Ultrastructural localization of SV40 viral DNA in cells, during lytic infection, by in situ molecular hybridization

Printed in Sweden Copyright Q 1974 by Academic Press, Inc. All rights of reproduction in any form reserved

Experimental Cell Research87 (1974) 175-185

ULTRASTRUCTURAL CELLS,

DURING

LOCALIZATION LYTIC

MOLECULAR

OF SV40 VIRAL

INFECTION,

DNA

IN

BY IN SITU

HYBRIDIZATION

M. GEUSKENS’ and E. MAY Institut de Recherches Scientifigues sur le Cancer, 948 00 Villejuif,

France

SUMMARY The in situ molecular hybridization method has been applied to the detection, at the electron microscope level, of SV40 viral DNA in permissively infected monkey kidney cell cultures. The observations suggest an important role of the host cell nucleolus during the lytic infection with sv40.

The in situ molecular hybridization method enables the detection by radioautography of hybrid molecules formed between radioactive polynucleotidic sequences in solution and the complementary DNA sequences located in cytological preparations. Since the publication of the first papers on this subject by Gall & Pardue [8, 191, the above method has been applied at the photon microscope level to the localization of specific DNA sequences in squashes, smears, paraffin-embedded tissue sections, sometimes after modification of the original technical conditions. As the usual molecular hybridization conditions allow the detection of only repetitive nucleotide sequences, the method has been principally used for localizing satellite DNAs in various biological materials (see references in [3]). 1 Permanent address: Laboratoire de Cytologie et d’Embryologie moleculaires, Universite libre de Bruxelles, 67, rue des Chevaux, Rhode-St-Genese, Belgium. 12-741816

The application of the method at the electron microscope level involves two main difficulties: (1) the labelled nucleic acid used for molecular hybridization must have a very high specific activity as the number of molecules hybridized at one site is low; (2) fixatives which preserve the fine structure of the cells may prevent molecular hybridization or at least prevent penetration of labelled nucleic acid molecules into cells. Nevertheless, two research groups who have used different technical conditions have published results obtained by application of the above method at the ultrastructural level [4, 141. The work of Croissant et al. [4] is an extension at the electron microscope level of in situ molecular hybridization experiments previously performed at the photon microscope level for studying the vegetative replication of viral DNA in tumours induced by the rabbit Shope papilloma virus [18]. Their results have shown that RNA complementary to viral DNA may be used for localizing DNA of this virus in host cells. Exptl Cell Res 87 (1974)

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Geuskens and Malt

Since then, the in situ molecular hybridization method has been used at the photon microscope level to recognize Epstein-Barr virus genomes in human tumour cells [23, 261, to detect adenovirus DNA in permissively and non-permissively infected cells [6, 171 and more recently to detect SV40 DNA in transformed rabbit cells of which a small proportion is spontaneously producing SV40 v41. We present here the first results of a work aiming at localizing at the ultrastructural level the sites of replication of SV40 DNA in permissively infected cells.

MATERIAL

AND METHODS

Viruses and cells. Simian virus 40 (SV40) (large plaque, SVl strain) [22] was grown and assayed for plaque-forming titre in CVl (monkey kidney) cells cloned by L. Montagnier [16] under 0.25 % agarose. Viral lysates were obtained by infection of CVl cultures with dilute viral preparations (0.01-O.1 PFU/ cell). Infection. CV 1 cells were grown in wells of Microtest II tissue culture plate (Falcon plastics) and used for infection when thev had reached confluence. In each well, the cells were infected with 0.020 ml of crude viral lvsate (titre 4 x lo8 PFU/ml). The virus was adsorbed to the cells at 37°C for 1 h In a CO2 incubator. The beginning of the adsorption period was considered as time zero of the infection. After adsorption, the cell cultures were washed twice and then covered with Eagle’s medium supplemented with 10 % Tryptose phosphate (Difco Labs) and 2 % calf serum. Under our experimental conditions, the viral DNA reolication was detected at 18 h after infection as judged by a Hirt extraction [12] of the viral DNA after a I h labelling of the cultures with 5 @X/ml of tritiated thymidine ([5-3H]methyl-thymidine; CEA, France; 12-20 Ci/mmol). At 44 h after infection nearly 100% of the cells were T antigen positive and approx. 50 % of the cells contained V antigen as judged by immunofluorescence tests. of superhelical SV40 DNA. Closed superhelical DNA molecules were separated by equilibrium sedimentation in CsCl containing ethidium bromide, according to Cuzin et al. [5]. Preparation

