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Replicative events in hepatitis A virus-infected MRC-5 cells
VIROLOGY
157,268-275
(1987)
Replicative
Events in Hepatitis
A Virus-infected
JOSE DE CHASTONAY Institute
for Hygiene
and Medical
Microbiology,
Received
September
AND
University 3, 1986;
GUNTER
of Bern, accepted
SIEGL’
Friedbilhlstrasse November
MRC-5 Cells
5 1, Bern
CH-30
10, Switzerland
17, 1986
Replication of hepatitis A virus (HAV) in MRC-5 cells was studied under one-step growth conditions. Viral replication neither interfered detectably with cellular DNA, RNA, and protein synthesis, nor could cytopathologic changes be recorded over a prolonged period of incubation. Synthesis of mature, infectious HAV particles could be detected as early as 2-4 days p.i. and occurred at a maximal rate around 8 days p.i., shortly before infectivity titers reached a plateau. Synthesis of total viral RNA, of positive-strand genomic RNA, of viral mRNA, as well as of negative-strand RNA followed the same pattern. By Day 14 p.i., when active HAV replication had developed into persistent infection, synthesis of viral RNA declined to background levels. The mechanism(s) guiding active HAV replication into a state of persistent infection could not be positively defined. Yet there exists the possibility that this is brought about by down regulation of viral RNA synthesis. Whether this is related to the appearance of a subgenomic viral RNA molecule about 2000 nucleotides in length and detected in association with ribosomes on Days 7 and 10 p.i. remains to be shown. 0 1997 Academic
Press,
Inc.
INTRODUCTION
processes of HAV. To do so, conditions must be elaborated to meet the requirements for a one-step growth curve. In a previous report (Siegl et a/., 1984b), it was shown that under such conditions, the HAV/cell relationship could be divided into three distinct phases: a short phase, characterized by an increase in titer, a second phase, in which pronounced synthesis of HAAg is detected although no further increase in infectivity is recorded, and from then on, a persistent type of infection, with but minor viral metabolic activity. Recently, similar results were reported by Wheeler and co-workers (1986) using another HAV strain as well as other cells. All results reported so far, however, yield no clue as to what mechanisms govern the establishment of and the switches between individual phases. As a first experimental approach to elucidate the functions operative in the development of the peculiar HAV/cell relationship we now report on the time course of the basic virus-specific synthetic events in infected cells. We provide evidence that, even under one-step growth conditions, replication of HAV in the human diploid fibroblast cell line MRC-5 is highly protracted. Moreover, the three phases revealed in previous studies have a counterpart at the level of viral RNA synthetic events.
Hepatitis A virus (HAV) has recently been classified within the genus Enferovirus of the family Picornaviridae (Melnick, 1982; Gust et a/., 1983). This classification, however, should not obscure the fact that many differences between HAV and enteroviruses do exist. For instance, it has not yet been possible to demonstrate primary replication of the virus in the gut of an infected host organism (Mathiesen et a/., 1978; Krawczynski et a/., 1981). HAV also exceeds the heat stability of poliovirus, a representative member of the enteroviruses, by a full 20” (Siegl et a/., 1984a). Furthermore, comparison of sequences of cloned HAV cDNA with sequence data for the genomes of picornaviruses revealed greatest homology with encephalomyocarditis virus of the genus Cardiovirus (Baroudy et al., 1985). This is in clear contrast to the extent of sequence conservation known to exist among the enteroviruses (Toyoda et al., 1984; Stalhandske et a/., 1984; Tracy et al., 1985; Kandolf and Hofschneider, 1985). Finally, HAV is distinguished from enteroviruses by its protracted replication in a vast majority of cells under in vitro conditions. This replication usually terminates in persistent rather than in acute Iytic infection (Provost and Hilleman, 1979; Frosner et a/., 1979; Gauss-MOller and Deinhardt, 1984; Siegl et al., 1984b; Bradley et al., 1984; Wang et a/., 1985; Friedman-Alvermann et a/., 1985). For both classification and practical purposes it appears to be essential to characterize the replicative ’ To whom 0042-6822/87
requests
for reprints
$3.00
Copyright 0 1907 by Academic Press. Inc. All rights of reproduction an any form reserved.
should
MATERIAL
AND
METHODS
Virus HAV strain GBM was originally isolated in MRC-5 cell cultures from fecal specimens collected during an outbreak of hepatitis A in Gormaringen, Federal Republic of Germany (Frosner et al., 1977). It was adapted
be addressed. 268
HEPATITIS
A VIRUS
REPLICATION
to replication at 32” and used to produce a virus stock in its fourth in vitro passage with an infectivity titer of 1 06.’ TCIDJml. Cells, conditions of cultivation, and mode of infection MRC-5 (human diploid embryonic lung) cells were cultivated in minimum essential medium (MEM), Eagle’s modified, with Earle’s salts supplemented with 1% nonessential amino acids, 2 m/l/l L-glutamine, and 25 mM HEPES buffer. For cultivation in closed containers, the pH of the culture medium was adjusted to 7.4-7.6; for incubation in the presence of 5% CO,, the pH was raised to 8.0. Fetal calf serum (FCS) was added to a final concentration of 10% (growth medium) or 5% (maintenance medium). Cultivation systems used in the outlined experiments were 25-cm* plastic cultivation flasks, petri dishes, as well as 24-well culture plates (Falcon) containing 10.5 X 10.5 mm coverslips. Cells were seeded into the culture units at a density allowing for the formation of intact monolayers within 4 to 5 days. Medium was changed weekly. Only cells grown to confluence were infected. For all kinetic studies, cells were infected at a multiplicity of 6-l 0. The inoculum was appropriately diluted in MEM without serum and adsorption was allowed to take place for 4 hr at 32”. Thereafter, it was replaced by maintenance medium containing 5% heat inactivated (56”, 30 min) FCS. Detection
of viral antigen
Hepatitis A antigen (HAAg) was detected and quantified in cellular extracts as described by Frdsner et al. (1977). Alternatively, HAAg was demonstrated in coverslip cultures after fixation with acetone (in situ RIA) (Siegl et al., 1984b). For the latter purpose, 0.1 ml of PBS and 1.5 ml of cold acetone (-20”) was added per coverslip and well. After 15 min at room temperature, the overlay was aspirated and the coverslips were airdried. Appropriately diluted ‘251-anti HAV in PBS and 0.1% bovine serum albumin was added and coverslips were incubated at room temperature over night. Following washing with distilled H20, bound radioactivity per individual coverslip was measured in a gamma counter (Packard). Titration
of HAV infectivity
Titration of HAV infectivity was based on the in situ RIA technique (Siegl et a/., 1984b). Briefly, MRC-5 monolayers were grown to confluence in multiwell dishes containing coverslips. Four wells were used per individual 1 O-fold dilution of the virus suspension and applied to the cells as a 0.5-ml inoculum. After adsorption, 1 ml of medium with 7.59/o heat-inactivated
IN MRC-5
CELLS
269
FCS was added to the wells. Maintenance medium was changed weekly. An in situ RIA was carried out on acetone-fixed coverslip cultures after 21 days of infection, and titers were calculated according to the method of Reed and Muench (1938). Precipitation
of radiolabeled
products
(a) Trichloroacetic acid (TCA) precipitation of cellular extracts. Aliquots of cellular extracts were diluted five times with PBS. An equal volume of 10% TCA was added and the tubes were kept on ice for 1 hr before precipitates were collected on Whatman microfiber filters (GF/B) equilibrated in 5% TCA. Filters were washed twice with ice-cold 5% TCA, rinsed with acetone, and dried at 50”. Bound radioactivity was measured in a Packard scintillation spectrometer. (b) TCA precipitation on coverslip cultures. An in situ TCA precipitation was carried out directly on coverslip cultures in multiwell dishes. At the time of harvest, coverslip cultures were washed twice with ice-cold 5%~ TCA in 0.2 R/I Na,P,O,, followed by incubation in the presence of 59/oTCA at 4” for at least 2 hr. Thereafter, wells were washed twice with 59’0 TCA and individual coverslips were dried and put into scintillation vials. The cellular material was dissolved by addition of 0.5 ml Soluene (Packard) at room temperature for 30 min. Incorporated radioactivity was measured in a scintillation counter. Isolation
and purification
of virus particles
At harvest, infected cell monolayers in 25-cm* plastic bottles were washed twice with PBS and then frozen in 5 ml PBS. Extracts were prepared by repeated (3X) freezing and thawing. After centrifugation at 3000 rpm for 20 min, RNase and DNase were added at a concentration of 50 pg/ml to the supernate and digestion was allowed to take place at 37” for 50 min. Virus was pelleted in a Beckman SW 50.1 rotor at 43,000 rpm for 160 min. The pellet was resuspended in 0.5 ml of 50 ml\/lTris-50 mlLl NaCI, pH 7.5 (TN buffer), 0.1% sodium laurylsarkosine (SLS), and layered onto a CsCl/sucrose step gradient consisting of a l-ml cushion of CsCl p = 1.5 g/ml, 4 ml CsCl p = 1.35 g/ml, and 4 ml of 30% (w/v) sucrose in an 1 l-ml polyallomer (SW41) tube. Centrifugation was carried out at 32,000 rpm at 15” for 16 hr. Thirty fractions of 0.3 ml were collected from the bottom of the tube. The CsCl and sucrose solutions used to make the gradients were made up in TN buffer containing 0.1 o/o SLS. Synthesis of viral positivenegative-strand RNA
