A cytological and immunological study of Tipula iridescent virus-infected Galleria mellonella larval hemocytes

VIROLOGY

61, 409-423 (1973)

A Cytological

and

Virus-Infected

Immunological Galleria

B. GILLIAN Department

of Biology,

Study

mellonella

YULE2 Carleton

AKD

Larval

PETER

University,

of Tipula

Ottawa,

iridescent

Hemocytes’

E. LEE Ontario,

Canada

Accepted ATovember 8, 1972 Viral structural protein was localized in Tipula iridescent virus (TIV) infected hemocytes of Galleria mellonella larvae by fluorescent antibody and immunoferritin techniques. Viral and cell DNA were located by acridine orange staining. Viral structural protein fluorescence was observed in the cytoplasm outside virus-induced viroplasms 1 day aft,er virus inoculation. The viroplasm did not normally fluoresce until the second day. The intensity of fluorescence increased during the next 5 days. Specific ferritin tagging appeared on virus occurring singly in the cell, on virus infecting or leaving the cells, around groups of particles within the cytoplasm and throughout the viroplasm. Viral DNA was located exclusively in the viroplasm. Morphological and immunological data permitted evaluation of hypotheses of TIV assembly and a proposal is presented for t,he development and assembly of the virion in hemocytes. INTRODUCTION

Tip& iridescent virus (TIV) induces cytoplasmic inclusion bodies or viroplasms in infected host cells (Xeros, 1954). With light microscopy, DP\;A was located in these areas by Feulgen staining (Bird, 1961) and by 3H-thymidine incorporation (Morris, 1970), suggesting viral DNA accumulates and probably is synthesized in the viroplasms. Viral protein antigen was detected by the fluorescent antibody technique in the cytoplasm of hemocytes of TIV-infected Pieris 0rassicae larvae 3 days after virus inoculation (Oliveira and Ponsen, 1966). It has not been determined whether accumulation and synthesis of viral prot’eins occur in the viroplasms. Virus assembly pools, visible in the electron microscope (Younghusband and Lee, 1969), correlate well with the Feulgen’ Research supported by a National Research Council Operating Grant (A-2911) and Capital Grant (E-2134). 2 Recipient of a National Research Council postgraduate scholarship.

staining regions observed in the light microscope. High-resolution DNA autorsdiography suggested viral DELTA synthesis occurs in the viroplasm S hr after virus inoculation (Younghusband and Lee, 1970). This substant.iates light microscope nutoradiographic evidence that viral DNA is synthesized in the viroplasm, although the unlikely possibility that viral DKA is synthesized elsewhere and rapidly transported to the viroplasm where it accumulates, canrlot be excluded. In t’he present study, fluorescent antibody and immunoferritin techniques were applied to TIV-infected Galleria mellonella larval hemocytes to localize viral structural protein in the cell. It was hoped that the site(s) and time of viral structural protein synthesis could be determined and correlated with the site and time of viral DNA synthesis. A second aspect of the study \vas concerned with virion assembly. Although the general cycle of TIV infection is known (Younghusband and Lee, 1969), the mode of particle formation is not. There are three 409

Copyright All rights

0 1973 by Academic Press, of reproduction in any form

Inc. reserved

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AND

main hypotheses: (a) formation of complete viral membranes (shells) prior to core development (Smith, 1956); (b) development of cores which are later coated with protein (Bird, 1961); (c) parallel formation of loose, irregular viral membranes and DNA fibrils in the virogenic stroma, with the membranes gradually assuming icosahedral shape while segregating the requisite amount of viral DNA from the stroma (Xeros, 1964). In the present study an attempt was made using electron microscope morphological data coupled with the immunoferritin technique to elucidate the sequence of events in TIV particle assembly. MATERIALS

AND

METHODS

Virus PuriJication, Antibody Production, and Preliminary Assays Virus-purification procedures were those of Younghusband and Lee (1969) with some modifications. Infected G. mellonella larvae were macerated in 0.01 1M sodium borat,e buffer, pH 7.3, and subjected to two cycles of low- and high-speed centrifugation at 2000g for 5 min and 35,000g for 30 min, respectively. The final high-speed pellet was dispersed in buffer, layered in 5ml quantities on lo-50% linear sucrose gradients, and centrifuged at 40,OOOg for 30 min. The main viral band in each gradient was removed by side puncture of the tube, pelleted, dispersed in buffer, and relayered and centrifuged through a new sucrose gradient, The single viral band obtained was removed, pelleted, and resuspended (2 mg/ml) in distilled water for antiserum production, or in an antibiotic solution containing 200 units penicillin, 200 fig streptomycin, and 5 pg Fungizone per ml (Grand Island Biological Co., NY) for larval infection. Two methods of rabbit inoculation were used. The first involved six weekly intramuscular injections of 1 ml TIV (2 mg/ml) emulsified with 1 ml Freund’s complete adjuvant. The second consisted of four weekly intravenous injections of 1 ml TIV (2 mg/ml) each. Rabbits were bled 1 week after the last injection in either case. Gamma globulins from antiserum and normal serum were purified by the ammo-

