). To

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AND HOLOWCZAK

in the cytoplasmic fraction prepared from virus-infected macrophages. The results of the experiments presented in Figs. 2 and 3

I

4 HOURS POST.ADSORPTION

FIG. 2. Cytoplasmic DNA synthesis in vaccinia virusinfected and mock-infected L-cells and thioglycollateelicited macrophages. Cell monolayers in 35-mm culture dishes were either mock-infected or infected with vaccinia virus, strain IHDJ (500 EBs/cell). After adsorption (60 min, 3’7”) the monolayers were washed and refed with medium. At the indicated times postadsorption the monolayers were pulse-labeled for 20 min with 5.0 &i [3H&hymidine/culture. The labeled monolayers were then washed, the cells lysed with nonionic detergent and the cell lysates were collected as described in legend, Fig. 2. The cell lysates were separated into a nuclear and cytoplasmic fraction by centrifugation @OOg,2 min). Aliquots of each cytoplasmic fraction were TCA precipitated, the precipitates collected on GF/C filters, washed, dried, and counted under a toluene based scintillator. Infected L-cells (O), mock-infected L-cells (0), infected thioglycollate-elicited macrophages (m), mock-infected thioglycollate-elicited macrophages (0).

exclude the possibility that, relative to virus-infected L-cells, DNA synthesis in macrophages was delayed, we labeled infected cells with rHjthymidine for 24 hr and then analyzed the cytoplasmic fraction for the presence of [3HJthymidine-labeled progeny virions. As shown in Fig. 3, 3Hlabeled progeny virions could be readily detected in the cytoplasmic fraction prepared from virus-infected L-cells but not

FIG. 3. Assembly of progeny virions containing [Q]thymidine-labeled DNA as detected by sedimentation analysis of infected cell extracts. L-cell or viruselicited macrophages (35-mm cultures) were mockinfected or infected with vaccinia virus, strain IHDJ (500 EB/cell) and then incubated in medium containing 5 aCi/ml of PHphymidine. Twenty to 2.4hr postinfection, cell cultures were harvested, frozen, and thawed (two cycles) and the lysates with the aid of a rubber policeman were transferred to a 15-ml conical centrifuge tube, sonicated, and centrifuged (3OOgfor 3 min, 4”) to remove large debris. The supernatant fraction was harvested, portions removed for titration on Vero cells and determination of total TCA-insoluble mhymidine-labeled material, and 1.2 X 10” purified virions, labeled with [“Cjthymidine or PSjmethionine were added to serve as internal sedimentation markers. The samples were sonicated (Holowczak, 1972) and layered onto preformed 20-40% (WW) sucrose gradients prepared in 1 mMphosphate buffer, pH 7.2. The gradients were centrifuged (Spinco SW 25.1 rotor; 15,000rpm; 45 min, 4’), fractionated, and portions of each fraction were removed and precipitated with TCA. Precipitates were collected by filtration on GF/ C glass filters, washed, dried, and counted under a toluene-based scintillator. To simplify the figure, the distribution of labeled virions added as sedimentation markers-labeled with [‘“Cjthymidine or [asS]methionine-is not shown (93% recovered in fractions 12-16). [sH]Thymidine-labeled material in the cytoplasm of virus-infected L-cells (m); virus-infected virus-elicited macrophages (0); and mock-infected L-cells o-rvirus-elicited macrophages (0).

