Restricted Replication of Human Hepatitis A Virus ... - Journal of Virology

Vol. 37, No. 1

JOURNAL OF VIROLOGY, Jan. 1981, p. 216-225 0022-538X/81/010216-10$02.00/0

Restricted Replication of Human Hepatitis A Virus in Cell Culture: Intracellular Biochemical Studies STEPHEN A. LOCARNINI,* ANTHONY G. COULEPIS,1 EDWIN G. WESTAWAY,2 AND IAN D. GUST' Virus Laboratory, Fairfield Hospital for Communicable Diseases, Fairfield, Victoria, 3078,1 and Microbiology Department, Monash Medical School, Monash University, Prahran, Victoria, 3181,2 Australia

When hepatitis A virus was inoculated into Vero cells, virus-specified protein and RNA synthesis was detected. Production of viral protein was detected by electrophoretic analysis in polyacrylamide gels by using a double-label coelectrophoresis and subtraction method which eliminated the contribution of host protein components from the profiles of virus-infected cytoplasm. Eleven virusspecified proteins were detected in the net electrophoretic profiles of hepatitis A virus-infected cells. The molecular weights of these proteins were very similar to those detected in cells infected with poliovirus type 1. Virus-specified protein synthesis could be detected at 3 to 6 h and continued for at least 48 h postinfection, but no significant effect on host-cell macromolecular synthesis was observed. Limited viral RNA replication occurred between 2 and 6 h postinfection. The genomic RNA of hepatitis A virus was extracted and shown to be capable of infecting cells and inducing the same set of proteins as intact virus, indicating that the RNA genome is positive stranded. Progeny virus was never detected in the supernatant fluids of infected cell cultures, and the cells showed no observable cytopathology, even though hepatitis A virus-specific proteins and antigens were being produced. The nature of the defect in the replicative cycle of hepatitis A virus in this system remains unknown.

Hepatitis A virus (HAV) is a 27- to 32-nm particle (4, 10) which, on the basis of morphological (10, 17), physicochemical (2, 17), and biochemical studies of its protein (2) and nucleic acid (20), appears to be a picornavirus. Recently, Provost and Hilleman (16) reported the successful propagation of HAV in cell culture, using a strain of virus which had been adapted by multiple passages through marmosets. This marmoset-adapted strain replicated in primary explant cell cultures of marmoset livers and in a fetal rhesus kidney cell line (FRhK-6). Although the virus appeared to grow quite well in FRhK6 cells, no cytopathology was observed. We inoculated fecal extracts containing HAV from patients with serologically confirmed hepatitis A (9, 11) into a variety of continuous cell lines of monkey kidney origin and looked for evidence of virus replication by direct immunofluorescence and solid-phase radioimmunoassay. Even though the cells appeared normal throughout the study period, evidence of limited replication was obtained by immunofluorescence in a number of cell lines, including Vero. Because of the ease of manipulation, Vero cells were chosen for further studies. This communication describes the intracellular biochemical events detected when human strains of HAV derived

from feces, and nucleic acid extracted from purified virions obtained from the same source, were inoculated into Vero cells. MATERIALS AND METHODS Cells and viruses. Vero cells were cultured in 60mm plastic petri dishes in medium 199 containing 15 mM HEPES (N-2-hydroxethylpiperazine-N'-2-ethanesulfonic acid) buffer and maintained in Eagle minimum essential medium containing 0.2% bovine serum albumin. Sufficient Vero cells were added so as to produce uniformly confluent monolayers in 24 or 48 h. HAV was obtained from fecal specimens (H1 to H4) collected from four patients with naturally acquired infections. Virus was identified by solid-phase radioimmunoassay and immune electron microscopy (9, 11) and then purified from each specimen by a process of differential centrifugation, chloroform extraction, column chromatography using agarose gel filtration, and isopycnic ultracentrifugation in cesium chloride (CsCl) as described previously (2, 8). During purification, the presence of HAV was monitored by solid-phase radioimmunoassay. The purity of each of the four HAV strains was assessed by direct and immune electron microscopy using human pre- and postinfection hepatitis A sera (9, 11), as well as sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (2, 8). The final purified product was shown to be free from extraneous material by direct electron microscopy, and immune electron microscopy revealed only 216

