- Email: [email protected]
Herpesvirus replication in vivo
VIROLOGY
49,
784-793
(1972)
Herpesvirus RICHARD
O’CALLAGHAN,’
Department of Microbiology,
Replication
CHARLES
University
in Viva
C. RANDALL,
of Mississippi
AND
GLENN
A. GENTRY
School of Medicine, Jackson, Mississippi
89.216
Accepted June 16, 1972 The replication of equine herpesvirus (equine abortion virus; EAV) provides a unique in vivo system characterized by hepatitis and viremia that permits quantitative molecular biological studies. The replication of infectious cell-associated virus precedes by a short time the onset of viremia, based on the rapid (less than 2 hr) appearance of significant amounts of labeled virus in the blood, after injection of 3H-thymidine. Analysis of thymidine metabolism revealed that peak viral synthesis took place at 8-9 hr postinfection. DNA-DNA hybridization and MAK column chromatography showed that, 85y0 or more of the newly synthesized DNA was viral. Host DNA synthesis, however, remained at a very low level throughout infection. The data show that EAV can replicate effectively in the absence of significant host DNA synthesis, a point recently in dispute. INTRODUCTION
Equine abortion virus is a member of the herpes group (Plummer, 1964), with a typical genome of double-stranded DNA of relatively high G + C content (Soehner et al., 1965). Its natural host is the horse, in which it causes a rhinopneumonitis, with abortion if the host is a pregnant mare (Doll et al., 1953). Although it grows well in various cell cultures, it appears to grow best in the young Syrian hamster, to which it was adapted a number of years ago (Doll et aZ., 1953). The effects on the hamster are dramatic. When inoculated with lo8 LDsO , the hamster develops hepatitis and dies about 12 hr after inoculation, with more than 10” virions per ml of blood (Randall and Bracken, 1957; Darlington and Randall, 1963). Histological evidence indicated that the infection consists solely of viral replication in parenchymal liver cell nuclei of which more than 95 % are infected in the typical growth curve. Subsequently the virus is released and accumulates in the blood. Although the hamsters are still growing at the age employed (21 days), only 1 Present address: Dept. of Microbiology, Louisiana State University LSU School of Medicine New Orleans, La.
a few liver parenchymal cells are in S phase at any time as judged by autoradiography with 3H-TdR (Gentry el al., 1962), and thus the infection apparently takes place in resting cells. It has been suggested recently (Lawrence, 1971) that EAV replicates only in cells in S phase. We have defined further the in vivo system and used it to study this question, and our results suggest that in the young hamster, EAV replicates independently of host cell DNA synthesis. MATERIALS
Virus passage and titration. The Kentucky A strain of equine abortion virus (EAV), adapted to hamsters by Doll et al., (1953) and maintained for more than 400 serial passages in this laboratory, was employed. The virus was passed as described in 35- to 40-g (about 14 days) Syrian hamsters (Lakeview Hamster Colony, Newfield, NJ), and the titers in liver and blood were determined by LDjo assay as described by Randall and Bracken (1957). Puri$cation of virus. Infected animals were decapitated and virus was purified from the heparinized blood as described by Darlington 784
Copyright All rights
@I 1972 by Academic Press, of reproduction in any form
Inc. reserved.
AND METHODS
HERPESVIRUS
REPLICATION
and Randall, 1963. Virus was separated from the filtered plasma by banding in linear sucrose density gradients @O-50%, w/v; 0.02 M phosphate buffer, pH 7.4) which were centrifuged in a Spinco SW 25.1 rotor for 1 hr at 20,000 rpm. The virus band was collected with a syringe and the sucrose removed by dilution with Hz0 and pelleting the virus under the same conditions of centrifugation. Labeling and extraction of liver DNA. Tritiated thymidine (3H-TdR, 100 ,.&Z/ml, lo-” M) was injected ip at a dosage of 2 &?i/g body weight into groups of hamsters at regular times during infection. One hour after the injection of isotope at least three animals were sacrificed and their livers were perfused in situ through the portal vein with physiological saline. Livers were then perfused with 10 % sucrose (pH 7.0) containing 0.1% trypsin (Difco 1: 250) and minced in sucrose containing 25 % polyvinylpyrrolidone (National Aniline Corp.). These small pieces of liver were disrupted further by forcing them through the orifice of a syringe connected to needles of decreasing diameter (17 through 20 gauge). Suspensions of isolated liver cells were then obtained by differential centrifugation and, after cell lysis with 2 % SLS, DNA was extracted with phenol as described by Soehner et al. (1965). DNA synthesis. To determine the specific activity of the labeled DNA, samples were hydrolyzed in hot trichloracetic acid (TCA, 5 %), radioactivity was counted in a Packard Tri-Carb liquid scintillation spectrometer, and the DNA was calorimetrically quantitated by the Burton modification of the diphenylamine reaction (Burton, 1956). Thymidine metabolism. Catabolism of 3HTdR in the hamster was monitored by measuring the conversion of 3H-TdR to 3Hz0. Control and infected hamsters injected at various times postinoculation with 3H-TdR (0.15 &i/g body wt, lo+ moles/animal) were sacrificed 1 hr after infection of isotope, and the pooled sera from the animals of each period were collected and frozen immediately. The serum water was separated from the solutes by lyophilization, and the amount of total radioactivity present as serum water was determined. Metabolism of TdR in liver extracts of
in viva
785
control and EAV-infected hamsters was estimated by measuring the enzymatic conversion of 3H-TdR to 3H-thymine (3H-T) and to 3H-thymidylate (3H-dTMP). Pooled livers were homogenized in 0.02 M Tris buffer (pH 7.8) containing 0.15 2M KC1 in a PotterElevejhem homogenizer at 0°C and centrifuged at 45,000 rpm for 2 hr in a Spinco No. 50 rotor. The supernatant fractions were frozen in small aliquots at - 22°C. The assays were conducted at 37°C by mixing a prewarmed liver extract with an equal volume of substrate mixture containing adenosine triphosphate (ATP), 6 X 10T3 M; 3-phosphoglycerate, 6 X low3 M; MgCIZ , 6 X 1O-3 M; Tris buffer, pH 7.8,6 X 10T3Al; 3H-TdR, 1 X 1O-2 M, 1.58 &Z/ml; dTMP, 8 X 10-j M; and NaF, 1.6 X low2 2M. At various times, 0.5-ml samples of the reaction mixture were mixed with 0.13 ml of 20% trichloracetic acid (TCA) at 0°C to stop the reaction. To separate and identify the substrate and products, ascending paper chromatography was employed. Samples (20 ~1) of the reaction mixture were spotted on Whatman No. 1 paper strips (1 X 12 in.) and chromatographed by the method of Adams and Fink (1966). The appropriate nonradioactive pyrimidines were co-chromatographed as standards for location of substrate and product by short-wave ultraviolet light. These spots were cut out and counted directly in a liquid scintillation spectrometer. The dTMP phosphatase activity was assayed in an analogous manner using 14C-dTMP as substrate. DNA-DNA hybridization. To determine the nature of the DNA synthesized in infected animals, DNA-DNA hybridization was carried out by the method of Takahashi et al. (1969). Briefly, high molecular weight DNA extracted from uninfected hamster liver cells, mouse L-M cells, and from purified EAV (18), was immobilized on filter membranes (40 pg/filter) and incubated with 22 kg of radioactive liver DNA purified from infected hamsters, at 60°C for 24 hr in 3X saline citrate (SSC, 0.15 &! NaCl, 0.02 M Na citrate). MAK column chromatography. The rates of host and viral DNA syntheses were measured individually by separating labeled in-
786
O’CALLAGHAN,
RANDALL,
fected-cell DNA by chromatography on columns of methylated albuminkeiselguhr (MAK) as described by Mandell and Hershey (1960). Columns were routinely loaded with 50-150 pg of DNA in 0.50 M D;aCl in 0.05 M phosphate buffer (pH 6.7) and DNA was eluted with a salt gradient of 0.50-0.70 M NaCl pumped through the column at a flow rate of either 0.25 ml/mm or 0.30 ml/ min. The salt concentration of various fractions was determined from the equation described by Mandell and Hershey (1960) and from the refractive index as described by Sueoka and Cheng (1967). To measure radioactivity, fractions were precipitated with 5 % TCA using bovine serum albumin (BSA, 400 pg/ml) as a coprecipitator, hydrolyzed in 5 % TCA, and counted as described above. To identifv the DNA in the column fractions, CsCl density gradients containing the unknown DNA and i4licrococcus lysocleikticus DNA were centrifuged to equilibrium in an ANK rotor of the Spinco model E ultracentrifuge (35,000 rpm for 42 hr). Buoyant densities of the DNA were calculated as described by Sueoka et al. (1959). Nitrocellulose column chromatography. The double-st’randedness of the newly synthe-
AND
GEIL‘TRY
sized Dr\‘A was studied by nitrocellulose column chromatography as described by Klamerth (1965). Nitro Cel S (Gallard-Schlesinger Chemical Manufacturing Company, New York), was packed tightly with a glass rod into 1 X 25-cm columns to a height of 10 cm. The packed column was washed with 2 X SSC at a flow rate of 0.3-0.4 ml/ min to remove any unbound DNA. This washing eluted approximately 98% of the native double-stranded DNA, but less than 5% of heat-denatured hamster DNA. The denatured DNA had been previously heated to 100°C in 1 X SSC for 15 min at a concentration of approximately 50 pg/ml and cooled slowly. The extent of denaturation was monitored by observing the hyperchromicity. To elute denatured Dh’A from the nitrocellulose columns, the columns were washed with 25 ml of 0.1 M NaOH and this wash eluted nearly 100 % of the bound DNA. RESULTS
In vivo growth. The kinetics of viral replication in hamster liver and its subsequent accumulation in blood were measured by LDjo assay, and the results are shown in Fig. 1. Although the size of the inoculum and the
o-0 LDso Units/g. Liver C--O LDso Units/ml. Blood
‘OSI
,,,.,,,. I
,. 2
3
4
5
6
7
8
9
IO
,
,
II
I2
HOURS POST INFECTION FIG. 1. Growth curve of EAV in hamsters. Hamsters (35-40 g) were injected intraperitoneally with l-2 X lo8 LD50 units of EAV. At regular intervals after injection, livers from three animals or blood from five animals were collected, weighed, and frozen at -22°C. Homogenates of the pooled samples were serially diluted under aseptic conditions and 1 ml per animal of the proper dilution wss injected intraperitoneally into at least four normal hamsters. The number of deaths was recorded and the LD60 titer was determined with the Reed-Muench method (Reed and Muench, 1938). Titers for liver were expressed as LDso units per gram of liver or per milliliter of whole blood.
