Site of viral RNA synthesis within the midgut cells of the silkworm, Bombyx mori, infected with cytoplasmic-polyhedrosis virus

JOUHNAL

OF

INVERTEBRATE

Site Cells

PATHOLOGY

of

Viral

of with

the

9,

480-487

RNA

( 1867)

Synthesis

Silkworm,

within

Bombyx

the

mori,

Cytoplasmic-Polyhedrosis

Midgut Infected

Virus1

HITOSHI WATANABE Laboratory

of Sericulture,

Faculty Bunkyo-ku,

Acceptecl

of Agriculture, Tokyo, Japan

October

25,

University

of

Tokyo,

1966

The pattern of nucleic acid synthetic activity within the midgut cells of healthy larvae of the silkworm, Bombyx mori, and of larvae infected with the cytoplasmicpolyhedrosis virus was demonstrated by means of autoradiography with labeled nucleic acid precursors. Five hours after injection of uridine-H’, the healthy cells generally incorporated the labeled material into cytoplasmic RNA and partly into nucleic RNA, whereas the diseased cells on the 2nd to the 5th day after virus inoculation incorporated much of the labeled uridine into nucleic RNA and some into cytoplasmic RNA. In the nuclei of virus-infected cells, the nucleic label appeared most densely over the nucleoli. Thus the distinct difference in the uptake of the RNA precursor between the healthy and infected cells indicated that the nucleolus of the infected cell may be a site of the viral RNA synthesis. Autoradiograms with thymidine-H3 revealed no essential difference in the pattern of DNA synthesis between healthy and diseased midguts, and only a few cells incorporated the labeled material into their nuclei. At the late stage of virus infection, however, when some infected midgut cells eventually degenerated, there was a slight increase in the nucleic label in the newly generated cells.

Histochemical changes in the midgut cells during the course of infection with the cytoplasmic-polyhedrosis virus in the silkworm, Bombyr nzori, were described by Iwashita and Aruga ( 1957). They found a pyroninophilic material which gave a positive polysaccharide reaction in surrounding the nucleus. the cytoplasm This indicated that some substances necessary for virus development were pro-

duced in the nucleus and diffused into the cytoplasm. Hayashi and Kawase (1965) ’ This work was supported in part by a Grant-inAid for Scientific Research from the Iapanese Ministry of Education. 480

chromatographically analyzed the subcellular distribution of ribonucleic acid (RNA) in the virus-infected midgut of the silkworm. They reported that the specific RNA for the virus in the nuclear, mitochondrial, and microsomal fractions of diseased midgut were 16.0%, 8.1%, and 5.3% of the total amount of RNA, respectively. Despite these investigations, the site of the synthesis of the RNA component of the cytoplasmic-polyhedrosis virus within the infected host-cell is still uncertain. By means of autoradiography and histochemical techniques carried out on the same preparation from virus-infected tissue, it is sometimes possible to establish both the

SITE

OF

VIRAL

RNA

SYNTHESIS

utilization of a radioactively labeled substance for the production of a particular viral or cellular component and also the intracellular locus at which the synthesis is effected (Benz, 1963; Bellett, 1965; Morris, 1966 ). The present paper describes the results of the autoradiographic research that was conducted subsequently to that reported previously (Watanabe, 1966) on the localization of RNA synthesis within the midgut cells of thp silkworm infected with the cytoplasmic-polyhedrosis virus. This report also presents the results of the study on the deoxyribonucleic acid (DNA) synthesis in the virus-infected ~~11s. \124TERI~LS

.4ND

METHODS

Fourth-instar larvae of several strains of the silkworm were used in this study. The virus emploved was a Bomhgx cytoplasmicpo1yhedrosi.i virus which formed polyhedra tetragonal in outline, and was known to contain RNA (Iwashita and Aruga, 1957; Aizawa and Iicla, 196rJ; Hayashi and Ka\5’asc, 1964 ) . Thr larvae were infected with the virus by feeding them for 20 hours on mulberry leaves smeared with a high concentration of the virus polvhedra suspension (5 >< 10” pol~hedra/ml) .’ Both control and infected larvae werp htAlc1 at 25°C throug’lout the experiment.