VACCINIA

VIRIONS

AND

confirmed the observations described in Table 1 where we showed by Hoechst staining that viral DNA replication occurred in 10% or less of the vaccinia virusinfected primary macrophages as well as the experimental results presented in Fig. 1 which indicated that the viral genomes, necessary for viral DNA replication, were degraded and released from virus-infected primary macrophages. Two experimental variables were examined in regard to the ability of primary macrophages to support viral DNA replication when measured as described above. First, it has been reported that macrophages in culture secrete thymidine into their culture fluids (Stadecker and Unanue, 1979).Excess cold thymidine could alter the uptake and labeling with CjHjthymidine. To test for such an effect, we transferred medium from macrophage cultures to virusinfected L-cells at the time they were pulsed with rH]thymidine to measure viral DNA synthesis (Stadeeker and Unanue, 1979). Incorporation of [aH]thymidine into cytoplasmic viral DNA was reduced by less than 15% in the infected L-cells fed with macrophage-medium as compared to virusinfected L-cells maintained in their own medium. When macrophages were refed with fresh medium or with medium harvested from L-cells at the time they were labeled, no change in the incorporation of rH]thymidine could be measured and upon analysis (data not shown) results like those presented in Fig. 2 were reproduced. Secondly, we supplemented the medium employed to feed the macrophages after infection with additional arginine (200 pg/ ml) to determine whether the depletion of arginine in the medium by arginase, secreted by the macrophages, could be responsible for the failure to detect vaccinia virus DNA synthesis (Obert et ak, 1971; Cooke and Williamson, 1973; Wildy et aL, 1982). No viral DNA replication could be measured in the infected macrophages fed with arginine-supplemented medium and, when assays like those described in Table 1 were conducted, no significant production of progeny virions could be measured. These results were in agreement with the

MACROPHAGE

INFECTION

363

observations of Wildy et al. (1982), in which macrophage-derived arginase was shown to be involved in the inhibition of herpesvirus and vaccinia virus replication in permissive cells cocultured with macrophages. This inhibition could be reversed by addition of arginine to the cultures. However, the addition of arginine to infected macrophage cultures failed to reverse the inhibition of herpesvirus replication within macrophages (Wildy et al, 1982).

Synthesis of viral proteins after ir4fection of primuw rnocroph,cqe cultures Following infection of primary macrophages with vaccinia virus, a rapid and almost complete inhibition of protein synthesis could be measured (data not shown). This inhibition of protein synthesis occurred more rapidly and was more complete following the infection of virus-elicited macrophages (87% at 6 hr p.i.) as compared to thioglycollateelicited macrophages (75% at 6 hr pi.). With the exception of the initial accelerated inhibition of protein synthesis in the vaccinia virus-infected macrophages, the overall kinetics of protein synthesis measured after infection of macrophages resembled that reported for permissive cells treated with cytosine arabinoside to inhibit viral DNA replication (Holowczak and Joklik, 1967). This suggested that “early” virus-specified protein synthesis might be curtailed and that no “late” virus-specified protein synthesis occurred in the vaccinia virus-infected, primary macrophages. To determine whether any virus-specified proteins were synthesized in the virusinfected primary macrophages, infected cultures labeled with [%]methionine from O-10 hr postinfection were harvested, extracts were prepared and immunoprecipitated as described under Materials and Methods. The immune complexes were analyzed by SDS-PAGE, and radiofluorograms were prepared and analyzed in a densitometer. The results from such analyses are summarized in Figs. 4 and 5. As compared to vaccinia virus-infected L-cells (lane M, Fig. 4) fewer virus-specified proteins could be immunoprecipitated from extracts of virus-infected thioglycollateelicited macrophages (lane L, Fig. 4) and a

