Complete nucleotide sequence of a cell culture-adapted variant of hepatitis a virus: Comparison with wild-type virus with restricted capacity for in vitro replication

VIROLOGY

163,

299-307

(1988)

Complete Nucleotide Sequence of a Cell Culture-Adapted Variant of Hepatitis A Virus: Comparison with Wild-Type Virus with Restricted Capacity for in vitro Replication ROBERT W. JANSEN,* Departments

Iof *Medicine

JOHN E. NEWBOLD,t

and tMicrobiology Chapel Received

September

AND

STANLEY

and Immunology, The University Hill, North Carolina 27599 28, 1987; accepted

December

M. LEMON*+’

of North

Carolina

a? Chapel

Hi//,

8. 1987

To determine the molecular changes associated with adaptation of hepatitis A virus (HAV) to growth in cell culture, the genome of a cell culture-adapted variant of HM175 strain HAV (~16 HM175, 16th in vitro passage level) was molecularly cloned and the complete nucleotide sequence of the virus was determined. Compared with wild-type virus, p16 HM175 replicates efficiently in monkey kidney (BS-C-1) cells (approximately 58 RNA-containing particles per one infectious unit, compared with 2.4 x 1 O5 for wild-type HM175). The nucleotide sequence of pl6 HM 175 revealed a total of 19 mutations from the wild-type genome, including 5 mutations in the 5’ nontranslated region, 1 mutation in the 3 nontranslated region, and 13 mutations predicting 8 changes in the amino acid sequences of HAV proteins. Only one amino acid substitution occurred among the capsid proteins (VP2), while others involved proteins 2A, 28,2C, VPg, and 3Dpol. When the sequence of p16 virus was compared with that reported previously for an independently isolated, cell culture-adapted v,ariant of HM175 virus (J. I. Cohen et a/., (1987). Proc. Nat/. Acad. Sci. USA 84, 2497-2501), there were three identical mutations in nontranslated regions of the RNA, and four mutations involving identical amino acids in proteins VP2, 2B, and 3Dpol. The distribution of these mutations within the genome suggests that changes in RNA replication may be of primary importance in adaptation of the virus to growth in vitro. These data are thus helpful in understanding the molecular basis of adaptation of HAV to cell culture and, since attenuation frequently accompanies adaptation of virus to growth in cell culture, may be of benefit in planning for attenuated vaccine development. o 1988 Academic

Press.

Inc.

INTRODUCTION

dented by a reduction in the ability of the virus to induce liver injury in both experimentally challenged primates and man (Provost et a/., 1982; R. A. Karron, R. Daemer, J. Ticehurst, E. D’Hondt, H, Popper, K. Mihalik, J. Phillips, S. Feinstone, and R. H. Purcell, manuscript submitted for publication). Extensively passaged virus may even be incapable of replication in viva, as no clinical evidence of infection and no measurable antibody response has followed parenteral challenge of humans with very extensively passaged virus (Provost et a/., 1986). However, because some virus variants that are well adapted to growth in cell culture retain nearly unaltered virulence in some primate species (e.g., owl monkeys) (Lemon et al., 1987b), attenuation and cell culture adaptation represent separate although closely related phenotypic characteristics of HAV. The molecular mechanisms underlying either adaptation of HAV to growth in cell culture or attenuation of HAV remain unknown. Because adaptation of HAV to growth in cell culture results in profound changes in the biologic characteristics of the virus and may be associated with a reduction in virulence in several different species of primates, we have cloned the genome of a cell culture-adapted variant of HM175 strain HAV and have determined its complete nucleotide sequence. We have compared the sequence of this virus

Hepatitis A virus (HAV) is a human picornavirus with a worldwide distribution (Lemon, 1985). At present, no vaccine is available for prevention of infection with this medically important virus, although attenuation of HAV has been shown to result from extended in vitro passage of virus and has been proposed as an approach to the development of attenuated vaccines (Provost et a/., 1982, 1986). Wild-type HAV, recovered from feces or liver of Infected prirnates, replicates slowly and to Iow titers in cell culture. Most strains of HAV do not induce cytopathic effeicts in cell cultures, and persistent infection is characteristic of this virus system. With successive passages, however, the virus becomes progressively adapted to growth in vitro (Binn et a/., 1984; Frijsner et a/., 1979; Provost and Hilleman, 1979). Adaptation to growth in cell culture results in a shortening of the interval between inoculation of cultures and maximum virus yield as well as increases in the yield of virus, and appears to be a multi-step process. Passage of virus in vitro has also been associated with attenuation of virulence as evi-

’ To whom cine/Infectious NC 27599.

requests for reprints Diseases, CB#7030,

should be addressed at: MediBurnett-Womack, Chapel Hill,

299

0042.6822/88

$3.00

CopyrIght 0 1989 by Academic Press. Inc. All rights of reproduction I” any form reserved.

300

JANSEN,

NEWBOLD,

to that reported previously for its wild-type parent (Cohen et a/,, 1987b), and have determined that adaptation of this virus variant to growth in vitro was associated with a limited number of mutations. MATERIAL

AND

METHODS

Cells HAV was propagated in vitro in continuous African green monkey kidney (BS-C-1) cells under conditions described previously (Binn et al., 1984).