VOL. 37, 1981

HAV-SPECIFIED PROTEINS AND RNA IN VERO CELLS

27-nm particles coated with anti-HAV. When analyzed by SDS-PAGE, the purified preparations were found to contain only the four structural polypAeptides recognized as typical of HAV (2, 8). The fouti specimens had particle counts in each electron microscope 400mesh grid square of approximately 10 (low-dose inoculum, Hi), 100 (medium-dose inoculum, H2), and over 1,000 (high-dose inocula, H3 and H4). Particle counts were determined as described previously (8). Samples of the four purified strains of HAV were then inoculated into duplicate tubes of primary monkey kidney, monkey embryonic kidney, and HeLa cells, examined for 3 weeks, and then passaged once more. No cytopathic agents were recovered. Stocks of poliovirus type 1 (Brunhilde strain) were obtained from Virgo Reagents (Electronucleonics Laboratories, Inc., Bethesda, Md.). Virus was grown in roller-bottle cultures of Vero cells and then purified on CsCl density gradients. The plaque titer of purified virus assayed on Vero cell monolayers under 0.7% agarose was 5.5 x 109 PFU/ml. Preparation and electrophoretic analysis of cytoplasm labeled with methionine. The experiments with poliovirus type 1 were carried out on the same day and under the same conditions as the HAV inoculation studies, but cells were infected, labeled, and processed in a separate laboratory. Confluent monolayers in 60-mm petri dishes were infected at 370C with each of the four purified HAV strains, as well as with poliovirus type 1, which was used at a multiplicity of infection of 30 PFU/cell. One hour later, all cells were fed with Eagle minimum essential medium plus bovine serum albumin and maintained at 370C. (i) Effect of virus infection on total protein synthesis. Mock-infected (control) and infected monolayers were exposed at various times after infection to methionine-deficient (one-tenth normal concentration) Eagle minimum essential medium plus bovine serum albumin, containing 30 MCi of [3H]methionine per ml, for 2 h. Incorporation was stopped by washing the monolayers twice in cold saline and dissolving the cells in 0.5 ml of 2% SDS per dish. Samples of the SDS-cytoplasm were transferred to Whatman GF/C filter disks for precipitation of macromolecules by trichloroacetic acid, and the incorporation of [3H]methibnine was measured in a liquid scintillation counter. (ii) Detection of virus-specified proteins in cells. Viral protein production was examined by SDSPAGE of infected cell lysates. Cells were infected as described above, and radioactive pulse-labeling of infected and control cells was performed as described by Westaway (26), using a double-label coelectrophoresis and subtraction method to eliminate host protein components from the electrophoretic profile of infected cytoplasm (29). At 3 h before the addition of radioisotopes, monolayers were fed methionine-deficient Eagle minimum essential medium plus bovine serum albumin containing 3 iLg of actinomycin D per ml. Infected cultures were then refed with similar medium that contained [3H]methionine (10 MCi/ml), and control (mock-infected) cultures were fed with medium containing [3S]methionine (5 AiCi/ml), for 3 h more. Medium was then removed, and the cell monolayers were

217

washed in cold saline and harvested in 0.5 ml of 2% SDS per dish. Proteins of virus-infected and control cells dissolved in SDS were analyzed by coelectrophoresis as described previously (26, 28) in 11% Laemmli (6) SDSdiscontinuous polyacrylamide gels (10 by 0.6 cm). Samples for analysis were brought to final concentrations of 1% dithiothreitol (Koch-Light Laboratories, England), 0.05 M Tris-hydrochloride (pH 6.8), 8% glycerol, and 0.005% bromophenol blue, and heated at 100°C for 3 min. The heated samples, in less than 100 pl, were applied directly to gels without dialysis and electrophoresed at 3 mA per gel for 4 h. Gels were removed from the glass tubes, frozen, and sliced into 1.5-mm-thick disks; the gel slices were transferred to vials, and 0.3 ml of water was added. After overnight incubation at 37°C, scintillation cocktail was added (Insta-gel; Packard Instruments Inc., Downers Grove, Ill.), and radioactivity was counted 24 h later in a double-channel liquid scintillation spectrometer (26, 28). Measurement of RNA synthesis in infected cells. Before infection, cells were treated for 4 h with medium 199 containing 2% fetal calf serum plus 3 ,ug of actinomycin D per ml. Cells were then infected as before, except the inocula contained 3 jg of actinomycin D per ml. The cells were then exposed at various times postinfection to the above medium containing [3H]uridine (3 ,uCi/ml) for 2 h. Samples of labeled cells dissolved in 2% SDS were then spotted onto squares (2 by 3 cm) of Whatman no. 1 chromatography paper, allowed to dry, and then washed seven times in cold 0.25 M perchloric acid and 9% sodium pyrophosphate solution. The papers were next washed twice in cold distilled water, cold ethanol, and finally in an ethanol-ether (1:1) mixture. The amount of incorporated [3H]uridine was then determined in a liquid scintillation counter. Analysis of labeled RNA species produced in cells. Nucleic acid was then extracted from each of the SDS-[3H]uridine-labeled cytoplasm samples (250 pl) described in the previous section by the phenolchloroform method as described by Tannock and Hierholzer (22), except that the aqueous phase was extracted three times, made 1% with respect to SDS to ensure complete deproteinization, and then extracted once more. The extracted nucleic acid was then concentrated by precipitation at -20°C for 16 h in the presence of 100 Mg of carrier RNA (yeast RNA, A

grade; Calbiochem-Behring, Sydney, Australia), 150 mM sodium acetate, and 2 volumes of cold ethanol (22). The nucleic acid was then pelleted by centrifugation at 120,000 x g for 15 min at -5°C, washed twice, and then suspended in NET buffer (10 mM EDTA, 100 mM NaCl, and 10 mM Tris-hydrochloride, pH 7.4). Each sample was applied to a 15 to 30% (wt/ vol in NET buffer, pH 7.4) sucrose gradient and centrifuged at 310,000 x g for 2 h and 40 min at 7°C. A parallel tube containing [3H]uridine-labeled rRNA was included in each run for approximate determination of sedimentation coefficients. After centrifugation, 20 fractions of 0.25 ml each were collected from the bottom of each tube, and each fraction was divided into two equal aliquots. To the first aliquot was added 5 pl of NET buffer, and to the second was added 5 Ml

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water.

method using a fluorescein isothiocyanate-conjugated chimpanzee anti-HAV serum kindly supplied by R. Purcell and S. Feinstone, National Institutes of Health, Bethesda, Md. After incubation at 20°C for 30 min and three 5-min washes in phosphate-buffered saline, the slides were mounted in a glycerol-phosphate-buffered saline medium and examined with a Zeiss standard microscope equipped with an incident light condensor and HBO 50 light source. Immunofluorescence results were graded on a scale of 1+ to 4+, depending on the brightness and quantity of intracellular fluorescence. The specificity of the immunofluorescent reaction was confirmed with blocking experiments using pre- and postinfection sera from a chimpanzee experimentally infected with HAV (3). For positive and negative controls, preinfection and acutephase liver biopsies from marmosets infected with HAV were employed. These tissues were kindly provided by R. Purcell and S. Feinstone. Reagents. L-[methyl-3H]methionine (87 Ci/mmol), L-[nS]methionine (910 Ci/mmol), and [5,6-3H]uridine (46 Ci/mmol) were purchased from the Radiochemical Centre, Amersham, England. Actinomycin D was a gift from Merck, Sharp and Dohme (Aust.) Pty. Ltd., Sydney, Australia.

