JOURNAL OF VIROLOGY, Apr. 1990, p. 1851-1854 0022-538X/90/041851-04$02.00/0 Copyright C) 1990, American Society for Microbiology

Vol. 64, No. 4

Defective Replication Units of Hepatitis B Virus PETER SCHRANZ,' HANSWALTER ZENTGRAF,1 IVAN F. LONCAREVIC,' MICHAEL NIEPMANN,2 AND CLAUS H. SCHRODER'* Institut fur Virusforschung, Deutsches Krebsforschungszentrum,' and Zentrum fur Molekulare Biologie, University of Heidelberg,2 D-6900 Heidelberg, Federal Republic of Germany Received 22 September 1989/Accepted 6 December 1989

Templates for the synthesis of defective hepatitis B virus RNA pregenomes were constructed. Viral sequences in these constructs were replaced by the neomycin resistance gene. Deletions spanned up to 80% of the genome and did not include the cohesive end region. The size of the defective replication units was reduced up to half of the wild-type unit length. After cotransfection with replication competent wild-type DNA, defective pregenomes became included into the pool of replicating viral nucleic acids. A natural template for a defective pregenome was derived from the integrated state in a hepatocellular carcinoma. Owing to a deletion, this unit was devoid of the hepatitis B virus enhancer. The hepatitis B virus (HBV) genome is a partially doublestranded DNA circle with neither strand covalently closed. The full-length minus strand is about 3.2 kilobases and has defined 5' and 3' ends. The plus strand is incomplete owing to variable 3' ends. The circular structure of the genome is maintained by cohesive overlaps at the 5' ends of the two strands. The cohesive end region is bracketed by an 11base-pair (bp) direct repeat, which is assumed to play a role in the replication of the genome, which involves reverse transcription of a terminally redundant RNA pregenome. This pregenome is transcribed from a completely doublestranded, covalently closed circular DNA which is formed following entry of the genome of an infecting virus into the nucleus of the host cell (7). The next steps in the replication of the viral genome, i.e., the formation of the long and short strands, are closely associated with virus particle formation. Transfection of cells with head-to-tail repeats of cloned HBV DNA has been successfully used to initiate replicative processes leading to the transient formation of mature virus particles (1, 3, 16, 21). In this type of experiment, the transfecting DNA substitutes for the covalently closed circular HBV DNA. In this paper we report on defective HBV DNA constructs which serve as templates for the synthesis of defective pregenomes. Various parts of the HBV genome except for the cohesive end region with the flanking 11-bp direct repeats, DRI and DRII, were replaced by nonviral DNA. Deletions introduced in this way constituted up to 80% of the total viral DNA sequences. Included into the study was a dimer of a defective HBV DNA unit that had been derived from the integrated state in a hepatocellular carcinoma. This unit carried a deletion comprising the viral enhancer element. Following cotransfection of cells with defective and replication-competent dimeric wild-type (wt) DNA, defective pregenomes became included in the pool of replicating viral nucleic acids. To identify viral DNA sequences which are dispensable for helper-dependent replication, were constructed three defective HBV DNA replication units in which extended parts of the HBV DNA genome were replaced by nonviral sequences. The structures of these plasmids, pddl, pdml, and pdd3, are outlined in Fig. 1A, 2A, and 3A, respectively. *