HERPESVIRUS
REPLICATION
titer of virus achieved resemble the growth curve reported by Randall and Bracken (1957) for EAV in hamster liver, the entire growth cycle ending in the death of the infected animals was 24 hr in the earlier study and only 12-14 hr in the present study. This difference probably reflects the adaptation of the virus to this experimental system achieved in the nearly 300 passages of virus elapsing between the two studies. It is apparent from the short latent period and the rapid increase in viral titer thereafter that the in viva growth curve is analogous to the in vitro growth of EAV in L-RI cells described by O’Callaghan et al. (1968b). By measuring the titer of virus in blood, it can be seen that the production of infectious virus in liver is followed shortly by the release and accumulation of virus in blood. Once release began, the rise in cell-free virus paralleled the production of virus in liver. Virus synthesis and release. Because studies of the virus growth cycle showed that infectious EAV appeared in blood soon after its detection in the liver, studies were undertaken to estimate the time required for 3H-TdR to be incorporated into viral DNA, encapsidated, and released from the liver cell. Analysis of plasma virus by sucrose gradients (Table 1) shows that during the time of active DNA synthesis (4-8 hr postinoculation) significant quantities of labeled virus appeared in the blood as early as 90 min after the injection of 3H-TdR, and continued to accumulate during the remainder of the growth cycle. This means that synthesis of DNA is closely coupled with encapsidation and release, for the entire process can be completed for a fraction of the DNA within 2 hr. DNA synthesis. Since earlier autoradiographic studies of liver nuclei from EAVinfected hamsters suggested changes in DNA synthesis during infection (Gentry et al., 1962), the incorporation of “H-TdR into total liver DNA was measured throughout the virus growth cycle. The results (Fig. 2) show that the incorporation of 3H-TdR into the DNA of uninfected hamster liver was low, during the early hours of infection, similar to that seen in controls. From 3 to 4 hr postinoculation, however, incorporation increased significantly, peaking at 7-X hr. This
in
vivo
787 TABLE
RELEASE
1
OF 3H-TdR-LansLsx HAMSTER PL.GMA
Time of injectiona of 3H-TdR (hr postinoculation) -
EAV
INTO
Cpm in EAV-virions purified from hamster plasma various times (hr) after injection of 3H-TdRb 0.5 1 -.__
4 6 8
131 73 64
G 3H-TdR
(2 &i/g
264 293 139
1.5 1,020 960 1,070 body
2 ______ 1,364 10,995 2,773
weight,
lo-”
animal) was injected intraperitoneally times indicated.
7 11,454 moles/
at the
b Aliquots (3 ml) of filtered hamster plasma, obtained from duplicate groups of animals sacrificed at the indicated times after injection of isotope, were layered onto linear sucrose density gradients (20-507”) and centrifuged for 1 hr at 20,090 rpm in an SW 25.1 rotor (Spinco). The fractions containing intact virions were collected, and the total radioactivity present in the acidinsoluble fraction was measured.
pattern is consistent with that reported earlier by Gentry et al. (1962), except that the onset, and peak of DNA synthesis occurs earlier in the present study in keeping with the compression of the growth cycle as described above. Thymidine metabolism. In any comparative study of in viva pulse labeling of DNA with 3H-TdR, the relatively large pyrimidine catabolic pathway should be studied to insure that changes in catabolism are not responsible for any differences in the incorporation of isotope into DNA-a decrease in catabolism may mean an increase in utilization, and a longer pulse time (Gentry et al., 1971). We, therefore, made preliminary measurements of the conversion in vivo of 3H-TdR to 3H20, and in liver extracts, of 3H-TdR to 3H-T and 3H-dTMP. Catabolism of 3H-TdR to 3Hz0 was very rapid, 7 X lo5 cpm/ml of serum water by 1 hr after the injection of approximately 5 X lo6 cpm of 3H-TdR. TdR phosphorylase similarly was active in all preparations, control as well as infected. Conversion of 3H-TdR to 3H-dTMP also was rapid, reaching equilibrium within 5 min. Because of this it was not possible to
788
O’CALLAGHAN,
RANDALL,
a 80oz % 60. \z
1 I
E 40. :: ,I
AND
GENTRY
;I
n
20. 0' 4-O l-l
n l-2
:
7-8 5-6 3.-4 HOURS POST INFECTED
II
II-12
9-10
FIG. 2. Incorporation of “H-TdR into the liver DNA of normal and EAV-infected hamsters. Hamsters (35-40 g) were injected with EAV (l-2 X lo8 LDso units per animal) at time zero. ZH-TdR (2 &i/g body wt, 10v6 moles per animal) was injected intraperitoneally into a group of three or more hamsters at 1 hr before the time of injection, and 1, 3, 5, 7, 9, and 11 hr afterward. The animals were sacrificed 1 hr after the injection of isotope and the liver DNA was extracted. After hydrolysis in hot 5yo trichloracetic acid, the DNA content and radioactivity were measured and expressed as counts per minute per microgram of DNA.
achieve linear kinetics, and TdR kinase could not be measured accurately, even when NaF (10d2 M) was added to reduce dTMP phosphatase activity (from 2 X 10eg moles/ hr/mg protein to 5 X lo-lo moles/hr/mg protein). It, therefore, appears that replication of EAV in the hamster liver does not greatly alter either anabolism or catabolism of 3H-TdR, and that the latter is much more active than the former. The studies on 3H-TdR metabolism demonstrate that the increased incorporation of this labeled precursor into DNA indeed reflects a net increase in DNA synthesis and not an alteration in TdR catabolism which might mimic the same effect . DNA-DNA hybridization. It was next important to determine the nature of the DNA being synthesized. Accordingly, DNA-DNA hybridization analysis was used to determine the nature of the DNA labeled 3-4 hr postinoculation (Table 2), the time when increased incorporation was first apparent. As seen in Table 2 the labeled DNA hybridized extensively to viral DNA and only slightly to hamster or mouse DNA. These data show that most if not all the DNA newly synthesized in the infected liver is viral and that stimulation of host cell DNA synthesis is not detected at this time. Chromatographic separation of viral and host DNA. To measure more accurately the
TABLE
2
OF 3H-TdR-LaeELsn LIVER CELL DNA FROM EAV-INFECTED HAMSTERS TO VARIOUS SPECIES OF DNA
HYBRIDIZATION
DNA imrnob$~;~ on
EAV Hamster* L-M cell5
input (cpm)
320 399 458
hybridized (cpm) 132 9 7
Efficiency of hybridization m 41.3 2.3 1.5
* Forty micrograms of the proper species of DNA was bound to each filter, and 22 pg of liver cell DNA from EAV-infected hamsters pulsed with 3H-TdR from +3 to +4 hr postinoculation, were hybridized to the filters for 24 hr. The filters were washed, dried, and the radioactivity was measured. Each value is the average of two independent measurements. The cpm hybridized were corrected by subtracting the radioactivity retained on the blank filters. * Uninfected.