LEE

nium sulfate method (Kendall, 1937). The procedure of Clark and Shepard (1963) was used for conjugation of r-globulin (lo-15 mg/ml) and fluorescent dye. Free fluorescein isothiocyanate (FITC) (Baltimore Biological Laboratory, Maryland) was separated from FITC-conjugated rabbit y-globulin by gel filtration on a G-50 Sephadex column (Zwaan and Van Dam, 1961). To reduce nonspecific staining the eluant was adsorbed twice with an acetone-dried powder of noninfected larvae using 100 mg powder/ml conjugate. Commercial FITClabeled anti-rabbit r-globulin (Mann Research Lab. New York) was used for the indirect method of antigen-antibody staining. Ferritin, 6 X crystallized, and cadmiumfree (Pentex Biochemicals, Kankakee, IL) was coupled to the FITC-labeled r-globulin fraction of antiserum induced by TIV, the FITC-labeled r-globulin fraction of normal serum, and FITC-labeled anti-rabbit y-globulin by means of the bifunctional reagent meta-xylylene diisocyanate (Hsu, 1963). The microprecipitin test was used to determine titer and specificity (van Slogteren, 1955) of induced y-globulin. Several antigens were titrated with dilutions of the yglobulin fraction of TIV antiserum. These antigens were (a) virus (0.2 and 0.02 mg/ ml); (b) the pellet from the first high-speed centrifugation of a noninfected larval preparation obtained in the same manner as that from infected larvae during virus purification; (c) that region of the sucrose density gradient of this noninfected larval preparation which corresponded to the main viral band. These antigens were also titrated with the r-globulin fraction of normal serum and with 0.85% NaCl, as a control. Microprecipitin tests were also used to verify that the FITC-labeled r-globulin would react specifically with antigen after conjugation with ferritin. In the first test, virus (2, 0.2, and 0.02 mg/ml) was titrated with (i) unlabeled TIV-induced y-globulin (10 mg/ml), (ii) fluorescein-ferritin-labeled TIV-induced y-globulin (10 mg/ml and 2.5 mg/ml), (iii) fluorescein-ferritin-labeled Y-

TZPULA IRIDESCENT globulin fraction of normal serum (10 mg/ml). In the second test, unlabeled TIVinduced y-globulin (10 and 2.5 mg/ml) was titrated with stock (10 mg/ml) and several dilutions (l/4, l/16, l/64, l/256) of fluorescem-ferritin doubly labeled anti-rabbit -r-globulin and with 0.85 % NaCl. Micro-double-diffusion reactions in 0.01 M sodium borate buffer, pH 7.3, containing 0.01% sodium aside were carried out on cellulose acetate membranes (Johnson et al., 1964). Virus (2 mg/ml) and the supernatant fractions from homogenates of infected and noninfected insects diffused against yglobulin fractions from TIV antiserum and normal serum. These homogenates were prepared by macerating 15 infected larvae at 5 days postinoculation and 15 noninfected larvae in 0.01 M sodium borate containing 0.01% sodium azide. The homogenates were filtered through cheesecloth and centrifuged at 980g for 5 min. Negative staining of virus-conjugate interactions was used to determine the specificity of fluorescein-ferritin-conjugated yglobulin. Antibody and antigen were combined as follows: Virus (0.2 and 0.02 mg/ml> was combined with (i) unlabeled TIV yglobulin (10 and 2.5 mg/ml), (ii) fluorescein-ferritin TIV y-globulin (10 and 2.5 mg/ml), (iii) fluorescein-ferritin y-globulin from normal serum, (iv) free ferritin, for 30 min, the resultant precipitate washed with 0.85% NaCl and centrifuged at 980s for 5 min. Doubly labeled anti-rabbit yglobulin (stock, 10 mg/ml, and l/4, l/16, l/64 dilutions) was added to (i) for 30 min and the precipitate washed with 0.85% NaCI. A small drop of the final suspension was applied by fine pipet to a carbonFormvar grid, and stained for 5 set with 2% phosphotungstic acid (PTA) buffered with 2 M KOH pH 6.7 (Brenner and Horne, 1959). Hemocyte Preparations and Direct Fluorescent Antibody Staining A larval proleg was clipped and hemolymph collected in freshly prepared 0.85% NaCl in plastic wells on glass slides (Chaudhary and Westwood, 1969) and the slides placed in moist chambers. Hemocytes from