Plasmid pddl contains a dimer of defective HBV DNA. Sequences spanning most of the core gene (positions 84 to 1796 [12]) and part of the surface gene were replaced by a fragment containing the Neor gene (Fig. 1A). The HBV-Neor construct of pddl is not deleted in the HBV enhancer (15, 17) and core gene promoter region and thus should be capable of directing the synthesis of a transcript with the same sequences at its 5' and 3' ends as are found on the viral pregenome (Fig. 1A) (4, 5, 20, 21). The second plasmid, pdml, contains a terminally redundant HBV genome. Sequences from map position 1004 within the pre-S region to position 2682 located at the 5' end of the X gene were replaced by Neor sequences (Fig. 2A). Regulatory sequences of the human metallothionein promoter were placed upstream of the transcription unit. The pregenomelike transcript potentially synthesized from plasmid pdml as a template is depicted in Fig. 2A. The HBV-Neor construct of the third plasmid, pdd3, combines the deletion of pddl and pdml. Viral sequences retained, thus spanning the genome from positions 2682 to 86, include the cohesive end region (Fig. 3A). Construct pdd3 does not contain HBV enhancer sequences or substituting nonviral regulatory sequences upstream of the defective HBV-Neor unit. To test whether transcripts of the defective HBV DNA constructs were reverse transcribed into DNA, HepG2 cells (2) were transfected (8) with either one of the defective constructs pddl, pdml, or pdd3 together with cloned HBV wt DNA, pd4al. pd4aI contains the dimerized XhoI monomer of HBV wt DNA derived from a hepatocellular carcinoma tissue in a head-to-tail orientation (9). Cytoplasmic core particles were isolated 3 to 4 days after cotransfection by immunoprecipitation (13), and viral progeny DNA contained in these particles was separated on 1% alkaline agarose gels (10). In subsequent Southern blot analyses with strand-specific Neor RNA as labeled probes, newly synthesized HBV minus-strand DNA was detected, with signals corresponding to sizes of 2.8, 2.7, and 1.8 kilobases, respectively (Fig. 1B, 2B, and 3B, lanes labeled neor -). The apparent lengths of these DNA strands were thus consistent with the size predicted for reverse transcripts from pddl, pdml, and pdd3 RNA pregenomes. In no case were similar signals obtained with a plus-strand-specific Neor probe (Fig. 1B, 2B, and 3B, lanes labeled neor +). In a third cycle of hybridization, labeled HBV DNA was used. Consistently, a band corresponding to the size of wt DNA could be visual-

Corresponding author. 1851

1852

J. VIROL.

NOTES

A

A II

EP

IB

PBE

S:

HHV- 14 s

11

IB

E

CP-

Ba

Ba l11

B/B

11 I8

E

I'

I

[l B

:00

pdd E

S

B/B E

111I

E'

Ba 11

Ba

ITxLCIcZr

HHV-2

pdni -d &IxTcIcicl-defective pregenome

defective pregenome

*

B

B

-_

-a a -I0

a-.

-0

a-

E -oI

-oE

E -%o

-a

-a-

-_.1 -aak -o3k-C-o0E -TaL-~~~~~~~~~~1-0_t ak -;

-10

a-.

qp

u.w~~~~~~~~~~

HBV

I -ne'(0r

FIG. 1. pddl. Template for a pregenome deleted in a region containing parts of the S and C genes. (A) Origin of sequences: pdl is a derivative of pSH14-3 (19; see reference 18 for HBV-14) in which the HBV sequences between the BglII restriction site at position 84 (numbering of nucleotide positions starts at the A of core ATG [12]) and position 1796 (PstI) were replaced by the 1,175-bp BglII-BamHI Neor fragment (pNeo; Pharmacia). pddl contains the dimeric template for a defective pregenome. (B) Reverse transcripts from immunoprecipitated core particles. Southern analysis after separation on a 1% alkaline agarose gel. At the top of individual lanes the DNAs used for transfection and cotransfection are indicated, respectively; at the bottom are shown the hybridization probes. HBV (total viral DNA) and neor - and neor + (Neorspecific probes [923-bp PstI fragment cloned in pGem-1; Promega Biotec] complementary to the minus and plus strands, respectively, of newly synthesized defective DNA). The unit-length DNA of HBV and the Neor-HBV constructs pddl and pdd3 served as size markers (3.2, 2.8, and 1.8 kilobases, respectively). Abbreviations (also used in Fig. 2 to 4): B, BglII; Ba, BamHI; B/B, BamHI-BgII; E, EcoRI; X, XhoI; Xb, XbaI; H, Hindlll; P, PstI; I and II, DRI and DRII, respectively; S, X, and C, surface, X, and core genes, respectively.

