Role of the host cell nucleus in the replication of African swine fever virus DNA

VIROLOGY

188, 637-649 (1992)

Role of the Host Cell Nucleus in the Replication of African Swine Fever Virus DNA R . GARCIA-BEATO, M . L . SALAS, E . VI11UELA, AND J . SALAS' Centro de Biologla Molecular, (CSIC-UAM), Facultad de Ciencias, Universidad Aut6noma, Cantoblanco, 28049, Madrid, Spain Receivedlune 17, 1991 ; accepted February 11 . 1992 An examination by autoradiography of African swine fever virus-infected alveolar macrophages pulse labeled with [3 H]thymidine showed that, at early times of viral DNA replication, the grains were localized exclusively in the nucleus in 20% of the cells, while in 45% the label was found in the cytoplasm . In the remaining 35%, newly synthesized DNA was detected in both the nucleus and the cytoplasm . At later times, the percentage of cells with grains in the nucleus decreased considerably . Pulse-chase experiments indicated that the DNA synthesized in the nucleus is then transported to the cytoplasm . The presence of virus-specific DNA sequences in the nucleus was confirmed by in situ hybridization of infected macrophages . Similar hybridization experiments with African swine fever virus-infected VERO cells followed byconfocal microscopy also indicated the existence of a nuclear stage in the localization of the viral DNA . These results suggest a mechanism for African swine fever virus DNA replication with an initial stage in the nucleus followed by a cytoplasmic phase . Specific nuclear forms associated with the hybridization signal have been observed in African swine fever virus-infected macrophages and VERO cells . The nuclear forms seen in macrophages are consistent with a mechanism for the egress of the viral DNA from the nucleus that involves initial budding at the nuclear membrane .

rA 1992 Academic Press, Inc .

INTRODUCTION

might be a site of viral DNA replication . In the present work, we have examined this possibility by autoradiography and in situ hybridization of swine alveolar macrophages infected with ASF virus . The results suggest a mechanism for ASF virus DNA replication in macrophages with an initial stage in the nucleus followed by a cytoplasmic phase . Similar hybridization experiments with infected VERO cells, followed by laser scan confocal microscopy, also indicate that virus-specific DNA sequences are localized within the nucleus after the onset of viral DNA replication . On the other hand, specific nuclear forms associated with the hybridization signal have been observed in ASF virus-infected macrophages and VERO cells . The possible significance of these structures in relation to the process of egress of the viral DNA from the nucleus is discussed .

African swine fever (ASF) virus is a large icosahedral deoxyvirus that shares a number of properties with the poxviruses . Thus, the double-stranded DNA molecule of about 170 kbp (Almendral et al ., 1984 ; Enjuanes et al ., 1 976b) contains hairpin loops and terminal inverted repetitions (Gonzalez et al., 1986 ; Sogo et al ., 1984), which resemble those of poxviruses (Baroudy et al., 1982 ; Wittek and Moss, 1980) . Furthermore, the ASF virus particles contain, as do those of poxviruses (Moss, 1990), the enzymatic machinery required for early RNA synthesis (Kuznar at al., 1980 ; Salas et al ., 1981, 1986) . However, morphologically ASF virus is indistinguishable from iridoviruses that infect vertebrates (Carrascosa et al., 1984) . Although ASF virus is thought to multiply in the cytoplasm of the infected cell (Breese and DeBoer, 1966), the virus does not grow in enucleated VERO cells nor is viral DNA synthesized (Ortin and Virluela, 1977) . However, the precise role of the host nucleus in ASF virus multiplication has not been determined so far . This nuclear dependence is not due to a requirement for the host cell RNA polymerase II in some stage of the virus growth cycle, since the toxin a-amanitin does not inhibit the production of infectious virus in VERO cells or porcine alveolar macrophages (Salas et al., 1988) . The finding that no virus DNA synthesis occurs in enucleated cells raises the possibility that the host nucleus

MATERIALS AND METHODS Cells and viruses The ASF virus isolate BA71 has been described (Enjuanes at a!., 1976a) . BA71V is the isolate BA71 adapted to grow in VERO cells (Enjuanes et al ., 1 976a ; Sanz et al ., 1985) . Pig alveolar macrophages were prepared by broncoalveolar lavage as previously reported (Carrascosa et al., 1982) . They were cultured in Dulbecco modified Eagle's medium (DMEM) supplemented with 30% swine serum . VERO cells were obtained from the American Type Culture Collection (ATCC CCL81) and were cultured in DMEM with 10%

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newborn calf serum . Swine poxvirus (SPV) was provided by the ATCC and was grown in swine testis (ST) cells (ATCC) with DMEM supplemented with 10% calf serum . The extracellular virus was layered on top of a 30% sucrose cushion in 10 mM NaPO„ pH 8 .5, and centrifuged at 4° in an AM 627 rotor for 30 min at 20,000 rpm .

siliconized coverslips . Hybridization was conducted overnight at 42° in a humidified chamber. The probe was total ASF virus DNA isolated from virions purified as previously reported (Carrascosa et al., 1985) . Before labeling, the DNAwas digested with Hindlll . The probe was labeled with digoxigenin-l1-dUTP (Boehringer) as described in the applications manual from Boehringer and was denatured by heating at 100° for 5 min .