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l- to 7-day-infected larvae which had been inoculated with 2 ~1 of virus (2 mg!ml), and from noninfected larvae were allowed to spread for 2-3 hr and then were washed with 0.85% NaCl and fixed in acetone for 1 min. Several dilutions of FITC-conjugated r-globulin (l/4, l/16, l/64), in addition to the stock concentration (10 mg/ml) were applied for 30 min. Slides were washed 4 hr in several changes of 0.85% NaCl, 10 min in distilled water, and mounted in glycerol. Acridine Orange Staining Acridine orange (0.01%) in either distilled water (final pH 3.8) or Mcllvain’s buffer pH 4.0, was applied for 3 min after acetone fixation, to samples of hemocytes from the same time series as that used in fluorescent antibody staining. As a control, some cells were treated with 0.4% DNAase in 0.003 M MgSOr for 45 min after acetone fixation and before staining. Electron Microscopy Techniques

and

Immunoferritin

Hemocytes were fixed in two stages with a combination of formaldehyde and glutaraldehyde and permeabilized with the detergent digitonin (Levinthal et al., 1969). Hemocytes were also fixed by routine methods (1.6 % buffered glutaraldehyde, Sabatini et at., 1963) for morphological comparison. Larval prolegs were clipped and hemolymph from infected insects at $5, 1, 2, 4, 8, 16, 24, 48, and 72 hr postinoculation and from noninfected insects collected in small cellulose nitrate tubes containing (a) 1.6% buffered glutaraldehyde [O.Ol M phosphatebuffered saline pH 7.3 containing 5 X lO+ M MgCl and 1 X 10m4M CaCl (PBSMgCa)] for one-half hour or (b) 0.01% formaldehyde (made with paraformaldehyde) plus glutaraldehyde in 0.01 M 0.008 % PBSMgCa pH 7.3 for 5 min (Levinthal et al., 1969). Digitonin (1.2 X lo-* M) was added to cells fixed in (b) for 30 set, followed by 1.0% formaldehyde plus 0.8 % glutaraldehyde in 0.01 M PBSMgCa for 5 1 hr in min. Samples were washed PBSMgCa, postfixed one-half hour in 1% osmic acid in PBSMgCa, washed 1 hr in

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PBSMgCa, dehydrated in ethanol, infiltrated with propylene oxide, and embedded in Epon Araldite (Mollenhauer, 1964). Variations on this procedure included bleeding larvae directly into digitonin or into 0.01 M PBSMgCa with subsequent digitonin treatment without prefixation, and into the low concentration of formaldehyde/glutaraldehyde followed by the high concentration without digitonin treatment. Sections were cut on a Reichert OmU2 microtome with a diamond knife and stained with uranyl acetate (Stempak and Ward, 1964) for 10 min or doubly stained with uranyl acetate and lead citrate (Reynolds, 1963) for 10 min each. Sections were mounted on carbon-Formvar grids and photographed in a Siemens 101 microscope at X0 kV. The indirect immunoferritin technique was used. Hemocytes from infected larvae at 35, 1, 2, 4, 8, 10, 12, 24, 48, and 72 hours postinoculation and from uninfected larvae were prepared with the two-stage fixation and intermediate digitonin treatment described. Samples were then washed 1 hr in 0.01 ~11PBSMgCa and suspended in 5 vol of a l/4 dilution of unlabeled TIV-specific y-globulin (2.5 mg/ml) for 1 hr. Cells were washed in 0.01 M PBSMgCa for 1 hr and 5 vol of doubly labeled fluorescein-ferritin anbi-rabbit y-globulin added for 1 hr. Samples were washed 1 hr in 0.01 1M PBSMgCa, postfixed in osmic acid, washed, dehydrated, and embedded as usual. Control preparations consisted of infected cells treated with the unlabeled r-globulin fraction of normal serum prior to treatment with doubly labeled anti-rabbit y-globulin

LEE

and infected cells treated directly with a fluorescein-ferritin-labeled r-globulin fraction of normal serum. In addition, uninfected cells were treated with unlabeled TN-induced r-globulin followed by fluerescein-ferritin-labeled anti-rabbit y-globulin. RESULTS

Both methods of antiserum production induced the same titer (64) of r-globulin when assayed by the microprecipitin test. This titration also indicated antiserum was specific for TIV since precipitate occurred only with purified virus. Microprecipitin tests suggested that the direct immunoferritin procedure could not be used since no specific precipitation occurred when virus reacted with fluoresceinferritin-labeled TIV r-globulin or fluorescein-ferritin-labeled r-globulin from normal serum. The indirect immunoferritin method was feasible since doubly labeled antiglobulin reacted strongly with its antigen (TIV-specific r-globulin). TIV y-globulin (10 and 2.5 mg/ml) combined with stock (10 mg/ml) or a l/4 dilution of doubly labeled antiglobulin was optimal. Precipitin lines formed in cellulose acetate membranes with purified virus or supernatant fractions of homogenized infected insects (Fig. la). No reaction occurred with any of the controls (Fig. lb). Negative staining of virus and conjugates suggested, as had the microprecipitin test, that only the indirect. immunoferritin reaction was feasible. Specific tagging of virus occurred when virus (0.2 or 0.02 mg/ml) was combined with unlabeled TIV-specific