LEMON

the detection of new clones containing p16 virus cDNA by colony hybridization. Additional probes were prepared subsequently from pl6 HM 175 cDNA clones as described below. 32P-labeled probes were synthesized from denatured insert DNA fragments by random priming with calf thymus oligodeoxynucleotides and E. co/i DNA polymerase I. Labeled DNA was separated from the reaction mixture by chromatography through Sephadex G-50 (Pharmacia, Piscataway, NJ), and used directly for hybridization. Quantitation

Virus Virus was initially isolated in primary African green monkey kidney cells (AGMK) from an homogenate of liver tissue obtained from a marmoset inoculated with wild-type HM 175 strain HAV (6th marmoset-passage), a gift of S. M. Feinstone and R. H. Purcell, Nationa! Institute of Allergy and Infectious Diseases (Bethesda, MD). The origin of this strain of HAV has been described (Gust et al., 1985). Virus was adapted to growth in vitro as described previously (Binn et al., 1984), and after 10 AGMK passages was passed once in continuous green monkey kidney cells (BS-C-1). Virus was then twice plaque-purified (Lemon and Jansen, 1985) and amplified at the 16th in vbo passage level (~16 HM175 virus) for isolation of viral RNA. Wild-type HM175 virus, employed for studies determining the efficiency of wild-type virus replication in vitro, was recovered from the feces of a New World owl monkey (Aotus trivirgatus) inoculated intravenously with a human fecal suspension containing HM175 strain HAV (LeDuc et al., 1983). Detection

and quantitation

of HAV

Viral antigen was detected in infected cell cultures by solid-phase radioimmunoassay (Lemon et al., 1982). Infectious, cell culture-adapted HAV was quantified by an indirect plaque assay (radioimmunofocus assay) (Lemon and Jansen, 1985). Results are presented as radioimmunofocus-forming units (RFU). cDNA

AND

hybridization

probes

Escherichia co/i cultures containing recombinant pBR322 plasmids pHAVLB1 307, pHAVLB228, and pHAVLB207 with inserts of cDNA complementary to portions of the wild-type (third marmoset passage) HM 175 strain genome were the gift of H. R. Ticehurst and R. H. Purcell of the National Institute of Allergy and Infectious Diseases (Ticehurst et a/., 1983). Pstl digestion fragments of plasmid DNA were prepared and purified as previously described (Jansen et a/., 1985), yielding six different cDNA fragments spanning various regions of the genome which were used as probes for

of viral RNA

Viral RNA was quantified by cDNA-RNA hybridization. Serial dilutions of virus were blotted to nitrocellulose paper and compared with serial dilutions of plasmid DNA of known concentrations (Jansen et al., 1985). Conditions included hybridization at 42’ in the presence of 50% formamide, 1 X Denhardt’s solution, 5X SSC, 0.1% SDS, and 100 /*g/ml denatured calf thymus DNA. Probe was boiled for 3 min immediately before dilution into hybridization solution (2 X 1 O6 cpm/ml). Hybridization was for 22-36 hr, following which the nitrocellulose was washed twice with 2X SSC, 0.1% SDS at room temperature for 15-30 min per wash, and twice with 0.1 X SSC, 0.1% SDS at 52” for 15-30 min per wash. Virus purification Chloroform-extracted pl6 HM175 virus was inoculated into four 890-cm2 roller bottles containing confluent cultures of BS-C-l cells. Media were replaced weekly. Twenty days after inoculation, media were removed, cell sheets were washed twice with 10 ml Hanks’ balanced salt solution (HBSS), and 25 ml of lysis buffer (10 mM Tris-HCI, pH 7.5, with 0.1% Nalaurylsarcosine) was added to each bottle. The cells were passed through three freeze-thaw cycles (-20 to 37”) and the resulting lysate was drawn three times through a 19-gauge needle to reduce viscosity. The lysate was clarified by centrifugation at 17,000 g for 1 hr at 4”, followed by centrifugation at 193,000 g for 16.5 hr at 4” in a Beckman 50.2 rotor (Beckman Instruments, Palo Alto, CA). Pellets were resuspended in water and briefly sonicated, and CsCl was added to bring the solution to a density of 1.32 g/ml. The CsCl suspension was centrifuged for 22 hr at 218,000 g at 4” in a Beckman SW40 rotot (5 ml/tube), and 0.125 ml fractions were collected from the bottom of the resulting gradients. Fractions containing virus detectable by radioimmunoassay (fractions 23-29) were pooled. The CsCl fractions were diluted in water, and virus was recovered by centrifugation at 218,000 g for 3 hr at 4”. The viral pellet was frozen at -70”.

CELL

CULTURE-ADAPTED

HAV RNA Gradient-purified virus was incubated at 37” for 3 hr in 20 mM Tris (pH 7.5) .with 10 mM EDTA, 50 mM NaCI, 1 mg/ml proteinase K (Boehringer-Mannheim, Chicago, IL), and 1% sodium dodecyl sulfate (SDS). The suspension was extracted twice with phenol/chloroform-isoamyl alcohol, and the aqueous phase was brought to 0.3 M sodium acetate. RNA was precipitated overnight by the addition of 2.5 vol ethanol at -7O”, followed by centrifugation at 108,000 g for 1 hr at 4”. All subsequent RNA manipulations employed diethyl pyrocarbonate (DEPC)-treated buffers and plasticware. Rate-zonal

centrifugation

of HAV RNA

RNA was resuspended in 25 ~199% DMSO, 10 mM Tris, pH 7.5, and heated at 37” for 5 min. After addition of 3 vol ETS buffer (1 mM EDTA, 10 mMTris, pH 7.5, 0.5% SDS), the RNA ‘was heated at 65” for 1 min, rapidly cooled to O”, and loaded onto a 4.5-ml gradient of 15-300~ sucrose in ETS with 0.1 M NaCI. Gradients were centrifuged at 1!30,000 g at 25” for 3 hr in a Beckman SW50.1 rotor, and 0.2 ml fractions were collected from the bottom. Aliquots of each fraction were examined by Northern blot analysis for HAV RNA following electrophoresis in a 1% agarose gel containing 2.2 M formaldehyde (Maniatis et al., 1982). RNA present in gradient frac:tions containing predominantly full-length RNA was precipitated with ethanol as above, and stored at -70”. cDNA