;2 series of larvae from both the control a virus-infected groups were collected at %-hour intervals. The larvae were slightly anesthetized with ether, and then injected with a solution of radioactive material at 20 ,kc/larva as follows: 1 me/ml uridine-H” (specific activity 1.76 c/mM, generallv labeled; Radiochemicll Centre, Amersham, Buckinghamshire, England ) as

IN

HOST

CELLS

481

an RNA precursor; I me/ml thymidineW ( specific act’ Ivi t y 6.0 c/mM; Schwarz BioResearch, Inc., Orangeburg, New York, U.S.A.) as a DNA precursor. Prior to each labeling experiment with uridine-Hz’, an excess of nonradioactive thymidine ( IO x ) was injected into both thts control and infected larvae in order to re-duce the incorporation of uridine-H” into DNA. The lengths of labeling periods werr 1, 2, 5, and 24 hours, respectively. Th(> midgut of the larvae treated with radioactive material \vas removed after each labeling period and fixed in CarnoIr’s Auitl for 2 hours, dehydrated, embedded in paraffin, and sectioned at 34 p thicknt,ss. The paraffin was dissolved from the scctio,ts with xylene and the sections were hs,drated. Then the sections \verc wash& with 2% perchloric acid at 4’ C for 20 minutes to remove the acid-soluble fractions of the incorporated label ( Woods, 1959 1. After 10 minutes in distilled water the slides were coated with Sakura NR-Vl-G nuclear emulsion ( Konishiroku Photo lnd. CO., Tokyo, Japan) (2 parts emulsion 1 part distilled water at 43 C 11.L\ftcr clxposure of L4 weeks at d”C, the autoradio-. grams were developed, and subsecluen t Iv stained with Ciemsa or hem~\tox\~lin-cosill. lZistopatltology To understand the biochemical features of the autoradiogram, the uncoatrtl SVCtions with nuclear cmulslon \v~‘rt~ staiiled c\tochemicallv. Sections chosen for this s&y were stained with Unna methyl green pyroninc. Fculgen light grecin. and 13% bromphc~nol blue.

i11

RESULTS

.4ND

~~scussros

The examination of the :tutoradiograms of healthy larvill midguts on the 2nd day of the fourth instz revc>aled that uriclinefl:’ after 5-hollr labeling was generally in-

WATANABE

FIG. 1. Autoradiographs demonstrating uridine-H” uptake in the healthy and diseased midguts at an early stage of infection with cytoplasmic-polyhedrosis virus. Fourth-instar larvae. B, body cavity; L, lumen of gut. (a) Healthy cells of the posterior portion of the midgut on the 2nd day of the fourth instar. Labeled for 5 hrs with uridine-H3. Stained with Giemsa. Magnification: 480 X. General incorporation of radioactive uridine into the cytoplasm. (b) Infected cells of the posterior portion of the midgut on the 2nd day after inoculation of the virus. Labeled for 5 hrs. Stained with Giemsa. Magnificatian: 480 X. The label is present mostly over the nucleus, particularly over the nucleolus. A much smaller number of radioactive grains are seen over the cytoplasm. (c) Infected epithelial cells on the 3rd day after inoculation of the virus. Labeled for 24 hrs prior to fixation. Stained with hematoxylin-eosin. Magnification: 660 X. Radioactive grains are present densely over both the nucleus and the cytoplasm.

SITE

OF

VIRAL

RNA

SYNTHESIS

corporated in the cytoplasm of numerous cells (Fig. la). The label appeared to be distributed more or less uniformly throughout most of the cytoplasm. Although no essential difference in the pattern of incorporation was observed among the l-, 2-, and S-hour labelings, some amount of uridine-H3 uptake into the nucleus alone occurred in the healthy cell within l-hour labeling. On the other hand, in the autoradiograms of the midgut cells in the early stage of virus infection, especially in the posterior portion of the gut, a high density of label was found over the nucleus but a much lower one over the cytoplasm (Fig. lb). Tn the nucleus, a comparatively high relative density of the label was always found over the nucleolus. An autoradiogram of a virus-infected cell laheled for 24 hours revealed that the radioactive grams were present densely over both the nucleus and the cytoplasm. The distribution of autoradiographic grains over the cytoplasm was apparently not uniform. The cytoplasmic radioactivity was mostlv localized around the nucleus as well as the apical region of the cell (Fig. 1~). A clear difference in the pattern of incorporation of uridine-H3 between healthy and virus-infected cells was also noted in the late stage of infection. In the infected cells, on the 5th day after inoculation of the virus when polyhedra had already developed in the cytoplasm, the concentration of radioactive grains was dense over the nucleus, particularly localized on the nucleoli, and some grains were distributed over the cytoplasm (Fig. 2b ). This pattern of distribution of radioactivity contrasted markedly with that of healthy cells where the grains were mostly distributed over the cytoplasm and yet the concentration was much lower than that of grains in the infected cells ( Fig. 2a ) . Histopathological observations of the early stage of virus infection revealed that the nucleoli became larger and were in-