FIG. 1. a, b. Micro-double-diffusion reactions in cellulose acetate strips, (a) TIV 2 mg/ml; (d) supernatant fraction from homogenate of noninfected larvae; (e) supernatant fraction from homogenate of 5.day-infected larvae. As, TIV-induced r-globulin, Ns, r-globulin from normal serum. FIG. 2. TIV particle treated indirectly with immunoferritin and negatively stained with PTA. x 190,000. FIG. 3. Typical TIV-infected hemocyte, 7 days postinoculation; note viroplasm V., nucleus N. X 2400. FIG. 4. a, b. Hemocyte, 5 days after TIV inoculation treated directly with FITC-labeled TIVspecific r-globulin. (a) photographed under phase-contrast optics, (b) photographed under uv optics. (arrow). N = Note fluorescence in the viroplasm V, and in other areas of the cytoplasm nucleus. X 1600. FIG. 5. a, b. Noninfected hemocytes treated directly with FITC-labeled TIV-specific r-globulin. (a) photographed under phase-contrast optics, (b) uv optics. X 1600.

TIPULA

IRIDESCENT

VIRUS

INFECTION

413

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YULE AND LEE

r-globulin (10 and 2.5 mg/ml) followed by doubly labeled anti-rabbit r-globulin at either stock (10 mg/ml) or l/4 dilution. Virus particles were covered and surrounded by ferritin (Fig. 2). Higher dilutions of doubly labeled anti-rabbit y-globulin resulted in a minimal tag of virus particles. TIV-speFluorescein-ferritin-conjugated cific r-globulin, doubly conjugated r-globulin from normal serum, and free ferritm, did not tag virus. Light Microscopy Viroplasms were visible in fibroblastic cells 1 day after virus inoculation. These increased in size with time from 3 days infection and by 5 days often filled the cytoplasm (Fig. 3). No similar structures were seen in noninfected cells. The l/4 dilution of FITC-conjugated, adsorbed TIV y-globulin produced bright specific and minimal nonspecific fluorescence (Fig. 4a, b). Faint fluorescence was observed as small spots in the cytoplasm of some cells on the first day after virus inoculation. The viroplasm did not normally fluoresce until the second day. By day 3, both the viroplasm and small areas in the cytoplasm were intensely fluorescent. The size of the cells and the viroplasm increased during the next 4 days. By day 5 the viroplasm occupied most of the cytoplasm. The intensity of fluorescence in all areas increased steadily throughout the test period. Specific fluorescence was absent in noninfected hemocytes stained with FITC-labeled TIV-specific y-globulin (Fig. 5a, b) and in infected and noninfected cells treated with a FITC-labeled y-globulin fraction of normal serum. Acridine orange staining confirmed that the viroplasm contains DNA. One day after virus inoculation the viroplasm and nucleus of infected cells fluoresced green indicating the presence of DNA. Cytoplasmic green fluorescence was confined to the viroplasm throughout the course of infection. Only one green center, the nucleus, was observed in noninfected cells against the red cytoplasmic background. With DNAase treatment prior to staining, green fluorescence was absent but red fluorescence was still apparent in the cytoplasm and nucleoli.

Electron Microscopy The fine structure of hemocytes fixed in 1.6 % glutaraldehyde was adequately preserved. Cells fixed in two stages with low and high concentrations of formaldehyde/ glutaraldehyde with intermediate permeabilization with digitonin were variable in appearance. A large number of cell profiles (J$ to g of the total) retained well-defined organelle structure (Fig. S), but there were cells within the same sample in which the fine structure was severely disrupted. Plasma membranes, nuclei, and viroplasms of disrupted cells often remained intact although the ground cytoplasm was disrupted. Hemolymph collected directly in 0.01 M PBSMgCa was indistinguishable from that collected and prefixed in formaldehyde/ glutaraldehyde. Hemolymph collected directly in digitonin had a greater percentage of disrupted cells than other treatments; however, many cells were sufliciently preserved. Control preparations consisting of cells fixed in two stages without digitonin treatment were indistinguishable from wellpreserved cells which had been permeabilized with digitonin. Specific tagging of intracellular and extracellular antigen occurred when hemocytes, prefixed in low concentrations of formaldehyde/glutaraldehyde or collected directly in 0.01 M PBSMgCa or in 1.2 X 10m4111 digitonin were treated indirectly with unlabeled TIV r-globulin followed by fluorescein-ferritin-labeled anti-rabbit r-globulin (Fig. 7). Digitonin treatment rendered the plasma membranes of approximately one half the cells sufficiently disrupted to allow penetration of ferritin. Specific ferritin tagging was observed in the following locations; the viro(i> Concentrated throughout plasm and around mature particles and developing forms of the particles in the virogenic stroma as early as 1.2hr after virus inoculation (Fig. 8). virus particles which (ii) Surrounding were either infecting or leaving the cell (Fig. 9). The tag on extracellular virus was generally heavier than that on intracellular virus, (Compare Fig. 9 with Fig. 7).