ized, similar to that observed following transfection with wt DNA alone (Fig. 1B, 2B, and 3B, lanes labeled HBV). Signals for DNA species with an apparently higher molecular weight in the Neor and in the HBV hybridization reflects the lasting presence of plasmid DNA used for transfection. This phenomenon was not consistently observed. The simultaneous detection of newly synthesized HBV-Neor hybrid

HBV I

neor

FIG. 2. pdml. Template for a pregenome deleted in a region from pre-S to the X gene. (A) Origin of sequences: pdml is a derivative of pMH-34/2922 (M. Niepmann, Ph.D. thesis, University of Heidelberg, Heidelberg, Federal Republic of Germany, 1989) with a 10% terminal redundancy (positions 2922 to 84) HBV-2 genome (18; Eco HBV DNA [see reference 6]) in which HBV sequences flanked by the BamHI sites at positions 1004 and 2682 were replaced by the 1,175-bp BglII-BamHI Neor fragment. MT indicates the human metallothionein promotor upstream element IIA from pMH-34/2922 (positions -280 to -34 upstream of HBV sequences; position 2922). (B) Reverse transcripts from immunoprecipitated core particles. HBV unit-length DNA and the monomer of pddl served as size markers in the lanes on the left of the panel. See also the legend to Fig. 1B.

and of wt DNA with an HBV probe occurred only in cotransfection experiments with pddl (Fig. 1B). The ratio of the respective unit-length DNAs, based on a densitometric scan of signal intensities, was determined to be about 1:5. In the cotransfection experiments with pd4al plus pdml and pd4aI plus pdd3, only unit-length DNA of the wt HBV could be identified (Fig. 2B and 3B). From the comparison of signal intensities for known amounts of hybrid marker DNA and newly synthesized hybrid DNA, a ratio of 1:25 for unitlength defective DNA to unit-length wt DNA was estimated in both cases. It can be concluded that transcripts of both defective HBV DNA constructs pdml and pdd3 were replicated, albeit in a ratio significantly lower than expected from the 1:1 mass ratio of the cotransfected plasmids.

VOL. 64, 1990

NOTES

A Ba 11

HB

'2 {FS

A

IB

:._X Xbl

1853

c

K I)

s

--

11I

Xb E

H

X

I'

-____S__

C

11

X

__

s.

X

.

. . .

. . .

H

-

11

p1I I . 11

Xb [loc

ti {3

!

11

Xb -

_ S=

-

X_C _:_S-

Xb

I

XX

x

defective pregenome_.

x

11

x

I

d .f.eetive progeno-m

n

c-

cd

p4111 a

-:s__

____ __

B ~

~

~

-

1

iJ 1-

!: '

so

-

-v,-

t':

,F, -it .

HBVI -

HBV

nerI

4+

neo'

FIG. 3. pdd3. Template for defective pregenomes combining the deletions of pddl and pdml. (A) Origin of sequences: the 584-bp BamHI-BglII fragment from HBV-2 (18; see reference 6 for Eco HBV DNA) was ligated to the BamHI-EcoRI Neor fragment excised from pdl and cloned in pGem-1. The flanking XbaI sites were used for dimerization (pdd3). (B) Reverse transcripts from immunoprecipitated core particles. For details, see the legend to Fig. 1B.

Finally, a defective DNA derived from the integrated state in a hepatocellular carcinoma was analyzed for its capacity to serve as a defective replication unit. It was excised with XhoI, an enzyme with a unique restriction site in HBV DNA, from an integrant termed K10 (Fig. 4A). Following dimerization of this unit in plasmid pIla, in vitro replication assays were carried out and evaluated as described above. The first hybridization probe served a subgenomic HBV DNA (AHBV), mapping within the bounderies of the 2107to-2633 deletion of plasmid pIla. Except for low-molecularweight HBV DNA, only DNA with the apparent length of the wt could be visualized (Fig. 4B, lane labeled AHBV). In a second hybridization cycle a subgenomic DNA probe was used, mapping in the internal region of the core gene (cHBV). This probe also recognized a DNA band of 2.7 kilobases, as expected for a defective progeny DNA of pdlla (Fig. 4B, lane labeled cHBV). In a densitometric scan the mass ratio of unit-length wt DNA to unit-length defective DNA was determined to be about 3:1. The construction and in vitro analysis of defective HBV DNA replication units in which extended parts of the viral DNA sequences have been replaced by nonviral procaryotic sequences aimed at the identification of sequences dispensable for helper-dependent transient replication. Naturally