364

NATUK

AND HOLOWCZAK

ABCDEFGHIJKLMN

93K66K-

43K31KZZK-

FIG. 4. Radiofluorograph of polyacrylamide gels in which polypeptides immunoprecipitated from [“Slmethionine-labeled extracts of mock-infected or vaccinia virus-infected primary macrophages or L-cells were analyzed. Thioglycollate-elicited, virus-elicited, or L-cell cultures (2 X lo6 cells/ culture) were either mock-infected or infected with vaccinia virus, strain IHDJ (500 EB/cell). After a 60-min adsorption period each culture was fed with medium containing 20% of the normal concentration of methionine and supplemented with 25 &i/culture with [%]methionine. Ten hours postadsorption, the cultures were harvested, detergent extracts were prepared (Gomer and Lazarides, 1983) and equal portions of the lysates were immunoprecipitated with preimmune or rabbit antivaccinia virus antiserum. Immune complexes were harvested, processed (Kessler, 1975; Soloski, et u& 1981) and analyzed by SDS-PAGE. Lanes A, B, and C: Polypeptides immunoprecipitated with preimmune serum from extracts of mock-infected, virus-elicited macrophages (lane A), mock-infected thioglycollate-elicited macrophages (lane B), or mock-infected L-cells (lane C). Lanes D, E, and F: Polypeptides immunoprecipitated with preimmune serum from extracts of virus-infected, viruselicited macrophages (lane D), virus-infected, thioglycollate-elicited macrophages (lane E), or virusinfected L-cells (lane F). Lanes H, I, and J: Polypeptides immunoprecipitated with anti-viral antiserum from extracts of mock-infected, virus-elicited macrophages (lane H), mock-infected thioglycollateelicited macrophages (lane I), or mock-infected L-cells (lane J). Lanes K, L, and M: Polypeptides immunoprecipitated with antiviral antiserum from extracts of vaccinia virus-infected, virus-elicited macrophages (lane K), vaccinia virus-infected thioglycollate-elicited macrophages (lane L), or vaccinia virus-infected L-cells (lane M). Lanes G and N: Purified pS]methionine-labeled vaccinia virions were analyzed (69-80 pg of protein as virus/sample).

subpopulation of the viral polypeptides expressed in thioglycollate-elicited macrophages could be immunoprecipitated from extracts of infected, virus-elicited macrophages (lane K, Fig. 4). When quantitated by densitometry, both the number as well as the amounts of the viral proteins synthesized in the infected primary macrophage cultures was reduced as compared to infected L-cells (Fig. 5). In the virus-infected thioglycollate-elicited macrophages, the major, [%]methionine-labeled, vaccinia virus-specified polypeptides detected had molecular mass of 88, 38, 37, 27, and 25 kDa (Figs. 4 and 5) while the major [35S]methionine-labeled, virusspecified polypeptides immunoprecipitated from extracts of infected, virus-elicited macrophages had molecular mass of 37,27, and 25 kDa (Figs. 4 and 5).

Expression of zlaminia virus-spec$ed antigem mathe plasma membrane: Lysis of irlfected rnacrophages by W-CTLS. The association with or expression of virus-specified antigens on the membranes of vaccinia virus-infected, primary macrophages was demonstrated by measuring the binding of ‘%I-protein A to virus-infected cells which had specifically bound anti-viral antibodies (Fig. 6). Analysis of this data (Table 2) indicated that compared to vaccinia virus-infected L-cells, the quantity and (or) the number of viral antigens expressed on the surfaces of infected macrophages was significantly reduced and never exceeded that present on the surfaces of virus-infected L-cells at 2 hr postinfection. To identify the virus-specified antigens which were synthesized and then expressed on the surfaces of vaccinia virus-infected

VACCINIA

VIRIONS

AND

I;-.rl L

FIG. 5. Densitometric analysis of radiofluorographs prepared from polyacrylamide gels in which the polypeptides synthesized in L-cells, thioglycollate-elicited macrophages, and virus-elicited macrophages infected with vaccinia virus were analyzed. Lanes C, D, and E from Fig. 7 were scanned using a Fiber Optic Scanner (see under Materials and Methods). (A) Infected Lcells. (B) Virus-infected thioglycollate-elicited macrophages. (C) Virus-infected virus-elicited macrophages. Arrows indicate molecular mass @Da) of some major virion polypeptides (Essani and Dales, 1979).

macrophages, [3H]leucine-labeled plasma membranes were prepared from vaccinia virus-infected primary macrophages and analyzed by SDS-PAGE. Radiofluorograms were prepared (inset, Fig. 7) and scanned in a densitometer. These analyses demonstrated that the synthesis of a number of macrophage plasma membrane proteins was significantly inhibited after viral infection (Fig. 7, compare A and B) and, polypeptides with molecular mass of 48,4546,40,36-37 (major), 32, and 25 kDa were uniquely expressed on the plasma membranes of vaccinia virus-infected, thioglycollate-elicited macrophages (inset, Fig. 7 and Fig. 7A). This represented a subset of the virus-specified antigens reported to be