TIPULA

IRIDESCENT

VIRUS

INFECTION

FIG. 6. TIV-infected hemocyte, 24 hr postinoculation, fixed in formaldehyde/glutaraldehyde, meabilized with digitonin, and postfixed in aldehyde and 0~04. Stained with uranyl acetate citrate. V = viroplasm. X 15,300. (iii) Around virus particles which appeared singly in the cell or in vacuoles similar to those reported by Younghusband and Lee (1969) at 4 hr postinoculation (Fig. 10, arrow). (iv) Membrane-bound groups of virus particles, probably lysosomes similar to those observed by Younghusband and Lee (1969), were tagged with ferritin (Fig. 11). (v) The plasma mcmbranc was often tagged with ferritin, probably nonspecifically. In some cells which had been adequately preserved ferritin was randomly distributed in the gound cytoplasm but there were no focal concentrations of this ferritin. At no time after infection was there retention of ferritin in the nuclei or mitochondria. Infected cells treated with doubly labeled y-globulin from normal serum or unlabeled y-globulin from normal serum followed by

415

per-

and lead

doubly labeled antiglobulins did not have ferritin-tagged virus. Noninfected hemocytes treated with unlabeled TIV y-globulin followed by doubly labeled antiglobulin showed minimal retention of ferritin molecules chiefly along the plasma membrane. Examination of numerous sections through the viroplasm of infected cells revealed virus particles in all stages of development and provided some answers to the question of virus assembly (Fig. 12). Developmental viral forms were observed in the virogenic stroma of cells fixed in two stages with formaldehyde/glutaraldehyde (Fig. 13) and in those cells subsequently treated with conjugated antibodies (Fig. 14). Various stages of shell assembly from onesided to five- or six-sided two-dimensional figures (depending on the axis of symmetry of the sectioned particle) were observed in the viroplasm (Figs. 12 and 13a-f). The

416

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FIG. 7. Portion of viroplasm from TIV-infected hemocyte, 48 hr postinoculation, treated indirectly with immunoferritin. Virus particles are tagged with ferritin. Arrow indicates ribosomes. X 108,000.

internal content of completed shells consisted of either (i) material indistinguishable from the viroplasmic matrix (Fig. 13f)

or (ii) varying

amounts of dense material

dots or (Fig. 13g, h) from small central threads (Fig. 13g) to full cores completely filling the shell (Fig. 13i). Some particles had core material attached to one end of

TZf’UL4

Ilbll>ESCI1:NT

VIRUS

FIG. 8. Ferritill-t,agged viroplasm of hemocyte -tS hr after plasmic cytoplasm is destroyed. Arrows indicate developing FIG. 9. Ferritin-tagged virus particle at cell surface, 2 hr membrane. X 119,000. FIG. 10. Ferritin-tagged virus particale (arrows) in vacuole ulation. X 88,000. FIG. Il. Group of ferrititl-tagged virus particles within 16 hr postinoculation. X 53,800.

the shell (Fig. 1Sg insert). Ferritin-tagged developmental forms can be seen in the sequence in Fig. 14 a-j. One, two, three, four, five, six sidw of the icosahedral partitle arc specifically tagged with ferritin (Fig. 14a-f). In thaw: stagw the particle is still in intimate cwntact with the viroplasm

417

INFECTIO;I;

virus inoculation. Most of the extravirovirus particles. X 50,000. postinoculation. Arrow indicates plasma in cytoplasm intracellular,

of hemocyte probably

4 hr postinoc-

lysosomal

vesicle,

(note Fig. 14b). Viroplasmic material can DC seen filling the inside of the partial shells (Fig. 14a, b, c). Complct’ed shells were also obwrwd with ferritin tagging on the inside of t,he shell (Fig. 14 f insert). Particles previously suggested to be hexagonalshapcxd viral cores (Figs. 13j, 14j) were

41s

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FIG. 12. Portion ot viroplasm of TIV-infected hemocyte part’cles in all stages of assembly (arrows). X 38,400.

tagged with ferritin (Fig. 14j). A proposal for TIV assembly is presented in the Discussion. Often groups of virus particle-: nveloped with a membrane from the host cell wern observed within the cell (Fig. 15). Single mature virus particles which appeared to have recently cscapcd from the host cell, probably by budding, \verc also frequently surrounded by a membrane (Fig. 16). DISCUSSION

A major part of the present investigation involved the detection of intracellular antigen at the light and electron microscope level. The nature of the virus-induced viroplasm has been examined closely at both these levels of resolution. Results indicate that viral structural protein accumulates exclusively in the viroplasm. There is clear evidence from acridine orange and Feulgen (Bird, 1961) staining and DNA autoradiography (Younghusband and Lee, 1970) that viral DNA also accumulates in these

24 hr postinoculation.