To enhance the infectivity of the viral RNA, the DEAE-dextran (DEAE-D) method (13, 14, 24) was modified as follows. The specimen of nucleic acid dissolved in distilled water was divided into two equal aliquots. One aliquot was made up to contain, in final concentration, 500 jig of DEAE-D (molecular weight of 2 x 106; Pharmacia Fine Chemicals, Uppsala, Sweden) per ml in Eagle basal medium (BME) buffered in Tris (pH 7.4). This BME-Tris-DEAE-D solution consisted of 3 parts of BME and 1 part of 0.2 M Trishydrochloride (pH 7.4), to which was added 0.01 volume of a 50-mg/mi DEAE-D solution in 0.2 M Trishydrochloride (pH 7.4). The second aliquot was treated with bovine pancreatic RNase A type III (20 pg/ml, final concentration) at 23°C for 30 min, then made up in BME-Tris-DEAE-D solution as described above. Both untreated and RNase-treated aliquots of extracted nucleic acid were then inoculated into Vero cells, pretreated for 1 min at 23°C with BME-TrisDEAE-D, for plaque assay of infectious RNA (poliovirus only) and for labeling of virus-specific proteins at 2 to 6 h postinfection by the double-label technique as described above (HAV only). The pretreatment of cells with DEAE-D had no effect on the efficiency of plating of poliovirus type 1 (12). Detection of HAAg by immunofluorescence. Tube monolayers of Vero ceUs were inoculated with 0.2 ml of each of the four purified HAV strains as described above. Duplicate inoculated and uninoculated phials were removed at various times and tested for hepatitis A antigen (HAAg) production by immunofluorescence. The cell monolayers were washed twice with Hanks balanced salt solution, after which 0.5 ml of a 0.125% trypsin-EDTA (1:5,000) mixture was added. The trypsinized cells were then deposited by low-speed centrifugation and suspended in 0.2 ml of sterile phosphate-buffered saline (pH 7.4). One drop of ceU suspension was spotted onto a microscope slide, air-dried, and then fixed in acetone. HAAg was detected in cells by the direct immunofluorescence

RESULTS Effect of virus infection on host cell shutoff. Three of the four purified preparations of HAV (Hi, H2, and H3) were used to infect cells at a low-, medium-, and high-dose inoculum (as determined by electron microscopy), and poliovirus was inoculated into cells at a multiplicity of infection of 30 PFU/cell. Total protein synthesis was measured in the mock-infected (control) and virus-infected cells by labeling with [3H]methionine for 2-h intervals, starting from 0 h and going through to 8 h postinfection as described in Materials and Methods. In poliovirus-infected cells, there was a marked inhibition of host cell protein synthesis (5), detectable at 2 to 4 h postinoculation, which steadily increased thereafter (results not shown). In contrast, incorporation of label by cells infected with the three strains of HAV was not appreciably altered throughout the study period as compared to the control cells (results not shown). Detection of HAAg in cells by immunofluorescence. Both inoculated and control cultures were tested for the development of HAAg. Three of the four specimens produced strong and specific fluorescence in infected cell cultures. Specimens H3 and H4 (high-dose inocula) produced 80 to 100% specific fluorescence in infected cells within 3 h, and specimen H2 (medium-dose inoculum) produced this much specific fluorescence within 12 h postinfection, but specimen Hi (low-dose inoculum) remained negative throughout the study period. The fluorescence was exclusively intracytoplasmic and usually appeared granular in nature. With time,

of bovine pancreatic RNase A, type III (Sigma Chemical Co., St. Louis, Mo.) to give a final concentration of RNase of 10 ,g/ml. The fractions were incubated at 23°C for 30 min and then spotted onto filter paper for precipitation with 0.25 M perchloric acid-9% sodium pyrophosphate and counting of radioactivity as above. Preparation and assay of infectious RNA from poliovirus type 1 and HAV. The two high-dose inocula, H3 and H4, of purified HAV were further concentrated by ultracentrifugation. Electron microscopic examination of this material revealed that each specimen contained in excess of 104 particles per electron microscope 400-mesh grid square (2, 8). Purified poliovirus type 1 was used at a titer of 5.5 x 109 PFU/ ml. Extraction and assay of infectious RNA was initially developed using poliovirus type 1 and subsequently applied to HAV. Nucleic acid was extracted from virions by using the phenol-chloroform method (22) described above, except that the material was extracted six times and SDS was present in the last extraction to ensure complete deproteinization of the nucleic acid. The extracted nucleic acid was washed and precipitated five times in ethanol and then suspended in distilled