MACROPHAGE

INFECTION

365

present on the plasma membrane of vaccinia virus-infected L-cells, early after infection (Mallon and Holowczak, 1985). To relate the expression of virus-specified proteins on the plasma membrane of virus-infected macrophages, to lysis by VVCTLs, 51Cr-labeled, vaccinia virus-infected macrophages were employed as targets for the in vitro assay of VV-CTL activity. The results of such an experiment are summarized in Table 3. Vaccinia virus-infected, resident, thioglycollate-elicited, and viruselicited macrophages were lysed by VVCTLs in a Class I, MHC restricted fashion (Table 3). The expression of the virus-specified antigens on the plasma membrane of vaccinia virus-infected, thioglycollateelicited macrophages described above (Fig. 7) were sufficient to render the virus-infected macrophages competent for lysis by VV-CTLs. DISCUSSION

The results of the experiments reported here confirm and extend the observations and conclusions reached by Silverstein (1970, 1975) and in agreement with other investigators (Nishmi and Bernkopf, 1958; Glasgow and Habel, 1963; Nishmi and Niecikowski, 1963) show that infection of primary peritoneal macrophages with vaccinia virus in vitro results in an abortive infection. It was possible to demonstrate that the initial stages of vaccinia virus infection in macrophages proceeded normally, partial uncoating of virions and, as evidenced by expression of some “early” virus-specified gene products, activation of the virion-associated DNA-dependent RNA polymerase (Munyon et cd, 1967; Kates and McAuslan, 1967; reviewed by Dales and Pogo, 1981), occurred in the infected macrophages. Subsequently, the replication cycle of vaccinia virus in primary macrophages was aborted, the final stages of uncoating were aberrent (Silverstein, 1970,1975), no “late” viral functions were expressed and progeny virions were not assembled. As compared to L-cells, there was a marked, accelerated develop-

366

NATUK

AND

HOLOWCZAK

6

TIME POST-ADSORFTION (HOURS)

FIG. 6. Expression of vaccinia virus antigens on the surfaces of infected cells as measured by binding of anti-viral antibody-?-labeled protein A. Confluent L-cell (A) and thioglycollate-elicited macrophage monolayers (B) were infected with vaccinia virus, strain IHDJ (600 EBs/cell). Unadsorbed virus was removed by aspiration, the monolayers were washed and refed with medium. At 0,2,4, and 8 hr postadsorption representative monolayers were removed, washed, and then incubated for 1 hr at 3’7” with one of the following: 1:lO dilution of rabbit anti-vaccinia virus antiserum (0); 1:lO dilution of rabbit preimmune antiserum (m), with PBS alone (A). The monolayer-s were washed and then incubated for 1 hr at 3’7” with 1161-protein A, washed, and solubilized with 0.3 N NaOH. The cell lysates were transferred to test tubes and counted in a gamma counter (see also legend, Table 2).

ment of CPE in the virus-infected primary macrophage cultures which culminated in the death of the infected macrophages. If similar events occur in v&o, the death of macrophages following infection with vaccinia virus could contribute to the removal of virus particles and curtail the spread of virus in an infected animal. While the exact mechanisms underlying the restriction of vaccinia virus replication in primary macrophages in vitro remain to be elucidated, there is growing evidence that the failure of viruses to replicate in explanted macrophages may be directly related to the exposure of macrophages to interferon(s) [and (or) other lymphokines] in wivo (Rama Roa et a& 1977; Haller et al, 1979, 1980; Haller, 1981; Belardelli, 1984). The experimental results presented here, concerning the replication of vaccinia virus in primary macrophages which showed (a) restriction of the virus proteins expressed to a portion of the “early” or “immediate