Viroplasm

contains

regions. Electron microscope morphological and immunological data has provided some insight into the question of virus assembly in these areas. In the light microscope well-developed viroplasm was visible in the cytoplasm of infected hemocytes 1 day after virus inoculaGon. Fluorescent antibody staining specific for viral structural protein was faintly dctcctablc in some cells at this time. Although viroplasms are well developed 24 hr after inoculation, this fluorescence generally occurred in tips of fingers of cytoplasm of cells which had spread considerably (e.g., Fig. 4). The viroplasm did not normally fluorrsco until the second day. Intensity of fluorescence and size of the viroplasm increased from 3-7 days. These observations do not entirely agree with an earlier report (Oliveira and I’onscn, 1966) that l- and a-day TIV-infected hemocytes of P. brassicae were indistinguishable from noninfected controls when both \verc treated directly \vit’h fluorescent antibody.

‘I’IPT:I,A

Il:II)ESCENT

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INFECTION

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FIG. 13. a-j. Electron micrographs of virus particle in proposed sequence of assembly. (a) twosided shell; (b) three-sided shell; (c) four-sided shell; (d] five-sided shell; (e) five-sided shell, arrow indicates point of attachment to viroplasm; (f) completed six-sided shell, containing material indistinguishable from viroplasmic matrix; (g, h) part,ially filled shells, arrow (insert, g) indicates attachmcut point of core to shell; (i) mature partictle; (j) particle probably glancing section through shell, masking core content, (arrow). X 107,OGO. FIG. 14. :t-j. Complementary micrographs of ferritin-t agged viral developmental stages iu proposed sequeuce of assembly. (a-d) successive stages in shell buildup with ferritiii inside shell (arrows); (e) completed six-sided shell with ferritin around shell; (f) ferritin-tagged completed shell containing material indistinguishable from the viroplasmic matrix; Insert: ferrit,irr tagging along inner edge of shell; (g, h) tagged particles with imreasiug amounts of core material; (i) tagged mature virus; (j) ferritintagged particale probably glancing section. X 107,000.

This discrepancy possibly rwults from the different host syst,(hrn, lower experimental temperatures, and less-critical virus purification procedurc~ used by tho above. In the present’ study, the time> factor involved in allowing hcmocytcs t’o spread on glass slides before t,reatment with fluoreswnt antibody probably contributed to the early detection of viral structural protein, since cells which have remained clumped or lvhich have not spread could have small sites of specific fluorcscencc ma&cd by nonspecific or background fluorescence. Acridinc orange staining and DiYAasc

treatment indicated th(l viroplasm is partially composed of viral DNA detectable 1 day after infection. Cytoplasmic DNA staining was confined to the viroplasm throughout the course of infection. An electron microscope autoradiographic study suggested TIV-DNA synthesis occurs as early as S hr in the viroplasm (Younghusband and Lee, 1970). Acridine orange staining and thymidine-3H incorporation in &ricesthis iridescent virus-DNA in Arctheraea eucalypti cells (Bellet,t, 1965) were observed in the same foci, substantiating the idea that DNA is synthesized in t.he acri-

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FIG. 15. Membrane-bound

group of virus particles 24 hr after virus inoculation. Arrow indicates edge of normal particle. X 115,300. FIG. 16. Virus particles at cell surface, probably budding. Lower particle has acquired extra membrane (arrow). X-69,200. outer

dine orange-stained viroplasm. Considered together the evidence from acridine orange staining, light and electron autoradiography suggests that TIV-DNA accumulates and probably is synthesized exclusively in the viroplasm, although the unlikely possibility that it is synthesized elsewhere and rapidly transported to the viroplasm where it accumulates cannot be excluded. Results of further investigations using the immunoferritin technique at the electron microscope level supported light microscope findings that viral structural protein accumulates in the viroplasm. Extracellular and intracellular virus in the following locations was specifically tagged with ferritin: (i) on virus either infecting or

leaving the cell, (ii) around particles occurring singly in the cell, (iii) around membran+bound groups of virus particles in the cytoplasm, (iv) in large concentrations throughout the viroplasmic matrix and around particles in the matrix. There was some ferritin associated with ribosomes in the ground cytoplasm but there were no focal concentrations of this ferritin. At no time was there retention of ferritin in the nuclei or mitochondria. Because of the asynchrony of TIV infection, a critical time study of viral structural protein synthesis was not feasible in the present investigation. Ferritin-tagged virogcnic stroma appeared as early as 12 hr after virus inoculation; however, the