IN

HOST

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483

tensely pyroninophilic, which indicated an increase in RNA synthesis. The cytoplasm around the nucleus as well as in the apical region of the cell was also strongly pyroninophilic and stained with bromphenol blue, which gives a protein-positive reaction. These pyroninophilic structures in the cytoplasm of the apical region apparently formed the virogenic stroma described by Xeros ( 1956, 1966) in the cvtoplasmic polyhedrosis of other species.’ In the late stage of infection, the polyhedra developed mainly in the cylindrical epithelial cells, and sometimes in the goblet cells. The polyhedra first developed in the apical region of the cytoplasm just bekm, the brush border where there was :\ greater staining intensity with pyroninc~. Later the polyhedra were formed at thc~ basal region. The cytochemical differences and the distinct differences in the pattern of tlie uptake of RN,4 precursor between infectrd and healthy cells indicate that the nuclrolus is the site of viral RNA synthesis within the midgut cells infected with the cvtoplasmic-polyhedrosis virus. Since the V&U and polyhedra have been ohserved with the electron microscope only in the cvtoplasm ( Hukuhara and Hashimoto, 1966) . the viral RNA produced in the nucleus apparently passes out into the cytoplasm where the complete virus with its protctin coat is formed. From the studies on the synthesis of virus components in animal cells. it has been suggested that RNA-containing ;rnima1 viruses, such as the encephalornyocarditis virus (Bellett and Burnrss, 1963), and influenza virus ( Bukrinskaya, 1964) . produce their RNA in the nuclei of irlfected cells. whereas Mengovirus RNA ( Franklin and Kosner, 1962) and poliovirus RNA (Baltimore and Franklin. 196.3) are synthesized in the cytoplasm. As far as the site of viral RNA svnthesis is <‘OILcemed. the Hom!qx cytoplasmicpolvhe-

FIG. 2. Autoradiographs a late stage of infection B, body cavity; L, lumen day of the fourth instar. terial into the cytoplasm. 5 hrs. Tetragonal-shaped tensely diseased midgut. these degenerating cells

demonstrating uridine-H3 uptake in the healthy and the diseased midguts at with cytoplasmic-polyhedrosis virus. Fourth-instar larvae. Stained with Giemsa. of gut; P, polyhedra. Magnification: 480 X. (a) Healthy cells on the 5th Labeled for 5 hrs with uridine-H’. General incorporation of the labeled ma(b) Infected cells on the 5th day after inoculation of the virus. Labeled for polyhedra developed in the cytoplasm, and the nucleoli heavily labeled. (c) InLabeled for 5 hrs. Infected epithelial cells discharged into the gut lumen, but still retained the radioactive uridine.

SITE

OF

VIRAL

RNA

SYNTHESIS

drosis virus seems to belong to the former virus group where the RNA is formed in the nucleus. At the advanced stage of the virus disease, the infected epithelial cells were discharged into the gut lumen. The Feulgen reaction of the nuclei of these degenerating cells was greatly reduced. The auto-

IN

HOST

CELLS

a5

radiograms with uridine-Ha, however, revealed that these degenerating cells still retained the radioactive material incorporated into the nucleus (Fig. 2~). Although it was difficult in this study to clearly distinguish viral RNA synthesis from hostcell RNA synthesis, a comparison of a series of autoradiograms between healthy

FIG. 3. An autoradiographic series demonstrating thymidine-H3 uptake in the healthy midgut and in the midgut infected with cytoplasmic-polyhedrosis virus. Fourth-instar larvae. Stained with Giemsa. B, body cavity: L, lumen of gut; P, polyhedra. Magnification: 380 X. (a) Healthy midgut of larva at: late stage of the fourth instar. Labeled for 5 hrs with thymidine-H”. (1~) Diseased midgut at the late stage of infection. Labeled for 5 hrs. (c) Heavily diseased midgut. Labeled for 5 hrs. Somcl rpithrlial ~11s discharged into the L-t lumen.