Detection of newly synthesized DNA by autoradiography

Confocal fluorescence microscopy

Porcine alveolar macrophages were seeded at a density of 2 X 10 6 cells/ml in plastic dishes (Nunc, Denmark) containing coverslips . The cells were preincubated for 24 hr with 2 mM sodium butyrate (Merck) and then mock-infected or infected with ASF virus (BA71) or SPV at a multiplicity of infection (m .o .i .) of 5 PFU per cell, maintaining the butyrate throughout the adsorption (1 hr) and infection periods . At different times postinfection, the cells were pulse-labeled with 30 pCi/ml of [ 3 H]thymidine (45 Ci/mmol ; Amersham, England) for 15 min and then fixed with acetic acid :ethanol (1 :3) . The coverslips were cemented to slides with Permount (Fisher Scientific, Fairlawn, NJ), dipped in Kodak NTB2 nuclear track emulsion, dried, and exposed at 4° in the dark for convenient periods of time . After exposure, the slides were developed in D-19 developer and fixer and then stained with Giemsa . In situ hybridization with virus-specific DNA probes labeled with digoxigenin The procedure for in situ hybridization using digoxigenin-labeled probes (including activation of the coverslips, prehybridization, hybridization, posthybridization, immunologic detection of the probe, and color reaction) was as described in the applications manual for nonradioactive DNA labeling and detection from Boehringer Mannheim Biochemica, with some modifications . Mock-infected or ASF virus-infected macrophages or VERO cells cultured on activated coverslips were fixed at different times postinfection with acetic acid :ethanol (1 :3) and, after the proteinase K digestion step, the samples were treated with 100 ug/ml of RNase A and 10 U/ml of RNase T, (Boehringer) as described by Brahic etal. (1981) to remove RNA . In some cases, the cells were then digested with 60 pg/ml of DNase I (Boehringer) for 30 min at 37° . Before hybridization, the DNA in the cells was denatured by placing the samples in a solution containing 95% freshly deionized formamide and 0 .1 X SSC at 65° for 15 min (Brahic et al., 1981) . Prehybridization was performed after this denaturation step . For the hybridization, 30 pl of hybridization solution containing 10 ng of denatured ASF virus DNA probe were applied per sample under

For confocal microscopy, the samples were incubated, after hybridization, for 1 hr at room temperature with 50 pg/ml of sheep antidigoxigenin Fab fragments conjugated to fluorescein (Boehringer) in PBS containing 0 .5% bovine serum albumin (Boehringer) and 1 % blocking reagent (Boehringer) . To label the cytoplasm, the cells were incubated for 1 hr at room temperature with 50 ul per sample of a 1 :200 dilution of a mouse monoclonal antitubulin antibody(Amersham) . The samples were then incubated for 1 hr at room temperature with 50 µl per sample of a 1 :20 dilution of a goat antimouse antibody conjugated to rhodamine (Tago, Inc ., United States) . The cells were viewed with a Zeiss laser scan microscope . RESULTS Effect of sodium butyrate on DNA synthesis in porcine alveolar macrophages Since the monocytes and macrophages are the primary target cells in natural infections by ASF virus, we initiated these studies with porcine alveolar macrophages . Although these cells do not divide in culture, they, however, synthesize DNA to a small extent . In an attempt to inhibit this synthesis, which might interfere with the interpretation of the data obtained by autoradiography in ASF virus-infected macrophages, we examined the effect of sodium butyrate on thymidine incorporation in uninfected macrophages, as this fatty acid has been shown to inhibit DNA synthesis in a variety of cultured cells (Daniell, 1980 ; Hagopian et al., 1977 ; Saemundsenetat, 1980) . Figure 1 shows that the rate of thymidine incorporation is strongly inhibited by butyrate in alveolar macrophages after 24 hr of exposure . At 2 mM butyrate, a 97% inhibition is obtained . Since macrophages incubated with this butyrate concentration for 48-72 hr did not show any toxic effects, as judged by microscopic examination (Fig . 2) and trypan blue exclusion, we used 2 mM butyrate in the following experiments . Time course of DNA synthesis in ASF virus-infected alveolar macrophages The kinetics of thymidine incorporation in ASF virusinfected macrophages in the presence and in the ab-



NUCLEAR PHASE IN ASF VIRUS DNA REPLICATION

6 39

butyrate does not reduce ASF virus production in alveolar macrophages (data not shown), as has been reported for adenovirus replication in butyrate-treated Hela cells (Daniell, 1980) .