In vitro svnthesis of SV40 complementary RNA (cRNA). Tiitiated &NA was synthesized in vitro

and purified by the method of Levine et al. [15]. Escherichia coli RNA polymerase was generously supplied by Dr Naono. 0.25 mCi of tritiated adenoExptl Cell Res 87 (1974)

sine, uridine and cytosine triphosphate and 0.10 mCi of tritiated guanosine triphosphate (NEN Chemicals, Frankfurt/Main) were pooled in the reaction tube and evaoorated to dryness to remove ethanol. The specific activities of A?P, UTP, CTP and GTP were resoectivelv 33.1, 26.9. 28.6 and 5.2 mCi!mmol. They w&e not-diluted wifh unlabelled nucleotides. The reaction mixture, when completed, was brought to a final volume of 125 1’1. After an incubation period of 2.5 h at 37 C, the reaction was stopped by adding 40 /Jg of deoxyribonuclease (15 min, 37°C). The sedimentation coefficient of the purified cRNA. after centrifugation in sucrose densitv gradient, spiead between 1%and 4S with a peak ai 4:5S. The tritiated cRNA had a soec. act. of 1.6 10” dpm/!rg as calculated from the ;ncorporated nucleotides. Calculation was based on the assumption that the four nucleotides were incorporated equally. Control and 20-, 30-, 44-h infected cultures were washed in phosphate-buffered saline (PBS) before being fixed in situ for 17 h at 4°C in a 4 % formaldehyde solution, freshly prepared from paraformaldehyde (Merck), in 0.1 M Sijrensen phosphate buffer pH 7.4. The cells were then washed for 48 h in the same buffer at 4°C. Subsequently, the DNA contained in this material was denaturated by incubating the cells in 50 “b formamide (Merck, chromatography grade) (V/V) in 0.1 SSC (SSC is 0.15 M NaCl; 0.015 M Na citrate) at 70°C for 15 min. After denaturation, the cells were washed twice in 0.1 SSC at WC and once in 2 SSC at 4°C. In each well containing a monolaver of control or infected cells were placid 50 /-cl of 3H-cRNA dissolved in 50 Ib formamide (v/v). . ., 2 SSC. 0.1 “b sodium dodecyl sulphate, 0.01 M TES (N-iris (hydroxymethyl) methyl-2-aminoethane sulphonic acid; Calbio, A grade) pH 7.4. 3H-cRNA was used at a concentration of 0.8 pg/ml. Each well was sealed with paraffin-sheet (Parafilm, Marathon) and incubated in a moist atmosphere for 45 h at 37°C.

In situ hybridization.

After the hybridization period, the cells were washed 4 times in 2 SSC at 37°C and subsequently treated with both oancreatic ribonuclease (Choav. incubated at 80°C ior 15 min) (20 pg/ml)‘ and ijbonuclease Tl (Sankyo Ltd., Tokyo, Japan) (10 U/ml) in 2 SSC for 1 h at 37°C. The cells were then washed 3 times in 2 SSC, 3 times in 5 % trichloroacetic acid at 4°C for a total of 50 min and 3 times in 0.1 M SGrensen buffer pH 7.4. Additional

fixations,

embedding and radioautography.

The four cell cultures were post-fixed in 1.6 % glutaraldehvde in uhosnhate buffer as above for 15 min at 4°C. After five washes in the same buffer, they were fixed a third time in 2 % osmium tetroxide in SGrensen buffer for 30 min. The cells were finally dehydrated in alcohol and embedded in Epon. After detachment from plastic wells, non-trimmed blocks were cut on their cell-containing edge with a

Localization of SV40 DNA in cells

177

MT 1 Sorvall microtome, using a diamond knife. Sections with gold interference colour were harvested onto carbon-Formvar-coated grids. Ilford L4 emulsion was applied on the grids by the loop method [ll]. Radioautograms were developed after being exposed for 2 to 5 months with Kodak D 19 developer made just before use with products for analysis. The sections were then contrasted with uranyl acetate and lead citrate according to Reynolds

WI.