and
RNA from two petri dishes (5 cm diameter) infected at m.o.i. 10 was harvested every other day by hot phe-
270
DE CHASTONAY
nol extraction (Scherrer, 1969). Total RNA was then precipitated with ethanol and resuspended in 100 ~1 of doubly distilled H20. Slot blots were made with 2% of the total RNA by adding an equal volume of formamide, heating to 65” for 10 min, diluting the sample with 50 vol of 5 M NaCI, and subsequently applying the mix to a GeneScreen-Plus membrane (New England Nuclear) by mild suction. Hybridization was carried out using SP6 polymerase generated 32P-labeled HAV RNA of either positive or negative polarity. For this purpose, HAV cDNA provided by Dr. J. Ticehurst, NIH Bethesda, MD (Ticehurst et a/., 1983) was recloned into the pGEM-2 vector. We used a 0.7-kb fragment representative for the 5’ end of HAV RNA (0.2 map units to 0.9 map units). Previous hybridization experiments with genomic RNA of a series of HAV strains adapted to cell culture indicated that this region of the HAV genome, as well as the region between 4.6 and 5.5 map units, contains highly conserved sequences among hepatitis A viruses (de Chastonay, Tracy, and Siegl, unpublished observation). Hybridization was carried out in the presence of formamide as recommended by New England Nuclear (NEN). For washing, however, SDS was added to a concentration of 2% and the temperature was raised to 70”. Isolation
of viral messenger
RNA
MRC-5 cells were grown to confluence in petri dishes (9 cm diameter) and infected at m.o.i. 10. Cellular and viral RNA in every dish was labeled by addition of 50 &i/ml of [3H]uridine for 24 hr prior to the harvest of the cells. At each point in time, each of four infected and two mock-infected cultures was washed twice with 5 ml PBS-A and 5 ml of PBS-A containing 100 pg/ml cycloheximide was added for 5 min at room temperature. The supernatant was then replaced by 4 ml of lysing buffer (0.1 mM Tris-HCI, pH 7.4, 0.1 mM KCI, 0.015 mM MgC12) supplemented with 50 mM NaCl and 100 pg/ml of cycloheximide. Cells were scraped off with a rubber policeman and pelleted (100 g, 5 min). The pellet was resuspended in 0.6 ml of lysing buffer, containing 100 pg/ml cycloheximide and 100 pg/ml DNase (RNase free, BRL), and incubated for 1 min at room temperature. To this, 0.6 ml lysing buffer containing 2% Triton X-l 00, 12 mM 2-mercaptoethanol, 100 pglml dextran sulfate, 200 pg/ml cycloheximide, and 80 pg/ml polyvinyl sulfate was added. The solution was supplemented with 0.3 ml of 20% Tween X-100, vortexed for 10 set, and then 0.36 ml of 4 M KCI, 40 mM MgC12 was added before the mix was passed five times through a 0.9 x 40-mm-gauge needle. After clarification (500 g, 4 min) the supernatant was layered onto a linear 40-109/o (w/v) sucrose gradient formed
AND
SIEGL
on top of a 0.5-ml 60% (w/v) sucrose cushion. The sucrose solutions were buffered in 0.1 MTris, pH 7.4, 5 mM MgCI,, and 0.5 M KCI. Centrifugation was carried out in a Beckman SW 27.1 rotor at 25,000 rpm for 18,500 W2t at 4’. Thirty fractions of 15 drops each were collected from the bottom of the tube. Of each fraction, 10 ~1 was assayed for TCA insoluble radioactivity. Intact, [14C]uridine-labeled poliovirus sedimenting at 160 S, [3H]thymidine-labeled Lu-Ill parvovirus sedimenting at 1 10 S, and Lu-Ill parvovirus capsids labeled with 3Hlabeled amino acids and sedimenting at 70 S served as markers. RNA aliquots were blotted directly onto GeneScreenPlus membranes. For this purpose, the RNA was suspended into 7.5% formaldehyde in 50 mM phosphate buffer, pH 7.0, heated to 60” for 15 min, and then blotted onto filters, preequilibrated with 1 OX SSC (1.5 M sodium chloride-O. 15 M sodium citrate). The membrane was then air-dried and baked for 2 hr at 80”. Electrophoresis
of RNA
RNA was denatured using deionized glyoxal/dimethylsulfoxide (DMSO) as described by Thomas (1980). Briefly, 2.7 PI of glyoxal, 8 ~1 of DMSO, and 5 ~1 of RNA in 100 mM phosphate buffer, pH 7.0, were incubated for 1 hr at 50”. Then, 4 ~1 of a solution of 0.25% bromphenol blue and 2 mM EDTA in 50% glycerol were added. Samples of 20 ~1 were loaded onto a 1% agarose gel (Sigma, Type 2) in 10 mM phosphate buffer, pH 7.0, and electrophoresis was carried out in a submarine, horizontal system at 60 V under constant buffer recirculation. The bromphenol blue marker was allowed to run for 6.5 cm. Using the protocol provided by NEN, RNA was transferred to GeneScreen-Plus filters and hybridization of dot blots and Northern transfers of encapsidated and ribosome-associated viral RNA were carried out without formamide as suggested by NEN, using 32P-nick translated HAV cDNA mapping in the 5’ region of the viral genome. The respective clone was kindly supplied by Dr. Klaus von der Helm, University of Munich, Federal Republic of Germany (von der Helm et a/., 1981). RESULTS Overall synthetic cell system
activity
in the HAV/MRC-5
An efficient interpretation of experimental data concerning replication of HAV over a period of several weeks is only possible if parameters such as cell numbers, synthesis of cellular DNA, RNA, and protein are easily controlled or remain largely constant. Our studies showed that in both infected and uninfected confluent
HEPATITIS
A VIRUS
REPLICATION
MRC-5 cultures, the cell number per square centimeter remained unchanged over a 40-day follow-up period. Absence of cell proliferation in such cultures was also indicated by the failure to incorporate significant quantities of [14C]thymidine into cellular DNA during labeling for 24 hr at various points in time. Between cell seeding and the establishment of a confluent monolayer, however, [14C]thymidine could be incorporated into the genome of MRC-5 cells to serve as a measure for cell number in subsequent experiments. On the basis of this standardization procedure it could be shown that HAV infection did not result in cell death over a period of at least 14 days. Likewise, incorporation of [3H]uridine (representative for total RNA synthesis) as well as of [3H]valine and [3H]leucine (representative for total protein synthesis) over multiple 24-hr periods reached practically identical values in both infected and uninfected cultures (results not shown). Synthesis and encapsidation of viral RNA The time course of synthesis and encapsidation of viral genomic RNA was followed by pulse-labeling experiments over a period of 16 days. Duplicate cultures in 25-cm2 flasks were infected at m.o.i. 6 and [3H]uridine (250 &i/flask) was added for 3-day intervals. After each interval, cell-associated virus was harvested and purified by gradient centrifugation as described under Material and Methods. In preliminary experiments, marker poliovirus and HAV were shown to accumulate in fractions 7 to 11, with peaks of HAV in
IN MRC-5
CELLS
271
fraction 8 and poliovirus in fraction 7, in accordance with previous observations (Siegl et al., 1981) of a slightly lower buoyant density for HAV (1.336 vs 1.34 g/ml). The amount of radioactivity accumulating in fraction 7 to 1 1 was considered as providing a measure for the amount of viral genomic RNA synthesized and encapsidated during the respective period. A steady increase in radioactivity was detected during the first three intervals, lasting to Day 10, after which a rapid decline of [3H]uridine-labeled particles could be recorded (Fig. 1). During Days 13-l 6, distribution of radioactivity in the gradient followed the pattern of mockinfected cultures. In Fig. 2, virus production is quantitated for each interval by presenting the integrated values of gradient fraction 7-l 1 of each labeling period. The infectivity titers as well as synthesis of total HAAg for this specific experiment were determined and also included. It can be concluded from these data, that encapsidation of newly synthesized viral RNA is limited to the phase of viral replication characterized by an active increase in infectivity titers. The plateau of infectivity present after Day 13 is therefore not maintained by a detectable active equilibrium between virus production and degradation. Much more, it appears that mature infectious virus particles remain stably cell associated. Synthesis of total viral RNA Encapsidation of viral RNA as illustrated in Fig. 2 may either cease due to a defect in morphogenic events or simply because synthesis of viral RNA stops. In order to elucidate the possible role of viral RNA synthesis as a rate-limiting step, synthesis of total viral RNA was followed over a period of 2 weeks. For this purpose, 8 pg/ml actinomycin D was added to duplicate infected and mock-infected cultures in multiwell dishes for 24 hr prior to harvest. One hour later, [3H]uridine (10 &i/ ml) was added and incubation continued for 23 hr. The quantity of labeled precursor incorporated per culture was determined by in situ TCA precipitation. The overall pattern of synthesis of total viral RNA (Fig. 3) coincides with the pattern of production of mature virus as illustrated in Fig. 2. The temporary decrease in viral RNA synthesis on Day 4 cannot yet be explained. Synthesis of negative- and positive-strand viral RNA
0
4
6
12
16
20
Fractions FIG. 1. Synthesis and encapsidation of viral genomic RNA in infected MRC-5 cells. HAV particles were labeled with [3H]uridlne over 3-day intervals, isolated as described, and centrifuged to equilibrium in &Cl-sucrose gradients. 0. Days 4-7 p.i.; 0, Days 7-10 p.i.; W, Days 1O-1 3 p.i.; . . . , control.