VOL. 37, 1981


larger clumps of fluorescence were detected which were discrete and often perinuclear. The amount of intracellular antigen increased markedly between 1 and 4 days, after which a steady but substantial decline was observed (N. I. Lehmann, R. C. Pringle, and I. D. Gust, unpublished data). Proteins specified by poliovirus type 1 and HAV during replication. Because of the suspected relationship of HAV to the picomaviruses, an electrophoretic profile of poliovirusspecified proteins was obtained as a basis for comparison with HAV. Poliovirus-infected cells were labeled with [3H]methionine and control cultures were labeled with [3S]methionine from 3 to 6 h postinfection. The virus-specified proteins were separated by electrophoresis in 11% SDS-discontinuous polyacrylamide gels (Fig. 1A). Coelectrophoresis with uninfected cells permitted the appropriate deduction (Fig. 1B) to eliminate the host cell protein component from the electrophoretic profile of the infected cells (26, 29). In Fig. 1A, virus-specified proteins represent 20% of the total incorporation of labeled methionine in infected cells and as much as 85% of the total counts in fractions containing the peaks of viral proteins. This value (20%) steadily increased over the next 6 h to 80%, by which time the cells were showing obvious cytopathology. Figure 1B shows the net profile obtained for poliovirus type 1 specified proteins and is in agreement with profiles obtained by using other approaches (1, 7, 27). Each of the four purified specimens of HAV was also inoculated into cells, and the production of virus-specified proteins was examined as described above for poliovirus. The results shown in Fig. 2 were obtained when the medium (H2)and high (H3)-dose specimens were used as inocula. The net electrophoretic profiles of the other two inocula were very similar to H2 (Fig. 2B) and H3 (Fig. 20). In Fig. 2A, virus-specified proteins represent 16% of the total labeled methionine incorporation in infected cells and as much as 85% of the total counts in gel slices containing the peaks of viral proteins. Similar values were obtained with the high-dose inocula, whereas only about 7% of the total labeled methionine incorporation was found for the lowdose inoculum. Eight virus-specified proteins were regularly observed in the net electrophoretic profiles (Fig. 2B and C) of HAV-infected cytoplasm, and they have been labeled Pl1O, P70, P51, P42, P39, P26, P24, and P14 according to their apparent molecular weights (x103) (1, 27). If electrophoresis was extended, three more proteins were resolved, P160, P34, and P29 (Fig. 3), but P14 was not detected, presumably because it ran off the gel with the dye marker.

219

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FIG. 1. PAGE of proteins synthesized at 3 to 6 h in poliovirus type 1-infected Vero cells (multiplicity of infection, 30 PFU/cell). Cells were treated with 3 pg of actinomycin D per ml for 3 h and then labeled for 3 h with 10 fCi of [3H]methionine per ml. Mockinfected control cultures were labeled with 5 liCi of [3SJmethionine per ml under the same conditions. Monolayers were then dissolved in 2% SDS, and samples of infected and uninfected SDS-cytoplasm were mixed and electrophoresed as described in the text (A). (B) Profiles of net poliovirus type 1 proteins were obtained by subtraction after an appropriate factor (in this case 1.4) was applied to the uninfected 35S counts per minute so as to match 3H counts per minute in fractions devoid of virus-specified proteins. Poliovirus type 1-specified proteins represented 20% of the total labeled methionine incorporation. Six proteins of poliovirus were detected and were identified by their electrophoretic mobility relative to 14Clabeled Kunjin virus proteins (26) and by a series of pulse-chase (1, 27) experiments (Locarnini and Westaway, unpublished data). The profiles of P110 (NCVPI), P79 (NCVP2), P58 (NCVP4), VPO, VP1+P31 (NCVPX), and VP3 are in accordance with other published reports of poliovirus-specified proteins (1, 7,27).

The molecular weights of the HAV-specified proteins were determined by coelectrophoresis with labeled polypeptides of poliovirus type 1 (1, 27) and Kunjin virus (26, 27). P100 and P24 of HAV comigrated with proteins NCVP 1 (110,000 daltons) and VP3 (24,000 daltons) of poliovirus, respectively (1). P70 and P51 comigrated with protein NV4 (70,500 daltons) and envelope protein V3 (51,300 daltons) of Kunjin

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0 20 30 40 FRACTION NUMBER FIG. 2. PAGE of proteins synthesized at 3 to 6 h in HA V-infected (H2; A and B) cells under the same conditions as described in Fig. 1. Eight apparently 10

virus-specified proteins, P110, P70, P51, P42, p39, P26, P24, and P14, were detected after subtraction, as described in Fig. 1, of the expanded uninfected 35S counts per minute (expansion factor, 3.35) from the HA V-infected 3H counts per minute, (A). HAV-specified proteins represent 16% of the total methionine incorporation in Vero cells. (C) PAGE of proteins specified in Vero cells by HA V (specimen H3) at 6 to