early” proteins expressed in L-cells, (b) the marked accelerated inhibition of protein synthesis and CPE development after infection of macrophages as compared to Lcells, and (c) the failure to detect significant formation of progeny virions after infection of primary macrophages, are in general agreement with the observations of a number of investigators who have examined the replication of vaccinia in murine cells treated with interferon (Joklik and Merrigan, 1966; Barban and Baron, 1968; Horak et al, 1971; Bodo et a& 1972; Suh et ak, 1974). Peritoneal macrophages have served as a convenient source of syngeneic, virus-infected cells to serve as targets for in vitro assays where the activity of VV-CTLs was measured (Zinkernagel and Doherty, 1977; Zinkernagel, 1976; Koszinowski et ah, 1976) and have proven useful in this regard for studies involving other viruses (Hosono and Katsura, 1982). Plasma membrane

VACCINIA

VIRIONS AND MACROPHAGE

367

INFECTION

TABLE 2 EXPRESSION OFVACCWIAVIRUS ANTIGENSONINFECTEDCELL SURFACESAS MEASUREDBY THE BINDING OFVIRAL ANTIBODY-‘251-P~~~~~ A COMPLEXES Percentage of maximum virus-specified cell surface antigen expression””

Cells infected” L-Cells Thioglycollate-elicited macrophages Vaccinia virus-elicited macrophages

1 hr postadsorption 45 34 10

8 hr postadsorption 100 45 10

’ Confluent cell monolayers (approximately 4 X lo5 cells/culture) were infected with vaccinia virus strain IHDJ, 500 EBs/cell. Infected L-cells, DBA/W, thioglycollate-elicited macrophages, and DBA/W vaccinia viruselicited macrophages (6 days after an intraperitoneal injection, 2.8 X lo6 EBs IHDJ/mouse) were employed in the experiments. * Maximum expression of vaccinia virus antigens on the surface of infected cells was determined to occur by 8 hr after infection of L cells. No significant increase in binding of antiviral antibodies could be measured after this time period. Sufficient antibody was employed at all time points analyzed to saturate the viral antigens expressed on 1 X lo6 virus-infected cells at 8 hr postinfection. ‘The expression of vaccinia virus antigens on cell surfaces was calculated as a percentage of antigen expression measured with infected L-cells at 8 hr postadsorption by the following formula: (CPMs infected - CPMs mock-infected) X 100% CPMs infected,, - CPMs mock-infected,, Where CPMs infected was the net difference of measured ‘%I bound to infected cell monolayers in the presence of immune serum minus that measured using preimmune serum. CPMs mock-infected, was the net difference of measured ‘“I bound to mock-infected cell monolayers in the presence of immune serum minus that measured using preimmune serum. CPMs infected,, was the net difference of measured ‘=I bound to infected L-cells (at 8 hr postadsorption) in the presence of immune serum minus that measured using preimmune serum. CPMs mock-infected,,, was the net difference of measured ‘9 bound in the presence of immune serum minus that measured using preimmune serum.

modifications appear to be among the earliest virus-specified events which can be measured following infection of cells with vaccinia virus and we were able to demonstrate that virus-specified antigens with molecular mass of 48,46,40,36-37,32, and 25 kDa were expressed on the plasma membranes of vaccinia virus-infected, thioglycollate-elicited macrophages but not uninfected macrophages. The antigens we detected would be available to associate with Class I, Major Histocompatibility Complex antigens to form the antigenic complexes with which VV-CTLs could interact (Doherty and Zinkernagel, 1974). While we were able to measure a significant

quantitative difference in the expression of virus-specified cell-surface antigens on virus-infected macrophages as compared to virus-infected L-cells (Fig. 6, Table Z), six of the eight viral antigens detected on cell surfaces “early” after infection of L-cells (Mallon and Holowczak, 1985; Mallon et aL, 1985) were expressed on the membranes of vaccinia virus-infected, thioglycollateelicited macrophages. As was found for vaccinia virus-infected L-cells (Mallon et aL, 1985) a virus-specified polypeptide with a molecular mass of 36-37 kDa was a major antigen expressed on the plasma membranes of vaccinia virus-infected, thioglycollate-elicited macrophages (Fig. 7) and