synthesis of host and viral DNA, the DNA extracted from infected liver was subjected to MAK column chromatography as shown in Fig. 3. Two peaks of radioactivity represented GFi-80% of the input radioactivity. These two peaks eluted at 0.60-0.61 M NaCl and 0.63-0.64 M NaCl, respectively, in repeated experiments using the same or altered salt gradients for elutions. The first peak (0.60-0.61 M NaCl) contained the ma-
HERPESVIRUS
REPLICATION
FRACTION
789
in vivo
NUMBER
3. Elution of EAV-infected hamster liver cell DNA from MAK columns. A total of 50 ~lg of DNA extracted from EAV-infected hamsters pulsed from 3 to 4 hr PI. with ‘H-TdR was loaded into a MAK column in 0.05 M PBS (pH 6.7, 0.05 M) at a concentration of 8 pg/ml. After washing the loaded column with 30 ml of 0.5 M NaCI, a salt gradient formed by pumping 0.9 M NaCl into a mixing chamber containing 100 ml of 0.5 2MNaCl at a flow rate of 0.25 ml/min was connected to the column. Fractions were automatically collected every 10 min and the radioactivity of the acid-insoluble portion of each fraction was measured by liquid scintillation counting. The molarity of NaCl in various fractions was calculated from the refractive index and from the formula described by Mandell and Hershey (1960). FIG.
jority of the radioactivity (85-90 %), but only a trace of the recovered DNA. The second peak of DNA (eluting at 0.63-0.64 M NaCl) contained only lo-15 % of the recovered radioactivity, yet contained greater than 90% of the recovered DNA. To fully identify the DNA in each peak, the two peaks were separately mixed with reference DNA and isopynically banded in C&l density gradients. As shown in Fig. 4, the 0.600.61 M NaCl peak contained DNA with a buoyant density of 1.716 g/cc which was identical to that of EAV DNA (Soehner et al., 1965). The 0.63-0.64 III NaCl peak contained DNA with a buoyant density of 1.702, which was identical to DNA similarly extracted from hamster liver cells, These results demonstrate that the chromatographic separation of EAV and hamster DNA is essentially complete and that during this labeling period EAV DNA is preferentially labeled. This later finding agrees with the DNA-DNA hybridization study which showed that in infected liver the newly synthesized DNA hybridized 20 times more efficiently with EAV DNA than with hamster DNA. Kinetics of DNA synthesis. Chromatographic separation of DNA extracted from
0.60 - 0.6 I M Peak 0.63 - 0.64 M Peak EAV DNA
Hamster DNA 1.702 I.716 I.731 g/Id. FIG. 4. Density gradient profile of DNA eluted from MAK columns. DNAs from the 0.690.61 M and the 0.63-0.64 M peaks eluting from MAK columns loaded with DNA extracted from EAVinfected hamster liver cells, were added to individual ultracentrifuge cells containing CsCl. M. Zysodeikticus DNA, p = 1.731 g ml, was added to the cells and they were centrifuged at 35,609 rpm for 41 hr in the ANK rotor in a Spinco Model E ultracentrifuge. Cells containing normal liver cell DNA and DNA from pure EAV particles were similarly prepared and centrifuged. Pictures of all cells at equilibrium were taken using ultraviolet optics and tracings were prepared with a densitometer. Buoyant densities were calculated according to the formula of Sueoka et al. (1959).
790
O’CALLAGIIAN.
RANDALL,
infected liver after 1-hr pulses of “H-TdR at various times after infection enabled the quantitation of the relative synthcscls of host and viral DNA (Fig. 5). These data show that, viral DNA synthesis begins at 3-4 hr postinfection (P.I.) and that the synthesis of EAV DNA accounbs for 8.5-95 %, of the D1\JA being synthesized after 3 hr P.I. The synthesis of host cell DNA prior t)o 3 hr P.I. proceeded at a ratmeof approximately 5 cpm of WTdR incorporated per Gg of DNA in a l-hr pulse. This rate remained const’ant throughout infection, but in relation to total DNA synthesis it decreased from 100 % prior to 3 hr P.I. to less than 15 % after 4 hr P.I. This marked decrease in the percentage of host to total synthesis reflects the synchronous initiation of viral DNA synt’hesis throughout the liver at 3-4 hr P.I. Once viral DlSA synthesis is initiated, the rate of viral DNA synthesis continued to increase unt’il8 hr P.I. when a maximal incorporation rate of 20 times normal liver was recorded. Thereafter, rates of synthesis of usual DNA decreased until the animals died at 12-14 hr P.I. The detection of low levels of host DNA synthesis throughout the infection was unexpected since EAV has been previously reported to inhibit host DNA synthesis in cell cultures (O’Callaghan et al., 1968a; Lawrence, 1971). It is possible t,hat the persistent synthesis of hamster DNA recorded here can
I
z
0
cpm/pg
hmp m
% Radioactivity % Radioactivity
AN11 GENTRY
be attributed to DSA synth& in nonparenchymal liver cells and to the presence of radioactive leukocytes trapped in t)hc numerous thrombi common in the blood supply of the liver as part of the pathology of EAV infection (Arhelger ef al., 1963). Nature oj EAV DNA. When the )IAB column elution profile of normal hamster liver DNA and liver cell DiYA extracted from EAV-infected liver were compared, no differences in clution profiles were noted-liver cell DNA eluted at 0.63-0.64 M NaCl in both cases (not shown). Similar comparisons between EAV DNA extracted from part,ially purified virions and EAV DNA extracted from infected liver cells revealed a gross difference between the two viral DNAs. Infected liver cell EAV DNA elutes at 0.60-0.61 M NaCl while virion-extracted DNA does not clute until 0.70-0.71 M NaCl is reached (Fig. 6). The elution of EAV virion-extract’ed DNA at this relatively high salt concentrat’ion was previously reported by Soehner et al. (1965), and confirmed by O’Callaghan et al. (1968a). The elution of a portion (7 %) of the intracellular EAV DNA extracted from infected L-N cells also occurred at 0.70-0.71 M NaCl (O’Callaghan et al., 1968a). In all cases, the DNA eluting at 0.70-0.71 M NaCl had a molecular weight of 90-100 X lo6 daltons, and this class of molecules having the proper
DNA in Hamster DNA in EAV DNA
20
b0 0
-1-o
l-2
3-4 HOURS
5-6
POST INFECTION
FIG. 5. The time course of EAV and hamster DNA syntheses. Composite of data obtained from MAK column chromatography experiments of DNA extracted from liver cells of normal and EAVinfected hamsters after a 1 hr pulse of 3H-TdR.
HERPESVIRUS
REPLICATION
791
in viva Heat -Denatured
DNA l -*
,
o-
105
100,
EAV
DNA
I
I2
-
3
4 Notive
/ 4
ttomster
8
I2 16 20 24 26 32 36 40 44 46
5
I2
3
4
DNA
.-•
Hamster
o-0 .-.
E AV lnfactad
Hamster
'052
FRACTION
FIG. 6. MAK column chromatography of normal hamster DNA and EAV DNA. MAK columns were loaded with W-labeled DNA (about 70 pg) extracted from pellets of pure EAV and other columns were loaded with aH-labeled DNA extracted from normal hamster liver cells. DNAs were added to the column in 0.05 M PBS at a concentration of 10 pg/ml. The material was eluted with a salt gradient formed by adding 0.9 M NaCl to a mixing chamber containing 100 ml of 0.5 M NaCl at a rate of 0.3 ml/min. The gradient was pumped through the column at this same rate and fractions were automatically collected every 10 min. The acid-insoluble portion of each fraction was measured by liquid scintillation counting and the NaCl concentration in various fractions was quantitated by the refractive index and according to the formula of Mandell and Hershey (1960). The horizontal bar (I---() in the figure denotes the elution position of EAV DNA when extracted from infected liver cells.
molecular weight (Soehner et al., 1965) is considered to be the EAV genome. The bulk (93 %) of the EAV DNA extracted from L-M cells eluted at a low salt molarity (0.43-0.45 Xl NaCl) and was shown to be highly fragmented EAV DEA molecules. Since the elution of EAV DNA from MAK columns at 0.60-0.61 M NaCl had not been observed, the properties of these tissue-extracted molecules as compared to the virionextracted molecules were determined. The density in CsCl of both DISA molecules as noted earlier (see Fig. 4) is very similar, indicating that the infected cell DNA is essentially a double-stranded molecule like the virion DNA. To confirm this point further, the ability was measured of both classes of
Fmction of 2 X SSC
Fmrthn of O.IN NoOH
7. Chromatography of EAV DNA and hamster DNA on nitrocellulose columns. DNA prepared from normal hamster liver, pure EAV particles, and EAV-infected liver cells (56 hr P.I.) was suspended in 2 X SSC at concentrations of approximately 20 pgg/ml and added separately to tightly packed nitrocellulose columns (Nitrocel S). Similar preparations of DNA were heated at 100°C in 1 X SSC, cooled slowly, adjusted to 2 X SSC, and added to similar columns. The columns were washed slowly (0.3 ml/min) with 2 X WC and five fractions (7 ml each) were collected. The columns were than washed with 0.1 N NaOH and four fractions (7 ml each) were collected. The acid-insoluble portion of each fraction was counted by liquid scintillation counting. FIG.