synthesis

and cloning

cDNA-RNA hybrid molecules were cloned into the Pstl site of plasmid pBR322 as described by Cann et a/. (1983) and Stanway et al. (1983) for other picornaviruses. First-strand cDNA was synthesized from sizeselected VII-al RNA using AMV reverse transcriptase under conc,itions described by Ticehurst (Ticehurst et a/., 1983). Both oligo(dT),n-,s and random calf thymus DNA oligor-lucleotide fragments were used as primers in separate reactions. cDNA-RNA hybrid molecules were extracted with phenol-chloroform, passed through a !$ephadex G-100 (Pharmacia) column, and precipitated in ethanol. Hybrid molecules were dCtailed with terminal deoxynucleotidyl transferase (New England Nuclear, Boston, MA), extracted with phenol-chloroform, and annealed to pBR322 DNA which had been linearized by restriction at the Pstl site and tailed at the 3’ ends with dG (dGN-pBR322-dGA”, New England Nuclear). Recombinant DNA was used to transform competent E. co/i HB 10 1 cells (Bethesda Research Laboratories, Gaithersburg, MD). Resulting tetracycline-resistant colonies were screened by in situ colony hybridization using a mixture of cDNA

HAV

301

probes. In several cloning experiments, over 300 E. co/i clones containing plasmids with stable HAV inserts were isolated. Characterization

of pl6

HM 175 cDNA

clones

cDNA inserts in selected clones were sized by agarose gel electrophoresis of plasmid DNA “mini-preps” (Maniatis et al., 1982) and assigned an approximate genomic location based on the results of slot blot hybridization with wild-type cDNA probes. These studies suggested the presence of substantial rearrangements (i.e., deletions) of cDNA in only one clone (as indicated by hybridization with probes derived from noncontiguous regions of the wild-type HM175 genome). Clones with overlapping inserts were further identified by hybridization of insert cDNAs, and by selected mapping of restriction endonuclease sites. Precise mapping of cDNA inserts was accomplished by dideoxynucleotide sequencing of plasmid DNA using pBR322 Pstl site-specific oligonucleotide primers (Pharmacia). cDNA

sequencing

Insert fragments from pl6 cDNA clones were subcloned into the phage vector M13mp8 or M13mp19 and subjected to rapid sequencing using the dideoxynucleotide method of Sanger @anger et a/., 1977). As physical mapping indicated few differences between pl6 virus cDNA and wild-type cDNA in terms of major restriction sites, a sequencing strategy was adopted which involved the subcloning and subsequent sequencing of overlapping restriction fragments. Where necessary, additional sequence information was obtained by direct sequence analysis of plasmid DNA using HA&specific oligonucleotide primers prepared in the laboratory of C. Hutchison of the University of North Carolina or provided as a gift by J. Cohen of the National Institute of Allergy and Infectious Diseases. The derived sequence for ~16 HAV cDNA was assembled with Micro-Genie software (Beckman) and compared with that reported by Cohen et al. for wildtype (3rd marmoset passage) HM 175 virus (Cohen et al., 1987b) and p35 chimpanzee-attenuated HM 175 (Cohen et a/., 1987a). Except where noted, mutations from the wild-type virus sequence were considered present only when found in at least two independent cDNA clones derived from pl6 virus. RESULTS Cell culture

adaptation

of p16 HM175

virus

The efficiency of replication of p16 HM175 virus in BS-C-l cells was compared with that of wild-type virus by determining the proportion of virus particles capa-

302

JANSEN,

NEWBOLD,

AND

LEMON

FIG. 1. HAV radroimmunofocus assay (Lemon and Jansen, 1985) of BS-C-1 cells inoculated 14 days prevrously with (A) pl6 HM175 virus, (B) wild-type HM175 virus (Ip owl monkey fecal virus), and (C) control, uninoculated cells. Quantitative cDNA-RNA hybridization indicated that the inoculum for (A) was approximately 1.2 X 1 O3 RNA-containing particles, and for (B) was approximately 3.3 X 10’ RNA-containing particles,

ble of inducing foci of infection detectable under agarose overlays 2 weeks after inoculation of cell cultures (Fig. 1). The wild-type virus used for this experiment was a suspension of feces obtained from an owl monkey (Aotus rrivirgatus) experimentally infected with HM 175 virus from human stool (LeDuc et al., 1983). The number of RNA-containing particles was estimated in both wild-type and pl6 cell culture inocula by quantitative cDNA-RNA hybridization (Jansen et al., 1985). Foci of viral replication, identified by staining infected cell sheets with radiolabeled polyclonal human antibody (Lemon and Jansen, 1985) were considerably larger in cells inoculated with pl6 virus than in cells inoculated with wild-type virus (Fig. l), indicating more efficient spread of the pl6 virus to adjacent cells. Moreover, cDNA-RNA hybridization results (not shown) indicated that the p16 virus inoculum contained approximately 58 genome copies (i.e., RNAcontaining particles) per RFU, compared with 2.4 X lo5 particles per RFU in the wild-type virus inoculum. Thus, there was an approximately 4000-fold difference in the ability of the cell culture-adapted (~16) virus and wild-type virus to initiate foci of replication in BS-C-1 cells. Molecular

