Precore and contiguous regions of hepatitis B virus in liver transplantation for end-stage hepatitis B

GASTROENTEROLOGY 1994;107:1774-1780

Precore and Contiguous Regions of Hepatitis B Virus in Liver Transplantation for End-Stage Hepatitis B TOMASZ LASKUS, JORGE and DAVID H. PERSING

RAKELA,

JEFFERY

L. STEERS,

RUSSEL

H. WIESNER,

Mayo Clinic and Foundation, Rochester, Minnesota

Background/Aims: Recurrent hepatitis B virus (HBV) infection is the leading cause of mortality and morbidity after otthotopic liver transplantation (OLT) for HBV-related liver disease, but the extent of viral genetic variation in this setting remains unknown. Methods: Eight patients who underwent 0l.T for HBV-related liver disease were studied; 7 had cirrhosis and 1 had fulminant hepatitis. Four patients received long-term hepatitis B immunoglobulin prophylaxis. A 240-base pair fragment (1742-1981) comprising the precore region of HBV was amplified by polymerase chain reaction from sera drawn before OLT and 6,12, and 24 months after OLT and analyzed. Results: All sera were positive by polymerase chain reaction. Nucleotide sequence variations were congruent within most patients before and after 0l.T; however, in one patient, substantial se quence variation was observed, suggesting infection with a new HBV strain. No sequence variation associated with a particular outcome could be identified. Two patients harbored HBV variants with a deletion or insertion upstream of the precore messenger RNA initiation site. Conclusions: Reinfection after OLT can occasionally be caused by HBV strains different from the one present before OLT. Changes within the sequenced region are not predictive of the outcome of reinfection.

0

rthotopic

liver transplantation

(OLT) is currently

the only effective therapy for fulminant hepatitis B; however, its use remains controversial in patients with chronic hepatitis B virus (HBV) infection caused by a

and is of potential

importance

in light of recent findings

that precore defective mutants are often associated with severe forms of hepatitis.“912 The aim of the present study was to define

the molecular

changes

within

the HBV

precore and adjacent regions in the setting of OLT and correlate these changes with outcome. We also wanted to determine

whether

is invariably

the viral strain present

responsible

OLT. Accordingly, in eight patients

for recurrent

we studied

HBV sequence

who underwent

disease. The precore,

proximal

sera obtained

after

variation

OLT for end-stage

HBV

core, and an essential

gion of the HBV core promoter quential

before OLT

infection

were determined

from these eight patients.

Materials

and

Methods

Patients Eight patients who underwent OLT for HBV-related liver disease at the Mayo Clinic between April 1989 and August 1992 and who survived for at least 1 month were subjects of the study. In seven cases, OLT was required for HBV-related chronic liver disease. In two of the patients, coinfection with hepatitis D virus was documented. In one case, liver failure was secondary to fulminant hepatitis B. Pertinent clinical, serological, and histological data on these patients are presented in Table 1. At the time of this report, 5 patients are still alive and 3 patients have died; 2 patients died of liver failure in the course of recurrent hepatitis, and 1 patient died of hepatocellular Standard

carcinoma

antirejection

corticosteroids,

(Table therapy

and azathioprine.

1). consisted

of cyclosporine,

Four patients

received hepa-

high rate of reinfection of the transplanted liver. Recurrent HBV infection is the leading cause of morbidity, mortality, and graft loss in patients undergoing OLT for HBV-related liver disease’,* and may be associated with

titis B immunoglobulin (Abbott Laboratories, Chicago, prophylaxis according to the following protocol: 10,000

rapid progression

antibody

of hepatitis

to cirrhosis2-5

or fibrosing

cholestatic hepatitis.6-8 The mechanisms behind various manifestations of recurrent HBV infection are poorly understood; however, in the case of fibrosing cholegatic hepatitis, there is evidence to suggest that it may be the result of cytopathic effect of uncontrolled viral replication.“” The extent of genetic variation of HBV after OLT is largely unknown