cHBV

IAHBV

FIG. 4. pdlla. Template for a pregenome carrying the deletion of a defective HBV DNA unit, excised from the integrated state in a hepatocellular carcinoma. (A) Origin of sequences: a cloned (Bluescript vector; Stratagene) Xhol unit, plla which was excised from integrated viral DNA, K10 (tumor 19 in reference 9), pdlla is a dimer of this excised Xhol unit. The dotted arrows indicate the inverted structure of the integrated DNA with a virus-virus junction at position 3132/710. Deletions are indicated by A and the respective map positions. (B) Reverse transcripts from immunoprecipitated core particles. Cloned core (Bglll, positions 84 to 499; cHBV) and cloned enhancer HBV DNA (Asull-SphIl, positions 2261 to 2546; AHBV) were used as hybridization probes. Marker DNA in the left lane is HBV unit-length DNA.

occurring defective HBV genomes have been isolated from serum samples of asymptomatic HBV carriers (11). Deletions in these genomes, which span sequences up to 1,000 bp in length, appeared to be confined to a region flanked by DRI and by the pre-Sl region of the HBs gene. With regard to the sequences deleted, the defective HBV DNA construct of plasmid pddl very closely resembles these in vivo defective genomes. In contrast, the HBV DNA of pdml and pdd3 are deleted in a region downstream of the deletions in natural

defective genomes. The transcription of a pregenome from pddl should be controlled by upstream sequences which are exclusively of

1854

J. VIROL.

NOTES

viral origin, whereas in pdml parts of these upstream secontaining the viral enhancer are replaced by a metallothionein enhancer. In pdd3 the viral enhancer is also deleted, but not replaced by foreign regulatory DNA sequences. The ratios of defective to wt progeny DNA determined in the described cotransfection experiments were different and depended on the defective DNA construct used. The low yield of defective progeny DNA in pdml and pdd3 may indicate the relative importance of viral sequences present in pddl that are not retained in pdml and pdd3. The defective HBV DNA element (Fig. 4A, pIla) that had been excised from the integrated state in a hepatocellular carcinoma (tumor 19 in reference 9) does not carry the viral enhancer. Its dimer (Fig. 4A, pdlla) specified a defective pregenome which appeared to be most efficiently reverse transcribed. The absence of the HBV enhancer in pdml and pdd3 thus does not explain the low fraction of newly synthesized defective DNA obtained with these constructs. The capacity of construct pdd3, which combines all deletions of the constructs used in this study, to become transcribed and reverse transcribed, shows that sequences from positions 84 to 2682, making up more than 80% of the viral genome, are dispensable for helper-dependent replication. It is conceivable that only sequences of the cohesive end region of the viral genome are essential for helper-dependent replication. The small fraction of defective progeny DNA that is obtained, however, may indicate that there are also sequences that influence the efficiency at which defective pregenomes are reverse transcribed. Hypothetically, deletions and substitutions could affect virion assembly, and this may indirectly interfere with reverse transcription. It may also lead to sensitivity to DNase of replicating intermediates in aberrant core particles (14). quences

We gratefully acknowledge the technical assistance of Claudia Ziegler. We thank Valerie Bosch and Heinz Schaller for valuable and fruitful discussions and the critical reading of the manuscript. This work was supported in part by a grant from the Stiftung Volkswagenwerk to C.H.S. We gratefully acknowledge financial support by H. Schaller (Deutsche Forschungsgemeinschaft, SFB 229).