cloning

of pl6

HM175

virus cDNA

The cDNA-RNA cloning strategy adopted permitted clones bearing stable HAV cDNA inserts to be obtained from minimal quantities of viral RNA. This approach was taken in order to limit the number of virus passages between plaque purification and isolation of viral RNA for molecular cloning. Clones derived from cDNA synthesized with oligo-dT,,-,, as primer were nearly always restricted to the 3’ terminus of the genome, while clones of cDNA synthesized with random oligonucleotide primers were randomly distributed within the genome. Several clones containing 5’ terminal sequences were identified, reflecting one advantage of the cloning of cDNA-RNA hybrid molecules (Cann et a/., 1983; Stanway et al., 1983). Clones with

overlapping inserts spanning the HAV genome were identified by hybridization, limited restriction mapping, and direct sequencing of plasmid DNA (Fig. 2) and selected cDNA insert fragments were subcloned into Ml 3 vectors for sequencing. The sequence of the complete genome was determined, with over 95% of the sequence confirmed in sequencing reactions involving both strands of cDNA. Mutations

in cell culture-adapted

pl6

HM175

A total of 19 mutations (20 base changes) was evident when the sequence of pl6 HM175 was compared with that previously reported for wild-type HM 175 virus (Cohen et al., 1987b), (Table 1). Except where indicated in Table 1, each of these mutations was documented in at least two independent cDNA clones derived from pl6 virus. Mutations clustered in the 5’ nontranslated region of the genome, and in the P2 and P3 regions (Fig. 3). Altogether, there were 13 mutations in the large open reading frame, predicting eight amino acid substitutions in the proteins of the pl6 virus (Table 1). (I’) 5’ nontranslated RNA. There were five mutations (six nucleotide changes) in the 5’ nontranslated RNA of the p16 virus genome. At the 5’ terminus, there was the addition of a U, confirmed in multiple cDNA clones, and between base positions 203-207 a UU deletion

5’ Q 119 112 le.6 194 I 227 2” l&l 2 172 0. 201 63 139 57 12

9

Q

Q!

4 3

2

P 4

Q 6

5

: 7kb

-3’

4 Q Q P -, P Q Patt Hindlll

FIG. 2. Physical HM 175 cDNA.

4 Sacl

9 Nail

map of pHAVcH

clones

containing

inserts

of pl6

CELL TABLE COMPARISON

OF WILD--rYPE

CULTURE-ADAPTED

1

AND

p16 HM175

CELL

CULTURE-ADAPTED

Amino

Region

wt pos

wt

~16

5’

0 8 152 203-7 687

G A ULJ U

U A G ddb G

964 1742

A G

G As

2A 28 28 28 2c 2c

3281 3711 3867 3889 4049 4426

A G U C C A

G A C” UB UB C

3A

5204 5255 6148 6216 6522

G A A U U

A U G C A

7430

A

G

Pas

acid

Wt

~16

54

LYS

Au

58 13

He

Met Asn

72

Ala

Val

144

LYS

Thr

11 67

Gln Asp

GIY

192

Ser

Thr

Pl VP2 VP3 P2

P3 w 3D~ol 3Dpol 3’

303

VP3 coding region (position 1742), but did not predict a change in the amino acid sequence of the capsid protein. This silent “mutation” has been found in all cell culture-adapted HM175 cDNA clones reported to date (Cohen et a/., 1987a; Ross et a/., 1986) and may in fact represent the true wild-type sequence, as the base in question has been determined in only a single wild-type cDNA clone (Cohen et al., 1987a). In addition, there was a mutation predicting a lysine to arginine substitution in amino acid residue 54 of VP2 (map position 964). This conservative substitution was the only predicted change in the capsid structure of the pl6 virion. (iii) P2 region. There were six mutations in the P2 region, predicting four amino acid substitutions. According to the HAV polyprotein cleavage sites proposed by Cohen et al. (1987b), these changes included one amino acid substitution in protein 2A, two in protein 2B and one in protein 2C (Table 1). (iv) P3 region. In the P3 region, there were five nucleotide substitutions predicting three amino acid substitutions. There were no predicted amino acid substitutions in proteins 3A or 3Cpro, the putative HAV protease. In the VPg (3B) protein (Weitz et a/., 1986) however, there was a glutamine to histidine substitution at amino acid 11. Within the proposed polymerase coding region there were three mutations, two of which predicted amino acid substitutions in 3Dpol. These substitutions included a change from aspartic acid to glycine at residue 67 and from serine to threonine at residue 192. (v) 3’ nontranslated RNA. A single mutation was present in the 3’ nontranslated RNA, involving an A + G substitution at map position 7430.

VIRUS

Nucleotide

HAV

His

a Identified In only one clone from pl6 virus: all other mutations confirmed in ;at least two clones. The mutation at 1742 has been confirmed by direct sequencing of viral RNA (unpublished data). b d, Deletion.

(map positions refer 1:o published wild-type HM175 sequence) (Cohen et a/., 1987b). Thus, the length of the 5’ nontranslated region was shortened by one base from that of the wild-type genome. The remaining changes included a G --, A at position 8, an A + G at position 152, and a U + G at position 687. (ii) Pl region. The capsid-encoding region was relatively free of mutations compared with other regions of the genome. One nuclleotide substitution from the reported wild-type sequence was present in the putative

Comparison of pl6 virus with adapted HM175 variants

other cell-culture

The complete sequence of an HM175 variant that had been adapted to growth in African green monkey kidney cells was recently reported by Cohen et al. (1987a). This p35 HM175 variant was isolated directly from human feces and adapted to growth in vitro

-PlpP2-P3-

2

I

VP3

3 VPI

4 124.