re-

in se-

during

the anhepatic

transplantation,

phase, every day for the first 7 days after

and

to hepatitis

Commercially tories) were used (HBsAg), hepatitis

IL) IU

then

on

a monthly

basis

whenever

B surface antigen levels were
available

immunoassays

(Abbott

Labora-

to test for hepatitis B surface antigen B e antigen, and antibody to hepatitis D

Abbreviations used in this paper: OLT, orthotoplc liver transplanta tion; PCR, polymersse chain reactlon. 0 1994 by the American Gastroenterologlcal Assoclatlon 0018-5085/94/$3.00

HBV

December 1994

Tabk~ 1. Clinical, Serological, HBsAg

and Histopathologic WVDNA

H&A8

PRECORE

REGION

IN LIVER

TRANSPLANTATION

1775

Data on Eight Patients with HBV Undergoing OLT

Hepetitis

Liver histopathol~/H6sAg

B

and HBcA6

in tissue

immunoglobulin (before/after

+/+

40/M

Before

+/+

+/+

+/+

57/M

prophylaxis

OLT)

4-6

OLT

mo

24 mo

18 mo

12 mo

Outcome

CAH+C

N/t

CPH/+

ND

CAH/+

Mild CAH 4 years

CAH+C+Ca

LB/+

CAH/+

ND

CAH/+

CAH tC

after OLT

+/-

-/+

_

3 years

aher OLT

44/M

+/+

+/+

-/+

48/M

+/+

+/+

t/t

52/M

+/-

+/-

51/F

+/-

+/-

CAH+C/t

CAHtC

CAH/+

CAH+C/ND

ND

Alive 30 mo after

_

CAH+Ca

PCH/t

ND

Ca

+/-

+

Submassive

N/-

N/-

Alive and well 16

+/-

+

CAH+CtCa

N/-

N/-

Alii

OLT Died 24 mo after OLT due to Ca necrosis

mo after OLT and wall 18

mo after DLT 62/M

+/-’

+/-

+

+/-

Severe

CAHD+C+Ca

Died 6%

mo

recurrent

after OLT due

hepatltls

to liver failure

B and D/+

8

t/p

42/M

+

+/-

+/-

CAHD

Died 12 mo tier

FCH/+

N/-

OLT due to liver failure SiBsAg

and HBV DNA recurrad

5%

SIBsAg

and HBV DNA recurred

11%

C, CirdWSiS; HBcAg,

Ca. hepatocellular

hepatitis

HEa&

virus, and the presence core antigen

mo after DLT.

cerclnoma;

B core antigen;

blot hybridization.

mo eher OLT. CAH.

chronic

hepatitis

active

B e antigen;

hepatltls

8; CAHD.

LB. lobular

of HBV DNA was determined

The presence

of HBsAg

chronic

hepatitis;

by dot-

and hepatitis

in the liver tissue was determined

actll

N, normal

B

by immunoper-

hepatitis

B and D; CPH.

or near nomtel;

sequencing

persistent

1814-1816,

of wild-type

HBV DNA

tion errors by Tuq polymerase,

24 months for HBV

after OLT in surviving DNA

extraction

patients.

and amplification

described.13 In brief, DNA was extracted and amplified

by polymerase

combinations

of primers

if no product

of the expected

immedi-

12 months,

The procedure was previously

from 100 PL of serum

chain reaction (PCR) using several

in conventional

(nonnested)

molecular

weight

reactions;

was found on

an agarose gel (as was the case in two sera), seminested cols were used (Tables used to prevent

2 and 3). Appropriate

PCR product

Direct cycle sequencing

and

proto-

measures

were

of the PCR products

according

to the Sanger chain termination

mercially

available kit (GIBCO/BRL,

On several occasions, products

was performed

method using a com-

Gaithenburg,

MD); the

Sequence

was routinely

ing with subsequent

sequencing

reaction.