virus, respectively (26). The molecular weights of the remaining proteins were determined relative to the migration of the other major polypeptides of poliovirus type 1 and Kunjin virus. Peak levels of HAV-specified incorporation of labeled methionine for three of the four inocula (H2, H3, and H4) were found to occur within 3 to 6 h postinfection. Incorporation could still be detected more than 48 h later, but at a much reduced (50%) rate (results not shown). Even though a constant rate of only about 7% of total incorporation into virus-specified proteins was found for the low-dose inoculum (Hi), the net protein profiles were identical to those obtained with the other inocula. Comparison of the net electrophoretic profiles obtained with each inoculum over the 48-h postinfection period did not reveal any apparent qualitative or quantitative changes in proteins specified at various times during infection, apart from a decrease of about 50% in synthesis of P51 between 6 and 48 h postinfection (data not shown). It is unlikely that the net electrophoretic profiles obtained are the result of contamination with an adventitious agent, because all four inocula were highly purified preparations of HAV which contained no foreign particulate material nor detectable cytopathic agents. To exclude the possibility that the protein profile shown in Fig. 2 represented host proteins induced subsequent to HAV inoculation, cells were treated with medium 199 plus fetal calf serum and actinomycin D for 4 h before infection. This treatment blocked host cell transcription (hence, induction of new mRNA) by reducing [3H]uridine incorporation into RNA by 99%. Cells were infected as before (except that the inocula contained actinomycin D) and then pulse-labeled, and the proteins were coelectrophoresed as described in Fig. 2. After the appropriate factor had been applied and the subtraction was made, the electrophoretic profile of HAV-specified proteins synthesized at 3 to 6 h postinfection was unchanged from Fig. 2 (results not shown). Omission of actinomycin D treatment of infected cells in the labeling experiments resulted in very poorly resolved profiles, presumably because actinomycin D partially suppresses the high background of host protein synthesis (results not

shown).

To further establish the specificity of the a sample of HAV (H3) incubated at 370C for 2 h with preinfection

HAV-induced proteins,

9 hpostinfection, under the same conditions as above. As with specimen H2, the eight HA V-specified proteins were detected after subtraction (expansion factor 2.5) and represented 12% of the total labeled methionine incorporation.


VOL. 37, 1981 A

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FIG. 3. PAGE of proteins synthesized at 3 to 6 h in HA V-infected (H3) cells, under the same conditions as described in Fig. 1, but with the electrophoresis period extended by 20 min to 4 h and 20 min. Three extra proteins, P160, P34, and P29, were resolved in the net profile, but P14 ran off the bottom of the gel. The appropriate factor in this case was 2.3, and HA V-specified proteins represented 14% of the total labeled methionine incorporation.

hepatitis A chimpanzee serum was inoculated into cells; this produced the same net virus-specified protein profile as seen in Fig. 2. This profile was not obtained after inoculation with a duplicate sample treated with convalescent-phase serum from the same chimpanzee (3). Viral RNA synthesis. Vero cells were treated with medium 199 plus fetal calf serum and actinomycin D for 4 h before infection to ensure that subsequent RNA synthesis was virus specified. Three of the four purified preparations of HAV (Hl, H2, and H3) were used as low-, medium-, and high-dose inocula. Poliovirus was fed to other cultures at a multiplicity of infection of 30 PFU/cell. Actinomycin D. was included in the medium throughout the infections. RNA synthesis in both control and infected cells was then evaluated by 2-h pulses with [3H]uridine for 2-h intervals from 0 to 10 h postinfection and

221

for one 5-h interval from 10 to 15 h postinoculation as described in Materials and Methods. In the poliovirus-infected cells, viral RNA synthesis was first evident 2 to 4 h postinfection; it increased as expected until the end of the 4- to 6-h postinfection period (5, 7) and subsequently declined (results not shown). In contrast, the three HAV-infected cell cultures showed no significant difference in incorporation of [3H]uridine from the control cells for any time interval over the 15-h postinoculation period (results not shown). Unique RNA species produced in HAVinfected cells. The [3H]uridine-labeled cytoplasm samples from the above experiment were analyzed further for the presence of small amounts of newly synthesized RNA which would not have been detected in the previous study. The labeled cytoplasm for the following four time intervals-(i) 0 to 2 h, (ii) 2 to 6 h (a pool of 2 to 4 h and 4 to 6 h), (iii) 6 to 8 h, and (iv) 8 to 15 h (a pool of 8 to 10 h and 10 to 15 h) postinfection-were extracted with phenol-chloroform and then processed by rate-zonal ultracentrifugation on sucrose gradients. The distribution of radioactivity in the untreated sucrose fractions for the time interval 2 to 6 h postinfection for each of the control, HAV-infected (highdose inoculum, H3), and poliovirus-infected cells is shown in Fig. 4. An apparently significant (2.5 times the control level) peak (fractions 7 to 11) of [3H]uridine-labeled material was observed in the sample from HAV-infected cells, and this sedimented as a 30S to 37S peak as compared to sedimentation of the 35S genome of poliovirus (21) and of ribosomal markers in parallel gradients. The 30S to 37S peak detected in the HAV-infected cells was sensitive to RNase, and an RNase-resistant peak was found (fraction 14) sedimenting at approximately 20S to 22S. Infection with the medium-dose inoculum (H2) produced similar results (data not shown). Similarly, the 35S peak of [3H]uridine-labeled poliovirus was also RNase sensitive, and the doublestranded (RNase-resistant) replicative form (22S) was also detected (7, 15). When the samples from the other time intervals were examined, no significant peaks of [3H]uridine-labeled material were detected in extracts from the HAV-infected cells. To further establish the specificity of the HAV-induced RNA, a sample of HAV (H3), incubated at 370C for 2 h with preinfection hepatitis A chimpanzee serum, was inoculated into cells; it produced the same RNA profile, as seen in Fig. 4B. This profile was not obtained after inoculation with a duplicate aliquot reacted with convalescent-phase serum from the same chimpanzee (3).