TZPGLA

IRII~ESCENT

fact’ that there was some scattered ferritin in the ground cytoplasm at t’his time makes hhe det,ermination of thr earliest appearance of viral structurA1 prot,cin difficult by this method. The concentration of ferritin on antigen? both extracellular and intracellular compared lvith the low number of ferritin particles attached to the plasma membrane and possibly some of the ribosomes outside the viroplasm is suflicient~ly impressive to give one confidence in the findings especially after examination of a large number of cells. Noninfected cells treated lvith TIV-specific y-globulin and fluorescein-ferritin-labeled antiglobulin or fluorescein-ferritinlabeled globulins from normal serum, showed minimal tagging, chiefly along the plasma membrane. This is probably nonspecific, although it is possible that r-globulins to host cell plasma membrane wcrc present in the TIV-induced r-globulin preparation, since virus particles may ncquirc :m extra mcmbranc from the host’ cell (Figs. 15, 16). In a purified virus preparation there could bc some virus particles surrounded by host cell membranes and -y-globulins against this nonviral membrane might bc induced. This seems unlikely, however, since neither cellulose acetate strip tests nor microprccipitin tests detected these antigens (Fig. 1). Infected cells treated with doubly labeled y-globulin from normal serum or unlabeled r-globulin from normal serum follo\ved by doubly labeled antiglobulin did not show specific tagging. The site of protein synthesis is still questionable. At both levels of resolution, accumulations of structural protein were found only in the viroplasm. It seems plausible to suggest that virus structural protein is synthesized in these centers as electron micrographs indicate ribosomes are prrsent (Fig. 14). In addition, morphological and immunological data reveal this is the region of virus maturation. Partially formed ferritin-tagged viral shells were observed forming within the dense ferritin-tagged stroma. The absence of any concentrated ferritin tagging in the extraviroplasmic cytoplasm substantiates this proposal. If viral protein

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were synthcsizrd in the cytoplasm and transport’cd to the viroplasm for shell assembly, presumably heavy ferritin tagging would be found near ribosomes in the cytoplasm. There was no evidence t,o support this at the electron microscope level, nlthough the occasional spots of fluorescence seen in tips of cells in the light microscope might suggest this possibility. Early fluorescence is probably caused by infecting virus particles which are contained in lysosomr-like structures (Younghusband and Lee, 1969). TIV-DKA synt’hesis is detectable S hr aft,er virus inoculation (Younghusband and Lee, 1970) and since mature ferritin-tagged virus progeny are visible in the viroplasm at 12 hr, protein synthesis presumably occurs between 8 and 12 hr. Viroplasms of infected hemocytes contained virus particles in all stages of assembly. Morphological and immunological data permitted critical evaluation of the t,hree main hypotheses for t’hc developmental sequence of the particle (Smith, 1956; Bird, 1961; Xeros, 1964). No morphological evidence to support Bird (1961) was found. His proposal that naked viral cores were formed prior to shell mat’erial and empty viral membranes were sectioning artifacts seems unreasonable since these membranes are numerous in the early stages of infection, and in the present study there is no reason t)o suspect artifact. Particles suggested t’o be naked viral cores (Younghusband and Lee, 1969) arc probably glancnig sections through the shell of the particle, masking core content. A hypothesis of viral assembly combining aspects of particle development suggested by Smith (19,56) and Xeros (1964) is proposed. This scheme is based mainly on cytological and immunological evidence and thus the hypothesis presented must be accepted with caution since a sequence of particle assembly is proposed on the above-mentioned lines of evidence. There is a sequential construction of the viral shell from the viroplasmic matrix and during this process all stages of shell assembly from one-sided to six-sided (two-dimensional) forms are visible. In sections, the five-sided or six-sided form, depending

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on the axis of symmetry of t,he sectioned particle, constitutes the completed shell. Once the shell is assembled, core mat,eriaI is introduced, perhaps through an opening left in the shell. This first appears as a small dot or thread and progressively enlarges until the shell is filled and mature particles are formed. Evidence that the shell and core are not assembled concurrently as proposed by Xeros (1964) is provided by numerous completed empty shells in the early stages of infection whose internal material is indistinguishable from the viroplasmic matrix. If sides of the particle mere formed and core material segregated at the same time, presumably partial shells would contain material of similar density to viral cores. That this core material is probably introduced through an opening left in the shell is substantiated by the appearance of virus particles with core material att.ached to one end of the shell (Fig. 13g, insert). Cross sections through this figure would not reveal the attachment point. Particles formerly thought to be hexagonal-shaped viral cores (Younghusband and Lee, 1969) are suggested t,o be glancing sections through the shell of the particle with core content occluded (Fig. 13j). All developmental stages of particle assembly from one-sided shells to fully mature particles were tagged with ferritin. Each stage in the construction of the shell was found to have ferritin tagging along the inner edge, indicating that shell and not core material is building up. In these stages the particle is still in intimate contact with the viroplasm (note Figs. 13e, 14~). The appearance of completed shells (two-dimensions) with ferritin tagging along the inner edge substantiates the suggestion that the entire shell is assembled face by face followed by the gradual development of core content. The possibility has not been overlooked that some six-sided forms could be cross sections through incomplete forms but from the number of completed shells observed it is unlikely that all are cross sections. It is on this point that the present proposal differs from Xeros (1964) who suggested the shell was formed in stages but that was segregated concominucleoprotein