NATUK

0

AND

5 DISTANCE (CM)

FIG. 7. Densitometric analysis of radiofluorograms prepared from polyacrylamide gels in which plasma membranes isolated from mock-infected or vaccinia virus-infected thioglycollate-elicited macrophages were analyzed. Thioglycollate-elicited macrophages were either mock infected or infected with vaccinia virus strain IHDJ. After adsorption for 50 min the monolayers were washed and refed medium containing 20% of the normal amount of leucine. The macrophages were cultured for 10 hr in the presence of [‘Hlleucine (25 &i/culture). Membranes were prepared and purified as described under Materials and Methods and analyzed by SDS-PAGE. Inset, (A) Radiofluorograph of gel in which [‘Hlleucine-labeled plasma membranes from mock-infected macrophages (lane A) or vaccinia virus-infected macropbages (lane B) were analyzed. Densitometer tracings were made from radiofluorographs. (A) Densitometer tracing of gel in which analysis of plasma membranes prepared

HOLOWCZAK

may play an important role in forming an antigenic complex recognized by W-CTLs. When we attempted to carry out a similar analysis with plasma membranes prepared from vaccinia virus-infected, viruselicited macrophages, we encountered experimental difficulties; the labeling of membranes components was significantly reduced and the yield of membranes inadequate for performing the required analyses. The rapid shut-off of host and virus-specified protein synthesis as well as the accelerated development of CPE which occurred after infection of virus-elicited macrophages undoubtedly contributed to the technical difficulties we encountered. We are presently testing various multiplicities of infection and labeling protocols in an effort to find conditions which will allow the isolation of labeled plasma membranes from infected, virus-elicited macrophages which will be suitable for analysis. We could detect only five virus-specified antigens in total extracts prepared from vaccinia virus-infected, virus-elicited macrophages (Fig. 4) and the expression of virus-specified cell-surface antigens on the plasma membranes of virus-infected viruselicited macrophages was reduced as compared to infected, thioglycollate-elicited macrophages (Table 2). The analysis of membranes prepared from vaccinia virusinfected, virus-elicited macrophages is important in that it may allow the identification of a minimum number of virusspecified antigens which, when expressed on vaccinia virus-infected cell surfaces, render the cells competent targets for VVCTLs. Further, these experiments may provide insights into the manner that macrophages process viral antigens and carry out their antigen-presenting function (Unanue, 1984) in vaccinia virus-infected animals.

from vaccinia virus-infected, tbioglycollate-elicited macrophage were analyzed. (B) Densitometer tracing of gel in which plasma membranes from mock-infected thioglycollate-elicited macrophage were analyzed. Arrows indicate molecular mass markers &Da).

N.D.

N.D. 25 f 4

N.D. 36 + 5

1+4

4+3

N.D.

3f3

Mock

33 + 3

Inf.

0*4 38 3~5

lf2

3+0

14*4

Inf.

1t3 28 + 5

4t1

o-to

6+3

Mock

Resident DBA/W maerophages’

PERITONEAL

N.D. 62f9

N.D.

O&6

19-11

Inf.

N.D. 29 f5

N.D.

Of3

2rl

Mock

Vaccinia virus-elicited DBA/W macrophagesC

N.D. 1823

N.D.

5rt4

69*6

Inf.

BY VV-CTLS

N.D. 21 f4

N.D.

10+5

422

Mock

4&l 34 +4

47 + 1

4*1

10+4

Inf.

320 24+3

8+0

O&l

llr2

Mock

L-Cells”

0*1 36-t3

28+2

4&l

2*1

Inf.

Ok0 21&2

1+1

4+0

3 +O

Mock

Resident C3H/HeJ macrophages’

target cells & SD”; E:T = 5O:l’

MACROPHAGES

P388Dl cells”

specific lysis of %r-labeled

VIRUS-INFECTED

Percentage

LYSIS OF VACCINIA

3

8&O N.D. 20 * 2

N.D. 68?3

N.D.

N.D. 47+1

N.D.’

Mock N.D.