EAV DNA, native and heat-denatured, to bind to nitrocellulose as shown in Fig. 7. Native DNA, hamster DNA, and infected DNA all pass through nitrocellulose columns without any significant binding, indicating that they all are double-stranded. Once heat-denatured, even if followed by slow cooling, the DNAs were bound tightly to the nitrocellulose. Thus, there were no apparent differences in the extent of singlestranded regions and crosslinked regions between liver cell-extracted and virion-extracted EAV-DNAs. DISCUSSION
The in viva replication of EAV in hamster liver as described in the present study is comparable to that of EAV in L-M cells as described by O’Callaghan et al. (1968b). In
792
O’CALLAGHAN.
RANDALL,
both systems the virus replicates in the nucleus of one cell type, intranuclear inclusions are formed early in the growth cycle, viral DNA synthesis occurs coincident with the inhibition or in the absence of host DNA synthesis, and infectious virus is detectable within the cell and is released into the medium shortly after assembly. Thus, a growth curve with a short latent period followed by a sharp increase in virus titer is common to both. The duplication of such growth-curve properties in an animal system is possible only when all the liver cells become infected at the same time. This synchrony is reflected likewise in the development of pathology in the liver and the nearly simultaneous death of the majority of infected animals. This ideal situation is dependent upon a rapid initiation of the infection and is achieved only when the amount of virus injected sufficiently saturates the susceptible cells. In fact, when smaller inocula are injected the resulting infections allow the animals to live far beyond 12-14 hr and the time of death varies among the population of infected animals. In the routine passage of virus, infectious virus was detectable in the cell-free state (in plasma) about 1 hr after initial increase in the level of infectious virus in liver. More detailed experiments indicated that infectious radioactive virus was released to plasma after 90 min from the time of the injection of isotope (see Table 1). These data reveal that some newly synthesized virus is capable of release from infected cells without prolonged exposure to the int,racellular environment, and they imply that a close temporal relationship exists between the synthesis of DNA and the encapsidation of DNA into infectious particles. A similar relationship was suggested previously (O’Callaghan et al., 1968) on the basis of the high percentage of intranuclear DNA which was resistant to extensive DNase digestion. This observation may be important in explaining the replication of EAV in L-M cultures contaminated with iMycoplasma hominis. Stock and Gentry (1969) have shown that Al. hominis grown in L-M cell cultures secretes a DNase capable of digesting intranuclear L-M cell DNA and, although the enzyme
AND
GENTRY
appclars ablr to digest chxtractcd EAV DSA molcculcs, it is quite capable of replicating in the nucleus of L->I cells contlaminatcd with M. hominis (Gentry and Evans, 1972). On the basis of these considerations, it is feasible to suggest that the process of the encapsidation of EAV DNA is rapid and is thus involved in protecting the viral genome. The minimum time required for encapsidation therefore appears to be somewhat less than the 2-3 hr reported by Olshcvsky et al. (1967) and Roizman et al. (1963) for herpes simplex virus. Although increased incorporation of tritiated thymidine has been previously associated with EAV infection of hamster liver (Gentry et al. 1962), it remained for the present study to establish t’hat a net increase in DNA synthesis occurs and that the newly syrlthesized DNA is predominantly viral. This last point is important for it proves that EAV DNA in liver replicates exclusively independent’ of host DSA synthesis. This finding clearly distinguishes the replication in liver from the unusual replication cycle of EAV recently report’ed in human KB cells by Lawrence (1971). Lawrence found that in synchronized cultures of KS cells EAV DiYA synthesis is initiated only when the host cell is in S phase. Once viral replication began, further rounds of host synthesis were inhibited. This dependency of EAV DNA synthesis upon KB cell synthesis was confusing for no such dependency has been previously reported for the well-studied herpes simplex and pseudorabies viruses (Kaplan, 1969)) and the only available studies on the biochemistry of EAV replication were performed on infected log phase L-M cell cultures (O’Callaghan et al., 1969b). These studies on log phase L-M cells clearly defined the kinetics of EAV-induced inhibition of L-M cell DNA synthesis shortly after infection, but no attempt was made to determine the relationship between viral DNA synthesis and the stage of cell cycle. Since no direct experimental evidence is yet available, the demonstration of EAV DNA synthesis in infected liver without any significant host DNA synthesis is clearly the best evidence of the replication of EAV independent of host DNA synthesis.
HERPESVIRUS
REPLICATION
ACKNOWLEDGMENTS This study was supported by USPHS Research Career Development Award 5K3 AI7021 (G.A.G.), Training Grant AI-69, and Research Grants AI-92632 and A1-09858. REFERENCES ADAMS, W., and FINK, K. (1966). Paper chromato-
graphic data for purines, pyrimidines, and derivatives in a variety of solvents. J. Chromatogf. 22, 118-129. ARHELGER, R., DARLINOTON, R. W., and RANDALL, C. C. (1963). An electron microscopic study of equine abortion virus infection in hamster liver. Amer. J. Pathol. 42, 703-713. BURTON, K. (1956). The conditions and mechanism of diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62,315323. DARLINGTON, R. W., and RANDALL, C. C. (1963). The nucleic acid content of equine abortion virus. Virology 19, 322-327. DOLL, E. R., RICHARDS, M., and WALLACE, M. (1953). Adaptation of the equine abortion virus to suckling Syrian hamsters. Cornell Vet. 43, 551-558. GENTRY, G. A., and EVANS, S. F. (1972). Herpesvirus replication resistant to heterologous DNase. Abstr. Annu. Meet. Amer. Sot. Microbial. p. 222. GENTRY, G. A., MORSE, P. A., JR., and DORSETT, M. T. (1971). In viva inhibition of pyrimidine catabolism by 5cyanouracil. Cancer Res. 31, 909-912. GENTRY, G. A., WALKER, B., and RANDALL, C. C. (1962). Correlated autoradiographic and biochemical study of DNA labeling in equine abortion virus hepatitis. Virology 16, 466-465. KAPLAN, A. S. (1969). Herpes simplex and pseudorabies viruses. Virol. Monogr., No. 5. SpringerVerlag, New York. pp. 44-47. KLAMIXRTH, 0. (1965). Separation of high molecular weight deoxyribonucleic acid and ribonucleic acid. Nature London 208,1318-1319. L.IWRENCE, W. (1971). Evidence for a relationship between equine abortion (herpes) virus deoxyribonucleic acid synthesis and the S phase of the KB cell mitotic cycle. J. Viral. ‘7.736-748. MANDELL, J., and HERSHEY, A. (1960). A fractionating column for analysis of nucleic acids. Anal. Biochem. 1, 66-77.
in uivo
793
D., CHEEVERS, W., and RANDALL, C. C. (1968a). Kinetics of cellular and viral DNA synthesis in equine abortion (herpes) virus infection of L-M cells. Virology 36, 104-114. O’CALLAGHAN, D., HYDE, J., GENTRY, G. A., and RANDALL, C. C. (1968b). Kinetics of viral deoxyribonucleic acid, protein, and infectious particle production and alterations in host macromolecular synthesis in equine abortion (herpes) virus-infected cells. J. Viral. 2,793-812. O’CALLAGHAN, R., JACKSON, L., RSNDALL, C. C., and GENTRY, G. A. (1968). Studies on the coating of equine herpes virus. Bacterial. Proc. 167-168. PLUMMER, G. (1964). Serological comparison of the herpes viruses. Brit. J. Ezp. Pathol. 45,125141. RANDALL, C. C., and BRACKI~N, E. C. (1957). Studies on hepatitis in hamsters infected with equine abortion virus. I. Sequential development of inclusions and the growth cycle. Amer. O’CALLAGHAN,
J. Pathol. 33,709-726. REED, L. J., and MUENCH,
H. (1938). A simple method of estimating fifty per cent endpoints. Amer. J. Hyg. 27,493-497. ROIZMAN, B., AURELIAN, L., and ROANE, P. R., JR. (1963). The multiplication of herpes simplex virus. I. The programming of viral DNA dnplication in HEp-2 cells. Virology 21,482-498. SOEHNER,R., GENTRY, G. A., and RANDALL, C. C. (1965). Some physico-chemical characteristics of equine abortion virus nucleic acid. Virology 26, 39445. STOCK, D., and GENTRY, G. A. (1969). Mycoplasma1 deoxyribonuclease activity in virus-infected L-cell cultures. J. Viral. 3, 313-317. SUEOKA, N., and CHENG, T. (1967). Fractionation of DNA on methylated albumin columns, In “Methods in Enzymology” S. Colowick and N. Kaplan (eds.), Vol. XII, pp 56‘2-566. Academic Press, New York. SUEOKA, N., MARMUR, J., and DOTY, P. (1959). Heterogeneity in deoxyribonucleic acids. II. Dependence of the density of deoxyribonucleic acids on guanine-cytosine content. Nature London 183,1429-1431. TAKAHASHI, M., O~INO, T., BABA, K., and ONAKA, M. (1969). Synthesis of deoxyribonucleic acid in human and hamster kidney cells infected with human adenovirus types 5 and 12. Virology 37, 513-520.