The main technical steps of the whole technique are diagrammatically summarized in fig. 1. Quantitative estimation of the label. One radioautogram exposed for 3 months was used for each cell culture to calculate the label density in each cell compartment. Every cell in all sections, including serial ones, were photographed at a magnification of 2000. They were printed at a magnification of 6000 for measurements. With the embedding and sectioning conditions used, we obtained crescentshaped sections containing about 25 cells. The nucleolus, nucleoplasm and cytoplasm of about 100 cells or cell parts were semi-automatically measured for each cell culture with a HewlettPackard 9810 A calculator using a digitaliser. The number of silver grains localized over these three cell compartments in each cell were counted in like manner for each culture. This data was registered by the calculator which produced the histograms of fig. 2.

RESULTS

Fig. 2. Histograms; ordinate: number of grains per 10 pm2 (density) abscissa: (from left to right) cyto-

plasmic (Is]), nucleoplasmic (I[) and nucleolar ( n ) compartment of control, 20-, 30- and 44-h infected cultures.

titative data (fig. 2), the number of grains per 10pm2 is a little greater for the nucleolus. The labelling of the control cells is superior to the background of the emulsion outside the sections. This background is negligible.

Control cell culture

20-h infected cell culture

The labelling is weak (fig. 3). Silver grains are occasionally seen localized over the three cellular compartments. According to quan-

The number of silver grains localized over the nucleoplasm of most cells is clearly greater than in the same cellular compartment of control cells (figs 4, 5). Some grains are localized over regions where chromatin is more condensed (fig. 5). Most nucleoli are hypertrophied. A preferential localization of silver grains over this organelle is frequently observed (fig. 4) and appears clearly from the histograms (fig. 2).

b

30-h infected cell culture

d

e

f

Fig. 1. Diagrammatic summary of the main technical steps. (a) cell cultures; (b) in vitro synthesis of cRNA;

(c) in situ molecular hybridization; (d) embedding; (e) sectioning; (.f) emulsion applying.

The ultrastructural aspect of the nucleoplasm and nucleolus in these cells have generally not changed with regard to those observed in cells of the 20-h infected culture. However, several nuclei contain many viral particles dispersed in the nucleoplasm (fig. 7). The nucleoplasmic labelling is heavy but, according to the quantitative analysis, not Exptl Cell Res 87 (1974)

significantly different from that in the cells of the 20-h infected culture (fig. 2). Silver grains are often observed over regions of more condensed chromatin. The nucleolar labelling is heavy (figs 6, 7). Sometimes, this label seems to be more particularly associated with the fibrillar region of the nucleolus (fig. 7). Quantitative results show that the nucleolar label density is as high as the one calculated for the 20-h infected culture (fig. 2). The cytoplasmic labelling is also equivalent to that observed in the 20-h infected culture. 44-h infected cell culture The nucleoplasm of most cells contain large zones filled with virus particles. Silver grains are frequently observed over chromatin clumps which persist between these zones (fig. 9). On the other hand, areas with virus particles are not labelled. Therefore, the density of the nucleoplasmic label is reduced with regard to those in the cells of the 20and 30-h infected cultures (fig. 2). The granular region of the nucleoli is generally more compact than in the cells infected for shorter periods. Intranucleolar areas of low electron density are fringed with fibrillar condensations (fig. 8). These areas often contain virus particles. The nucleolar labelling is heavy (fig. 8). However, according to the quantitative analysis, the nucleolar label density is reduced with regard to the one calculated for the cells of the 20- and 30-h infected cultures (fig. 2).

The cytoplasmic labelling is equivalent to that observed in the same cellular compartment in the two other infected cultures. DISCUSSION The large disparity of labelling between cells of control and infected cultures, as well as the preferential localization of silver grains over the nucleoplasm and particularly over the nucleoli of infected cells, show that specific RNA/DNA hybrids can be formed in situ under our experimental conditions. The incubation in the presence of a cRNA of small molecular weight of a cell monolayer fixed in formaldehyde allows thus cRNA penetration into cells. The presence of formamide in the denaturation and hybridization mediums has allowed us to carry out these technical steps at the respective temperatures of 70 and 37°C and to damage only to a slight extent the fine structure of the cells. Moreover, hybridization in the presence of formamide increases the efficiency of the method [I]. The labelling of cells of the control culture varies from 3.5 grains per 10 pm2 for the nucleolus to 1 grain per 10 prnZ for the cytoplasm after 3 months’ exposure of the radioautograms. This latter label is weak but is nevertheless greater than the emulsion background. Some of these silver grains can result from the presence of residual 3H-cRNA degradation products which are trapped in the cell constituents even after many washes and particularly in cold dilute trichloroacetic acid.