1261

:

GTC A$;

T2::(2

:

0 A* NV

2C

A\ I

h\\ \I !i WT PI6

5

i .

‘Gt ::

FIG. 3. Genomic location of mutations present in pl6 HM 175 virus. The sequence of pl6 HM 175 virus was compared with that reported

CA T C :

6

7

1343QroI

A

G A AT a Ii

Putative protein assignments for wild-type HM 175 (three

kb

3Dpl

\ I

ATT CCA E

3’

lt

:

are as described by Cohen marmoset passages).

et a/. (1987a).

304

JANSEN.

NEWBOLD.

HM175 human feces Gust et al.. 1965 I \

rnain~~ei

p6

h(ploy&, p17 (17 AGMK)

FIG. 4. Passage history and relation between variants of HM 175 virus for which partial or complete nucleotide sequences have been determined. Variants for which the sequence is known are enclosed in boxes. Literature citations for these HM 175 variants are as follows: wild-type HM175 (human feces) Gust et a/. (1985); wild-type HM 175 (3p marmoset) Ticehurst ef al. (1983) Cohen et al. (1987b); p16 HM175, this paper; p35 HM175. Cohen et a/. (1987a); p59 HM175, Ross eta/. (1986).

without prior marmoset passage. It has been shown to be attenuated in chimpanzees, and to a lesser degree in marmosets (R. A. Karron, R. Daemer, J. Ticehurst, E. D’Hondt, H. Popper, K. Mihalik, J. Phillips, S. Feinstone, and R. H. Purcell, manuscript submitted for publication). It is likely that this virus is significantly more attenuated than the pl6 variant described in this paper (see Discussion). In addition, the partial sequence of a third variant (p59) of HM175 virus has been reported by Ross et al. (1986). The p59 virus had been adapted to growth in monkey kidney cells following six marmoset passages, in vivo passages shared in common with the wild-type virus (first three marmoset passages) cloned by Ticehurst et al. (Cohen et al., 198713) and the pl6 virus (all six marmoset passages). The passage history of these three cell cultureadapted HM175 variants, each of which has been independently adapted to growth in primary or continuous monkey kidney cells, is depicted in Fig. 4. Mutations common to these independently isolated viruses can be expected to have special relevance to the ability of virus to replicate in cell culture. A comparison of the available sequence data shows that HM175 variants sequenced at the 16th, 35th, and 59th passage levels have 6, 7, and 8 base substitutions, deletions, or additions respectively, within the 5’ nontranslated region of the viral genome. (The 5’terminal 29 bases have not been reported for the p59 virus,) The p16 and p35 sequences share a common mutation at base position 152 (A + G) and a common U deletion between 203 and 207. This latter change is also found in p59 virus, and is the only 5’ mutation common to all three variants. The U + G substitution

AND

LEMON

found at map position 687 in pl6 virus is also present in the p59 variant, but is not found in p35 virus. Therefore, this mutation may have occurred during the last three marmoset passages which the pl6 and p59 viruses shared in common (Fig. 4). The region representing map positions 124 through 207 appears especially prone to develop mutations during in vitro passage of the virus (Jansen et al., 1988). Within this region, 3.6% of base residues are altered in pl6 virus, as are 8.4% in the p35 sequence, and 2.4% in the p59 sequence. This mutational “hot spot” also contains two of the three 5’ region mutations (map positions 152 and 203-207) common to two or more variants. The full-length sequences of the p16 and p35 HM 175 variants are remarkably concordant in terms of the distribution of mutations within the genome and share a striking number of identical or very similar mutations In addition to the two identical mutations in the 5’ nontranslated region, there are four mutations involving identical amino acids in the proteins of pl6 and p35 viruses (Table 2). This represents 50% of the predicted amino acid substitutions present in pl6 virus, and 33% of those present in p35 virus. In the Pl region, the single mutation (lysine --* arginine) in VP2 of pl6 virus is also present in p35 virus; a second mutation found in VP1 of p35 virus is not present in p16 HM175. In the P2 region, each variant has one mutation in protein 2A, and two in protein 28 (including a common alanine --* valine substitution at residue 72). The greatest difference between the two viruses is in the 2C protein, in which there is one predicted amino acid substitution in pl6 virus and four substitutions in the p35 variant. Neither virus has amino acid substitutions in proteins 3A or 3Cpro, but each virus has one substitution (at different sites) among the 23 amino acids of VPg (protein 3B). Of particular interest, there are two predicted amino acid substitutions in 3Dpol of both pl6 and p35 viruses. These substitutions involve identical amino acid residues, including an identical substitution (serine + threonine) at residue 192, and different mutations involving aspartic acid 67. In ~16, a mutation at map position 6148 predicts a substitution of this residue with glycine, while in p35 virus a mutation in the adjacent nucleotide (6 147) predicts that this amino acid is replaced with asparagine. Last, a single, common mutation (A --, G at position 7430) is present in the 3’ nontranslated region of both variants. With the possible exception of an apparent silent mutation at map position 1742 that is also present in both cell culture-adapted viruses (not shown in Table 2), these changes do not reflect errors in the reported sequence of wild-type HM175. The wild-type sequence at the seven other sites of common mutations indicated in Table 2 was confirmed by Cohen e2 al. in at least two

CELL TABLE

CULTURE-ADAPTED

2

MUTATIONS IN INDEPENDENTLY ISOLATED CELL CULTURE-ADAPTED HM 175 VARIANTS

Region 5'