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Intracellular localization of replicating ASF virus DNA determined by autoradiography

0

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[SODIUM BUTYRATE], MM

Fir . 1 . Effect of sodium butyrate on DNA synthesis in swine alveolar macrophages . The macrophages were seeded in plastic plates at a density of 2 X 10 6 cells/ml, allowed to adhere to the plates for 24 hr, and then incubated with the indicated sodium butyrate concentrations for another 24 hr . At the end of this period, the cells were labeled for 1 .5 hr with 5 riCi/ml of [3 Hlthymidine . The incorporation into acid-insoluble material in the absence of butyrate was 2900 cpm per 1 X 10 6 cells .

sence of 2 mM sodium butyrate was determined . The cells were pretreated or not with butyrate for 24 hr, infected with ASF virus (BA71) at a multiplicity of 5 PFU per cell or mock-infected, and, at different times postinfection, pulse-labeled for 15 min with [ 3 H]thymidine . The results are shown in Fig . 3 . It can be seen that the synthesis of viral DNA does not take place in a single wave, but two peaks are observed in the kinetics, the first of which is considerably smaller than the second . These two peaks are found both in the absence and in the presence of butyrate, which inhibits the synthesis of viral DNA only by 30-40% . On the other hand, 2 mM

To examine the intracellular localization of newly synthesized ASF virus DNA, the macrophages were infected and pulse-labeled as described and, at the end of the labeling period, were fixed and processed for autoradiography (see Materials and Methods) . As a control, we carried in parallel swine alveolar macrophages infected with swine poxvirus (SPV), since it is known that the replication of poxvirus DNA occurs only within the cytoplasm of the infected cell (see Moyer, 1987 for a review) . The grain distribution in ASF virusor SPV-infected macrophages is presented in Fig . 4 . Mock-infected cells showed significant label in the nucleus over background grains only in about 0 .2% of the cells and are not represented in the figure . In ASF virus-infected macrophages pulse-labeled at 4 hr postinfection, which is an early time of viral DNA synthesis, 20% of the cells showed grains exclusively in the nucleus, while in 45% the label was localized in the cytoplasm . In the remaining 35% of the cells, newly synthesized DNA was found both in the nucleus and in the cytoplasm . At 6 hr the percentage of cells with grains only in the nucleus or in both the nucleus and the cytoplasm decreased to 3 and 9%, respectively . This pattern of predominantly cytoplasmic distribution was still maintained at 8 hr postinfection (not shown) . In contrast with these results, the percentage of SPV-Infected macrophages with grains only in the nucleus

FIG . 2 . Swine alveolar macrophages cultured with or without sodium butyrate . The macrophages were cultured as indicated in the legend to Fig . 1 . (A) Cells without butyrate . (B) Cells incubated with 2 mM butyrate for 48 hr . Magnification, X320 .



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FIG . 3. DNA synthesis in swine alveolar macrophages infected with ASF virus in the presence and in the absence of 2 TIM butyrate . The cells were cultured as indicated in the legend to Fig . 1, pretreated or not with 2 TIM butyrate for 24 hr, and then mock-infected or infected with ASF virus (BA71) at a m .o .i . of 5 PFU per cell . At the indicated times, the cells were pulse-labeled for 15 min with 30 iCi/ ml of ['H]thymidine . (0) Infected cells without butyrate ; (A) infected cells with butyrate ; (0) mock-infected cells without butyrate ; (A) mock-infected cells with butyrate .

was, as expected, less than 0 .5 96 between 4 and 6 hr postinfection, when the wave of SPV DNA synthesis occurs in swine macrophages (not shown) . The percentage of these cells with grains confined to the cytoplasm was always higher than 90 96 . The findings with ASF virus-infected macrophages suggest that, soon after the onset of viral DNA replication, the host nucleus is a site of virus DNA synthesis, while, at later times, the replication occurs mainly within the cytoplasm . Examples of autoradiographies of mock-infected and ASF virus- or SPV-infected macrophages are shown in Fig . 5 . The low unspecific background of mock-infected cells is illustrated in Fig . 5A . SPV-infected cells labeled at 4 hr postinfection are shown in Fig . 5B . Usually, multiple foci are found in the cytoplasm . Figure 5C is an autoradiograph of ASF virus-infected macrophages . Cells with label localized only in the nucleus or in the cytoplasm or in both sites can be seen at 4 hr . Most of the cells show a single focus . When the label is in the nucleus, the grains are not distributed throughout it, but are usually restricted to a region in proximity to the nuclear membrane . Fate of the newly synthesized nuclear DNA in ASE virus-infected macrophages The results on the location of replicating ASF virus DNA raised the possibility that the DNA synthesized in