Synthesis of positive-strand genomic viral RNA can be assumed to proceed via a negative-strand intermediate. Accumulation of this type of molecule was used as an additional means to determine active replication of HAV RNA. As indicated by the blot hybridization assay with a positive-strand RNA probe, nega-
272
DE CHASTONAY
AND
SIEGL
14
r7
-14
12 2 cx I~~
- 12
loq ‘c
8-
SLO x ‘2 $ ,o ‘Z Ob E-8 C .
6-
P 0
4-
B .
-6g
T - lOr*! ? 8_ -8
z=
x
2-
-5
6E -4
& I z
-2; -0
o1
4 Days
7 post
10 infection
13
16
FIG. 2. Encapsidation of newly synthesized genomic vRNA (0) accumulation of infectious HAV (m), and rate of HAAg synthesis (A) in MRC5 cells infected as described. The integral amount of radioactivity accumulating in fractions 7-l 1 in the CsCI-sucrose gradient shown in Fig. 1 was taken as a measure for the amount of virus produced during the respective interval. Infectious virus was assayed by in situ RIA and HAAg by sandwich RIA. Accumulation of infectious HAV is plotted on a logarithmic scale; the other parameters are indicated in a linear scale.
tive-strand RNA could be recorded clearly as of Day 4 p.i. (Fig. 4). Its presence peaked on Day 8 p.i. and decreased to background values between Days 10 and 14, suggesting relatively rapid degradation of such viral RNA in the course of infection. By a similar experimental approach, accumulation of positive-strand RNA was also observed as of Day 4 p.i.; yet, after reaching maximum concentrations on Day 8 p.i., it remained associated with the cells in significant concentrations, seemingly reflecting its presence in mature progeny virus particles.
Presence
of viral mRNA
Some viral antigen synthesis is still recorded after cessation of viral RNA synthesis (Fig. 2). Therefore, viral messenger RNA may be assumed to have a prolonged half-life time. To investigate this aspect of HAV replication, we devised a method for isolation of RNA from MRC-5 cells which allowed us to distinguish encapsidated viral RNA from viral ribosome-associated RNA. For this purpose, polysomes were isolated and, in the course of extraction, disrupted into monosomes. Ultracentrifugation through a linear 40-l 0% sucrose gradient then allowed for separation of monosome-associated HAV RNA from mature HAV particles. Figure 5 shows the profile of the radiolabeled RNA in such a gradient. Almost all TCA precipitable radioactivity is days C
pos. viral
Days FIG. 3. Total
post
2
post 4
infection 6
strand RNA
neg.
strand
viral
RNA
c =
uninfected
8
10
14
+ I
-
m*,llciwmrr
CI r*c i~mvrrub-r
r)
I*
II
infection
synthesis of viral RNA per 24 hr in MRC-5 cells infected as described. RNA was labeled with [3H]uridine in the presence of actinomycin D. Viral RNA synthesis was estimated by subtracting TCA insoluble radioactivity incorporated in mock-infected cultures from that incorporated in infected cultures.
cells
FIG. 4. Presence of positive (upper)and negative (lower)-strand vRNA in cell extracts harvested at different times p.i. Both forms of RNA were detected by hybridization with radioactive RNA of complementary polarity as described.
HEPATITIS
+HA”
day
4
+HAV
day
7
day
13
+HAV
160s
REPLICATION
Pools
-
r-l-
A VIRUS
110s
70s
20s
I
I
4
g” 0 -2 X
E CL 1 0 0 5
10
15
20
25
Fractions FIG. 5. Association of vRNA with ribosomes. The cytoplasmic extracts of infected cells were harvested 4, 7, and 13 days p.i. The monosome profile obtained upon sedimentation in 1 O-40% sucrose gradients is shown as TCA-insoluble [3H]uridine-labeled material. Fractions were pooled as indicated, half of the material was blotted, and viral RNA sequences were detected by hybridization with HAVcDNA. Equal amounts of an infected (+HAV) and mock-infected (-HAV) culture was blotted at each harvesting point.
present in fractions 12-l 5. Judged by the positions of 160 S poliovirus and of 1 10 and 70 S parvovirus Lu-Ill particles in such gradients, cellular RNA sediments at 80 S, as expected for monosomes.
160 4
Polio
7
CELLS
ri bosome associated co
273
Blot hybridization analysis of the different pools derived from the gradients of harvests carried out at different times p.i. yielded a positive HAV signal in the 160 S pools as of Day 4 p.i. The intensity of the signal rose especially from Day 4 to 7 p.i. HAV RNA could be detected in the 80 S pools of Days 4 and 7 and structures containing HAV-specific RNA sequences were also present in the bottom fractions of the gradients harvested on Day 13 p.i. All other pools derived from HAV-infected cells could not be shown to contain HAVspecific RNA. This also holds true for those pools made from material sedimenting at 70 S and less and therefore expected to contain HAV replicative intermediate RNA molecules. This suggests a loss of such RNA molecules in the course of that particular extraction procedure. Gel electrophoresis and Northern blotting (Fig. 6) detected genome-sized HAV RNA in samples derived from the 160 S gradient pools on Days 7, 10, and 13. Genome-sized HAV RNA was also found in association with ribosomes on Day 10. In addition, the latter pools derived from harvests of Days 7 and 10 contained an apparently HAV-specific, subgenomic RNA molecule of about 2000 bases. Attempts to further characterize this uncommon molecule (e.g., with respect to the kinetics of its appearance after HAV infection or by hybridization with cDNA probes derived from genome regions other than the 5’terminal region), failed repeatedly due to the exceptionally low concentration of virus-specific RNA extractable from HAV-infected MRC-5 cells with that particular method. Additional experiments, however, are in progress.