to direct sequencing,

was performed

ladder from direct sequencing

whenever

“strong

stop,”

of different

products

were found on an agarose gel. In addition,

PCR products PCR products

of primer

or if multiple

from sequential

characterize

Corporation,

the presence

lengths

evolution

extension

closely spaced PCR

sera drawn

an unusual

the

showed more than one

products

indicating

PCR

clones analyzed. Clon-

we cloned

from patient of his HBV

were cloned using a TA cloning

2 to strain.

kit (Invitrogen

San Diego, CA). Several clones were analyzed from

each PCR reaction; plasmid DNA was extracted by boiling, amas described

above.

and Sequencing of

5’-3’

HBVl

CTGAGTGCTGTATGGTGAGGTGA

HBV2

GACCllGAGGCATAlTKAC

HBV3

AGGAGAlTAGGTWATGGTCTTTGT GTAACTCCACAGMGCTCCA GACCl-rGAGGCATAlTTCMAGACTGT ACACAATAGCTTGCCTGAGTGCTGT GGGAGGAGAlTAGGllAATGGTClTTG GAGGCTGTAGGCATAAAITGGTCTGTKAC

HBV4 HBVB HBV6 HBV? HBV8

direct sequencing

in addition

plified by PCR, and sequenced

Table 2. Primers Used for Amplification HBV DNA

190 1- 1903) and compared strains. To rule out incorpora-

were cloned and the individual

sequence

codon

and the core starting

repeated for each serum from a new amplification

better

contamination.‘3

240 nucle-

site; the precore starting

to nucleotides

to nucleotides

months,

hepatitis;

system is from the unique &RI codon corresponds

from sera drawn

cholestatic

1742 to 1981 (the numbering

with the sequences

was amplified

PCH. fibmsing

primers are listed in Table 2. Altogether,

Amplificationand Sequencingof HBV DNA ately before OLT and then at 4-6

Designation

hepatitis;

otides were read from position corresponds

oxidase staining.

chronic

ND, not done.

Location

2067-2045 1692-1715 1747-1771 1946-1922 1692-1718 2081-2057 1744-1770 1772-1806

TaMe 3. Primers Used for Amplification HBV DNA

and Sequencing of

Outer primers HBVl

Inner primers

and HBM

HBVl and HBV3 HBV2 and HBV4 HBV5 and HBV4 HBV6 and HBV7

HBV5 and HBV6

NOTE. HBV8.

Primers

used

for sequencing

are HBVl,

HBVS,

HVB4,

and

1776

LASKUS

ET AL.

GASTROENTEROLOGY

Resutts

tides

HBV DNA sequences and amplified obtained

were successfully

from all serum

from patients

munoprophylaxis

who received

with hepatitis

were HBsAg-negative were identical

samples,

B immunoglobulin

wild-type

by the sequence

ladder) of wild-type

All nucleotide

substitutions

evolution amino

and predicted

patient

(patient

quences

in only

(patient

7); all

and variant

strain;

varia-

(as evidenced

compared

wild-type

acid sequences

sequences

HBV strains

or a mixture

imand

viral strains with sequence

tion in at least one position

known

those

postoperative

from the same patient

other samples harbored

closely related

including

in serum. The amplified

to known

three sera drawn

recovered

with their

strains. the most

sequential

effects on the precore and core are described

2), we were unable

in all posttransplant

serum

only precore and core promoter

in Table

4. In one

to recover core sesamples;

sequences

therefore,

were analyzed.