the nucleus might be transported later to the cytoplasm to complete replication . To see this, pulsechase experiments followed by autoradiography were performed . ASF virus-infected macrophages were pulse labeled for 15 min with [ 3 H]thymidine at 4 .5 and 5 hr postinfection and then chased for 2, 3, and 4 hr in the presence of 2 mM thymidine . These chase conditions were found to be completely efficient in preventing [3 H]thymidine incorporation and, on the other hand, 2 mMthymidine does not inhibit viral DNA synthesis as determined by dot-blot hybridization (data not shown) . The results of a pulse-chase experiment are shown in Table 1 . It can be seen that the percentage of cells with grains in the nucleus or both in the nucleus and the cytoplasm detected at the end of the labeling period decreased by B0-90 96 after a 4 hr chase, indicating that the DNA synthesized in the nucleus is transported to the cytoplasm, Detection of ASF virus DNA in infected macrophages by in situ hybridization Although the results described suggest the existence of a nuclear stage in the replication of ASF virus DNA in macrophages, the possibility exists in principle that the label detected in the nucleus in infected cells might be due to cellular DNA synthesis induced by the infection . This possibility, however, seems unlikely because the cells are infected in the presence of sodium butyrate, a strong inhibitor of cell DNA synthesis . Also, the results from the pulse-chase experiments argue against this possibility as well as previous results from our laboratory which indicated that the synthesis of cell DNA is not induced in ASF virus-infected porcine macrophages (Enjuanes et al., 1 976b) . Nevertheless, we examined the possible presence of virus-specific DNA sequences in the nucleus of ASF virus-infected macrophages by in situ hybridization using an ASF virus DNA probe labeled with digoxigenin (see Materials and Methods) . We found that, at 5 hr postinfection, a strong hybridization signal was localized in the nucleus in 26 96 of the infected cells, while mock-infected macrophages showed no significant nuclear staining (see below) . At later times, the number of infected cells with nuclear signal decreased to 7 96 . Infected cells hybridized at 4 hr postinfection were also examined, but the signal in the nucleus was rather weak and might not be significant . It should also be mentioned that, if after the treatment with RNases, the samples are digested with DNase I (see Materials and Methods) no hybridization signal was found, indicating that the hybridization conditions used are specific for DNA detection . Figure 6 illustrates the hybridization of mock-infected and infected macrophages . Mock-infected cells (Fig . 6A)



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FIG. 4. Grain distribution in ['H]thymidine-labeled alveolar macrophages infected with ASF virus or swine poxvirus (SPV/ . The cells were seeded on dishes containing coverslips, preincubated with 2 mM butyrate, and then mock infected or infected with ASE virus or SPV at a m .o .i . of 5 PFU per cell . At the indicated times, the cells were pulse-labeled for 15 min with 30 eCi/ml of ['H]thymidine, then fixed with acetic acid :ethanol (1 :3), and processed for autoradiography as described under Materials and Methods . The percentage of ASF virus-infected cells showing significant label was 35% at 4 hr and 50% at 6 hr . Each point is the mean of three separate experiments with the standard deviation . Between 300 and 500 labeled cells were counted at each point of the three experiments . The data are expressed as percentages from the total number of cells with label . C . N, and N and C stand for cells with label in the cytoplasm, in the nucleus, or in both the nucleus and the cytoplasm, respectively.

show only background staining, with the nucleus usually without stain . Figure 6B illustrates the intense hybridization signal that is found in the nucleus of ASF virus-infected cells . The signal is restricted to an area of the nucleus nearthe nuclearmembrane (Fig . 6C) in a similar way to the label detected by autoradiography . The results from these in situ hybridization experiments with ASF virus-infected macrophages indicate that virus-specific DNA molecules are present in the nucleus predominantly at early times of virus DNA synthesis, which is in line with the data obtained by autoradiography . Nuclear and cytoplasmic forms associated with the hybridization signal in ASF virus-infected macrophages In the course of these hybridization experiments with infected macrophages, we have observed with the light microscope specific nuclear structures that are associated with the hybridization signal . The detection and preliminary characterization of these structures was facilitated by the fact that the nuclear membrane is easily identifiable in the hybridized cytopreparations . These nuclear forms, which are predominantly seen at the times when the percentage of infected macrophages with nuclear labeling is maximal, are consistent with a pathway for the egress of the viral DNA from