LITERATURE CITED 1. Acs, G., M. A. Sells, R. H. Purcell, P. Price, R. Engle, M. Shapiro, and H. Popper. 1987. Hepatitis B virus produced by transfected HepG2 cells causes hepatitis in chimpanzees. Proc. Natl. Acad. Sci. USA 84:4641-4644. 2. Aden, D. P., A. Fogel, S. Plotkin, I. Damjanov, and B. Knowles. 1979. Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature (London) 282:615-616. 3. Chang, C., K. Jeng, C. Hu, C. J. Lo, L. P. Ting, C. K. Chou, S. Han, E. Pfaff, J. Salfeld, and H. Schaller. 1987. Production of hepatitis B virus in vitro by transient expression of cloned HBV DNA in a hepatoma cell line. EMBO J. 6:675-680. 4. Enders, G. H., D. Ganem, and H. E. Varmus. 1985. Mapping the major transcripts of ground squirrel hepatitis virus: the presumptive template for reverse transcriptase is terminally redun-

dant. Cell 42:297-308. 5. Enders, G. H., D. Ganem, and H. E. Varmus. 1987. 5'-Terminal sequences influence the segregation of ground squirrel hepatitis virus RNAs into polyribosomes and viral core particles. J. Virol. 61:35-41. 6. Galibert, F., E. Mandart, F. Fitoussi, P. Tiollais, and P. Charnay. 1979. Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli. Nature (London) 281:646-650. 7. Ganem, D., and H. E. Varmus. 1987. The molecular biology of the hepatitis B viruses. Annu. Rev. Biochem. 56:651-693. 8. Graham, F. L., G. Veldhuisen, and N. M. Wilkie. 1973. A new technique for the assay of infectivity of human adenovirus S DNA. Virology 52:456-467. 9. LonEarevic, I. F., P. Schranz, H. Zentgraf, H.-H. Liang, G. Herrmann, Z.-Y. Tang, and C. H. Schroder. 1990. Replication of hepatitis B virus in a hepatocellular carcinoma. Virology 174:158-168. 10. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 11. Okamoto, H., F. Tsuda, and M. Mayumi. 1987. Defective mutants of hepatitis B virus in the circulation of symptom-free carrieres. Jpn. J. Exp. Med. 57:217-221. 12. Pasek, M., T. Goto, W. Gilbert, B. Zink, H. Schaller, P. MacKay, G. Leadbetter, and K. Murray. 1979. Hepatitis B virus genes and their expression in E. coli. Nature (London) 282: 575-579. 13. Radziwill, G., H. Zentgraf, H. Schaller, and V. Bosch. 1988. The duck hepatitis B virus polymerase is tightly associated with the viral core structure and unable to switch to an exogenous template. Virology 163:123-132. 14. Raimondo, G., R. D. Burk, H. Lieberman, J. Muschel, S. J. Hadziyannis, H. Will, M. C. Kew, M. Dusheiko, and D. A. Shafritz. 1988. Interrupted replication of hepatitis B virus in liver tissue of HBsAg carriers with hepatocellular carcinoma. Virology 166:103-112. 15. Shaul, S., W. J. Rutter, and 0. Laub. 1985. A human hepatitis B viral enhancer element. EMBO J. 4:427-430. 16. Sureau, C., J. L. Romet-Lemonne, J. I. Mullins, and M. Essex. 1986. Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. Cell 47:37-47. 17. Treinin, M., and 0. Laub. 1987. Identification of a promoter element located upstream from the hepatitis B virus x gene. Mol. Cell. Biol. 7:545-548. 18. Will, H., R. Cattaneo, H. G. Koch, G. Darai, and H. Schaller. 1982. Cloned HBV DNA causes hepatitis in chimpanzees. Nature (London) 299:740-742. 19. Will, H., C. Kuhn, R. Cattaneo, and H. Schaller. 1982. Structure and function of the hepatitis B virus genome, p. 237-247. In M. Miwa and S. Nishimura (ed.), Primary and tertiary structure of nucleic acids and cancer research. Japan Scientific Societies

Press, Tokyo. 20. Will, H., W. Reiser, T. Weimer, E. Pfaff, M. Buscher, R. Sprengel, R. Cattaneo, and H. Schaller. 1987. Replication strategy of human hepatitis B virus. J. Virol. 61:904-911. 21. Yaginuma, K., Y. Shirakata, M. Kobayashi, and K. Koike. 1987. Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA. Proc. Natl. Acad. Sci. USA 84:2678-2682.