Inf.

Vaccinia virus-elicited C3H/HeJ macrophages”

“In vitro “Cr-release assays were performed and the percentage specific lysis calculated as described previously (Byrne et aL, 1983). The percentage specific lysis measured at an effector:target (E:T) ratio of 5O:l are presented. When various E:T ratios were employed and the results were analyzed as described (Perlmann and Cerottini, 1979), the specific lysis measured at E:T = 5O:l fell on the linear portion of the dose-response curve. Effecters were incubated with targets for 10 hr. b Splenic effector cell populations were prepared from unmanipulated mice or from animals 6 days after intraperitoneal injection of 2 X lo6 PFU of purified vaccinia virus, strain IHDJ. Specific lysis of virus-infected L-cells by splenic effecters (E:T was 5O:l) from virus-infected C3H mice was 57 * 2 [before treatment with monoclonal Thy-l antibodies (New England Nuclear, Mass.) plus complement]; 8 f 3 (after treatment with Thy-l antibodies plus complement) (Hutcher and Kuhn, 1982). ’ Macrophages were prepared (see under Materials and Methods) from the PEC population elicited 6 days after intraperitoneal injection of thioglycollate or vaccinia virus, strain IHDJ (2 X 10s PFU/dose). Target cells (various primary macrophages) labeled with %r were infected (inf.) with vaccinia virus, strain IHDJ (500 EB/cell) or mock-infected (mock) and then employed as targets for ia vitro ‘%r-release assays. d SR-Spontaneous release of ‘iCr from mock-infected and virus-infected target cells measured in a series of three experiments (Byrne et al, 1983). Note the particularly significant spontaneous release values recorded after infection of vaccinia virus-elicited macrophages which may have reflected, in part, the rapid development of CPE in such cultures. “N.D.-Not done.

Vaccinia virusinfected Unmanipulated % SRd

C3H/HeJ

from b

Vaccinia virusinfected Unmanipulated

Effecters

DBA/W

Mouse strain

Thioglycollateelicited DBA/W macrophages’

SPECIFIC

TABLE

i2

2

g 2n

ii

8 g

E

& u

g (I)