the nucleus as shown in Fig . 7 . It is frequently seen that the region of the nucleus where the hybridization signal is localized appears to be slightly prominent (Fig . 7A) . These nuclear regions might become more pronounced (Fig . 7B) and eventually develop into forms resembling budding processes (Fig . 7C) . These forms, which consist of the hybridization signal apparently surrounded by the nuclear membrane, can reach a large size and, at this stage, a thin membrane at the confluency of the nucleus and the structure is frequently seen (Figs . 7D and 7E) . This membrane follows the contour of the nucleus and might conceivably be a regenerating nuclear membrane . Complete reconstitution of this membrane might lead to the release of the bud-like structure . Consistent with this process, the cytoplasmic signal is always surrounded by an apparent membrane, which might thus be of nuclear origin (Fig . 7F) . Localization of ASF virus DNA in infected VERO cells determined by in situ hybridization The nuclear replication of ASF virus DNA in macrophages raised the possibility that in VERO cells infected with adapted virus (BA71 V) a similar stage in the synthesis of the viral DNA might also exist . Previous results from our laboratory obtained in autoradiographic studies on the replication of ASF virus DNA in



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%

.1

FIG . 5 . Autoradiographs of mock-infected and ASF virus or SPV-infected macrophages . (A) Mock-infected cells labeled with [ 3 H]thymidine at 4 hr postinfection . (B) SPV-infected macrophages labeled at 4 hr . Multiple foci are seen in the cytoplasm of a cell (c) . (C) ASF virus-infected cells labeled at 4 hr . Cells with grains in the cytoplasm (c), in the nucleus (n), or in both the nucleus and the cytoplasm (n . c) can be seen . The statistical analysis of grain distribution in infected cells at 4 hr was shown in Fig . 4 . Magnification, x600 .

VERO cells indicated the existence of a cytoplasmic replicative phase at 8-10 hr postinfection followed by a perinuclear stage from 12 to 18 hr (A . L. Carrascosa, unpublished results) . Because of the extensive cellular DNA synthesis in VERO cells, it was not possible in those studies to determine whether the viral DNA also replicates in the nucleus . Since sodium butyrate was found to inhibit cell DNA synthesis in VERO to a lesser extent than in macrophages, we approached this ques-

tion by determining the intracellular localization of the virus DNA by in situ hybridization of infected VERO cells . To this end, VERO cells grown on activated coverslips (see Materials and Methods) were mock-infected or infected with adapted ASF virus at a m .o .i . of 5 PFU per cell and hybridized with a virus DNA probe labeled with digoxigenin as described for macrophages . Figure 8 shows the distribution of the hybridization signal in infected VERO cells as a function of the time postinfec-



NUCLEAR PHASE IN ASF VIRUS DNA REPLICATION TABLE 1 GRAIN DISTRIBUTION IN ASF VIRUS-INFECTED MACROPHAGES AFTER PULSE LABEL AND CHASE Labeled cells (%) 4 .5 hr p .i .

5 hr p .i .

Chase

Cytoplasm Nucleus Nucleus and cytoplasm

Pulse 15 min

2 hr

3 hr

50 15

72 8

B2

35

20

Chase 4 hr

Pulse 15 min

2 hr

3 hr

4 hr

5

91 1

62 10

76 7

82 2

97 1

13

8

28

17

16

2

Note. ASF virus-infected macrophages were pulse-labeled for 16 min with 30 Mci/mI of [3H]thymidine at 4 .5 and 5 hr postinfection . At the end of the labeling period, the medium was removed and the cells were washed three times with PBS . Fresh medium containing 2 mM thymidine was then added and the cultures were further incubated for 2, 3, and 4 hr. The percentage of labeled cells was determined by autoradiography at the end of the pulse and pulse-chase periods . Between 500 and 1500 labeled cells were counted at each point . The data are expressed as percentages from the total number of cells with label .

tion . Mock-infected cells had no significant signal (see below) and are not shown . At 8 hr postinfection, a time when the synthesis of virus DNA was already under way (see insert in Fig . 8), approximately 70% of the infected cells showed the hybridization signal localized exclusively in the nucleus . This percentage fell to 25% at 9 hr and then continued to slowly decrease . Cells with signal confined to the cytoplasm were also observed, the percentage of these cells reaching a maxi-