49,
784-793
(1972)
Herpesvirus RICHARD
O’CALLAGHAN,’
Department of Microbiology,
Replication
CHARLES
University
in Viva
C. RANDALL,
of Mississippi
AND
GLENN
A. GENTRY
School of Medicine, Jackson, Mississippi
89.216
Accepted June 16, 1972 The replication of equine herpesvirus (equine abortion virus; EAV) provides a unique in vivo system characterized by hepatitis and viremia that permits quantitative molecular biological studies. The replication of infectious cell-associated virus precedes by a short time the onset of viremia, based on the rapid (less than 2 hr) appearance of significant amounts of labeled virus in the blood, after injection of 3H-thymidine. Analysis of thymidine metabolism revealed that peak viral synthesis took place at 8-9 hr postinfection. DNA-DNA hybridization and MAK column chromatography showed that, 85y0 or more of the newly synthesized DNA was viral. Host DNA synthesis, however, remained at a very low level throughout infection. The data show that EAV can replicate effectively in the absence of significant host DNA synthesis, a point recently in dispute. INTRODUCTION
Equine abortion virus is a member of the herpes group (Plummer, 1964), with a typical genome of double-stranded DNA of relatively high G + C content (Soehner et al., 1965). Its natural host is the horse, in which it causes a rhinopneumonitis, with abortion if the host is a pregnant mare (Doll et al., 1953). Although it grows well in various cell cultures, it appears to grow best in the young Syrian hamster, to which it was adapted a number of years ago (Doll et aZ., 1953). The effects on the hamster are dramatic. When inoculated with lo8 LDsO , the hamster develops hepatitis and dies about 12 hr after inoculation, with more than 10” virions per ml of blood (Randall and Bracken, 1957; Darlington and Randall, 1963). Histological evidence indicated that the infection consists solely of viral replication in parenchymal liver cell nuclei of which more than 95 % are infected in the typical growth curve. Subsequently the virus is released and accumulates in the blood. Although the hamsters are still growing at the age employed (21 days), only 1 Present address: Dept. of Microbiology, Louisiana State University LSU School of Medicine New Orleans, La.
a few liver parenchymal cells are in S phase at any time as judged by autoradiography with 3H-TdR (Gentry el al., 1962), and thus the infection apparently takes place in resting cells. It has been suggested recently (Lawrence, 1971) that EAV replicates only in cells in S phase. We have defined further the in vivo system and used it to study this question, and our results suggest that in the young hamster, EAV replicates independently of host cell DNA synthesis. MATERIALS
Virus passage and titration. The Kentucky A strain of equine abortion virus (EAV), adapted to hamsters by Doll et al., (1953) and maintained for more than 400 serial passages in this laboratory, was employed. The virus was passed as described in 35- to 40-g (about 14 days) Syrian hamsters (Lakeview Hamster Colony, Newfield, NJ), and the titers in liver and blood were determined by LDjo assay as described by Randall and Bracken (1957). Puri$cation of virus. Infected animals were decapitated and virus was purified from the heparinized blood as described by Darlington 784
Copyright All rights
@I 1972 by Academic Press, of reproduction in any form
Inc. reserved.
AND METHODS
HERPESVIRUS
REPLICATION
and Randall, 1963. Virus was separated from the filtered plasma by banding in linear sucrose density gradients @O-50%, w/v; 0.02 M phosphate buffer, pH 7.4) which were centrifuged in a Spinco SW 25.1 rotor for 1 hr at 20,000 rpm. The virus band was collected with a syringe and the sucrose removed by dilution with Hz0 and pelleting the virus under the same conditions of centrifugation. Labeling and extraction of liver DNA. Tritiated thymidine (3H-TdR, 100 ,.&Z/ml, lo-” M) was injected ip at a dosage of 2 &?i/g body weight into groups of hamsters at regular times during infection. One hour after the injection of isotope at least three animals were sacrificed and their livers were perfused in situ through the portal vein with physiological saline. Livers were then perfused with 10 % sucrose (pH 7.0) containing 0.1% trypsin (Difco 1: 250) and minced in sucrose containing 25 % polyvinylpyrrolidone (National Aniline Corp.). These small pieces of liver were disrupted further by forcing them through the orifice of a syringe connected to needles of decreasing diameter (17 through 20 gauge). Suspensions of isolated liver cells were then obtained by differential centrifugation and, after cell lysis with 2 % SLS, DNA was extracted with phenol as described by Soehner et al. (1965). DNA synthesis. To determine the specific activity of the labeled DNA, samples were hydrolyzed in hot trichloracetic acid (TCA, 5 %), radioactivity was counted in a Packard Tri-Carb liquid scintillation spectrometer, and the DNA was calorimetrically quantitated by the Burton modification of the diphenylamine reaction (Burton, 1956). Thymidine metabolism. Catabolism of 3HTdR in the hamster was monitored by measuring the conversion of 3H-TdR to 3Hz0. Control and infected hamsters injected at various times postinoculation with 3H-TdR (0.15 &i/g body wt, lo+ moles/animal) were sacrificed 1 hr after infection of isotope, and the pooled sera from the animals of each period were collected and frozen immediately. The serum water was separated from the solutes by lyophilization, and the amount of total radioactivity present as serum water was determined. Metabolism of TdR in liver extracts of
in viva
785
control and EAV-infected hamsters was estimated by measuring the enzymatic conversion of 3H-TdR to 3H-thymine (3H-T) and to 3H-thymidylate (3H-dTMP). Pooled livers were homogenized in 0.02 M Tris buffer (pH 7.8) containing 0.15 2M KC1 in a PotterElevejhem homogenizer at 0°C and centrifuged at 45,000 rpm for 2 hr in a Spinco No. 50 rotor. The supernatant fractions were frozen in small aliquots at - 22°C. The assays were conducted at 37°C by mixing a prewarmed liver extract with an equal volume of substrate mixture containing adenosine triphosphate (ATP), 6 X 10T3 M; 3-phosphoglycerate, 6 X low3 M; MgCIZ , 6 X 1O-3 M; Tris buffer, pH 7.8,6 X 10T3Al; 3H-TdR, 1 X 1O-2 M, 1.58 &Z/ml; dTMP, 8 X 10-j M; and NaF, 1.6 X low2 2M. At various times, 0.5-ml samples of the reaction mixture were mixed with 0.13 ml of 20% trichloracetic acid (TCA) at 0°C to stop the reaction. To separate and identify the substrate and products, ascending paper chromatography was employed. Samples (20 ~1) of the reaction mixture were spotted on Whatman No. 1 paper strips (1 X 12 in.) and chromatographed by the method of Adams and Fink (1966). The appropriate nonradioactive pyrimidines were co-chromatographed as standards for location of substrate and product by short-wave ultraviolet light. These spots were cut out and counted directly in a liquid scintillation spectrometer. The dTMP phosphatase activity was assayed in an analogous manner using 14C-dTMP as substrate. DNA-DNA hybridization. To determine the nature of the DNA synthesized in infected animals, DNA-DNA hybridization was carried out by the method of Takahashi et al. (1969). Briefly, high molecular weight DNA extracted from uninfected hamster liver cells, mouse L-M cells, and from purified EAV (18), was immobilized on filter membranes (40 pg/filter) and incubated with 22 kg of radioactive liver DNA purified from infected hamsters, at 60°C for 24 hr in 3X saline citrate (SSC, 0.15 &! NaCl, 0.02 M Na citrate). MAK column chromatography. The rates of host and viral DNA syntheses were measured individually by separating labeled in-
786
O’CALLAGHAN,
RANDALL,
fected-cell DNA by chromatography on columns of methylated albuminkeiselguhr (MAK) as described by Mandell and Hershey (1960). Columns were routinely loaded with 50-150 pg of DNA in 0.50 M D;aCl in 0.05 M phosphate buffer (pH 6.7) and DNA was eluted with a salt gradient of 0.50-0.70 M NaCl pumped through the column at a flow rate of either 0.25 ml/mm or 0.30 ml/ min. The salt concentration of various fractions was determined from the equation described by Mandell and Hershey (1960) and from the refractive index as described by Sueoka and Cheng (1967). To measure radioactivity, fractions were precipitated with 5 % TCA using bovine serum albumin (BSA, 400 pg/ml) as a coprecipitator, hydrolyzed in 5 % TCA, and counted as described above. To identifv the DNA in the column fractions, CsCl density gradients containing the unknown DNA and i4licrococcus lysocleikticus DNA were centrifuged to equilibrium in an ANK rotor of the Spinco model E ultracentrifuge (35,000 rpm for 42 hr). Buoyant densities of the DNA were calculated as described by Sueoka et al. (1959). Nitrocellulose column chromatography. The double-st’randedness of the newly synthe-
AND
GEIL‘TRY
sized Dr\‘A was studied by nitrocellulose column chromatography as described by Klamerth (1965). Nitro Cel S (Gallard-Schlesinger Chemical Manufacturing Company, New York), was packed tightly with a glass rod into 1 X 25-cm columns to a height of 10 cm. The packed column was washed with 2 X SSC at a flow rate of 0.3-0.4 ml/ min to remove any unbound DNA. This washing eluted approximately 98% of the native double-stranded DNA, but less than 5% of heat-denatured hamster DNA. The denatured DNA had been previously heated to 100°C in 1 X SSC for 15 min at a concentration of approximately 50 pg/ml and cooled slowly. The extent of denaturation was monitored by observing the hyperchromicity. To elute denatured Dh’A from the nitrocellulose columns, the columns were washed with 25 ml of 0.1 M NaOH and this wash eluted nearly 100 % of the bound DNA. RESULTS
In vivo growth. The kinetics of viral replication in hamster liver and its subsequent accumulation in blood were measured by LDjo assay, and the results are shown in Fig. 1. Although the size of the inoculum and the
o-0 LDso Units/g. Liver C--O LDso Units/ml. Blood
‘OSI
,,,.,,,. I
,. 2
3
4
5
6
7
8
9
IO
,
,
II
I2
HOURS POST INFECTION FIG. 1. Growth curve of EAV in hamsters. Hamsters (35-40 g) were injected intraperitoneally with l-2 X lo8 LD50 units of EAV. At regular intervals after injection, livers from three animals or blood from five animals were collected, weighed, and frozen at -22°C. Homogenates of the pooled samples were serially diluted under aseptic conditions and 1 ml per animal of the proper dilution wss injected intraperitoneally into at least four normal hamsters. The number of deaths was recorded and the LD60 titer was determined with the Reed-Muench method (Reed and Muench, 1938). Titers for liver were expressed as LDso units per gram of liver or per milliliter of whole blood.