S 10
IN MRC-5
4
7
10
pools co
days
-
p. i
-
HAV
-
23s
-
16s
-a rRNA
-b
rRNA
-
FIG. 6. Electrophoretic analysis of RNA associated with the 160 S pool and the ribosome pool on Days 4, 7, and 10 p.i. The mock-infected culture (Co) was harvested on Day 10. After electrophoresis, the RNA was transferred to Gene Screen-Plus filters and HAV specific sequences were detected by hybridization as described. On the left-hand side, the positions of poliovirus and of Escherichia co/i rRNAs are indicated. The right-hand side points to the migration position of genome sized HAV RNA and gives the sedimentation values of the rRNAs. Arrow “a” indicates the position of RNA sequences detected in the ribosome pool of both infected and mock-infected cells. Arrow “b” points to the position of a subgenomic vRNA molecule apparently only present in material from infected cultures.
274
DE CHASTONAY
DISCUSSION The virus-specific molecular events underlying the three tentatively defined phases of HAV replication in MRC-5 cells have in part been elucidated. Most evidently, the synthesis of total viral RNA (i.e., negativestrand template RNA, genomic RNA, and viral mRNA), as well as synthesis and encapsidation of genomic RNA occur in parallel to the appearance and accumulation of progeny infectious virus. These events slow down rapidly (phase 2) and can hardly be detected in the course of persistent infection (phase 3). On the basis of these observations, the cycle of active HAV replication can be redefined as extending from the start of infection to the time major synthetic events (e.g., synthesis and encapsidation of vRNA) again became undetectable. Approximately 13 days elapse before this occurs. Such a time course contrasts sharply to the 7hr replication cycle described for poliovirus (for references, see Koch and Koch, 1985). In poliovirus-infected cells, termination of viral replication is identical with death and lysis of the infected host cell. This is initiated by shutoff of cellular metabolic activity relatively early in infection (2 hr). As indicated by the lack of impact of HAV replication on the MRC5 cell metabolism over a prolonged period of time, shutoff of cellular RNA and protein synthesis apparently plays no important role in regulation of the described HAV/cell relationship. At the present time, we can only speculate on the nature of factor(s) or event(s) which may be operative in controlling transition of the HAV/MRC-5 cell relationship from phase 1 to phase 2 and, finally, into the state of persistent infection. The block may be sought in replication of viral genomic RNA or in the production of viral mRNA, because both events appear to cease with the end of phase 1. Cessation of RNA synthesis may be brought about by a block in RNA translation and protein processing toward the end of phase 1. Such a block would not necessarily affect synthesis of viral structural proteins during phase 2 but might specifically concern translation of virus specific protease and RNA polymerase. In analogy to the organization of a standard picornavirus genome, these essential enzymes are coded for far downstream from the structural proteins and their translation might well be negatively affected by the outstandingly high proportion of secondary structure (1 O-20016) present in the HAV genome (Siegl eta/., 1981). This line of thought is supported by the existence of a translational barrier in the central region of the encephalomyocarditis (EMC) virus genome (Svitkin and Agol, 1983) and probably also due to secondary structure in this region. Such a translational barrier could also be expected to lead to an over-
AND
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production of capsid proteins as has indeed been observed in the late stage of mengovirus infection (LucasLennard, 1974; Paucha et a/., 1974). Another factor which may contribute to the efficient down regulation of HAV replication subsequent to phase 1 may be related to the occurrence of a subgenomic RNA about 2000 nucleotides in length closely related to the region of the HAV genome coding for the structural proteins. Subgenomic viral RNAs with similar characteristics have been found in cells infected with caliciviruses (Ehresmann and Schaffer, 1977) and cowpea mosaic virus (Franssen et al., 1984). The role of these molecules in virus replication, however, has not been resolved. In the rare instance where subgenomic RNA molecules have been detected in picornavirus systems (e.g., Domingo et al., 1985), the molecules resembled the highly deleted genomes of defective interfering (DI) particles which tend to lack the region coding for structural proteins (Huang, 1973). Due to the quantitative problems lined out under Results, we were so far not able to further characterize the subgenomic RNA in HAV-infected MRC-5 cells. Hence, we are not yet in the position to decide whether this RNA represents the genome of a distinct class of DI particles, plays a special, necessary role in HAV replication, or controls the development of the persistent type of HAV infection in MRC-5 cells. ACKNOWLEDGMENTS Thanks are due to Drs. K. von der Helm (Munich) and J. Ticehurst (Bethesda, MD) for kindly providing us with their HAV cDNA clones. We also thank J. Nijesch (Bern) for recloning such cDNA from the pBR 322 into the pGEM vector. This study was supported in part by Grant 3.813.83 from the Swiss National Science Foundation.
REFERENCES BAROUDY, B. M., TICEHURST, J. R., MIELE, T. A., MAIZEL, J. V., PURCELL, R. H., and FEINSTONE, S. M. (1985). Sequence analysis of hepatitis A virus cDNA coding for capsid proteins and RNA polymerase. Proc. Natl. Acad. Sci. USA 82, 2 143-2 147. BLACK, D., and BROWN, F. (1975/76). A major difference in the strategy of the calici- and picornavirus replication and its significance in classification. lntervirology 6, 57-60. DOMINGO, E., SOBRINO, F., MARTINEZ-SALES, E., DAVILA, M.. DELATORRE, J. C., VILLANUEVA, N., NEGRO, P., and ORTIN, I. (1985). “Genetic Heterogeneity of Foot-and-Mouth-Disease Virus.” Proceedings of the Fourth Meeting of the European Group of Mol. Biol. of Picornaviruses. F9. EHRESMANN, D.. and SCHAFFER, F. (1977). RNA synthesized in calicivirus infected cells is atypical of picornaviruses. J. Viral. 22, 572576. FRANSSEN, H., LEUNISSEN. J., GOLDBACH, R., LOMONOSSOFF, G., and ZIMMERN, D. (1984). Homologous sequences in non-structural proteins from cowpea mosaic virus and picornaviruses. fi%YO J. 3, 855861.
HEPATITIS
A VIRUS
REPLICATION
FRIEDMAN-ALVERMANN, B., MOLLER, D., and SPIES, K. (1985). Isolation and propagation of hepatitis A virus in hepatoma cell cultures. Acta Viral. 29, 482-486. FROSNER, G. G., DEINHARDT, F., SCHEID, R., GAUSS-MULLER, V., HOLMES, N., MESSELBERGER, V., SIEGL, G., and ALEXANDER, J. J. (1979). Propagation of human hepatitis A virus in a hepatoma cell line. lnfecfion 7,303-306. FR~SNER, G. G., OVERBY. L. R., FLEHMIG, B., GERTH, H. J., HAAS, H.. DECKER, R. H., LING, C. M., ZUCKERMAN, A. J., and FR~SNER. H. R. (1977). Seroepidemiological investigation of patients and family contacts in an epidemic of hepatitis A. J. Med. Viral. 1, 163-l 73. GAUSS-MOLLER, V., and DEINHARDT, F. (1984). Effect of hepatitis A virus infection on cell metabolism in vitro. Proc. Sot. fxp. Biol. Med. 175, 10-15. GUST, J., COULEPIS, A. G.. FEINSTONE, S. M.. LOCARNINI, S. A., MORITSUGU, Y., NAIERA, R., and SIEGL, G. (1983). Taxonomic classification of hepatitis A virus. Intervirology 20, l-7. HUANG, A. S. (1973). Defective interfering viruses. Annu. Rev. Microbiol. 27, 101-117. KANDOLF, R., and HOFSCHNEIDER, P. H. (1985). Molecular cloning of the genome of a cardiotropic coxsackie 83 virus: Full length reverse transcription recombinant cDNA generates infectious virus in mammalian cells. Proc. Nat/. Acad. Sci. USA 82, 4818-4822. KOCH, F., and KOCH, G. (1985). “The Molecular Biology of Poliovirus.” Springer-Verlag, Vienna/New York. KRAWCZYNSKI, K. K., BRADLEY, D. W., MURPHY, B. L., EBERT, J. W.. ANDERSON, T. E.. DOTO, I. L., NOWOS~AWSKY, A., DUERMEYER. W., and MAYNARD, J. E. (1981). Pathogenic aspects of hepatitis A virus infection in enterally inoculated marmosets. Amer. /. C/in. Pathol. 76, 698-706. LUCAS-LENARD, J. (1974). J. Viral. 14, 261-269.
Cleavage
of mengovirus
polyproteins
in vivo.