64 3

mum (70%) at 10 hr. Finally, hybridization signal localized in the perinuclear region was found in a rather low percentage of cells between 8 and 10 hr but increased at 15 hr and was maximal at 17 hr (67%) . Two classes of hybridization signal were found in infected VERO cells : a diffuse purple stain similar to that detected in macrophages and a granular signal consisting of intensely blue grains (see Fig . 9) . These two signals have been previously observed in cells hybridized with digoxigenin-labeled probes (Samoszuk and Nansen, 1990) . In VERO cells, the diffuse and granular signals coexist when the hybridization signal is strong, although the diffuse stain partially conceals the grains . On the other hand, fading of the diffuse signal occurs in 24-48 hr, while the grains persist for at least 15 days . Hybridized mock-infected' and infected VERO cells are illustrated in Fig . 9 . Mock-infected cells show a low background, but the nuclear membrane is stained, as in macrophages, and is therefore easily identifiable (Fig, 9A) . Infected cells hybridized at 3 hr postinfection, before the onset of the viral DNA synthesis (see insert in Fig . 8), show no significant signal, which is probably due to the fact that the hybridization conditions used are not sensitive enough to detect the input DNA (Fig . 98) . Infected cells hybridized at 8 hr showing a predominantly granular signal in the nucleus are illustrated in Fig . 9C . Figure 9D is a micrograph of an infected cell at higher magnification, showing the localization of the nuclear signal in proximity to the nuclear membrane, as is also seen the signal in macrophages (see Fig . 6C) . The cytoplasmic signal separated from the nucleus at 9-10 hr postinfection is shown in Fig . 9E . In contrast to macrophages, this sig-

FIG. 6 . In situ hybridization of mock-infected or ASF virus-infected macrophages with an ASF virus DNA probe labeled with digoxigenin . The cells were seeded in dishes containing activated coverslips, treated with butyrate, and mock-infected or ASF virus-infected as before . After fixation with acetic acid :ethanol (1 :3), the cells were hybridized with an ASF virus DNA probe labeled with digoxigenin as described under Materials and Methods . (A) Mock-infected macrophages. (B) ASF virus-infected cells . Arrows indicate signal localized in the nucleus, which was found in 26% of the stained cells at 5 hr postinfection (about 600 cells were counted) . (C) ASF virus-infected macrophages at higher magnification with signal localized in the nucleus in proximity to the nuclear membrane . Magnification in (A) and (B), X425 ; in (C), x1063 .

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FIG . 7 . Nuclear and cytoplasmic forms associated with the hybridization signal in ASF virus-infected macrophages . (A) Nucleus of infected macrophage hybridized at 5 hr postinfection, showing a slight prominence in proximity to the signal (s) . (B, c) Nuclear bud-like structures (s) in infected cells hybridized at 5 hr . (D, E) Infected cells hybridized at 6 hr . The nucleus and the structure containing the signal are indicated by n and s, respectively. The membrane surrounding the signal is apparently continuous with the nuclear membrane . Notice also the membrane at the confluency of the nucleus and the structure with the signal (arrowhead) . (F) infected cell at 6 hr with cytoplasmic signal surrounded by a membrane (m). The nucleus is indicated by n . About 300 cells with nuclear signal were examined . The structures shown were found in 30% of the cells . Magnification, x1250 .

nal is not surrounded by a membrane . At 17 hr, the stain is localized in the perinuclear region in most of the cells (Fig . 9F) . Infected VERO cells hybridized with the virus-specific probe were also analyzed by confocal fluorescence microscopy, using an antitubulin antibody as a cytoplasmic marker . The binding sites in the cell of this antibody were visualized through secondary labeling with a rhodamine-coupled antibody (see Materials and Methods) . The labeling of the cytoplasm with the antitubulin antibody allows to ascertain the location of the nucleus relative to the viral signal, detected with an antidigoxigenin antibody conjugated to fluorescein . The overlaid confocal images of the fluorescein and rhodamine patterns (Fig . 10) show that the nuclearsignal detected in the hybridized cytopreparations is indeed localized within the nucleus . The results obtained by in situ hybridization of ASF virus-infected VERO cells indicate the existence of three successive sites of localization of the viral DNA : a first stage in the nucleus followed by a cytoplasmic

phase in which the signal is separate from the nucleus and a final perinuclear stage .

Nuclear structures in infected VERO cells We have observed in infected VERO cells hybridized at 8-9 hr postinfection, nuclear structures in which the nuclear membrane appears to be partially dissociated into two thin membranes, which might be the inner and outer layers of this membrane (Fig . 11) . The hybridization signal in these structures is localized between the two thin membranes in what might be a dilated perinuclear space .