The observed nucleotide sequence variations were congruent within a given patient before and after OLT with the exception

of patient

2. In this

patient,

sequences

recovered before and after OLT differed significantly, suggesting an infection with a new HBV strain during the posttransplant period. To better study the sequential HBV

molecular

changes

amplicons

were cloned

sequenced

(Figure

in this patient,

the respective clones

were

1). Eleven clones were analyzed

and the individual

from

the initial serum sample, whereas 16 and 40 clones were analyzed from sera drawn after 5 and 24 months, respec-

1777 and 1778, which

of the precore transcription 7 and Figure

the X open reading

sequences

amplified

related,

strain. The patient tive throughout A mixture sistently

from patient

remained

in one sample

6; these products

Snucleotide

2B). This deletion protein

serum (Figure

patient

experienced

a bout

of hepatitis

3 months

after

OLT with histopathologic changes compatible with lobular hepatitis. The cause of this hepatitis could not be determined, and the patient was found to be negative for HCV, cytomegalovirus, and Epstein-Barr virus infections at that time. During and immediately after OLT, he received massive transfusions of blood and blood products (14 U of red blood cells, 10 U of fresh frozen plasma, 10 U cryoprecipitate, and 6 U of platelets) derived from 45 donors. All I6 clones obtained from serum drawn 5 months after OLT, as well as a consensus sequence in serum drawn 12 months after OLT, had a G to A transition mutation at nucleotide 1896, turning the penultimate codon of the precore region into a stop codon (Figure 1, lines 2-6). Twenty-four months after OLT, this mutation could no longer be identified in the consensus sequence and was found only in 2 of 40 HBV DNA clones (5 %) derived from this serum sample. However, all other clones (95%) now harbored a 12-nucleotide insertion between nucleo-

sizes was con-

from patient

5 and one

were analyzed

after clon-

5 was found to harbor

variant

with an

1763 - 1770;

Figure

was only found in the initial,

and was predicted 2B). The deletion

to truncate

the X

was found in 5 of 12

clones (42%) from this serum. Patient mutant

6 harbored

a mixture

with a 7-nucleotide

1969) in serum drawn

of wild-type

deletion

12 months

HBV and a

(nucleotides

translation

mutation

product

resulted

in the core region; the predicted

would

contain

coded by the core open reading amino

1963-

after OLT. This dele-

tion, which was found in 5 of 10 clones (50%), in a frameshift

21 amino

frame followed

acids and a stop codon (Figure

acids enby seven

3).

Discussion In the current the precore,

proximal

study, we amplified

in sequential

been accepted

sera from eight patients

core

undergo-

liver disease. It has generally

that HBV reinfection

by the same strain that was present few studies

and sequenced

core, and part of the HBV

ing OLT for HBV-related

This

after OLT.

(nucleotides

initial

substitutions.

of the same

B e antigen-nega-

HBV and a viral variant

deletion

pre-OLT

2A). All

after OLT were

of different

vector. Patient

of wild-type

1, line

was predicted

evolution

hepatitis

the follow-up

ing into a plasmid

site (Figure

frame (Figure

with

No. 6

just upstream

variant

this patient

of PCR products

found

a mixture

from

consistent

promoter

by 12- 14 nucleotide

initiation

2A). This insertion

tively. Four closely related HBV strains were found in serum drawn 5 months after OLT; all differed from the strain

was located

to disrupt closely

Vol. 107,

have been performed.14

after OLT is caused before OLT; however, Our

study

showed

that this is usually the case; there may be occasional exceptions as shown by patient 2 in whom pre-OLT and post-OLT

HBV sequences

1). In support

differed

significantly

of de novo HBV infection

(Figure

in this patient

is the observation that he experienced an unexplained bout of clinical hepatitis 3 months after OLT with histological changes compatible with lobular hepatitis in the liver biopsy specimen. The source of this infection was unclear; immediately after OLT, the patient received blood and blood products from 45 donors. All blood donors as well as the organ donor were antibody to hepatitis B core antigen negative at the time of donation; however, individuals without serological markers of HBV may occasionally transmit infection.‘5@ First-time HBV infection in OLT transplants is not unusual; Chazouilleres et al.” recently reported on 20 patients who were initially HBsAg-negative but who showed HBV