HERPESVIRUS
REPLICATION
titer of virus achieved resemble the growth curve reported by Randall and Bracken (1957) for EAV in hamster liver, the entire growth cycle ending in the death of the infected animals was 24 hr in the earlier study and only 12-14 hr in the present study. This difference probably reflects the adaptation of the virus to this experimental system achieved in the nearly 300 passages of virus elapsing between the two studies. It is apparent from the short latent period and the rapid increase in viral titer thereafter that the in viva growth curve is analogous to the in vitro growth of EAV in L-RI cells described by O’Callaghan et al. (1968b). By measuring the titer of virus in blood, it can be seen that the production of infectious virus in liver is followed shortly by the release and accumulation of virus in blood. Once release began, the rise in cell-free virus paralleled the production of virus in liver. Virus synthesis and release. Because studies of the virus growth cycle showed that infectious EAV appeared in blood soon after its detection in the liver, studies were undertaken to estimate the time required for 3H-TdR to be incorporated into viral DNA, encapsidated, and released from the liver cell. Analysis of plasma virus by sucrose gradients (Table 1) shows that during the time of active DNA synthesis (4-8 hr postinoculation) significant quantities of labeled virus appeared in the blood as early as 90 min after the injection of 3H-TdR, and continued to accumulate during the remainder of the growth cycle. This means that synthesis of DNA is closely coupled with encapsidation and release, for the entire process can be completed for a fraction of the DNA within 2 hr. DNA synthesis. Since earlier autoradiographic studies of liver nuclei from EAVinfected hamsters suggested changes in DNA synthesis during infection (Gentry et al., 1962), the incorporation of “H-TdR into total liver DNA was measured throughout the virus growth cycle. The results (Fig. 2) show that the incorporation of 3H-TdR into the DNA of uninfected hamster liver was low, during the early hours of infection, similar to that seen in controls. From 3 to 4 hr postinoculation, however, incorporation increased significantly, peaking at 7-X hr. This
in
vivo
787 TABLE
RELEASE
1
OF 3H-TdR-LansLsx HAMSTER PL.GMA
Time of injectiona of 3H-TdR (hr postinoculation) -
EAV
INTO
Cpm in EAV-virions purified from hamster plasma various times (hr) after injection of 3H-TdRb 0.5 1 -.__
4 6 8
131 73 64
G 3H-TdR
(2 &i/g
264 293 139
1.5 1,020 960 1,070 body
2 ______ 1,364 10,995 2,773
weight,
lo-”
animal) was injected intraperitoneally times indicated.
7 11,454 moles/
at the
b Aliquots (3 ml) of filtered hamster plasma, obtained from duplicate groups of animals sacrificed at the indicated times after injection of isotope, were layered onto linear sucrose density gradients (20-507”) and centrifuged for 1 hr at 20,090 rpm in an SW 25.1 rotor (Spinco). The fractions containing intact virions were collected, and the total radioactivity present in the acidinsoluble fraction was measured.
pattern is consistent with that reported earlier by Gentry et al. (1962), except that the onset, and peak of DNA synthesis occurs earlier in the present study in keeping with the compression of the growth cycle as described above. Thymidine metabolism. In any comparative study of in viva pulse labeling of DNA with 3H-TdR, the relatively large pyrimidine catabolic pathway should be studied to insure that changes in catabolism are not responsible for any differences in the incorporation of isotope into DNA-a decrease in catabolism may mean an increase in utilization, and a longer pulse time (Gentry et al., 1971). We, therefore, made preliminary measurements of the conversion in vivo of 3H-TdR to 3H20, and in liver extracts, of 3H-TdR to 3H-T and 3H-dTMP. Catabolism of 3H-TdR to 3Hz0 was very rapid, 7 X lo5 cpm/ml of serum water by 1 hr after the injection of approximately 5 X lo6 cpm of 3H-TdR. TdR phosphorylase similarly was active in all preparations, control as well as infected. Conversion of 3H-TdR to 3H-dTMP also was rapid, reaching equilibrium within 5 min. Because of this it was not possible to
788
O’CALLAGHAN,
RANDALL,
a 80oz % 60. \z
1 I
E 40. :: ,I
AND
GENTRY
;I
n
20. 0' 4-O l-l
n l-2
:
7-8 5-6 3.-4 HOURS POST INFECTED
II
II-12
9-10
FIG. 2. Incorporation of “H-TdR into the liver DNA of normal and EAV-infected hamsters. Hamsters (35-40 g) were injected with EAV (l-2 X lo8 LDso units per animal) at time zero. ZH-TdR (2 &i/g body wt, 10v6 moles per animal) was injected intraperitoneally into a group of three or more hamsters at 1 hr before the time of injection, and 1, 3, 5, 7, 9, and 11 hr afterward. The animals were sacrificed 1 hr after the injection of isotope and the liver DNA was extracted. After hydrolysis in hot 5yo trichloracetic acid, the DNA content and radioactivity were measured and expressed as counts per minute per microgram of DNA.
achieve linear kinetics, and TdR kinase could not be measured accurately, even when NaF (10d2 M) was added to reduce dTMP phosphatase activity (from 2 X 10eg moles/ hr/mg protein to 5 X lo-lo moles/hr/mg protein). It, therefore, appears that replication of EAV in the hamster liver does not greatly alter either anabolism or catabolism of 3H-TdR, and that the latter is much more active than the former. The studies on 3H-TdR metabolism demonstrate that the increased incorporation of this labeled precursor into DNA indeed reflects a net increase in DNA synthesis and not an alteration in TdR catabolism which might mimic the same effect . DNA-DNA hybridization. It was next important to determine the nature of the DNA being synthesized. Accordingly, DNA-DNA hybridization analysis was used to determine the nature of the DNA labeled 3-4 hr postinoculation (Table 2), the time when increased incorporation was first apparent. As seen in Table 2 the labeled DNA hybridized extensively to viral DNA and only slightly to hamster or mouse DNA. These data show that most if not all the DNA newly synthesized in the infected liver is viral and that stimulation of host cell DNA synthesis is not detected at this time. Chromatographic separation of viral and host DNA. To measure more accurately the
TABLE
2
OF 3H-TdR-LaeELsn LIVER CELL DNA FROM EAV-INFECTED HAMSTERS TO VARIOUS SPECIES OF DNA
HYBRIDIZATION
DNA imrnob$~;~ on
EAV Hamster* L-M cell5
input (cpm)
320 399 458
hybridized (cpm) 132 9 7
Efficiency of hybridization m 41.3 2.3 1.5
* Forty micrograms of the proper species of DNA was bound to each filter, and 22 pg of liver cell DNA from EAV-infected hamsters pulsed with 3H-TdR from +3 to +4 hr postinoculation, were hybridized to the filters for 24 hr. The filters were washed, dried, and the radioactivity was measured. Each value is the average of two independent measurements. The cpm hybridized were corrected by subtracting the radioactivity retained on the blank filters. * Uninfected.