DISCUSSION The data obtained by autoradiography of ASF virusinfected alveolar macrophages in pulse label and pulse-chase experiments as well as by in situ hybridization suggest a mechanism for viral DNA replication



NUCLEAR PHASE IN ASF VIRUS DNA REPLICATION

2

100

x

0

5

10 15 20 TIME,h/O P

U

p 50 J

0 J

0 N I TI TIME AFTER INFECTION, h FIG. 8 . Distribution of the hybridization signal in ASF virus-infected VERO cells as a function of the time postinfection . VERO cells were grown in plastic dishes containing activated coverslips and, when the cultures reached a density of 80,000 cells/cm', they were mockinfected or infected with adapted virus (BA71V) at a m .c .i . of 5 PFU per cell . The cells were not pretreated with butyrate before infection . At the indicated times, the cells were fixed with acetic acid : ethanol (1 :3) and hybridized with a virus DNA probe labeled with digoxigenin (see Materials and Methods) . The percentage of infected cells showing a hybridization signal was 30-40% at B hr and 80-90% at 15-17 hr. Between 500 and 1000 stained cells were counted at each point . The data are expressed as percentages from the total number of cells with hybridization signal . N, C, and P stand for cells with signal localized in the nucleus, in the cytoplasm, or in the perinuclear region, respectively . The insert shows the time course of DNA synthesis in the cytoplasmic fractions, prepared as described elsewhere (Moreno et al., 1978), of mock-infected and ASF virus- infected VERO cells pulse-labeled for 30 min with 30 µCi/ml of ['Hthymidine . (0) Infected cells : (0) mock-infected cells .

in two phases : an initial stage in the nucleus and a second phase in the cytoplasm . It is noteworthy that the synthesis of virus DNA in infected macrophages does not occur in a single wave, but two peaks are partially resolved when the time course of thymidine incorporation is determined . This biphasic kinetics is also observed with a different virus isolate (CC83) and when peripheral blood monocytes instead of macrophages are used (J . C . Gonzalez, personal communication) . The second large peak in the kinetics probably represents the cytoplasmic stage of replication, since at the times when this peak is observed the newly synthesized DNA is found by autoradiography mainly in the cytoplasm . On the other hand, the first and minor peak might correspond, at least in part, to the nuclear phase of virus DNA replication, as the maximal percentages of cells with nuclear labeling coincide with the initial part of this peak .

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In VERO cells infected with adapted ASF virus, in situ hybridization experiments followed by confocal microscopy also indicate the existence of a nuclear stage in the localization of the viral DNA, which is followed by cytoplasmic and perinuclear phases . The times at which these two last phases occur are approximately the same as those of the cytoplasmic and perinuclear stages detected by autoradiography of infected VERO cells (A . L . Carrascosa, unpublished results) . Thus, it is likely that the stages in the cytoplasm and perinuclear region observed by in situ hybridization in the present work represent replicative phases and not a mere localization of replicated DNA molecules . On the other hand, the fact that the input viral DNA can not be detected in infected VERO cells under our hybridization conditions suggests that the signal found in the nucleus of these cells might be due to de nova synthesis of virus DNA . In relation to the existence of a nuclear phase in the synthesis of ASF virus DNA in macrophages, it is interesting to mention that the replication of the iridovirus frog virus 3, an icosahedral DNA-containing virus, occurs in two stages, the synthesis of the viral DNA being initiated in the nucleus and completed in the cytoplasm (Goorha, 1982 ; Goorha et al., 1978) . The replication of ASF virus DNA in macrophages might thus be similar to that of frog virus 3 in the two-stage mechanism, even though the structure of the ASF virus genome (Gonzalez et al., 1986 ; Sogo et al ., 1984) is different from that of frog virus DNA (Goorha and Murti, 1982 ; Murti et al., 1982) and therefore the molecular mechanisms of DNA synthesis are probably also different . It is now well established that the replication of vaccinia virus DNA occurs only within the cytoplasm of the infected cell (see Moyer, 1987 for a review) . It is therefore striking that poxviruses and ASF virus, which share a number of properties, including similar DNA structures that suggests a common DNA replication mechanism, might differ in the requirement of the host nucleus for DNA synthesis . The need for a nuclear protein(s), perhaps involved in the initiation of DNA synthesis and not codified by the virus genome, might explain the nuclear requirement in ASF virus DNA replication . A collateral but interesting finding in the course of these studies was that high thymidine concentrations, which are inhibitory to host DNA synthesis, did not inhibit the synthesis of ASF virus DNA as determined by dot-blot hybridization . A possible explanation for this result is that the virus-codified ribonucleotide reductase (Boursnell et at, 1991) might be insensitive to allosteric inhibition by dTTP, as has been shown for the enzyme induced by herpes viruses (Averett et al., 1983 ; Huszar and Bachetti, 1981 ; Langelier and Buttin, 1981 ; Lankinen et al., 1982) . We are currently ex-