December 1994

Table 4. Mutations

No.

in the Core Promoter,

GenBank accession number of most closely related wild-type strain

of HBV Recovered

OLT

1809 1812 1862 1888 1809 1812 1821 1862 1888

G-T C-T G-T” GA G-T C-T T-Aa G-T” G-A

2b

VO0866 for initial serum and JO2203 for follow-up sera

3 4

VOO866 VOO866

5

X70185

1753 T-C 1981 A-C Deletion 17631770” 1913 C-A/C”

6

X70185

1913 C-A/C’

1981 A-C

Identical to wild-type 1753 T-C 1762 A-T” 1764 GAa 1810 C-A=

From Eight Patients

Undergoing

with respect to wild-type strain 12 mo

24 mo

Identical to initial serum

Identical to initial serum

Identical to initial serum

1753 T-C 1762 GA 1763 GA 1764 G-A 1773 C-T 1802 C-T 1803 G-T 1819 A-Ta 1845 C-T” 1896 GA (stop) 1899 G-A” Identical to initial serum Identical to initial serum

1762 1764 1773 1802 1803 1845 1896 1899

Insertion 12 nucleotide between nucleotide 1777-1778 1773 C-T 1802 C-T 1803 G-T 1845 C-T”

1809 G-T 1812 C-T 1862 G-T” 1888 G-A 1913 C-A/C’ Identical to initial serum

Identical to previous serum

4-6

Initial serum

X70185

X70185 JO2203

and Core Regions

1777

Nucletide substitutions

1

7 8

Precore,

HBV PRECORE REGION IN LIVER TRANSPLANTATION

mo

A-T GA C-T C-T G-T C-T” G-A (stop) G-A”

Identical to initial serum ND

Identical to initial serum

1809 G-T 1812 C-T 1862 G-T” 1888 GA 1913 C-A/C” Deletion 1963-196gd Identical to wild-type’ Identical to initial serum

Identical to wild-type Identical to initial serum

NOTE. All sequences were determined by direct sequencing of PCR products; in addition, several specimens from patient 2, initial specimen from patient 5, and final specimen from patient 6 were sequenced after cloning. Wedicted to change deduced amino acid sequence within the precore or core open reading frame. bSequential changes of HBV recovered from this patient are shown in Figure 1, whereas the effect of found insertion on X open reading frame is shown in Figure 2. “Deletion was found in 5 of 12 clones (42%) from this serum: localization and predicted effect on X open reading frame are presented in Figure 2. dDeletion was found in 5 of 10 clones (50%); its predicted effect on core open reading frame is shown in Figure 3. “This serum was drawn 6% mo after OLT. A/C, Two-chain termination products detected.

infection

after OLT.

In the majority

of these patients,

the source of infection was not identified. We were unable to correlate specific changes

of viral variants the precore

in the

sequenced region that would explain various manifestations of recurrent hepatitis B in liver transplants, such as rapid acceleration of liver disease2-4 and fibrosing cholestatic hepatitis6-’ (Tables 1 and 4). Five of our 8 patients showed few changes within the sequenced region; however, in the remaining 3, extensive sequence variations were observed. No specific clinical patterns were associated with these variations (Tables 1 and 4). Particularly interesting was the finding in two patients

mixture

with a deletion

region.

of wild-type

or insertion

upstream

of

(patient

5) harbored

a

One patient HBV

strain

and a strain

with

a

deletion before OLT, whereas another (patient 2) converted from a hepatitis B e antigen-negative mutant to an HBV insertion variant during the follow-up period (Figure 1). The meaning of these molecular changes is unclear; however, they both affect a genetic region previously shown to be essential for core promoter activity” and are located just upstream of the precore messenger RNA initiation sites.‘9.20 It is possible that the observed rearrangements might reduce or eliminate the initiation

1778

LASKUS ET AL.

VOO866 JO2203 1. 2. 3. 4. 5. 6. I.

