Characterization of hepatitis A virus structural proteins

VIROLOGY

155,732-736(1986)

Characterization

of Hepatitis A Virus Structural Proteins

VERENAGAUSS-MULLER, *J FRIEDRICH LOTTSPEICH,~

AND FRIEDRICH

DEINHARDT*

*Max vm Pettenkqfer Institute for Hygiene and Medical Microbiology, University of Munich, Pellenkofestrasse 9a D8000 Munich 2, and TGew Center, University of Munich, D-8033 Martirwied bei Munich, West Germany Received March 14, 1986;accepted August 30, 1986 HAV particles isolated from infected cells banded at buoyant densities of 1.42,1.32, and 1.20 g/ml, and distinctive protein patterns were established by gel electrophoresis and reverse phase high performance liquid chromatography. The relatively higher amounts of p30 in particles with lower buoyant densities suggest that this protein is VP0 and is part of the immature picornavirion. The protein elution profiles obtained by HPLC were virtually identical for all the HAV strains examined but differed from those of other picornaviruses. The N-terminal amino acid sequence of VP1 and VP2 was determined and aligned to the nucleotide sequence. Sequencing VP0 and VP3 was not possible, probably because the amino termini are blocked. VPl, VP3, and VP0 induced specific antibodies in rabbits. 6 1986 Academic Press, Inc.

The morphology, stability, and biophysical properties of hepatitis A virus (HAV) have led to its classification as enterovirus 72, but its biological behavior in cell culture is markedly different to other well-studied picornaviruses such as polio, Coxsackie, and encephalomyocarditis (EMC) virus (I). HAV particles of different densities (1.42, 1.32, and 1.27 g/ml, respectively) have been isolated from patients’ stools and from infected cell cultures (2). As no comparison of the peptide patterns of heavy and light particles has been made, it is still controversial whether particles of low density correspond to the defective and interfering poliovirus particles containing incomplete RNA or whether the lower density is due to an altered capsid structure. We report here the protein analysis of HAV particles of different densities using gel electrophoresis and reverse phase high performance liquid chromatography (RP-HPLC). The latter technique was also used to prepare highly purified viral proteins for amino acid sequence analysis and for use as immunogens. In order to determine the protein patterns of HAV particles of different buoyant 1 TO whom correspondence should be addressed.

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densities HAV was grown in a cell factory (Nunc) and purified by polyethylene glycol precipitation, pelleting through a sucrose cushion by ultracentrifugation, and separation in an isopycnic gradient (3). Typically the viral particles banded at densities 1.42 (I), 1.32 (II), and 1.20 g/ml (III), respectively, as is shown in Fig. 1. Each particle pool was subsequently concentrated by a second pelleting through a sucrose cushion. The proteins of each pool were separated on a 12.5% SDS gel containing 4 M urea and visualized by Coomassie blue and silver staining (inset Fig. 1). The major proteins of particle pools I and II migrated with apparent molecular masses of 33,29, and 2’7kDa. In pool II an additional protein of 30 kDa could be detected; this protein was also present, in proportionally greater amounts, in pool III. Proteins ~33, ~30, and ~2’7 were found in almost equal amounts in pool III. Apart from these prominent polypeptides, some larger molecular weight polypeptides and (in some virus preparations) proteins of 24,22, and 18 kDa were also detected. On the assumption that pool III consists of immature virions which (as in other picornaviruses) are composed of VPl, VP3, and the precursor VPO, the HAV proteins were assigned as follows: p33 to VPl, p30 to VPO, p29 to VP2, and ~27 to

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FIG. 1. Isopycnic banding of HAV (strain HM 175) in a self-forming CsCl gradient and protein composition of three particle pools analyzed on SDS-PAGE. Antigen was detected in each fraction by radioimmunoassay. Bars indicate the fractions pooled to yield particle pools I, II, and III of average densities of 1.4!2,1.32, and 1.20 g/ml, respectively. The particles were concentrated by pelleting. An aliquot of the pellets was subjected to SDS-PAGE and the proteins were visualized by silver staining. The position of HAV proteins is marked on the right.