synthesis of host and viral DNA, the DNA extracted from infected liver was subjected to MAK column chromatography as shown in Fig. 3. Two peaks of radioactivity represented GFi-80% of the input radioactivity. These two peaks eluted at 0.60-0.61 M NaCl and 0.63-0.64 M NaCl, respectively, in repeated experiments using the same or altered salt gradients for elutions. The first peak (0.60-0.61 M NaCl) contained the ma-
HERPESVIRUS
REPLICATION
FRACTION
789
in vivo
NUMBER
3. Elution of EAV-infected hamster liver cell DNA from MAK columns. A total of 50 ~lg of DNA extracted from EAV-infected hamsters pulsed from 3 to 4 hr PI. with ‘H-TdR was loaded into a MAK column in 0.05 M PBS (pH 6.7, 0.05 M) at a concentration of 8 pg/ml. After washing the loaded column with 30 ml of 0.5 M NaCI, a salt gradient formed by pumping 0.9 M NaCl into a mixing chamber containing 100 ml of 0.5 2MNaCl at a flow rate of 0.25 ml/min was connected to the column. Fractions were automatically collected every 10 min and the radioactivity of the acid-insoluble portion of each fraction was measured by liquid scintillation counting. The molarity of NaCl in various fractions was calculated from the refractive index and from the formula described by Mandell and Hershey (1960). FIG.
jority of the radioactivity (85-90 %), but only a trace of the recovered DNA. The second peak of DNA (eluting at 0.63-0.64 M NaCl) contained only lo-15 % of the recovered radioactivity, yet contained greater than 90% of the recovered DNA. To fully identify the DNA in each peak, the two peaks were separately mixed with reference DNA and isopynically banded in C&l density gradients. As shown in Fig. 4, the 0.600.61 M NaCl peak contained DNA with a buoyant density of 1.716 g/cc which was identical to that of EAV DNA (Soehner et al., 1965). The 0.63-0.64 III NaCl peak contained DNA with a buoyant density of 1.702, which was identical to DNA similarly extracted from hamster liver cells, These results demonstrate that the chromatographic separation of EAV and hamster DNA is essentially complete and that during this labeling period EAV DNA is preferentially labeled. This later finding agrees with the DNA-DNA hybridization study which showed that in infected liver the newly synthesized DNA hybridized 20 times more efficiently with EAV DNA than with hamster DNA. Kinetics of DNA synthesis. Chromatographic separation of DNA extracted from
0.60 - 0.6 I M Peak 0.63 - 0.64 M Peak EAV DNA
Hamster DNA 1.702 I.716 I.731 g/Id. FIG. 4. Density gradient profile of DNA eluted from MAK columns. DNAs from the 0.690.61 M and the 0.63-0.64 M peaks eluting from MAK columns loaded with DNA extracted from EAVinfected hamster liver cells, were added to individual ultracentrifuge cells containing CsCl. M. Zysodeikticus DNA, p = 1.731 g ml, was added to the cells and they were centrifuged at 35,609 rpm for 41 hr in the ANK rotor in a Spinco Model E ultracentrifuge. Cells containing normal liver cell DNA and DNA from pure EAV particles were similarly prepared and centrifuged. Pictures of all cells at equilibrium were taken using ultraviolet optics and tracings were prepared with a densitometer. Buoyant densities were calculated according to the formula of Sueoka et al. (1959).
790
O’CALLAGIIAN.
RANDALL,
infected liver after 1-hr pulses of “H-TdR at various times after infection enabled the quantitation of the relative synthcscls of host and viral DNA (Fig. 5). These data show that, viral DNA synthesis begins at 3-4 hr postinfection (P.I.) and that the synthesis of EAV DNA accounbs for 8.5-95 %, of the D1\JA being synthesized after 3 hr P.I. The synthesis of host cell DNA prior t)o 3 hr P.I. proceeded at a ratmeof approximately 5 cpm of WTdR incorporated per Gg of DNA in a l-hr pulse. This rate remained const’ant throughout infection, but in relation to total DNA synthesis it decreased from 100 % prior to 3 hr P.I. to less than 15 % after 4 hr P.I. This marked decrease in the percentage of host to total synthesis reflects the synchronous initiation of viral DNA synt’hesis throughout the liver at 3-4 hr P.I. Once viral DlSA synthesis is initiated, the rate of viral DNA synthesis continued to increase unt’il8 hr P.I. when a maximal incorporation rate of 20 times normal liver was recorded. Thereafter, rates of synthesis of usual DNA decreased until the animals died at 12-14 hr P.I. The detection of low levels of host DNA synthesis throughout the infection was unexpected since EAV has been previously reported to inhibit host DNA synthesis in cell cultures (O’Callaghan et al., 1968a; Lawrence, 1971). It is possible t,hat the persistent synthesis of hamster DNA recorded here can
I
z
0
cpm/pg
hmp m
% Radioactivity % Radioactivity
AN11 GENTRY
be attributed to DSA synth& in nonparenchymal liver cells and to the presence of radioactive leukocytes trapped in t)hc numerous thrombi common in the blood supply of the liver as part of the pathology of EAV infection (Arhelger ef al., 1963). Nature oj EAV DNA. When the )IAB column elution profile of normal hamster liver DNA and liver cell DiYA extracted from EAV-infected liver were compared, no differences in clution profiles were noted-liver cell DNA eluted at 0.63-0.64 M NaCl in both cases (not shown). Similar comparisons between EAV DNA extracted from part,ially purified virions and EAV DNA extracted from infected liver cells revealed a gross difference between the two viral DNAs. Infected liver cell EAV DNA elutes at 0.60-0.61 M NaCl while virion-extracted DNA does not clute until 0.70-0.71 M NaCl is reached (Fig. 6). The elution of EAV virion-extract’ed DNA at this relatively high salt concentrat’ion was previously reported by Soehner et al. (1965), and confirmed by O’Callaghan et al. (1968a). The elution of a portion (7 %) of the intracellular EAV DNA extracted from infected L-N cells also occurred at 0.70-0.71 M NaCl (O’Callaghan et al., 1968a). In all cases, the DNA eluting at 0.70-0.71 M NaCl had a molecular weight of 90-100 X lo6 daltons, and this class of molecules having the proper
DNA in Hamster DNA in EAV DNA
20
b0 0
-1-o
l-2
3-4 HOURS
5-6
POST INFECTION
FIG. 5. The time course of EAV and hamster DNA syntheses. Composite of data obtained from MAK column chromatography experiments of DNA extracted from liver cells of normal and EAVinfected hamsters after a 1 hr pulse of 3H-TdR.
HERPESVIRUS
REPLICATION
791
in viva Heat -Denatured
DNA l -*
,
o-
105
100,
EAV
DNA
I
I2
-
3
4 Notive
/ 4
ttomster
8
I2 16 20 24 26 32 36 40 44 46
5
I2
3
4
DNA
.-•
Hamster
o-0 .-.
E AV lnfactad
Hamster
'052
FRACTION
FIG. 6. MAK column chromatography of normal hamster DNA and EAV DNA. MAK columns were loaded with W-labeled DNA (about 70 pg) extracted from pellets of pure EAV and other columns were loaded with aH-labeled DNA extracted from normal hamster liver cells. DNAs were added to the column in 0.05 M PBS at a concentration of 10 pg/ml. The material was eluted with a salt gradient formed by adding 0.9 M NaCl to a mixing chamber containing 100 ml of 0.5 M NaCl at a rate of 0.3 ml/min. The gradient was pumped through the column at this same rate and fractions were automatically collected every 10 min. The acid-insoluble portion of each fraction was measured by liquid scintillation counting and the NaCl concentration in various fractions was quantitated by the refractive index and according to the formula of Mandell and Hershey (1960). The horizontal bar (I---() in the figure denotes the elution position of EAV DNA when extracted from infected liver cells.
molecular weight (Soehner et al., 1965) is considered to be the EAV genome. The bulk (93 %) of the EAV DNA extracted from L-M cells eluted at a low salt molarity (0.43-0.45 Xl NaCl) and was shown to be highly fragmented EAV DEA molecules. Since the elution of EAV DNA from MAK columns at 0.60-0.61 M NaCl had not been observed, the properties of these tissue-extracted molecules as compared to the virionextracted molecules were determined. The density in CsCl of both DISA molecules as noted earlier (see Fig. 4) is very similar, indicating that the infected cell DNA is essentially a double-stranded molecule like the virion DNA. To confirm this point further, the ability was measured of both classes of
Fmction of 2 X SSC
Fmrthn of O.IN NoOH
7. Chromatography of EAV DNA and hamster DNA on nitrocellulose columns. DNA prepared from normal hamster liver, pure EAV particles, and EAV-infected liver cells (56 hr P.I.) was suspended in 2 X SSC at concentrations of approximately 20 pgg/ml and added separately to tightly packed nitrocellulose columns (Nitrocel S). Similar preparations of DNA were heated at 100°C in 1 X SSC, cooled slowly, adjusted to 2 X SSC, and added to similar columns. The columns were washed slowly (0.3 ml/min) with 2 X WC and five fractions (7 ml each) were collected. The columns were than washed with 0.1 N NaOH and four fractions (7 ml each) were collected. The acid-insoluble portion of each fraction was counted by liquid scintillation counting. FIG.