Nucleotide or amino acid positiOns

wt

~18

0 8 124 131-134 152 203-20.7 687

G U UUUG A uu U

U A

G dd” G

P35

C dddd Gb dUb

Pl VP2 VP1

54 273

LYS Glu

2A 2A 28 28 28 2c 2c 2c 2c 2c

30 58 13 72 82 29 64 76 144 190

Asn Ile Asp Ala GIY LYS Glu Phe LYS Val

VP!3 m 3Dpol 3Dpol

4 11 67 192

His Gln

His

Asp Ser

GIY Thr

Asnb Thr’

A

G

Gb

Arg

Argb Val

P2 Ser Met Asn Val

Valb Ala Met LYS Ser

Thr Ile

P3

3'

7430

Tyr

a Based on polyprotein cleavage sites proposed by Cohen et al. (1987a). * Similar or identical mut,stions (only mutations in 5’ and 3’ nontranslated RNA, and nonsilent mutations in the translated regions of the genome, are listed). The p35 sequence was determined by Cohen et a/. I 1987). cd. Deletion.

independent 1987a).

wild-type

cDNA

clones

(Cohen

et a/.,

DISCUSSION The mutations we identified in the p16 variant of HM 175 virus provide information that will be useful in understanding the molecular basis of adaptation of HAV to cell culture. This variant is highly adapted to replication in monkey kidney cells in vitro, as evidenced by a substantially lower particle to infectious unit ratio (58 vs 2.4 X 1O5 for wild-type virus) and larger replication foci in monolayers of BS-C-1 cells when compared with wild-type virus (Fig. 1). However, the degree of attenuation that may have accompanied the adaptation of p16 virus to cell culture has not been

HAV

305

determined by primate challenge, making the relevance of these mutations to attenuation less certain. Attenuation of HAV is at best a poorly defined and inadequately characterized property of the virus. HAV variants have been considered to be “attenuated” if they demonstrate an impaired ability to induce acute hepatocellular injury in man or any of several available primate models of acute hepatitis A. The degree of hepatocellular injury may be measured by quantitation of serum liver-derived enzymes following experimental challenge (Provost et al., 1982, 1986) or by direct histopathologic examination. The severity of liver injury accompanying challenge with cell culture-passaged virus not only varies with the virus variant tested, but may also be affected by the route of administration, the species of primate (i.e., humans chimpanzees, marmosets, or owl monkeys), and possibly the age (and perhaps other unknown attributes) of the infected individual (Provost et al., 1982, 1986; Lemon, 1985; Purcell, 1987; R. A. Karron, R. Daemer, J. Ticehurst, E. D’Hondt, H. Popper, K. Mihalik, J. Phillips, S. Feinstone, and R. H. Purcell, manuscript submitted for publication). While HAV variants that are attenuated for humans have clearly been derived (Provost et al., 1986), attenuation is difficult to quantify and no reliable in vitro markers of attenuation have yet been developed. Experimental infection of New World owl monkeys with a neutralization-resistant variant of HM 175 virus that is closely related by passage history to pl6 virus has recently demonstrated nearly unaltered virulence at the 22nd in vitro passage level (Lemon et al., 1988). This suggests that the mutations present in pl6 have not resulted in significant attenuation for owl monkeys. At present, it is difficult to relate these findings to the question of attenuation for either chimpanzees or man, as there are no data available addressing the relative susceptibility of these species and owl monkeys to disease following challenge with cell culture-passaged virus. Of overriding importance, however, is the fact that an understanding of the molecular basis of attenuation of HAV is likely to be dependent on understanding the events accompanying adaptation of the virus to cell culture, as this has been the first step in the development of all attenuated variants examined to date (Provost et al., 1982, 1986; R. A. Karron, R. Daemer, J. Ticehurst, E. D’Hondt, H. Popper, K. Mihalik, J. Phillips, S. Feinstone, and R. H. Purcell, manuscript submitted for publication). The lack of mutations predicting changes in the surface structures of the p16 virus argues against alterations in virus receptor-binding activity as a mechanism of adaptation to growth in vitro. The mutation in VP2 that is common to both p16 and p35 variants appears unlikely to have induced changes in the re-

306

JANSEN,

NEWBOLD,

ceptor-binding activity of the virus, given that the position of this residue is probably deep within the capsid structure according to computer-generated alignments derived by A. Palmenberg (personal communication) and the known crystal structure of mengovirus (Luo et a/., 1987; M. Rossmann, personal communication). However, the precise location of this VP2 residue in the HAV capsid and the possible functional relevance of this mutation must await crystallographic determination of the HAV capsid structure. This change in VP2 does not appear to be essential for adaptation of virus to growth in cell culture, however, as it is not present in the cell culture-adapted p59 variant of HM175 (Ross et a/., 1986). Similarly, the absence of mutations in the putative protease, 3Cpro, or at proposed cleavage sites for polyprotein processing in HAV (Cohen et a/., 1987b), suggests that changes in post-translational processing do not play major roles in adaptation of virus to cell culture. Mutations within the 5’ and 3’ nontranslated regions of the genomes of p16 and p35 viruses could affect priming or initiation of both positive- and negativestrand RNA replication. In addition, since guanidine resistance of poliovirus maps to the 2C protein of that virus (Pincus et al., 1986), mutations present in the analogous HAV protein may also affect RNA replication. Last, the coevolution of related mutations in the putative polymerase (3Dpol) proteins of both pl6 and p35 viruses (Table 2) argues that changes in polymerase function and hence RNA replication may be central to efficient growth in vitro. Such mutations could result in more efficient interactions between viral proteins, viral RNA, and cell-specific proteins suspected to be involved in replication of picornaviral RNA (Andrews et al., 1986; Morrow et a/., 1985). This interpretation is consistent with evidence suggesting that the restrictive event in HAV replication in vitro may be replication of the viral RNA (Anderson et a/., 1987). Host cell factors necessary for replication of viral RNA in cultured monkey kidney cells may be different from those available to the virus in the human hepatocyte, the cell type within which virus replication normally occurs in v&o. If so, mutations occurring during adaptation of virus to growth in cell culture and permitting more efficient utilization of host cell factors in vitro might be expected to lead to a reduction in the ability of virus to replicate within the hepatocyte in viva. This hypothesis provides a possible explanation for the attenuation of HAV that has been associated with passage of the virus in cell cultures. It is consistent with the observation that attenuated HAV (specifically, the F’ variant of CR326 strain HAV) replicates only to low levels in nonimmune humans, as evidenced by delayed and relatively low level antibody responses (Provost et a/., 1986; S. M. Lemon and P. Provost, unpub-