VP3. Other authors have observed a reversed migration of VP2 and VP3, which might be explained by the use of another gel system not containing urea (4). In all particle pools, the ratio of the amounts of proteins p33 and ~2’7remained almost constant, whereas the amount of p29 was less and that of p30 was greater in the particle pools with lower buoyant densities. This could indicate a precursor-product relationship between p30 and p29. Following the method of Heukeshoven (5) each particle pool was further analyzed by RP-HPLC, making use of the tech-

nique’s rapid separation capacity combined with a high recovery rate (Fig. 2). Primary disintegration of HAV particles was achieved by incubation in 4 M guanidinum hydrochloride (GuHCl) prior to injection. For optimal separation of viral polypeptides, an acetonitrile gradient of 20-40s in 60% formic acid was applied. Under these conditions, particle pool I was resolved into two protein peaks (21.5 and 22.6). As shown by SDS-PAGE, peak 21.5 contained both VP1 (~33) and VP2 (p29) which were identified by amino acid sequence analysis (see below). The protein of peak 22.6 was VP3

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FIG. 2. RP-HPLC separation of HAV (strain MBB) proteins of particle pools I, II, and III and protein composition of the peak fractions. Virus particles of different buoyant densities (see Fig. 1) were prepared from one HAV-infected cell factory and disintegrated by incubation in 4 M GuHCl and 1 mMdithiothreito1 for 10 min at 50°. The inset shows the protein analysis of an aliquot of the peak fractions by SDS-PAGE in 4 Murea. Numbers give time of peak elution after injection. Start of the gradient was 5 min after sample injection. The column was developed with a linear gradient from 20-4055, acetronitrite in 60% formic acid over 30 min. AUF& absorption unit full scale.

(~27) which was proven immunologically (see below). Proteins of particle pool II also eluted in two major peaks. As is shown in the inset, the peak eluting at 20.9 min consists of two proteins, VP1 (~33) and VP2

(p29). Peak 22.0 is made up of VP3 (~27). The minor peak at 24.0 min is VP0 (p30), peak 18.6 is ~72 and ~24, peak 15.7 contains p22 and p40. Similarly, all HPLC fractions of particle pool III were tested by SDS-

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PAGE. Peaks 20.6,21.7, and 23.7 were designated as VP1 (p33), VP3 (p27), and VP0 (p30), respectively. Sometimes, VP0 was contaminated by VP3. In contrast to the first peaks of Fig. 2, showing particle pools I and II, peak 20.6 of pool III contains almost exclusively VP1 and no VP2. All three proteins of pool III were eluted in almost equal amounts which agrees well with the 1:l:l stoichiometry of picornaviral proteins VPl, VP3, and VPO. The comparison of the RP-HPLC protein elution profile of different HAV (MBB, GBM, HM175, Janda) strains revealed an almost identical pattern among the strains tested which was markedly different from that of polio and EMC virus (not shown). The similarity of the chromatographic pattern of the various HAV strains we examined supports the notion that they are members of the same serogroup. Preparative amounts of the individual viral peptides could be obtained by RPHPLC and all peak fractions of particle pools II and III in Fig. 2 were lyophilized and subjected to gas phase amino acid analysis and sequencing (6). The proteins of peak 21.7 (VPO) and 23.7 (VP3) of pool III could not be sequenced, presumably because they are blocked at the N terminus. The N-terminal sequence of eight amino acids for the protein of peak 20.6 (pool III) is VGDDSGGF which had already been shown to be the N-terminal sequence of VP1 (7). When peak 20.9 of pool II was used for sequencing, an additional sequence (DIEEEQMI) which represents that of VP2 was detected next to the N-terminal sequence of VPl. The N-terminal sequence of VP2 could be determined only in a mixture with VP1 because VP2 coelutes with VP1 under the applied reverse-phase conditions. Based on our sequence information as well as on published nucleotide sequence data, a small peptide ought to be localized at the 5’ end of the coding sequence (7-9). In the genomes of other picornaviruses this domain represents VP4. In our virus preparations, analyzed by RP-HPLC or SDSPAGE, no peptide corresponding to VP4 could be detected. Since neither tyrosine nor tryptophan (amino acids responsible

for the absorption of proteins at 278 nm) is coded for by this part of the HAV genome, it is not surprising that this peptide escaped detection by HPLC. Furthermore, the peptide is too small to be identified by the applied SDS-PAGE conditions since the resolution is not sufficient for peptides of this size. For the preparation of pure protein fractions as immunogens, light virus particles (density 1.20 g/ml) of strain MBB (see Fig. 2111)were disintegrated and the proteins VPl, VP3, and VP0 were separated by RP-HPLC. The particle pools I and II were unsuitable for antibody production because VP1 and VP2 eluted in the same peak. Approximately 25 pg each of protein VPl, VP3, and VP0 were lyophilized and injected into rabbits following a standard immunization scheme. In addition, complete native virions (pool II) were injected into rabbits. To test for specific antibodies, an immunoblot assay was performed (Fig. 3) in which purified viral proteins were separated on 12.5% SDS-gels containing 4 M urea and subsequently transferred to nitrocellulose (IO). The antiserum raised against VP1 and diluted 1:lOOOrecognized ~33, anti-VP0 antiserum reacted with p30 and p29, and anti-VP3 antiserum with ~27 (minor reaction with ~30). The reactivity