s s3

2 8 z s

370

NATUK

AND HOLOWCZAK

ACKNOWLEDGMENTS

DALES, S. (1965). Penetration of animal viruses into cells. Prog. Med Viral 7,1-43. DALES, S., and Poco, B. G. T. (1981). Biology of poxviruses. Fir01 Monogr. 18.1-109. DOHERTY,P. C., and ZINKERNAGEL,R. M. (1974). Tcell mediated immunopathology in viral infections. Transplant. Rev. 19,89-120. DVORAK,H., and DVORAK,A. M. (1982). Immunohistological Characterization of Inflammatory Cells. In “Tumor Immunity in Prognosis” (S. Haskill, ed.), pp. 279-307. Dekker, New York. EDELSON,P. J., and COHN,A. (1976). Purification and REFERENCES cultivation of monocytes and macrophages. In “In Vitro Methods in Cell-Mediated and Tumor ImunADAMS, D. O., and HAMILTON, T. A. (1984). The cell ity” (B. R. Bloom and J. R. David, eds.), pp. 333biology of macrophage activation. Annu. Rev. Im340. Academic Press, New York. mund 2,283-318. ALLISON, A. C. (1974). On the role of mononuclear ESSANI, K., and DALES, S. (1979). Biogenesis of vacphagocytes in immunity against viruses. Prog. Mea! cinia: Evidence for more than 100 polypeptides in the virion. V+ology 95,385-394. vi?-01 18, 15-31. ALLISON, A. C. (1976). Fluorescence microscopy of ESTEBAN,M. (1977). Rifampicin and vaccinia DNA. J. lymphocytes and mononuclear phagocytes and the Viral 21, 796-801. J. A. (1977).Replication use of silica to eliminate the latter. In “In Vitro ESTEBAN,M., and HOLOWCZAK, of vaccinia DNA in mouse L-cells. I. In tivo DNA Methods in Cell-Mediated and Tumor Immunity” synthesis. virology 78,57-75. (B. R. Bloom and J. R. David, eds.), pp. 395-404. GLASGOW,L. A., and HABEL, K. (1963). Interferon Academic Press, New York. production by mouse leukocytes in vitro and in vivo. BARBAN,S., and BARON,S. (1968). Differential inhibJ. Exp. Med 117,149-160. itory effects of interferon on deoxythymidine kinase induction of vaccinia-infected cell cultures. Proc GODING,J. W. (1978). Use of staphylococcal protein A as an immunological reagent. J. Immunol MethSot. Exp. Biol Med 127,160-164. ods 20,241-253. BELARDELLI,F., VIGNAUX,F., PROLETTI,E., and GRESSER. I. (1984). Injection of mice with antibody to GOMER,R. H., and LAZARIDES,E. (1983). Switching of filamin polypeptides during myogenesis in vitro. J interferon renders peritoneal macrophages perCell Biol 96,321-329. missive for vesicular stomatitis virus and encephalomyocarditis virus. Proc. NatL Acad Sci USA 81, HALLER, 0. (1981). Inborn resistance of mice to orthomyxoviruses. Cum-. Top. Microbial Immund 92, 602606. 25-52. BODO, G., SCHEIRER, W., SUH, M., SCHULTZE,B., HORAK, I., and JUNGWIRTH,C. (1972). Protein syn- HALLER, O., ARNHEITER,H., GRESSER,I., and LINDERMANN, J. (1979). A genetically determined, interthesis in pox-infected cells treated with interferon. feron-dependent resistance to influenza virus in virology 50, 140-147. mice. J. Exp. Med 149,601-602. BRUNETTE,D. M., and TILL, J. E. (1971). A rapid method for the isolation of L-cell surface mem- HALLER, O., ARNHEITER, H., LINDERMANN, J., and GRESSER,I. (1980). Host gene influences sensitivity branes using an aqueous two-phase polymer system. to interferon action selectively for influenza virus. J. Membr. Biol 5,215~224. Nature (London) 283,660-662. BUCHMEIER,N. A., GEE, S. R., MURPHY, F. A., and RAWLS,W. E. (1979).Abortive replication of vaccinia HOLOWCZAK,J. A. (1972). Uncoating of poxviruses. I. virus in activated rabbit macrophages. In&e& ImDetection and characterization of subviral particles in the uncoating process. Virology 50,216-232. mun 26,328-338. BYRNE, J. A., SOLOSKI,M., and HOLOWCZAK,J. A. HOLOWCZAK,J. A. (1976). Poxvirus DNA. I. Studies on the structure of the vaccinia genome. virdogy (1983). Immune responses of DBA/2 mice bearing 72,121-133. melanoma tumors: Cell-mediated immune responses after challenge with vaccinia virus. Cancer Immu- HOLOWCZAK,J. A., and JOKLIK, W. K. (1967). Studies on the structural proteins of vaccinia virus. II. Kino1 Immunother. 16,81-87. netics of the synthesis of individual groups of CAIRNS,J. (1960). The initiation of vaccinia infection. structural proteins. lrirology 33, 726-739. virology 11,603-623. COOKE,B. C., and WILLIAMSON,J. D. (1973). Enhanced HORAK, I., JUNGWIRTH,C., and BODO,C. (1971). Poxvirus specific cytopathic effect in interferon treated utilization of citrulline in rabbitpox virus-infected L-cells. Virology 45,456-462. mouse sarcoma 180 cells. J. Gen ViroL 21,339-348.

The expert technical assistance of Ms. Domenica Bucolo is gratefully acknowledged. This investigation was supported by a US Public Health Research Grant (CA-11027), National Research Service Award Institutional Grant (CA-09069) from the National Cancer Institute, a grant from the American Cancer Society (ACS-IM-169), and research support from the Foundation of the University of Medicine and Dentistry of New Jersey.

VACCINIA

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HOSONO, M., and KATSURA, Y. (1982). The use of

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