EAV DNA, native and heat-denatured, to bind to nitrocellulose as shown in Fig. 7. Native DNA, hamster DNA, and infected DNA all pass through nitrocellulose columns without any significant binding, indicating that they all are double-stranded. Once heat-denatured, even if followed by slow cooling, the DNAs were bound tightly to the nitrocellulose. Thus, there were no apparent differences in the extent of singlestranded regions and crosslinked regions between liver cell-extracted and virion-extracted EAV-DNAs. DISCUSSION
The in viva replication of EAV in hamster liver as described in the present study is comparable to that of EAV in L-M cells as described by O’Callaghan et al. (1968b). In
792
O’CALLAGHAN.
RANDALL,
both systems the virus replicates in the nucleus of one cell type, intranuclear inclusions are formed early in the growth cycle, viral DNA synthesis occurs coincident with the inhibition or in the absence of host DNA synthesis, and infectious virus is detectable within the cell and is released into the medium shortly after assembly. Thus, a growth curve with a short latent period followed by a sharp increase in virus titer is common to both. The duplication of such growth-curve properties in an animal system is possible only when all the liver cells become infected at the same time. This synchrony is reflected likewise in the development of pathology in the liver and the nearly simultaneous death of the majority of infected animals. This ideal situation is dependent upon a rapid initiation of the infection and is achieved only when the amount of virus injected sufficiently saturates the susceptible cells. In fact, when smaller inocula are injected the resulting infections allow the animals to live far beyond 12-14 hr and the time of death varies among the population of infected animals. In the routine passage of virus, infectious virus was detectable in the cell-free state (in plasma) about 1 hr after initial increase in the level of infectious virus in liver. More detailed experiments indicated that infectious radioactive virus was released to plasma after 90 min from the time of the injection of isotope (see Table 1). These data reveal that some newly synthesized virus is capable of release from infected cells without prolonged exposure to the int,racellular environment, and they imply that a close temporal relationship exists between the synthesis of DNA and the encapsidation of DNA into infectious particles. A similar relationship was suggested previously (O’Callaghan et al., 1968) on the basis of the high percentage of intranuclear DNA which was resistant to extensive DNase digestion. This observation may be important in explaining the replication of EAV in L-M cultures contaminated with iMycoplasma hominis. Stock and Gentry (1969) have shown that Al. hominis grown in L-M cell cultures secretes a DNase capable of digesting intranuclear L-M cell DNA and, although the enzyme
AND
GENTRY
appclars ablr to digest chxtractcd EAV DSA molcculcs, it is quite capable of replicating in the nucleus of L->I cells contlaminatcd with M. hominis (Gentry and Evans, 1972). On the basis of these considerations, it is feasible to suggest that the process of the encapsidation of EAV DNA is rapid and is thus involved in protecting the viral genome. The minimum time required for encapsidation therefore appears to be somewhat less than the 2-3 hr reported by Olshcvsky et al. (1967) and Roizman et al. (1963) for herpes simplex virus. Although increased incorporation of tritiated thymidine has been previously associated with EAV infection of hamster liver (Gentry et al. 1962), it remained for the present study to establish t’hat a net increase in DNA synthesis occurs and that the newly syrlthesized DNA is predominantly viral. This last point is important for it proves that EAV DNA in liver replicates exclusively independent’ of host DSA synthesis. This finding clearly distinguishes the replication in liver from the unusual replication cycle of EAV recently report’ed in human KB cells by Lawrence (1971). Lawrence found that in synchronized cultures of KS cells EAV DiYA synthesis is initiated only when the host cell is in S phase. Once viral replication began, further rounds of host synthesis were inhibited. This dependency of EAV DNA synthesis upon KB cell synthesis was confusing for no such dependency has been previously reported for the well-studied herpes simplex and pseudorabies viruses (Kaplan, 1969)) and the only available studies on the biochemistry of EAV replication were performed on infected log phase L-M cell cultures (O’Callaghan et al., 1969b). These studies on log phase L-M cells clearly defined the kinetics of EAV-induced inhibition of L-M cell DNA synthesis shortly after infection, but no attempt was made to determine the relationship between viral DNA synthesis and the stage of cell cycle. Since no direct experimental evidence is yet available, the demonstration of EAV DNA synthesis in infected liver without any significant host DNA synthesis is clearly the best evidence of the replication of EAV independent of host DNA synthesis.
HERPESVIRUS
REPLICATION
ACKNOWLEDGMENTS This study was supported by USPHS Research Career Development Award 5K3 AI7021 (G.A.G.), Training Grant AI-69, and Research Grants AI-92632 and A1-09858. REFERENCES ADAMS, W., and FINK, K. (1966). Paper chromato-
graphic data for purines, pyrimidines, and derivatives in a variety of solvents. J. Chromatogf. 22, 118-129. ARHELGER, R., DARLINOTON, R. W., and RANDALL, C. C. (1963). An electron microscopic study of equine abortion virus infection in hamster liver. Amer. J. Pathol. 42, 703-713. BURTON, K. (1956). The conditions and mechanism of diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62,315323. DARLINGTON, R. W., and RANDALL, C. C. (1963). The nucleic acid content of equine abortion virus. Virology 19, 322-327. DOLL, E. R., RICHARDS, M., and WALLACE, M. (1953). Adaptation of the equine abortion virus to suckling Syrian hamsters. Cornell Vet. 43, 551-558. GENTRY, G. A., and EVANS, S. F. (1972). Herpesvirus replication resistant to heterologous DNase. Abstr. Annu. Meet. Amer. Sot. Microbial. p. 222. GENTRY, G. A., MORSE, P. A., JR., and DORSETT, M. T. (1971). In viva inhibition of pyrimidine catabolism by 5cyanouracil. Cancer Res. 31, 909-912. GENTRY, G. A., WALKER, B., and RANDALL, C. C. (1962). Correlated autoradiographic and biochemical study of DNA labeling in equine abortion virus hepatitis. Virology 16, 466-465. KAPLAN, A. S. (1969). Herpes simplex and pseudorabies viruses. Virol. Monogr., No. 5. SpringerVerlag, New York. pp. 44-47. KLAMIXRTH, 0. (1965). Separation of high molecular weight deoxyribonucleic acid and ribonucleic acid. Nature London 208,1318-1319. L.IWRENCE, W. (1971). Evidence for a relationship between equine abortion (herpes) virus deoxyribonucleic acid synthesis and the S phase of the KB cell mitotic cycle. J. Viral. ‘7.736-748. MANDELL, J., and HERSHEY, A. (1960). A fractionating column for analysis of nucleic acids. Anal. Biochem. 1, 66-77.
in uivo
793
D., CHEEVERS, W., and RANDALL, C. C. (1968a). Kinetics of cellular and viral DNA synthesis in equine abortion (herpes) virus infection of L-M cells. Virology 36, 104-114. O’CALLAGHAN, D., HYDE, J., GENTRY, G. A., and RANDALL, C. C. (1968b). Kinetics of viral deoxyribonucleic acid, protein, and infectious particle production and alterations in host macromolecular synthesis in equine abortion (herpes) virus-infected cells. J. Viral. 2,793-812. O’CALLAGHAN, R., JACKSON, L., RSNDALL, C. C., and GENTRY, G. A. (1968). Studies on the coating of equine herpes virus. Bacterial. Proc. 167-168. PLUMMER, G. (1964). Serological comparison of the herpes viruses. Brit. J. Ezp. Pathol. 45,125141. RANDALL, C. C., and BRACKI~N, E. C. (1957). Studies on hepatitis in hamsters infected with equine abortion virus. I. Sequential development of inclusions and the growth cycle. Amer. O’CALLAGHAN,
J. Pathol. 33,709-726. REED, L. J., and MUENCH,
H. (1938). A simple method of estimating fifty per cent endpoints. Amer. J. Hyg. 27,493-497. ROIZMAN, B., AURELIAN, L., and ROANE, P. R., JR. (1963). The multiplication of herpes simplex virus. I. The programming of viral DNA dnplication in HEp-2 cells. Virology 21,482-498. SOEHNER,R., GENTRY, G. A., and RANDALL, C. C. (1965). Some physico-chemical characteristics of equine abortion virus nucleic acid. Virology 26, 39445. STOCK, D., and GENTRY, G. A. (1969). Mycoplasma1 deoxyribonuclease activity in virus-infected L-cell cultures. J. Viral. 3, 313-317. SUEOKA, N., and CHENG, T. (1967). Fractionation of DNA on methylated albumin columns, In “Methods in Enzymology” S. Colowick and N. Kaplan (eds.), Vol. XII, pp 56‘2-566. Academic Press, New York. SUEOKA, N., MARMUR, J., and DOTY, P. (1959). Heterogeneity in deoxyribonucleic acids. II. Dependence of the density of deoxyribonucleic acids on guanine-cytosine content. Nature London 183,1429-1431. TAKAHASHI, M., O~INO, T., BABA, K., and ONAKA, M. (1969). Synthesis of deoxyribonucleic acid in human and hamster kidney cells infected with human adenovirus types 5 and 12. Virology 37, 513-520.