AND

LEE

tantly. Later stages of assembly involving the buildup of the core content can be seen in Fig. 14g-i. Ferritin tagging of the forms previously believed to be naked cores (Younghusband and Lee, 1969) substantiates the idea that these are glancing sections t’hrough the shell of the particle (Fig. 14j). The present proposal is an elaboration of Smith’s (1956) idea that empty shells are early stages in particle devclopment and particles with varying amoums of core material, later stages. Since TIV particles were often surrounded by extra membranes from the host cell and particles which appeared to have recently budded were similarly enveloped, it appears particles may easily and often become enveloped in extra membranes. This phenomenon occurs with Xericesthis iridescent virus (Bellett and Rlercer, 1964) and other morphologically similar viruses (Lymphocystis virus, Midlige and Mnlsberger, 1968). REFERENCES BELLETT, A. J. D. (1965). The multiplication of Sericesthis iridescent virus in cell cultures from Antheraea eucalypti Scott. III. Quantitative Experiments. Virology 26, 132-141. BELLETT, A. J. D., and MERCER, E. H. (1964). The multiplication of Sericesthis iridescent virus in cell cultures from Anlheraea eucalypti Scott. I. Qualitative experiments. Virology 24, 645-653. BIRD, F. T. (1961). The development of ‘I%pula iridescent virus in the crane fly, ‘I’ipula and t,he wax moth, Galleria paludosa Meig., mellonella L. Can. J. IVicrobiol. 7, 827-830. BRENNER, S., and HORNE, R. W. (1959). A negative staining method for high resolution electron microscopy of viruses. BiochinL. Riophys. Ada 34, 103-110. CHAUDHARY, R. K., and WESTIVOOD, J. C. N. (1969). Plaque assay of poliovirus in plastic chambers. Can. J. Microbial. 15, 1301-1303. CLARK, F. H., and SHEPARD, C. C. (1963). A dialysis technique for preparing fluorescent antibody. Virology 20, 642-644. Hsu, K. C. (1963). Protocol of labelling globulin with fluorescein and/or ferritin. Dept. of Microbiology. Columbia Univ. Coll. Phys. and Surgeons, NY. JOHNSON, C. M., WEST\VOOD, J. C. N., and BEAULIEU, M. (1964). A continuous flow, microdouble-diffusion technique with cellulose acetate. Nature (London) 204, 1321-1322.

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KITXDALL, F. E. (1937). Studies on serum proteins.

I. Identification of a single serum globulin by immunological means. J. Clin.. Invest. 16, 921931. LEVINTHAI,, J. D., DUNNEUACKE, T. H., and WILLIAMS, R. C. (1969). Study of poliovirlls infection of human and monkey cells by immunoferritin technique. Virology 49, 211-223. MIDIJGIC, F. H., and MALSDERGEII, R. G. (1968). In vitro morphology and maturation of Lymphocystis virus. J. Viral. 2, 833-836. MOI~LENHAUER, H. H. (1964). Plastic embedding mixtures for use in electron microscopy. Stain Techiaol. 39, 111-114. MORRIS, 0. N. (1970). Metabolic changes in diseased insects. III. Nucleic acid metabolism in Lepidoptera infected by densonucleosis and Tipula iridescent viruses. J. Invertebr. Pathol. 16, 180-186. OLIVEIRA, A. R., and POSSIZN, M. B. (1966). The development of a viral antigen in the hemocytes of Pieris brassicae inoculated with Tipula iridescent virus. Neth. J. Plant Pathol. 72 259-264. Rk:~\or,ns, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell. Bid. 17, 20&212. SIKVPINI, D. D., BENSCH, K. R., and BRANIGTT, I:. J. (1963). Cytochemistry and electron mi-

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croscopy. The preservation of cellular ultrastructure and enzyme activity by aldehyde fixat,ion. J. Cell. Biol. 17, 19-58. SMITH, K. M. (1956). The structure of insect virus particles. J. Biophys. Riochem. Cytol. 2, 301-306. S’PICMPAK,

J. C;., and WARD, R. T. (1964). An improved staining method for electron microscopy. J. Cell. Biol. 22, 697-701. VAN SLWTEREN, D. H. M. (1955). Serological microreaction with plant viruses under paraffin oil. Proc. Conj. Potato Viruses Dis. 2, Lisse, Wageningen 1954, 51-54. Xmas, N. (1954). A second virus disease of the leatherjacket, Tip& paludosa. Nature (London) 174, 562-563. X~;ROS, N. (1964). Development of the Tip&a iridescent virus (TIV). J. Insect Pathol. 6, 261-283. YOUNGHUSBAND, H. B., and LEE, P. E. (1969). Virus cell studies of Tipula iridescent virus in Galleria mellonella (L.). Virology 38, 247-254. YOUNGHUSBAND, H. B., and LEE, P. E. (1970). Cytochemistry and autoradiography of Tip&a iridescent virus in Gnlleria melloraella. Virology 40, 757-760. Zwah~, J., and VAN DAM, A. F. (1961). Rapid separation of fluorescent antisera and unconjugated dye. Acta Histochem. 11, 306-308.