MATHIESEN, L. R., DRUCKER, J., LORENZ, D., WAGNER, J. A., GEREN, R. J., and PURCELL, R. H. (1978). Localization of hepatitis A antigen in marmoset organs during acute infection with hepatitis A virus. J. Infect. Dis. 138, 369-377. MELNICK. J. (1982). Classification of hepatitis A virus as enterovirus type 72 and of hepatitis B virus as hepadnavirus 1. Intervirology 18, 105-106. PAUCHA, E., SEEHAFER. J.. and COLTER, J. S. (1974). Synthesis of viralspecific polypeptides in mengovirus infected L cells: Evidence for the asymetric translation of the viral genome. Virology 61, 315326. PROVOST, P. J., and HILLEMAN, M. R. (1979). Propagation of human
IN MRC-5
CELLS
275
hepatitis A virus in cell culture in vitro. Proc. Sot. fxp. i3iol. Med. 160, 213-221. REED, L. J., and MUENCH, H. (1938). A simple method of estimating fifty percent endpoints. Amer. J. Hyg. 27, 493-497. SCHERRER, K. (1969). Isolation and sucrose gradient analysis of RNA. /n “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, Eds.), pp. 413-432. Academic Press, New York/London. SIEGL, G., FR~SNER, G. G., GAUSS-MULLER, V., TRATSCHIN, J. D., and DEINHARDT, F. (1981). The physicochemical properties of infectious hepatitis A virions. f. Gen. Viral. 57, 331-341. SIEGL, G., WEITZ. M., and KRONAUER, G. (1984a). Stability of hepatitis A virus. Intervirology 22, 218-226. SIEGL, G., DE CHASTONAY. J., and KRONAUER. G. (198413). Propagation and assay of hepatitis A virus in vitro. 1. Viral. Methods 9, 53-67. STALHANDSKE, P. 0. K., LiNDaERG. M., and PE~ERSSON, U. (1984). Replicase gene of coxsackie B3 virus. 1. Viral. 51, 742-746. SVITKIN, Y. V., and AGOL, V. I. (1983). Translation barrier in central region of encephalomyocarditis virus genome modulation by elongation factor 2 (eEF 2). fur. J. Biochem. 133, 145-154. THOMAS, P. S. (1980). Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Nat/. Acad. Sci. USA 77, 5201-5205. TICEHURST, J. R., RACANIELLO, V. R., BAROUDY, B. M.. BALTIMORE. D., PURCELL, R. H., and FEINSTONE, S. M. (1983). Molecular cloning and characterization of hepatitis A virus cDNA. Proc. Nat/. Acad. Sci. USA 80, 5885-5889. TOYODA, H.. KOHARA, M., KATOAKA, Y.. SUGANUMA, T., OMATA, T., IMURA, N., and NOMOTO, A. (1984). Complete nucleotide sequences of all three poliovirus serotype genomes: Implications for genetic relationship, gene function and antigenic determinants. /. Mol. Biol. 174, 561-585. TRACY, S., LIU, H. L., and CHAPMAN, N. M. (1985). Coxsackievirus 83: Primary structure of the 5’ noncoding and capsid protein coding regions of the genome. Virus Res. 3, 263-270. VON DER HELM, K., WINNACKER, E. L.. DEINHARDT. F., FROSNER, G., GAUSSMUELLER, V., BAYERL, B., SCHEID, R.. and SIEGL. G. (1981). Cloning of hepatitis A virus genome. 1. Viral. Methods 3, 37-42. WANG, K. Cl., NIELSEN, C. M., and VESTERGAARD, B. F. (1985). Isolation and adaptation characteristics of hepatitis A virus in african green monkey kidney cells: Production of antigen useful for ELISA serology. Intervirology 24, 99-l 07. WHEELER, C. M., FIELDS, H. A., SCHABLE, C. A., MEINKE, W. J., and MAYNARD, J. E. (1986). Adsorption, purification and growth characteristics of hepatitis A virus strain HAS-1 5 propagated in fetal rhesus monkey kidney cells. J. C/in. Microbial. 23, 434-440.
157,268-275
(1987)
Replicative
Events in Hepatitis
A Virus-infected
JOSE DE CHASTONAY Institute
for Hygiene
and Medical
Microbiology,
Received
September
AND
University 3, 1986;
GUNTER
of Bern, accepted
SIEGL’
Friedbilhlstrasse November
MRC-5 Cells
5 1, Bern
CH-30
10, Switzerland
17, 1986
Replication of hepatitis A virus (HAV) in MRC-5 cells was studied under one-step growth conditions. Viral replication neither interfered detectably with cellular DNA, RNA, and protein synthesis, nor could cytopathologic changes be recorded over a prolonged period of incubation. Synthesis of mature, infectious HAV particles could be detected as early as 2-4 days p.i. and occurred at a maximal rate around 8 days p.i., shortly before infectivity titers reached a plateau. Synthesis of total viral RNA, of positive-strand genomic RNA, of viral mRNA, as well as of negative-strand RNA followed the same pattern. By Day 14 p.i., when active HAV replication had developed into persistent infection, synthesis of viral RNA declined to background levels. The mechanism(s) guiding active HAV replication into a state of persistent infection could not be positively defined. Yet there exists the possibility that this is brought about by down regulation of viral RNA synthesis. Whether this is related to the appearance of a subgenomic viral RNA molecule about 2000 nucleotides in length and detected in association with ribosomes on Days 7 and 10 p.i. remains to be shown. 0 1997 Academic
Press,
Inc.
INTRODUCTION
processes of HAV. To do so, conditions must be elaborated to meet the requirements for a one-step growth curve. In a previous report (Siegl et a/., 1984b), it was shown that under such conditions, the HAV/cell relationship could be divided into three distinct phases: a short phase, characterized by an increase in titer, a second phase, in which pronounced synthesis of HAAg is detected although no further increase in infectivity is recorded, and from then on, a persistent type of infection, with but minor viral metabolic activity. Recently, similar results were reported by Wheeler and co-workers (1986) using another HAV strain as well as other cells. All results reported so far, however, yield no clue as to what mechanisms govern the establishment of and the switches between individual phases. As a first experimental approach to elucidate the functions operative in the development of the peculiar HAV/cell relationship we now report on the time course of the basic virus-specific synthetic events in infected cells. We provide evidence that, even under one-step growth conditions, replication of HAV in the human diploid fibroblast cell line MRC-5 is highly protracted. Moreover, the three phases revealed in previous studies have a counterpart at the level of viral RNA synthetic events.
Hepatitis A virus (HAV) has recently been classified within the genus Enferovirus of the family Picornaviridae (Melnick, 1982; Gust et a/., 1983). This classification, however, should not obscure the fact that many differences between HAV and enteroviruses do exist. For instance, it has not yet been possible to demonstrate primary replication of the virus in the gut of an infected host organism (Mathiesen et a/., 1978; Krawczynski et a/., 1981). HAV also exceeds the heat stability of poliovirus, a representative member of the enteroviruses, by a full 20” (Siegl et a/., 1984a). Furthermore, comparison of sequences of cloned HAV cDNA with sequence data for the genomes of picornaviruses revealed greatest homology with encephalomyocarditis virus of the genus Cardiovirus (Baroudy et al., 1985). This is in clear contrast to the extent of sequence conservation known to exist among the enteroviruses (Toyoda et al., 1984; Stalhandske et a/., 1984; Tracy et al., 1985; Kandolf and Hofschneider, 1985). Finally, HAV is distinguished from enteroviruses by its protracted replication in a vast majority of cells under in vitro conditions. This replication usually terminates in persistent rather than in acute Iytic infection (Provost and Hilleman, 1979; Frosner et a/., 1979; Gauss-MOller and Deinhardt, 1984; Siegl et al., 1984b; Bradley et al., 1984; Wang et a/., 1985; Friedman-Alvermann et a/., 1985). For both classification and practical purposes it appears to be essential to characterize the replicative ’ To whom 0042-6822/87
requests
for reprints
$3.00
Copyright 0 1907 by Academic Press. Inc. All rights of reproduction an any form reserved.