486

WATANABE

and diseased midguts has resulted in an interpretation that host RNA and viral are individually synthesized in RNA slightly infected cells, but in heavily infected cells only the viral RNA synthesis takes place. Uptake

of Thymidine-H”

Autoradiograms with thymidine-HZ of healthy midguts of larvae at any stage of the fourth instar demonstrated that only a few cells incorporated labeled material into the nucleus ( Fig. 3a ) . The incidence of cells containing labeled nucleus was about the same as that of diseased midguts (Fig. 3b). Histochemically, the nucleus of a cell at an early stage of infection gave a Feulgen-positive reaction of the same intensity as that of a healthy cell, but the reaction became less intense towards the end of polyhedra formation. At the late stage of the virus disease when most of the infected epithelial cells were discharged into the gut lumen, the regenerative cells located in nidi at the basal region of the midgut developed into cylindrical cells by squeezing between the goblet cells and the old infected cells that were filled with polyhedra. Autoradiograms of the midgut at this stage of infection showed that the uptake of thymidineH3 into the nucleus slightly increased in the newly developed cylindrical cells (Fig. 3~). This acceleration of DNA synthesis apparently has no direct relation with the virus multiplication but is concerned with the regenerative development of cells. ACKNOWLEDCMENTS The author wishes to thank Professor H. Aruga for encouragement during the course of this work. The author is also deeply indebted to Dr. Y. Tanada, Department of Entomology and Parasitology, University of California for reading and correcting the original manuscript.

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AND IIDA, S. 1963. Nucleic acids exfrom the virus polyhedra of the silkBombyx mori (Linnaeus). J. Insect 5, 344-348.

BALTIMORE, D., AND FRANKLIN, R. M. 1963. A new ribonucleic acid polymerase appearing after mengovirus infection of L-cells. J. Biol. Chem., 238, 3395-3400. BELLE=, A. J. D. 1965. The multiplication of Sercesthis iridescent virus in cell culture from Antheraea eucalypti Scott. III. Quantitative experiments. Virology, 26, 132-141. BELLETT, A. J. D., AND BURNESS, A. T. H. 1963. Intracellular sites of encephalomyocarditis virus components in Krebs-2 ascites tumour cells. J. Gen. Microbial., 30, 131-140. BENZ,

G. 1963. Physiopathology istry. In “Insect Pathology” ed.), Vol. 1, pp. 299338. New York.

and histochem(E. A. Steinhaus, Academic Press,

BUKRINSKAYA, A. G. 1964. Resistance of synthesis of influenza virus ribonucleic acid to the action of actinomycin D. Nature, 204, 205-206. FRANKLIN, R. M., AND ROSNER, J. 1962. Localization of ribonucleic acid synthesis in mengovirus-infected L-cells. Biochim. Biophys. Acta, 55, 240-241. H~YASHI, Y., AND KAWASE, S. 1964. Base paring in ribonucleic acid extracted from the cytoplasmic polyhedra of the silkworm. Virology, 23, 612-614. HAYASHI, Y., AND KAWASE, S. 1965. Studies on the RNA in the cytoplasmic polyhedra of the silkworm, Bombyx mori L. IV. Subcellular distribution. J. Sericult. Sci. Japan, 34, 171175 (in Japanese with English summary). HUKUHARA, T., AND HASHIMOTO, Y. 1966. Studies of two strains of cytoplasmic-polyhedrosis virus. 3. Invertebrate Pathol., 8, 184-192. I~ASHITA, Y., AND ARUGA, H. 1957. Mechanism of resistance to virus diseases in the silkworm, Bombyx mori. III. Histological studies on the polyhedroses in the silkworm. J. Sericult. Sci. Japan, 26, 323-328 (in Japanese with English summary). MORRIS, 0. uridine

N. 1986. Incorporation into the RNA of

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OF

J. Itwertebrate

H. 1986. Localization acid synthesis in the midgut with cytoplasmic-polyhedrosis silkworm, Bomhyx mori L. Romhycidae) . AII)II. Entomol. 15.5.

WATANABE,

VIRAL

RNA

SYNTHESIS

Pathol., 8,

of ribonucleic cells infected virus in the (Lepidoptera: Zonl., 1, 154-

WOODS,

IN

P.

HOST

s.

487

CELLS

195%

RNA

in

nUCkar-CytOphSnliC

interaction. Brookhacen Symp. Bid., 12, lS:i174. SEROS, N. 1956. The virogenic stroma in nuclear and cytoplasmic polyhedroses. Nutuw, 178, 412-413. ?IEROS, N. 1966. Light microscopy of the virogeuic stromata of cytopolyl~edros~,s. j. Int;ertebrfltc~ Pathol., 8, X-87.