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FIG. 4. Sucrose gradient analysis of cell extracts of poliovirus, HAV, and control RNA, synthesized from 2 to 6 h postinfection. RNA was extracted from the [3H]uridine-labeled cytoplasm by the phenolchloroform method, carrier RNA was added, and all RNAs were precipitated with ethanol (22). Each precipitate was dissolved in 0.5 ml of NET buffer and analyzed by ultracentrifugation in sucrose gradients as described in the text. (A) Comparison of the untreated sucrose gradient fractions from the [3H]uridine-labeled cytoplasms of the control (0) and HAVinfected (0) cells. (B) Sensitivity of the [3H]uridinelabeled HA V RNA (0) to pancreatic RNase (0). (C) Sensitivity of the [3H]uridine-labeledpoliovirus type 1 RNA (0), synthesized at 2 to 6 h postinfection, to pancreatic RNase (0). The relative mobilities of peaks of 3H-labeled acid-insoluble radioactivity were determined by comparison with externally run [3H]uridine-labeled 28S and 18S rRNA (22).

HAV-specified protein synthesis in cells infected with HAV RNA. Comparative infectivity assays of poliovirus type 1 and its extracted RNA yielded plaque titers of 5.5 x 109 and 9 x 105 PFU/ml, respectively. No plaques were observed in assays with the RNase-treated samples of extracted poliovirus RNA. RNA was extracted from HAV and inoculated into cells in the same manner as for the poliovirus RNA. These cells were labeled with [3H]methionine, and the mock-infected cells were labeled with [35S]methionine, at 2 to 6 h

postinfection, and the cell extracts were analyzed by coelectrophoresis as described in Fig. 2. The results of the infectious nucleic acid experiment using specimen H4 are shown in Fig. 5. The net protein profile of the untreated HAV RNA was very similar to that obtained with whole virus, shown in Fig. 2. HAV RNA-specified proteins represented 31% of the total labeled methionine incorporation and as much as 65% of the total counts in those gel slices containing the peaks of viral proteins. The typical profile of HAV-specified proteins was also detected with RNA extracted from the H3 specimen, but as with the H4 RNA, one extra protein, P45, was detected. In previous experiments, a prominent peak in a similar position in the gross profiles (Fig. 1, fraction 15; Fig. 2A, fraction 12; Fig. 2C, fraction 12: and Fig. 3, fraction 14) was removed by subtraction. This protein is probably host cell actin, which is apparently less depressed in synthesis after infection with extracted nucleic acid than after infection with intact virus. Confirmation that the net profiles obtained with HAV RNA (Fig. 5) were HAV specified was obtained by a control experiment in which the same RNA was treated with RNase before inoculation into cells and the synthesis of viralcoded proteins was drastically reduced (Fig. 5). However, some continuing virus-specified proNET PROTEINS

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40

50

FRACTION NUMBER FIG. 5. The net electrophoretic profiles ofproteins synthesized at 2 to 6 h in cells infected with untreated HAV RNA (0) and with RNase-treated HAV RNA (0), under the same conditions as described in Fig. 2. Seven virus-specified proteins, P110, P70, P51, P39, P34, P24, and P14, were detected, as well as one other protein, P45. This last protein probably represents host cell actin whose synthesis was not switched off when nucleic acid was used as inoculum instead of intact virions. The appropriate expansion factor for the profile obtained with untreated HA V RNA was 1.7, and the profile obtained with RNase-treated HAVRNA was 3.1. HA V-specified proteins obtained with the untreated RNA represented 31% of the total labeled methionine incorporation.

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tein synthesis appeared to occur, at least for P24. A similar result was obtained with specimen H3. The net electrophoretic profile 2 to 6 h postinfection, obtained after whole virions were inoculated into the BME-Tris-DEAE-D pretreated cells, was identical to that in Fig. 2B (results not shown). Because of insufficient material, only a limited number of experiments could be performed with the extracted RNA. Electron microscopic examination of each RNA preparation did not reveal any particulate material, and both HAV RNA specimens were negative for HAV antigen by solid-phase radioimmunoassay. DISCUSSION This report provides the first biochemical description of HAV-specified proteins synthesized in primate cells and the first evidence that the HAV genome can function as mRNA in vitro. At least 11 virus-specified proteins (P160, P110, P70, P51, P42, P39, P34, P29, P26, P24, and P14) were regularly observed in the net electrophoretic profiles of HAV-infected cytoplasm. These profiles were remarkably similar to those of poliovirus type 1 and other picornaviruses (19). Interestingly, P34, P26, P24, and P14 showed molecular weights almost identical to those of VP1 (34,000), VP2 (25,500), VP3 (23,000), and VP4 (14,000), respectively, of HAV, determined previously in this laboratory (2; Coulepis, Locarnini, Gust, unpublished data). P39 had almost the same molecular weight as VP2+VP4 of HAV (1, 2, 19). Confirmation and identification of these proteins as structural proteins will require tryptic peptide mapping. There can be little doubt regarding the specificity of the results obtained. Virus was prepared from each of the four specimens by a method known to produce material of great purity. The final product was free from extraneous material by direct electron microscopy, and immune electron microscopy revealed only 27-nm particles complexed with anti-HAV. When analyzed by SDS-PAGE, the purified inocula were found to contain only the four structural polypeptides now recognized as typical of HAV (2, 8). No cytopathic agents were recovered from either the crude fecal samples or highly purified specimens. The same protein profile was obtained with the four different specimens of HAV purified from the feces of unrelated patients, and the results were reproducible from specimen to specimen and in repeat tests carried out on the same specimen. Polypeptide and RNA production could be prevented by preincubating the inocula with convalescent-phase sera from a chimpanzee experimentally infected with HAV, but not with sera collected before infection of the animal (3).