should
MATERIAL
AND
METHODS
Virus HAV strain GBM was originally isolated in MRC-5 cell cultures from fecal specimens collected during an outbreak of hepatitis A in Gormaringen, Federal Republic of Germany (Frosner et al., 1977). It was adapted
be addressed. 268
HEPATITIS
A VIRUS
REPLICATION
to replication at 32” and used to produce a virus stock in its fourth in vitro passage with an infectivity titer of 1 06.’ TCIDJml. Cells, conditions of cultivation, and mode of infection MRC-5 (human diploid embryonic lung) cells were cultivated in minimum essential medium (MEM), Eagle’s modified, with Earle’s salts supplemented with 1% nonessential amino acids, 2 m/l/l L-glutamine, and 25 mM HEPES buffer. For cultivation in closed containers, the pH of the culture medium was adjusted to 7.4-7.6; for incubation in the presence of 5% CO,, the pH was raised to 8.0. Fetal calf serum (FCS) was added to a final concentration of 10% (growth medium) or 5% (maintenance medium). Cultivation systems used in the outlined experiments were 25-cm* plastic cultivation flasks, petri dishes, as well as 24-well culture plates (Falcon) containing 10.5 X 10.5 mm coverslips. Cells were seeded into the culture units at a density allowing for the formation of intact monolayers within 4 to 5 days. Medium was changed weekly. Only cells grown to confluence were infected. For all kinetic studies, cells were infected at a multiplicity of 6-l 0. The inoculum was appropriately diluted in MEM without serum and adsorption was allowed to take place for 4 hr at 32”. Thereafter, it was replaced by maintenance medium containing 5% heat inactivated (56”, 30 min) FCS. Detection
of viral antigen
Hepatitis A antigen (HAAg) was detected and quantified in cellular extracts as described by Frdsner et al. (1977). Alternatively, HAAg was demonstrated in coverslip cultures after fixation with acetone (in situ RIA) (Siegl et al., 1984b). For the latter purpose, 0.1 ml of PBS and 1.5 ml of cold acetone (-20”) was added per coverslip and well. After 15 min at room temperature, the overlay was aspirated and the coverslips were airdried. Appropriately diluted ‘251-anti HAV in PBS and 0.1% bovine serum albumin was added and coverslips were incubated at room temperature over night. Following washing with distilled H20, bound radioactivity per individual coverslip was measured in a gamma counter (Packard). Titration
of HAV infectivity
Titration of HAV infectivity was based on the in situ RIA technique (Siegl et a/., 1984b). Briefly, MRC-5 monolayers were grown to confluence in multiwell dishes containing coverslips. Four wells were used per individual 1 O-fold dilution of the virus suspension and applied to the cells as a 0.5-ml inoculum. After adsorption, 1 ml of medium with 7.59/o heat-inactivated
IN MRC-5
CELLS
269
FCS was added to the wells. Maintenance medium was changed weekly. An in situ RIA was carried out on acetone-fixed coverslip cultures after 21 days of infection, and titers were calculated according to the method of Reed and Muench (1938). Precipitation
of radiolabeled
products
(a) Trichloroacetic acid (TCA) precipitation of cellular extracts. Aliquots of cellular extracts were diluted five times with PBS. An equal volume of 10% TCA was added and the tubes were kept on ice for 1 hr before precipitates were collected on Whatman microfiber filters (GF/B) equilibrated in 5% TCA. Filters were washed twice with ice-cold 5% TCA, rinsed with acetone, and dried at 50”. Bound radioactivity was measured in a Packard scintillation spectrometer. (b) TCA precipitation on coverslip cultures. An in situ TCA precipitation was carried out directly on coverslip cultures in multiwell dishes. At the time of harvest, coverslip cultures were washed twice with ice-cold 5%~ TCA in 0.2 R/I Na,P,O,, followed by incubation in the presence of 59/oTCA at 4” for at least 2 hr. Thereafter, wells were washed twice with 59’0 TCA and individual coverslips were dried and put into scintillation vials. The cellular material was dissolved by addition of 0.5 ml Soluene (Packard) at room temperature for 30 min. Incorporated radioactivity was measured in a scintillation counter. Isolation
and purification
of virus particles
At harvest, infected cell monolayers in 25-cm* plastic bottles were washed twice with PBS and then frozen in 5 ml PBS. Extracts were prepared by repeated (3X) freezing and thawing. After centrifugation at 3000 rpm for 20 min, RNase and DNase were added at a concentration of 50 pg/ml to the supernate and digestion was allowed to take place at 37” for 50 min. Virus was pelleted in a Beckman SW 50.1 rotor at 43,000 rpm for 160 min. The pellet was resuspended in 0.5 ml of 50 ml\/lTris-50 mlLl NaCI, pH 7.5 (TN buffer), 0.1% sodium laurylsarkosine (SLS), and layered onto a CsCl/sucrose step gradient consisting of a l-ml cushion of CsCl p = 1.5 g/ml, 4 ml CsCl p = 1.35 g/ml, and 4 ml of 30% (w/v) sucrose in an 1 l-ml polyallomer (SW41) tube. Centrifugation was carried out at 32,000 rpm at 15” for 16 hr. Thirty fractions of 0.3 ml were collected from the bottom of the tube. The CsCl and sucrose solutions used to make the gradients were made up in TN buffer containing 0.1 o/o SLS. Synthesis of viral positivenegative-strand RNA
and
RNA from two petri dishes (5 cm diameter) infected at m.o.i. 10 was harvested every other day by hot phe-
270
DE CHASTONAY
nol extraction (Scherrer, 1969). Total RNA was then precipitated with ethanol and resuspended in 100 ~1 of doubly distilled H20. Slot blots were made with 2% of the total RNA by adding an equal volume of formamide, heating to 65” for 10 min, diluting the sample with 50 vol of 5 M NaCI, and subsequently applying the mix to a GeneScreen-Plus membrane (New England Nuclear) by mild suction. Hybridization was carried out using SP6 polymerase generated 32P-labeled HAV RNA of either positive or negative polarity. For this purpose, HAV cDNA provided by Dr. J. Ticehurst, NIH Bethesda, MD (Ticehurst et a/., 1983) was recloned into the pGEM-2 vector. We used a 0.7-kb fragment representative for the 5’ end of HAV RNA (0.2 map units to 0.9 map units). Previous hybridization experiments with genomic RNA of a series of HAV strains adapted to cell culture indicated that this region of the HAV genome, as well as the region between 4.6 and 5.5 map units, contains highly conserved sequences among hepatitis A viruses (de Chastonay, Tracy, and Siegl, unpublished observation). Hybridization was carried out in the presence of formamide as recommended by New England Nuclear (NEN). For washing, however, SDS was added to a concentration of 2% and the temperature was raised to 70”. Isolation
of viral messenger
RNA
MRC-5 cells were grown to confluence in petri dishes (9 cm diameter) and infected at m.o.i. 10. Cellular and viral RNA in every dish was labeled by addition of 50 &i/ml of [3H]uridine for 24 hr prior to the harvest of the cells. At each point in time, each of four infected and two mock-infected cultures was washed twice with 5 ml PBS-A and 5 ml of PBS-A containing 100 pg/ml cycloheximide was added for 5 min at room temperature. The supernatant was then replaced by 4 ml of lysing buffer (0.1 mM Tris-HCI, pH 7.4, 0.1 mM KCI, 0.015 mM MgC12) supplemented with 50 mM NaCl and 100 pg/ml of cycloheximide. Cells were scraped off with a rubber policeman and pelleted (100 g, 5 min). The pellet was resuspended in 0.6 ml of lysing buffer, containing 100 pg/ml cycloheximide and 100 pg/ml DNase (RNase free, BRL), and incubated for 1 min at room temperature. To this, 0.6 ml lysing buffer containing 2% Triton X-l 00, 12 mM 2-mercaptoethanol, 100 pglml dextran sulfate, 200 pg/ml cycloheximide, and 80 pg/ml polyvinyl sulfate was added. The solution was supplemented with 0.3 ml of 20% Tween X-100, vortexed for 10 set, and then 0.36 ml of 4 M KCI, 40 mM MgC12 was added before the mix was passed five times through a 0.9 x 40-mm-gauge needle. After clarification (500 g, 4 min) the supernatant was layered onto a linear 40-109/o (w/v) sucrose gradient formed
AND
SIEGL