Fig. 3. Cells of the control culture. Radioautogram exposed for 2 months. N, nucleoplasm; FU, nucleolus. x8400. Figs 4, 5. Cells of the 20-h infected culture. Radioautograms exposed for 5 months. N, nucleoplasm; nu, nu-

cleolus. Fig. 4, x 11 500; fig. 5, y 11 100. Figs 6, 7. Cells of the 30-h infected culture. Radioautograms exposed for 2 (fig. 6) or 5 (fig. 7) months. N, nucleoplasm; nu, nucleolus; U, virus. Fig. 6, :( 11 600; fig. 7, x 17 000. Figs 8, 9. Cells of the 44-h infected culture. Radioautograms exposed for 5 (fig. 8) or 2 (fig. 9) months. N, nucleoplasm; nu, nucleolus; c/z, chromatin; o, virus. Fig. 8, x 9 600; fig. 9, x 20 000. Exptl Cell Res 87 (1974)

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Localization of SV40 DNA in cells

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Localization of SV40 DNA in cells 183

Exptl Cell Res 87 (1974)

184 Geuskens and May Concerning the nucleolar and nucleoplasmic labelling, it cannot be excluded that part of this label results from a molecular hybridization between the short sequences of SV40 cRNA and partial complementary cellular DNA sequences. In this case, an equivalent part of the nuclear label in infected cells also results from such hybridization. In all the infected cells the cytoplasmic labelling is greater than that found in the control cells. It is likely that most of this label results from the formation of molecular hybrids between SV40 cRNA and infecting uncoated viral DNA released into the cytoplasm. The presence of intact infecting particles in the host cell nucleus and their intranuclear uncoating are well established [2, 131, but it cannot be excluded that virus uncoating is also carried out in the cytoplasm [2]. It cannot either be ruled out that molecular hybrids may occur between 3H-cRNA and viral messenger RNA localized in the cytoplasm. The localization of silver grains over nucleoli and chromatin clumps of infected cells is clear-cut. Many viral DNA molecules are localized in nucleoli of cells of the 20-h infected culture. This observation is compatible with the fact that SV40 DNA replication is detectable as soon as 18 h after infection. Previous electron microscope studies concerning the SV40 infectious cycle [lo] and combined radioautographical and cytochemical studies of nucleic acid synthesis at the ultrastructural level during the eclipse phase of SV40 virus [9] have already suggested that the nucleolus has an important role during the lytic infection with SV40: early hypertrophy of the nucleoli goes with an increase of intranucleolar chromatin bands and nucleolar DNA synthesis is increased at 10 h post-infection. Weil et al. [25] have also reported results suggesting that the nucleolus takes an important part during the lytic inExptl Cell Res 87 (1974)

fection of primary mouse kidney cell cultures with polyoma virus. Our present results demonstrate that unencapsidated viral DNA molecules are preferentially localized in the nucleolar body. More experiments are necessary to determine the exact part taken by this organelle in the process of lytic infection with SV40. The role of chromatin in this process is suggested from the association of the label with condensed chromatin. It has already been suggested that during the lytic infection of mouse cells with polyoma virus, a sitespecific association takes place between the viral DNA and the cellular DNA replication apparatus [7, 211. It is surprising that the number of silver grains per nucleus does not increase significantly later than 20 h post-infection, but we have to keep in mind that only the form II (circular relaxed) and the replicative form of SV40 viral DNA are sure to be denatured without subsequent renaturation under our denaturing conditions. The question now under investigation is to determine if SV40 DNA molecules which are in the process of replication (or of being transcribed) are preferentially detected by in situ molecular hybridization under our experimental conditions. Isolation of the nucleoli of infected cells and characterization of the associated viral DNA molecules could possibly give information about the role of this organelle during the lytic infection with SV40.

The authors wish to express their gratitude to Dr W. Bernhard and Dr P. May for their interest in this work and encouragement. They are also indebted to Dr 0. Croissant for helpful discussions. Warm thanks are due to G. Moyne for programmation of the calculator and help during the measurements. The authors acknowledge with gratitude the excellent technical assistance of M. J. Rurglen. M. G. is a qualified research worker of the Belgian National Fund for Scientific Research.

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Received December 11, 1973

Exptl Cell Res 87 (1974)