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The validity of the double-label and subtraction method to obtain net electrophoretic profiles of virus-specified proteins has been tested previously using Vero cells infected with flaviviruses, which, like HAV, do not switch off host protein synthesis (26); the same net profiles were obtained (i) at different times late in infection and (ii) when another cell line was used. In the experiments with HAV in Vero cells, net profiles were unchanged after treatment of cells with actinomycin D before infection or when the radioactive labels were reversed (i.e., [3H]methionine for mock-infected and [3S]methionine for infected cells; Locarnini and Westaway, unpublished data), thereby excluding the possibility of an induced response from the Vero cells. In addition, the development of specific HAAg in infected cells as demonstrated by immunofluorescence correlated very well with the detection of virus-directed protein and RNA synthesis for the same time intervals after virus inoculation. Depending on the dose, the majority of cells showed evidence of infection with HAV as a function of time postinoculation. Finally, cells infected with RNA extracted from HAV produced virtually the same net profile of virusspecified proteins as that obtained with intact HAV; again this synthesis was blocked if the RNA was pretreated with RNase. The 30S to 37S sensitive peak of HAV RNA and the 20S to 22S RNase-resistant RNA detected at 2 to 6 h postinfection are very similar to the single- and double-stranded RNAs, including the replicative complex, of the picomavirus group (7, 15, 19). Similarly, all these intracellular events were detected in the presence of actinomycin D, further strengthening the evidence that HAV is a picornavirus. The small net amount of virus-specified protein synthesis detected in cells inoculated with the RNasetreated HAV RNA could be explained if it is assumed that (i) the genetic maps of HAV and other picornaviruses are similar and (ii) P24 is VP3 of HAV. The genes coding for the structural proteins of picornaviruses are located at the 5' end of the mRNA, where translation is initiated (18). It is possible that the profile obtained from the RNase-treated sample represents translation from a protected fragment of the 5' end of the HAV genome taken up by the Vero cells. The virus-cell interaction existing between HAV and Vero cells remains undefined. Although supernatant fluids of infected cells have always been found to be negative for HAV antigens by solid-phase radioimmunoassay (N. I. Lehmann, R. C. Pringle, and I. D. Gust, unpublished data), virus-specified antigens can be detected within cells up to 7 weeks after inoculation and virus-directed protein synthesis can be

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detected up to 48 h (the last interval studied). Labeled methionine was incorporated into virusspecified proteins early postinfection (3 to 6 h), but the rate of incorporation had declined to a constant low level by 12 h. During the 48-h study period, the electrophoretic profiles of the four HAV strains did not alter significantly. However, there was a detectable shift in the amount of label in P51; i.e., the amount of P51 produced at 3 to 6 h postinfection had declined to 50% by 12 to 15 h and stayed at this level through to 48 h postinfection. The stability of the rate of virusspecified protein synthesis occurring throughout most of the 48-h study period indicates that the small amount of labeled 30S to 37S RNA detectable by analysis of cells only at 2 to 6 h (Fig. 4) probably represents synthesis of stable HAV RNA. Attempts to isolate strains of HAV in tissue culture have yielded varied results. Two patterns have so far emerged. In our studies and those by workers in the Soviet Union (M. S. Balayan, personal communication), there has been a very early appearance of HAAg, whereas other investigators (16; S. M. Feinstone and R. H Purcell, personal communication; G. Frosner and F. Deinhardt, personal communication), using different cell culture systems, have reported a gradual and late appearance of HAAg. At this stage the reasons for the discrepancies between the different research groups are unknown, but these discrepancies could reflect differences in the virus strains and cells used or the detection of different phenomena. Because penetration and true eclipse occur after HAV infection in Vero cells (cell supernatants were negative for HAAg by solid-phase radioimmunoassay after inoculation of all four specimens), it is possible that an intracellular restriction is operating (23). Studies of mengovirus restriction in MDBK cells demonstrated that the early steps of infection are efficient (absorption, penetration, and uncoating) in the restricted cell (25). However, by 4 h postinfection, there appears to be a cessation of viral RNA accumulation (25). Taylor and Chinchar (23) have suggested that the viral RNA is poorly used as a template for replication, or is not capable of being translated efficiently into viral proteins, or both. Whatever or wherever the block or defect in HAV replication is, further studies are required to identify and resolve this problem. The HAV-Vero cell infection described in this report appears to provide the first readily available system for analysis of some aspects of HAV replication and for the study of control mechanisms in restricted replication by an albeit atypical picornavirus.

J. VIROL. ACKNOWLEDGMENTS This work was supported by grants from the National Health and Medical Research Council of Australia. We acknowledge the technical services of Ruhina Patel and wish to thank Peter Cooper and Gregory Tannock for helpful discussions. We are grateful for the cooperation of the medical, nursing, and laboratory staff of Fairfield Hospital, and we thank Loris Brenton for typing the manuscript.