on top of a 0.5-ml 60% (w/v) sucrose cushion. The sucrose solutions were buffered in 0.1 MTris, pH 7.4, 5 mM MgCI,, and 0.5 M KCI. Centrifugation was carried out in a Beckman SW 27.1 rotor at 25,000 rpm for 18,500 W2t at 4’. Thirty fractions of 15 drops each were collected from the bottom of the tube. Of each fraction, 10 ~1 was assayed for TCA insoluble radioactivity. Intact, [14C]uridine-labeled poliovirus sedimenting at 160 S, [3H]thymidine-labeled Lu-Ill parvovirus sedimenting at 1 10 S, and Lu-Ill parvovirus capsids labeled with 3Hlabeled amino acids and sedimenting at 70 S served as markers. RNA aliquots were blotted directly onto GeneScreenPlus membranes. For this purpose, the RNA was suspended into 7.5% formaldehyde in 50 mM phosphate buffer, pH 7.0, heated to 60” for 15 min, and then blotted onto filters, preequilibrated with 1 OX SSC (1.5 M sodium chloride-O. 15 M sodium citrate). The membrane was then air-dried and baked for 2 hr at 80”. Electrophoresis
of RNA
RNA was denatured using deionized glyoxal/dimethylsulfoxide (DMSO) as described by Thomas (1980). Briefly, 2.7 PI of glyoxal, 8 ~1 of DMSO, and 5 ~1 of RNA in 100 mM phosphate buffer, pH 7.0, were incubated for 1 hr at 50”. Then, 4 ~1 of a solution of 0.25% bromphenol blue and 2 mM EDTA in 50% glycerol were added. Samples of 20 ~1 were loaded onto a 1% agarose gel (Sigma, Type 2) in 10 mM phosphate buffer, pH 7.0, and electrophoresis was carried out in a submarine, horizontal system at 60 V under constant buffer recirculation. The bromphenol blue marker was allowed to run for 6.5 cm. Using the protocol provided by NEN, RNA was transferred to GeneScreen-Plus filters and hybridization of dot blots and Northern transfers of encapsidated and ribosome-associated viral RNA were carried out without formamide as suggested by NEN, using 32P-nick translated HAV cDNA mapping in the 5’ region of the viral genome. The respective clone was kindly supplied by Dr. Klaus von der Helm, University of Munich, Federal Republic of Germany (von der Helm et a/., 1981). RESULTS Overall synthetic cell system
activity
in the HAV/MRC-5
An efficient interpretation of experimental data concerning replication of HAV over a period of several weeks is only possible if parameters such as cell numbers, synthesis of cellular DNA, RNA, and protein are easily controlled or remain largely constant. Our studies showed that in both infected and uninfected confluent
HEPATITIS
A VIRUS
REPLICATION
MRC-5 cultures, the cell number per square centimeter remained unchanged over a 40-day follow-up period. Absence of cell proliferation in such cultures was also indicated by the failure to incorporate significant quantities of [14C]thymidine into cellular DNA during labeling for 24 hr at various points in time. Between cell seeding and the establishment of a confluent monolayer, however, [14C]thymidine could be incorporated into the genome of MRC-5 cells to serve as a measure for cell number in subsequent experiments. On the basis of this standardization procedure it could be shown that HAV infection did not result in cell death over a period of at least 14 days. Likewise, incorporation of [3H]uridine (representative for total RNA synthesis) as well as of [3H]valine and [3H]leucine (representative for total protein synthesis) over multiple 24-hr periods reached practically identical values in both infected and uninfected cultures (results not shown). Synthesis and encapsidation of viral RNA The time course of synthesis and encapsidation of viral genomic RNA was followed by pulse-labeling experiments over a period of 16 days. Duplicate cultures in 25-cm2 flasks were infected at m.o.i. 6 and [3H]uridine (250 &i/flask) was added for 3-day intervals. After each interval, cell-associated virus was harvested and purified by gradient centrifugation as described under Material and Methods. In preliminary experiments, marker poliovirus and HAV were shown to accumulate in fractions 7 to 11, with peaks of HAV in
IN MRC-5
CELLS
271
fraction 8 and poliovirus in fraction 7, in accordance with previous observations (Siegl et al., 1981) of a slightly lower buoyant density for HAV (1.336 vs 1.34 g/ml). The amount of radioactivity accumulating in fraction 7 to 1 1 was considered as providing a measure for the amount of viral genomic RNA synthesized and encapsidated during the respective period. A steady increase in radioactivity was detected during the first three intervals, lasting to Day 10, after which a rapid decline of [3H]uridine-labeled particles could be recorded (Fig. 1). During Days 13-l 6, distribution of radioactivity in the gradient followed the pattern of mockinfected cultures. In Fig. 2, virus production is quantitated for each interval by presenting the integrated values of gradient fraction 7-l 1 of each labeling period. The infectivity titers as well as synthesis of total HAAg for this specific experiment were determined and also included. It can be concluded from these data, that encapsidation of newly synthesized viral RNA is limited to the phase of viral replication characterized by an active increase in infectivity titers. The plateau of infectivity present after Day 13 is therefore not maintained by a detectable active equilibrium between virus production and degradation. Much more, it appears that mature infectious virus particles remain stably cell associated. Synthesis of total viral RNA Encapsidation of viral RNA as illustrated in Fig. 2 may either cease due to a defect in morphogenic events or simply because synthesis of viral RNA stops. In order to elucidate the possible role of viral RNA synthesis as a rate-limiting step, synthesis of total viral RNA was followed over a period of 2 weeks. For this purpose, 8 pg/ml actinomycin D was added to duplicate infected and mock-infected cultures in multiwell dishes for 24 hr prior to harvest. One hour later, [3H]uridine (10 &i/ ml) was added and incubation continued for 23 hr. The quantity of labeled precursor incorporated per culture was determined by in situ TCA precipitation. The overall pattern of synthesis of total viral RNA (Fig. 3) coincides with the pattern of production of mature virus as illustrated in Fig. 2. The temporary decrease in viral RNA synthesis on Day 4 cannot yet be explained. Synthesis of negative- and positive-strand viral RNA
0
4
6
12
16
20
Fractions FIG. 1. Synthesis and encapsidation of viral genomic RNA in infected MRC-5 cells. HAV particles were labeled with [3H]uridlne over 3-day intervals, isolated as described, and centrifuged to equilibrium in &Cl-sucrose gradients. 0. Days 4-7 p.i.; 0, Days 7-10 p.i.; W, Days 1O-1 3 p.i.; . . . , control.
Synthesis of positive-strand genomic viral RNA can be assumed to proceed via a negative-strand intermediate. Accumulation of this type of molecule was used as an additional means to determine active replication of HAV RNA. As indicated by the blot hybridization assay with a positive-strand RNA probe, nega-
272
DE CHASTONAY
AND
SIEGL
14
r7
-14
12 2 cx I~~
- 12
loq ‘c
8-
SLO x ‘2 $ ,o ‘Z Ob E-8 C .
6-
P 0
4-
B .
-6g
T - lOr*! ? 8_ -8
z=
x
2-
-5
6E -4
& I z
-2; -0
o1
4 Days
7 post
10 infection
13
16
FIG. 2. Encapsidation of newly synthesized genomic vRNA (0) accumulation of infectious HAV (m), and rate of HAAg synthesis (A) in MRC5 cells infected as described. The integral amount of radioactivity accumulating in fractions 7-l 1 in the CsCI-sucrose gradient shown in Fig. 1 was taken as a measure for the amount of virus produced during the respective interval. Infectious virus was assayed by in situ RIA and HAAg by sandwich RIA. Accumulation of infectious HAV is plotted on a logarithmic scale; the other parameters are indicated in a linear scale.
tive-strand RNA could be recorded clearly as of Day 4 p.i. (Fig. 4). Its presence peaked on Day 8 p.i. and decreased to background values between Days 10 and 14, suggesting relatively rapid degradation of such viral RNA in the course of infection. By a similar experimental approach, accumulation of positive-strand RNA was also observed as of Day 4 p.i.; yet, after reaching maximum concentrations on Day 8 p.i., it remained associated with the cells in significant concentrations, seemingly reflecting its presence in mature progeny virus particles.