AND

LEMON

lished data). Attenuation of HAV, like attenuation of poliovirus (Sabin, 1985), may thus be related to mutations in several regions of the genome (Minor et al., 1986; Omata et al., 1986). An effect on ribosome recognition or initiation of translation cannot be ruled out for the nucleotide changes found in the 5’ nontranslated region of the genomes of cell culture-adapted HAV variants, however, as the functions of the nontranslated RNA of picornaviruses remain poorly defined. Nor can the influence of mutations on viral assembly, or on the efficiency of virus uncoating following penetration, be ascertained. The recent availability of an infectious molecular construct derived from the p35 variant of HM 175 (Cohen et a/., 1987c) should, however, allow more precise mapping of viral functions involved in adaptation of virus to in vitro growth and may possibly shed light on their relevance to attenuation. ACKNOWLEDGMENTS This work was supported in part by Contract DAMD17-85C-5272 from the U.S. Army Medical Research and Development Command, and by Public Health Service Grant l-ROl-AI-22279-02 from the National Institute of Allergy and Infectious Diseases. We thank J. Ticehurst, J. Cohen, and R. H. Purcell of the NIAID for their generous gifts of f. co/i strains bearing wild-type cDNA inserts of HAV and HM175-strain specific oligonucleotide primers, as well as for providing us with sequence data in advance of publicafion, and C. Hutchlson for the synthesis of other oligonucleotides. We thank P. Murphy for excellent technlcal assistance, and R. Knight for preparation of the manuscript.

REFERENCES ANDERSON, D. A., LOCARNINI, S. A., Ross, B. C., COULEPIS, A. G., ANDERSON, B. N., and GUST, I. D. (1987). Single-cycle growth kinetics of hepatitis A virus in BSC-1 cells. In “Positive Strand RNA Viruses” (M. A. Bnnton and R. R. Rueckert, Eds.). pp. 497-507. A. R. Liss, New York. ANDREW% N. C., and BALTIMORE, D. (1986). Purification of a terminal uridylyltransferase that acts as host factor in the in vitro poliovirus replicase reaction. Proc. Nat/. Acad. Sci. USA 83, 22 l-225. BINN. L. N., LEMON, S. M., MARCHWICKI, R. H., REDFIELD, R. R.. GATES, N. L., and BANCROFI, W. H. (1984). Primary isolation and serial passage of hepatitis A virus strains in primate cell cultures. J. C/in, Microbial. 20, 28-33. CANN, A. J.. STANWAY, G., HAUPTMAN, R., MINOR, P. D., SCHILD, G. C., CLARKE, L. D., MOUNTFORD, R. C.. and ALMOND, J. W. (1983). POliovirus type 3: Molecular cloning of the genome and nucleotide sequence of the region encoding the protease and polymerase proteins. Nucleic Acids Res. 11, 1267-l 28 1. COHEN, J. I., ROSENBLUM, 8.. TICEHURST, J. R., DAEMER, R. J., FEINSTONE. S. M., and PURCELL, R. H. (1987a). Complete nucleotide sequence of an attenuated hepatitis virus: Comparison with wildtype virus. Proc. Natl. Acad. Sci. USA 84, 2497-2501. COHEN, J. I., TICEHURST, J. R., FEINSTONE, S. M., ROSENBLUM, B., and PURCELL, R. H. (1987c). Hepatitis A virus cDNA and its RNA transcripts are infectious in cell culture. J. Viral. 61, 3035-3039. COHEN, J. I., TICEHURST, J. R., PURCELL, R. H., BUCKLER-WHITE, A., and BAROUDY. B. M. (198713). Complete nucleotide sequence of wildtype hepatitis A virus: Comparison with different strains of hepatitis A virus and other picornaviruses. /. Viral. 61, 50-59.