ADDENDUM IN PROOF The residue of P24 in the net protein profile obtained after inoculation of RNase-treated HAV! RNA (Fig. 5) has subsequently been shown to be due to an apparent, limited transfer of the 3H-methyl group (from the [methyl-3H]methionine) to tRNA (molecular weight, approximately 25,000) by internal methylation (A. K. Banerjee, Microbiol. Rev. 44:175-205, 1980); this methyltransferase reaction is unaffected by actinomycin D. The experiments described in the legends to Fig. 2 and 5 were repeated with double-label leucine (L-[4,5-3H]leucine and L-[U-'4C]leucine), and the net protein profiles were identical to those obtained with double-label methionine, except P24 was not detected after RNase treatment of HAV RNA. However, P24 was still prominent in the other net protein profiles. LITERATURE CITED 1. Abraham, G., and P. D. Cooper. 1975. Poliovirus polypeptides examined in more detail. J. Gen. Virol. 29:199213. 2. Coulepis, A. G., S. A. Locarnini, A. A. Ferris, N. L. Lehmann, and I. D. Gust. 1978. The polypeptides of hepatitis A virus. Intervirology 10:24-31. 3. Dienstag, J. L., S. M. Feinstone, R. H. Purcell, J. H. Hoofnagle, L. F. Barker, W. T. London, H. Popper, J. M. Peterson, and A. Z. Kapikian. 1975. Experimental infection of chimpanzees with hepatitis A virus. J. Infect. Dis. 132:532-545. 4. Feinstone, S. M., A. Z. Kapikian, and R. H. Purcell. 1973. Hepatitis A: detection by immune electron microscopy of a virus-like antigen associated with acute illness. Science 182:1026-1028. 5. Helentjaris, T., and E. Ehrenfeld. 1977. Inhibition of host cell protein synthesis by UV-inactivated poliovirus. J. Virol. 21:259-267. 6. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 7. Levintow , L. 1974. The reproduction of picornaviruses. Compr. Virol. 2:109-169. 8. Locarnini, S. A., A. G. Coulepis, A. A. Ferris, N. I. Lehmann, and I. D. Gust. 1978. Purification of hepatitis A virus from human feces. Intervirology 10:300308. 9. Locarnini, S. A., A. G. Coulepis, A. M. Stratton, J. Kaldor, and I. D. Gust. 1979. Solid-phase enzymelinked immunosorbent assay for detection of hepatitis A-specific immunoglobulin M. J. Clin. Microbiol. 9:459465. 10. Locarnini, S. A., A. A. Ferris, A. C. Stott, and I. D. Gust. 1974. The relationship between a 27 nm viruslike particle and hepatitis A as demonstrated by immune electron microscopy. Intervirology 4:110-118. 11. Locarnini, S. A., S. M. Garland, N. L. Lehmann, R. C. Pringle, and I. D. Gust. 1978. Solid-phase enzymelinked immunosorbent assay for detection of hepatitis A virus. J. Clin. Microbiol. 8:277-282. 12. Oppermann, H., and G. Koch. 1973. Kinetics of poliovirus replication in HeLa cells. Biochem. Biophys. Res. Commun. 52:635-640.

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13. Pagano, J. S. 1970. Biologic activity of isolated viral nucleic acids. Prog. Med. Virol. 12:1-48. 14. Pagano, J. S., and A. Vaheri. 1965. Enhancement of infectivity of poliovirus RNA with diethylaminoethyldextran (DEAE-D). Arch. Gesamte Virusforsch. 17: 456-464. 15. Perez-Bercoff, R. 1979. The mechanism of replication of picornavirus RNA, p. 293-318. In R. Perez-Bercoff (ed.), The molecular biology of picornaviruses. Plenum Press, New York. 16. Provost, P. J., and M. R. Hilleman. 1979. Propagation of human hepatitis A virus in cell culture in vitro. Proc. Soc. Exp. Biol. Med. 160:213-221. 17. Provost, P. J., B. S. Wolanski, W. J. Miller, 0. L. Ittensohn, W. J. McAleer, and M. R. Hilleman. 1975. Physical, chemical and morphological dimensions of human hepatitis A virus strain CR326. Proc. Soc. Exp. Biol. Med. 148:532-539. 18. Rekosh, D. 1972. Gene order of the poliovirus capsid proteins. J. Virol. 9:479-487. 19. Sangar, D. V. 1979. The replication of picornaviruses. J. Gen. Virol. 45:1-13. 20. Siegl, G., and G. G. Frosner. 1978. Characterization and classification of virus particles associated with hepatitis A. II. Type and configuration of nucleic acid. J. Virol. 26:48-53.

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21. Tannock, G. A., A. J. Gibbs, and P. D. Cooper. 1970. A re-examination of the molecular weight of poliovirus RNA. Biochem. Biophys. Res. Commun. 38:298-304. 22. Tannock, G. A., and J. C. Hierholzer. 1977. The RNA of human coronavirus OC-43. Virology 78:500-510. 23. Taylor, M. W., and V. G. Chinchar. 1979. Host restriction of picornavirus infection, p. 337-348. In R. PerezBercoff (ed.), The molecular biology of picornaviruses. Plenum Press, New York. 24. Vaheri, A., and J. S. Pagano. 1965. Infectious poliovirus RNA: a sensitive method of assay. Virology 27:435-436. 25. Wall, R., and M. W. Taylor. 1970. Mengovirus RNA synthesis in productive and restrictive cell lines. Virology 42:78-86. 26. Westaway, E. G. 1973. Proteins specified by group B togaviruses in mammalian cells during productive infections. Virology 51:454-465. 27. Westaway, E. G. 1977. Strategy of the flavivirus genome: evidence for multiple internal initiation of translation of proteins specified by Kunjin virus in mammalian cells. Virology 80:320-335. 28. Westaway, E. G., and B. M. Reedman. 1969. Proteins of the group B arbovirus Kunjin. J. Virol. 4:688-693. 29. Zweerink, H. J., and W. K. Joklik. 1970. Studies on the intracellular synthesis of reovirus specified proteins. Virology 41:501-518.