Presence
of viral mRNA
Some viral antigen synthesis is still recorded after cessation of viral RNA synthesis (Fig. 2). Therefore, viral messenger RNA may be assumed to have a prolonged half-life time. To investigate this aspect of HAV replication, we devised a method for isolation of RNA from MRC-5 cells which allowed us to distinguish encapsidated viral RNA from viral ribosome-associated RNA. For this purpose, polysomes were isolated and, in the course of extraction, disrupted into monosomes. Ultracentrifugation through a linear 40-l 0% sucrose gradient then allowed for separation of monosome-associated HAV RNA from mature HAV particles. Figure 5 shows the profile of the radiolabeled RNA in such a gradient. Almost all TCA precipitable radioactivity is days C
pos. viral
Days FIG. 3. Total
post
2
post 4
infection 6
strand RNA
neg.
strand
viral
RNA
c =
uninfected
8
10
14
+ I
-
m*,llciwmrr
CI r*c i~mvrrub-r
r)
I*
II
infection
synthesis of viral RNA per 24 hr in MRC-5 cells infected as described. RNA was labeled with [3H]uridine in the presence of actinomycin D. Viral RNA synthesis was estimated by subtracting TCA insoluble radioactivity incorporated in mock-infected cultures from that incorporated in infected cultures.
cells
FIG. 4. Presence of positive (upper)and negative (lower)-strand vRNA in cell extracts harvested at different times p.i. Both forms of RNA were detected by hybridization with radioactive RNA of complementary polarity as described.
HEPATITIS
+HA”
day
4
+HAV
day
7
day
13
+HAV
160s
REPLICATION
Pools
-
r-l-
A VIRUS
110s
70s
20s
I
I
4
g” 0 -2 X
E CL 1 0 0 5
10
15
20
25
Fractions FIG. 5. Association of vRNA with ribosomes. The cytoplasmic extracts of infected cells were harvested 4, 7, and 13 days p.i. The monosome profile obtained upon sedimentation in 1 O-40% sucrose gradients is shown as TCA-insoluble [3H]uridine-labeled material. Fractions were pooled as indicated, half of the material was blotted, and viral RNA sequences were detected by hybridization with HAVcDNA. Equal amounts of an infected (+HAV) and mock-infected (-HAV) culture was blotted at each harvesting point.
present in fractions 12-l 5. Judged by the positions of 160 S poliovirus and of 1 10 and 70 S parvovirus Lu-Ill particles in such gradients, cellular RNA sediments at 80 S, as expected for monosomes.
160 4
Polio
7
CELLS
ri bosome associated co
273
Blot hybridization analysis of the different pools derived from the gradients of harvests carried out at different times p.i. yielded a positive HAV signal in the 160 S pools as of Day 4 p.i. The intensity of the signal rose especially from Day 4 to 7 p.i. HAV RNA could be detected in the 80 S pools of Days 4 and 7 and structures containing HAV-specific RNA sequences were also present in the bottom fractions of the gradients harvested on Day 13 p.i. All other pools derived from HAV-infected cells could not be shown to contain HAVspecific RNA. This also holds true for those pools made from material sedimenting at 70 S and less and therefore expected to contain HAV replicative intermediate RNA molecules. This suggests a loss of such RNA molecules in the course of that particular extraction procedure. Gel electrophoresis and Northern blotting (Fig. 6) detected genome-sized HAV RNA in samples derived from the 160 S gradient pools on Days 7, 10, and 13. Genome-sized HAV RNA was also found in association with ribosomes on Day 10. In addition, the latter pools derived from harvests of Days 7 and 10 contained an apparently HAV-specific, subgenomic RNA molecule of about 2000 bases. Attempts to further characterize this uncommon molecule (e.g., with respect to the kinetics of its appearance after HAV infection or by hybridization with cDNA probes derived from genome regions other than the 5’terminal region), failed repeatedly due to the exceptionally low concentration of virus-specific RNA extractable from HAV-infected MRC-5 cells with that particular method. Additional experiments, however, are in progress.
S 10
IN MRC-5
4
7
10
pools co
days
-
p. i
-
HAV
-
23s
-
16s
-a rRNA
-b
rRNA
-
FIG. 6. Electrophoretic analysis of RNA associated with the 160 S pool and the ribosome pool on Days 4, 7, and 10 p.i. The mock-infected culture (Co) was harvested on Day 10. After electrophoresis, the RNA was transferred to Gene Screen-Plus filters and HAV specific sequences were detected by hybridization as described. On the left-hand side, the positions of poliovirus and of Escherichia co/i rRNAs are indicated. The right-hand side points to the migration position of genome sized HAV RNA and gives the sedimentation values of the rRNAs. Arrow “a” indicates the position of RNA sequences detected in the ribosome pool of both infected and mock-infected cells. Arrow “b” points to the position of a subgenomic vRNA molecule apparently only present in material from infected cultures.
274
DE CHASTONAY
DISCUSSION The virus-specific molecular events underlying the three tentatively defined phases of HAV replication in MRC-5 cells have in part been elucidated. Most evidently, the synthesis of total viral RNA (i.e., negativestrand template RNA, genomic RNA, and viral mRNA), as well as synthesis and encapsidation of genomic RNA occur in parallel to the appearance and accumulation of progeny infectious virus. These events slow down rapidly (phase 2) and can hardly be detected in the course of persistent infection (phase 3). On the basis of these observations, the cycle of active HAV replication can be redefined as extending from the start of infection to the time major synthetic events (e.g., synthesis and encapsidation of vRNA) again became undetectable. Approximately 13 days elapse before this occurs. Such a time course contrasts sharply to the 7hr replication cycle described for poliovirus (for references, see Koch and Koch, 1985). In poliovirus-infected cells, termination of viral replication is identical with death and lysis of the infected host cell. This is initiated by shutoff of cellular metabolic activity relatively early in infection (2 hr). As indicated by the lack of impact of HAV replication on the MRC5 cell metabolism over a prolonged period of time, shutoff of cellular RNA and protein synthesis apparently plays no important role in regulation of the described HAV/cell relationship. At the present time, we can only speculate on the nature of factor(s) or event(s) which may be operative in controlling transition of the HAV/MRC-5 cell relationship from phase 1 to phase 2 and, finally, into the state of persistent infection. The block may be sought in replication of viral genomic RNA or in the production of viral mRNA, because both events appear to cease with the end of phase 1. Cessation of RNA synthesis may be brought about by a block in RNA translation and protein processing toward the end of phase 1. Such a block would not necessarily affect synthesis of viral structural proteins during phase 2 but might specifically concern translation of virus specific protease and RNA polymerase. In analogy to the organization of a standard picornavirus genome, these essential enzymes are coded for far downstream from the structural proteins and their translation might well be negatively affected by the outstandingly high proportion of secondary structure (1 O-20016) present in the HAV genome (Siegl eta/., 1981). This line of thought is supported by the existence of a translational barrier in the central region of the encephalomyocarditis (EMC) virus genome (Svitkin and Agol, 1983) and probably also due to secondary structure in this region. Such a translational barrier could also be expected to lead to an over-
AND
SIEGL
production of capsid proteins as has indeed been observed in the late stage of mengovirus infection (LucasLennard, 1974; Paucha et a/., 1974). Another factor which may contribute to the efficient down regulation of HAV replication subsequent to phase 1 may be related to the occurrence of a subgenomic RNA about 2000 nucleotides in length closely related to the region of the HAV genome coding for the structural proteins. Subgenomic viral RNAs with similar characteristics have been found in cells infected with caliciviruses (Ehresmann and Schaffer, 1977) and cowpea mosaic virus (Franssen et al., 1984). The role of these molecules in virus replication, however, has not been resolved. In the rare instance where subgenomic RNA molecules have been detected in picornavirus systems (e.g., Domingo et al., 1985), the molecules resembled the highly deleted genomes of defective interfering (DI) particles which tend to lack the region coding for structural proteins (Huang, 1973). Due to the quantitative problems lined out under Results, we were so far not able to further characterize the subgenomic RNA in HAV-infected MRC-5 cells. Hence, we are not yet in the position to decide whether this RNA represents the genome of a distinct class of DI particles, plays a special, necessary role in HAV replication, or controls the development of the persistent type of HAV infection in MRC-5 cells. ACKNOWLEDGMENTS Thanks are due to Drs. K. von der Helm (Munich) and J. Ticehurst (Bethesda, MD) for kindly providing us with their HAV cDNA clones. We also thank J. Nijesch (Bern) for recloning such cDNA from the pBR 322 into the pGEM vector. This study was supported in part by Grant 3.813.83 from the Swiss National Science Foundation.
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