GASTROENTEROLOGY Vol. 107, No. 6

-------_-___----_-_-----------------

. . . . . . . . . . ..------------

-__-__-_____-__-____----------------

------_~~~~~~---~~~~~~~~-~~~~~~~~~~~

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

..------------

.

..------------

Figure 1. Nucleotide sequence of the basic core promoter and precore regions (nucleo tides 1742-1900) of HBV in patient 2 compared with the most closely related wildtype strains (GenBank accession numbers VO0866 and JO2203). Sequences of HBV DNA were PCR-amplified and -sequenced directly and after cloning. (1) Sequences recovered from initial serum; all 11 clones and consensus sequences were identical. (2-5) Sequences of HBV DNA recovered from se rum drawn 5 months after OLT; 4 slightly differing sequences were found among 16 clones. (6) Sequence 12 months after OLT; in this case, PCR product was directly se quenced only. (7) Sequence recovered 24 months after OLT; consensus sequence as well as 38 of 40 clones (95%) showed 12nucleotide insertion between nucleotides 1777 and 1778, whereas 2 clones (5%) were identical to sequence 4.

VOO866 JO2203 1. 2. 3. 4. 5. 6. 7.

VOO866 JO2203 1. 2. 3. 4. 5. 6. 7.

of transcription providing

of the precore

an alternate

antigen-negative ported

messenger

pathway

phenotype.

by the observation

RNA,

thus

type virus,

B e

necessarily

for the hepatitis

This

speculation

that patient

is sup-

2, whose virus

switched from precore nonsense mutant to a core promoter insertion variant, remained hepatitis B e antigennegative

throughout

the nonsense 5 harbored

his course despite

mutation

at position

a core promoter

the reversion

of

1896. Because patient

deletion

mutant

and wild-

negative

his hepatitis

B e antigen

status

reflect the presence of a hepatitis Both

mutant.

liver disease,

patients

2 and

and it is possible

that

would

not

B e antigen5 had active

the emergence

of

these HBV variants was the result of immunologic selection pressure, a process that would be similar to that postulated

for the emergence

of precore stop codon

21

mutants. The deletion

and insertion

A

found in the HBV sequence

Precore start 1800

1742

+

+rv

n-+w ~~~A~~A~ATTA~~TT-~~T-TA*A-~T~TA-AT-T~~TC~~T~A~~-~~~~~C*TT Gly Glu Glu Ile Arg Leu Lys Val Phe Val Leu Gly Stop 124

B

Precore

l-r+il-e ~~OM~AWMIATTMCITTAUI~CIT~~\TOITATTA--TGTA-*T-~~~~~~*~~A~A~~A~-CTTT Gly

starl

1800

1742

Glu

Glu

Ile

Arg Leu

ASP’

&I-r?

Ile’ Arg’ Arg’ Leu Stop

124

2. Deletion and insertion in the core promoter region of HBV and its predicted effect on X open reading frame in patients with HBV undergoing OLT. Substituted amino acids are marked by an asterisk. The sequence is numbered from the unique EcoRl site; in this system, the precore open reading frame starting codon corresponds to nucleotides 1814-1816. The arrows mark the starting sites of precore and pregenomic transcripts. Precore ATG is underlined. Deleted or inserted regions are boxed. (A) Insertion found in patient 2. (r3) Deletion found in patient 5. Flgure