CELL

CULTURE-ADAPTED

FROSNER, G. G., DEINHARCIT, F., SCHEID, R., GAUSS-MOLLER, V., HOLMES, N., MESSELBERC~ER, V., SIEGL, G., and ALEXANDER, J. 1. (1979). Propagation of human hepatitis A virus in a hepatoma cell Itne. Infect/on 7, 303-3O!j. GUST, I. D., LEHMANN, N. I , CROWE, S., MCCRORIE, M., LOCARNINI, S. A., and LUCAS, C. R. (‘1985). The origin of the HM175 strain of hepatitis A virus. J. Infect. Dis. 151, 365-367. JANSEN, R. W., NEWBOLD, 1. E., and LEMON, S. M. (1985). Combined immunoafflnity cDNA-RNA hybridization assay for detectlon of hepatitis A virus in clin cal specimens. f. C/in. Microbial. 22, 984-989. JANSEN. R. v1’., NEWBOLD, J. I!., and LEMON, S. M. (1988). Mutations in the capsid-encoding and 5’ nontranslated regions of the hepatitis A virus genome associated with adaptation of virus to growth in cell culture. In “Viral Hepatitis and Liver Disease” (A. Zuckerman, Ed.), pp. 36-42. A. R. Liss, Inc.. New York. LEDUC, J. W LEMON, S. M., KEENAN, C. M., GRAHAM, R. R., MARCHWICKI, R. H., and BINN, L. N. (1983). Experimental infection of the New World owl monkey (Aotus frivirgatus) with hepatitis A virus. Infect. Immun. 40, 766-772. LEMON, S. M (1985). Type A viral hepatitis: New developments in an old diseas,e. N. Engl. J. Med. 313, 1059-1067. LEMON, S. IV., CHAO, S-F., JANSEN, R. W., BINN, L. N., and LEDUC, J. W. (198?a). Genomic heterogeneity among human and nonhuman strains of hepatitis A virus. /. Viral. 61, 735-742. LEMON, S. M., and JANSEN, R. W. (1985). A simple method for clonal selectlon Iof hepatitis A virus based on recovery of virus from radloimmunofocus overlays. J. Viral. Methods 11, 17 l-l 76. LEMON, S. M., LEDUC, J. W., BINN. L. N., ASCAJADILLO, A., and ISHAK, K. G. (19E,2). Transmission of hepatitis A virus among recently captured Panamanian ovvl monkeys. f. Med. Viral. 10, 25-36. LEMON, S. M., STAPLETON, J. T., LEDUC, J. W., TAYLOR, D., MARCHWICKI, R., and BINN, L. N. (1988). Cell-culture-adapted vanant of hepatitis A virus selected for resistance to neutralizing monoclonal antibody retains virulence in owl monkeys. In “Viral Hepatitis and Liver Disease” (A Zuckerman, Ed.), pp. 70-73. A. R. Liss, Inc., New York. Luo, M., VF:IEND, G., KAMER, G., MINOR, I., ARNOLD, A., ROSSMANN, M. G., BOEGE, U., SCRABA, D. G., DUKE, G. M., and PALMENBERG, A. C. (1987). The atomic structure of mengo virus at 3.0 A resolution. Science 235, 182-l 89. MANIATIS, T., FRISSCH, E. F., and SAMBROOK, J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MINOR, P. D., SCHILD, G. C , CANN, A. J., DUNN, G., EVANS, D. M. A., FERGUSON M., STANWAY, G., WESTROP, G., and ALMOND, J. W. (1986). Studies on the molecular aspects of antlgenic structure

HAV

307

and virulence of poliovirus. Ann. Inst. PasteWVirol. 137 E. 107-125. MORROW, C. D., LUBINSKI, J., HOCKO, I., GIBBONS, G. F., and DASGUPTA, A. (1985). Punflcation of a soluble template-dependent rhinovirus RNA polymerase and Its dependence on a host cell protein for viral RNA synthesis. /. Viral. 53, 266-272. OMATA. T., KOHARA. M.. KUGE. S., KOMATSU, T., ABE, S., SEMLER, B. L., KAMEDA, A., ITOH. H.. ARITA, M., WIMMER, E., and NOMOTO, A. (1986). Genetic analysis of the attenuation phenotype of poliovirus type 1. I. Viral. 58, 348-358. PINCUS, S. E.. DIAMOND, D. C., EMINI, E. A., and WIMMER. E. (1986). Guanidine-selected mutants of poliovirus: Mapping of point mutations to polypeptide 2C. /. Vkol. 57, 638-646. PROVOST, P. J., BANKER, F. S.. GIESA, P. A., MCALEER, W. J., BUYNAK. E. B.. and HILLEMAN, M. R. (1982). Progress toward a live, attenuated human hepatitis A vaccine. Proc. Sot. fxp. Biol. Med. 170, 8-14. PROVOST, P. J., BISHOP, R. P.. GERETY, R. J., HILLEMAN. M. R., McALEER, W. J., SCOLNICK, E. M.. and STEVENS, C. E. (1986). New findings in live, attenuated hepatitis A vaccine development. 1. Med. viral. 20, 165-175. PROVOST. P. J., and HILLEMAN, M. R. (1979). Propagation of human hepatitis A virus in cell culture in vifro. Proc. Sot. Exp. Biol. Med. 160, 213-221. PURCELL, R. H. (1987). Status of hepatitis A vaccines. In “Positive Strand RNA Viruses” (M. A. Brinton, and R. R. Rueckert, Eds.), pp. 565-574. A. R. Liss, New York. Ross, B. C., ANDERSON, B. N., COULEPIS, A. G., CHENOWETH, M. P., and GUST, I. D. (1986). Molecular cloning of cDNAfrom hepatitis A virus strain HM-175 after multiple passages in viva and in vitro. /. Gen. Viral. 67, 1741-l 744. SABIN, A. B. (1985). Oral poliovirus vaccine: History of its development and use and current challenge to eliminate poliomyelitis from the world. J. infect. Dis. 151, 420-436. SANGER, R.. NICKLEN, S., and COULSON, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Nat/. Acad. Sci. USA. 74, 5463-5467. STANWAY, G., CANN, A. J., HAUPTMAN. R., HUGHES, P., and ALMOND, J. W. (1983). The nucleotide sequence of poliovlrus type 3 Leon a,b: Comparison with poliovirus type 1. Nucleic Acids Res. 11, 5629-5643. 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. Nafl. Acad. Sci. USA 80, 5885-5889. WEITL, M.. BAROUDY, B. M., MALOY, W. L., TICEHURST, J. R.. and PURCELL, R. H. (1986). Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs. J. Viral. 60, 124-l 30.