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C

NUCLEAR PHASE IN ASF VIRUS DNA REPLICATION

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A

B

C s i

Flc . 10. Optical sections through infected VERO cells hybridized with the ASF virus DNA probe and double-labeled with antidigoxigenin and antitubulin antibodies . The virus DNA probe was detected by confocal microscopy, using antidigoxigenin Fab fragments conjugated to fluorescein, as indicated under Materials and Methods . The cells were then incubated with a mouse monoclonal antitubulin antibody, which was detected with a goat anti-mouse antibody labeled with rhodamine . The panels from left to right show optical sections at 1-µm intervals through infected VERO cells obtained with the confocal microscope . (A) Fluorescein staining pattern, argon laser 488 nm . (B) Rhodamine labeling, HeNe laser 543 am . (C) Overlaid images of (A) and (B) . The fluorescent viral signal indicated by arrows is localized within the area of the nucleus in proximity to the boundary of the nucleus with the cytoplasm, as is also seen the signal detected by the color reaction . The weak nuclear signal seen in the first panel of the rhodamine pattern is probably due to cross-detection of the intense fluorescein focus . Magnification, x529 .

ploring this possibility by testing the effect of dTTP and dATP on the ribonucleotide reductase activity in extracts from ASF virus-infected macrophages . A question that poses the nuclear synthesis of ASF virus DNA in infected macrophages is the mechanism by which the replicating viral DNA exits the nucleus . The signal detected by in situ hybridization, which is not disperse throughout the nucleus but localized as a single focus in proximity to the nuclear membrane, is apparently too large to be released through the nuclear pores . The nuclear bud-like forms seen in infected mac-

rophages at early times of viral DNA replication might provide some insight into this question . These structures, which contain the hybridization signal apparently surrounded by the entire nuclear membrane, are consistent with a mechanism for the egress of the viral DNA from the nucleus by budding through the nuclear membrane . In line with this possibility, the cytoplasmic signal is enveloped in a membrane, which might have been acquired in the budding process . On the other hand, we have not detected in ASF virus-infected VERO cells nuclear forms similar to those found in mac-

Fla . 9. In situ hybridization of mock-infected and ASF virus-infected VERO cells . (A) Mock-infected cells . (B) Infected cells hybridized at 3 hr postinfection . No significant signal is observed . (C) Infected cells hybridized at 8 hr showing a predominantly granular signal in several nuclei (arrows) . (D) Infected cell at higher magnification . A diffuse signal is localized in the nucleus near the nuclear membrane (arrow) . (E) Infected cell at 9 hr with granular cytoplasmic signal (arrow) . (F) At 17 hr, the signal is found in the perinuclear region in most of the cells (arrows) . The statistical analysis of signal distribution in infected cells at the different times postinfection was shown in Fig . 8 . Original magnification in (A), (B), (C), and (F), X410 ; in (D) and (E), X1025 .

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ACKNOWLEDGMENTS We thank Carmen Martinez and Jesus Garcia del Moral for advice on the confocal microscope . This work was supported by grants from the Comis16n Interministerial de Ciencia y Tecnologia and the Consejerfa de Agriculture de )a Junta de Extremadura and by an institutional grant of Fundaci6n Ram6n Areces .

REFERENCES

FIG . 11 . Nuclear structures in hybridized VERO cells infected with ASF virus . Notice the two thin membranes in the region of the nuclear membrane indicated by an arrow. The hybridization signal appears to be localized between these membranes . This structure was found in 1696 of the cells with nuclear signal examined (400) .

rophages and the cytoplasmic signal does not seem to be surrounded by a membrane . Instead, the hybridization signal appears to be localized between the inner and outer nuclear membranes . In relation to this finding, it may be of interest to mention that Whealy at al. (1991) have recently reported that, in PK 15 cells infected with pseudorabiesvirus in the presence of brefeldin A, a specific inhibitor of export to the Golgi apparatus, enveloped virions accumulate between the inner and outer layers of the nuclear membrane and in the lumen of the rough endoplasmic reticulum after egress of capsids from the nucleus by budding through the inner nuclear membrane, A similar accumulation of enveloped nucleocapsids in the perinuclear space of brefeldin-treated L cells infected with herpes simplex virus has been described by Cheung et al. (1991) . In the multistep pathway for pseudorabiesvirus particle assembly and egress proposed by Whealy et al. (1991), the capsids would then be transported through the endoplasmic reticulum and released into the cytoplasm in proximity to the trans-Golgi . It is possible that the hybridization signal apparently located in the perinuclear space in ASF virus-infected VERO cells might likewise represent an intermediate step in the release of the viral DNA from the nucleus . If the nuclear forms observed in infected macrophages and VERO cells are indeed intermediate stages in the pathway of egress of the viral DNA from the nucleus, then this pathway might be different in these two cell systems . Electron microscopic analysis will be needed for a more precise definition of these structures as well as of the possible steps in the release of the viral DNA from the nucleus in macrophages and VERO cells .

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