HBV PRECORE REGION IN LIVER TRANSPLANTATION

December 1994

core

from patients

the X open reading (Figure

MetAspI1eAspProTyrLysGluPheQlyAlaThrValGluLeuLeuSerPhe

tuted

the majority

consistent

frame and truncate

ing an X helper deletion

function

variants

tients

with chronic

hepatitis

ment

of such variants

by coinfection and other

for this defective with wild-type

patients

significance

will

in pa-

the developthat

Alternatively,

the

virus could be provided

be necessary

study of this

to determine

the

was found to harbor a mixture

and an HBV variant with a deletion

the core open reading virus requires tein. Extensive

frame. Obviously,

wild-type

“helper”

within

of

disrupting

such a defective

virus to provide

in frame deletions

Furthermore,

our calculations

were based on a relative

short stretch

of HBV genome,

and it is likely conserved

C pro-

and whether

with severe liver disease remains Based on the changes and using

the method

within

of Gojobori

they are associated studied

and Yokoyama,24

tide position

per year, which

is similar

range of 4 X lo-‘, whereas Okamoto et a1.26 estimated the mutation rate of HBV to be about 1.4-3.2 X 10P5 base substitutions. site-' . yr-‘. However, the studies on HBV mutation rate were performed on chronically infected asymptomatic individuals with probably inactive liver disease for which the effects of immune selection

rate can occasionally

In summary,

Therefore,

approach

it

that encoun-

to which hepadnaviridae

our results suggest

OLT may occasionally ent from that present precore and adjacent

are closely

mutations region remains

before OLT. Changes (core promoter

we described with alteration

in patients

that reinfection

after

be caused by an HBV strain differ-

are not predictive

In addition,

of the outcome the presence within

within

and proximal

the core)

of reinfection.

of a class of HBV

the HBV core promoter

with OLT, the significance

of which

to be determined.

References 1. Samuel D, Muller R, Alexander G, Fassati L, Ducot B, Benhamou

2.

3.

4.

to that for 28

patients with chronic hepatitis (7.8 X 10-j) (unpublished observations). This mutation rate is much higher than calculated by some other investigators; Orito et a1.25 calculated the substitution rate for HBV to be in the

liver disease.

related.27

we

calculated the mutation rate of this region of the HBV genome after OLT to be around 7.4 X 10e3 per nucleo-

rate was also high

which may also be due to the fact

had active

tered in retroviruses2*

undetermined. the sequence

group,

the mutation

tants predicted to disrupt the core open reading frame are probably rare.23 Whether these variants represent a form of escape mutants

of the virus are less

than others. The mutation

that our controls

the core open

reading frame were reported to exist in a large proportion of patients with chronic and active hepatitis B, but mu-

that some regions

seems that at least for some regions of the HBV genome,

regions

of this class of mutations.

One of our patients wild-type

these

indicator

virus. Further

to be weaker.

in our control

is supply-

prevalent

B infection,22

has occurred.

2 is

the possibil-

may be an early

integration

helper function

in patient

If so, because

are highly

are likely

to disrupt

in hepatocytes

in trans.

6

virus consti-

including

HBV DNA

or insertion

HBV DNA

No.

the X protein

virions

with several scenarios,

_______________-__-----_---_________--------___-_----____-_______-_--__-_-------

6

X70185

that the mutant

of circulating

ity that integrated

No.

2 and 5 are predicted

1). The observation

codon

ATQGACATTGACCCTTATAAAQAATTTQQAGCTACTGTQQAGTTACTCTCGTTT

X70185 figure 3. Nucleotide sequence of the proximal core region in patient 6 and the predicted effect of the found deletion on translation of the core open reading frame compared with the most closely related wild-type strain (Gen Bank accession number X70185). HBV sequence was recovered from serum drawn 12 months after OLT; PCR product was cloned. In 5 of 10 clones (50%) a 7-nucleotide deletion disrupting core open reading frame was found.

recovered

start

1779

5.

6.

7.

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Received February 11, 1994. Accepted July 22, 1994. Address requests for reprints to: Jorge Rakela, M.D., Division of Transplantation Medicine, 301 Lhormer Building, 200 Lothrop Street, Pittsburgh, Pennsylvania 152132582. Supported by grant CR20 (to J.R.) from the Mayo Clinic and Foundation and in part by Public Health Service grants A132403, AR41497, and Al30548 from the National Institutes of Health (to D.H.P.).

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