a

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FIG. 3. Immunoblot analysis of sera of rabbits immunized with RP-HPLC purified VP1 (a), VP0 (b), VP3 (c), and with complete virions (d) (HAV strain MBB). Purified HAV was separated by SDS-PAGE and the proteins were transferred to nitrocellulose membranes. After incubation of the individual strips with the respective antisera dilutions, specific binding was probed with horseradish peroxidase-conjugated anti-rabbit Ig immunoglobulin and a color reaction with diaminobenzidin and HzOz.

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of the antisera with whole virus was tested in a competition assay (HAVAB kit, Abbott Laboratories). When the serum samples were assayed under a high competition ratio (20 ~1of test serum to 180 ~1of iodinated human anti-HAV), no significant response of the monospecific antisera to whole virus could be detected. A weak reactivity of anti-VP1 was measured when a competition ratio of 1:l was used. Furthermore, all three antisera reacted positively in an immunofluorescence assay with HAV-infected cells but none had neutralizing potency in cell culture assays. The antibody response which was elicited in rabbits by inoculation of complete virions, however, was positive both in the competition assay and in the neutralization assay whereas presera were always negative. Other reports of neutralizing activity of either monoclonal antisera against VP1 or polyclonal antisera directed against VPl, VP2, and VP3 (II,&?) used other animal species and elicited antibody responses of varying quality, as shown as reactivity in the immunoblot, immunoprecipitation, neutralization, and competition assay. Although VP1 and VP2 were not separated by RPHPLC, this method yielded preparative amounts of individual viral proteins which were suitable for amino acid sequence determination and for immunization. At present experiments are under way to separate VP1 and VP2, to obtain VP2 in a pure form. ACKNOWLEDGMENTS The authors thank Dr. Peter Miiller for his generous help in running the HPLC and for helpful discussions.

We are grateful to Ms. I. J%ckel for excellent technical assistance and to Ms. S. Stefanitsch for preparation of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft Grant Ga 304/1-l. REFERENCES 1. FR~SNER, G. G., DEINHARDT, F., SCHEID, R., GAUSSMILLER, V., HOLMES, N., MESSELBERGER, V., SIEGL, G., and ALEXANDER, J. J., Infection 7, 303-306 (1979). 2. COULEPIS, A. G., LOCARNINI, S. A., WESTAWAY, E. G., TANNOCK, G. A., and GUST, I. D., Intwvirology 18,107-12’7 (1982). 3. SIEGL, G., and FR~SNER, G. G., J. ViroL 26,40-47 (1978). 4. WHEELER, C. M., ROBERTSON,B. M., VAN NEST, G., DINA, D., BRADLEY, D. W., and FIELDS, H. A., J. Viral. 58,307-313 (1986). 5. HEUKESHOVEN, J., and DERNICK, R., Chrmatc+ gruphia 19,95-100 (1985). 6. LOTTSPEICH, F., J. Chromatogr. 26,321-327 (1985). 7, LINEMEYER, D. L., MENKE, J. G., MARTIN-GALLARDO, A., HUGHES, J. V., YOUNG, A., and MITRA, S. W., J. fir01 26,247-255 (1985). 8. BAROUDY, B. M., TICEHURST, J. R., MIELE, T. A., MAIZEL, H. J. V., PURCELL, R. H., and FEINSTONE, S. M., Proc. Natl. Ad Sci. USA 32, 2143-2147 (1985). 9. NAJARIAN, R., CAPUT, D., GEE, W., POTTER, J. J., RENARD, A., MERRYWEATHER, J., VAN NEST, G., and DINA, D., Proc. Natl. Auui Sci USA 82, 2627-2631 (1985). 10. TOWBIN, H., STAEHLIN, T., and GORDON, J., Proc Nat1 Acad. Sci USA 76,4350-4354 (1979). 11. HUGHES, J. V., STANTON, L. W., TOMASSINI, J. E., LONG, W. J., and SCOLNICK, E. M., .J. viral. 52, 465-473 (1984). 12. HUGHES, J. V., BENNET, C., STANTON, L., LINEMEYER, D. L., and MITRA, S. W., In “Vaccines ‘85,” pp. 255-259. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1985.