The hepadnaviral DNA genome is synthesized by a viral-encoded reverse transcriptase, but the nature of this protein(s) in vivo remains obscure. We have previously described studies in which activity gel assays identified multiple DNA polymerase (DNAp) activities associated with highly purified duck hepatitis B virus (DHBV) core particles. We now report that virions isolated from viremic sera are associated with DNA-dependent DNAp activities which are nearly identical to major DNAp activities detected with highly purified DHBV core particles. These results suggest that the virion-associated polymerases are the same as those which are detected with core particles and are likely to represent DHBV pol gene products involved in replication of the genome

Newbold, John E.

Oberhaus, Stephanie M.

English

Carolina Digital Repository

VIROLOGY 226, 132–134 (1996)ARTICLE NO. 0636SHORT COMMUNICATIONPreparations of Duck Hepatitis B Virions Contain Multiple DNA Polymerase ActivitiesSTEPHANIE M. OBERHAUS1 and JOHN E. NEWBOLD2Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill,Chapel Hill, North Carolina 27599-7290Received June 11, 1996; revised September 13, 1996; accepted September 18, 1996The hepadnaviral DNA genome is synthesized by a viral-encoded reverse transcriptase, but the nature of this protein(s)in vivo remains obscure. We have previously described studies in which activity gel assays identified multiple DNA polymer-ase (DNAp) activities associated with highly purified duck hepatitis B virus (DHBV) core particles. We now report that virionsisolated from viremic sera are associated with DNA-dependent DNAp activities which are nearly identical to major DNApactivities detected with highly purified DHBV core particles. These results suggest that the virion-associated polymerasesare the same as those which are detected with core particles and are likely to represent DHBV pol gene products involvedin replication of the genome. q 1996 Academic Press, Inc.For hepadnaviruses, synthesis of the virion DNA occurs ml of serum taken from DHBV congenitally infected Pekinducks. Viremic serum contains two kinds of viral parti-primarily in the liver within cytoplasmic core particles con-cles, infectious virions, and surface antigen particlestaining the pregenomic RNA and viral polymerase mole-which are present in excess of virions. Surface antigencules. The RNA pregenome templates synthesis of theparticles are composed of viral S and pre-S proteins, asvirion DNA by reverse transcription (1). This process in-well as host-derived lipids, but do not contain viral nu-cludes polymerization of minus-strand DNA (via RNA-de-cleic acids, core, or polymerase proteins. Virions andpendent DNA synthesis), degradation of the pregenomesurface antigen particles have different densities, but arefrom an RNA:DNA heteroduplex (by a putative RNase Hdifficult to separate completely by banding in buoyantactivity) as minus-strand DNA synthesis proceeds, andCsCl density gradients. A 40-ml sample from the crudesynthesis of plus-strand DNA from the minus-strand DNAvirion preparation (900 ml) was mixed with CsCl in coretemplate (via DNA-dependent DNA synthesis). Enzymaticbuffer (CB) containing Triton X-100 and 2-mercaptoetha-activities responsible for these steps are encoded by thenol as has been described for banding DHBV core parti-viral polymerase gene. Sequence analyses (2, 3) and mu-cles (8). By using this procedure we hoped to isolatetational studies have indicated four domains within thecore particles derived from virions stripped of envelopepolymerase open reading frame: an amino terminal(S and pre-S) proteins and to solubilize surface antigenprimer, a spacer region, a reverse transcriptase (RT), and aparticles. Fifty-one (100-ml) fractions were collected fromcarboxy terminal RNase H (4–7). However, little is knownthe gradient and tested for the presence of virus particlesregarding expression of this viral gene and the nature ofusing the endogenous DNAp assay and scintillationthe protein(s) in vivo which carry out the enzymatic func-counting (8). Fractions exhibiting endogenous DNAp re-tions required during reverse transcription. Our previousactivity (EPR) were pooled and banded again in a secondstudies using activity gel assays identified multiple DNA-CsCl density gradient. Fractions (100-ml) were collectedand RNA-dependent DNA polymerase (DNAp) activitiesand analyzed for the presence of EPR and viral DNA byassociated with highly purified duck hepatitis B virusSouthern blotting and hybridization with a radiolabeled(DHBV) core particles (8). In this report we show that multi-plus-stranded RNA probe (9) and then diluted with CBple DNA-dependent DNAp (DDDP) activities are also de-and centrifuged. Pellets were dissolved in sample buffertected in preparations of virions.and tested in the DDDP activity gel assay. The DDDPA crude preparation of virions was made (9) from 100activity gel assay was done as described previously (8)with a modified washing protocol (10). After electrophore-1 Current address: Department of Neurology, University of Colorado sis, each gel was incubated at 47 on a shaker with 1-Health Sciences Center, Campus Box B182, 4200 East Ninth Avenue,liter buffer changes as follows: 1 hr with two changes ofDenver, CO 80262.buffer 1:50 mM Tris–HCl (pH 8.0), 2 mM dithiothreitol2 To whom correspondence and reprint requests should be ad-dressed. and 20% glycerol; then 16 hr with two changes of buffer1320042-6822/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.AID VY 8242 / 6a21$$$621 11-01-96 11:29:59 vira AP: Virology133SHORT COMMUNICATIONFIG. 1. Fractions from the second CsCl gradient in which serum-derived virions were banded and tested for the presence of nucleocapsids usingthe endogenous DNAp reaction (EPR) and Southern blotting (S. blot). Products of the EPR were analyzed by agarose gel electrophoresis (A). DHBVDNA was detected using a radiolabeled plus-stranded RNA probe (/ probe) (B). Fractions were numbered from the bottom of the gradient to thetop. Open circular (OC) and single-stranded linear (SSL) molecules are designated by arrows.2:buffer 1 with the addition of 70 mM KCl and 10 mM DDDPs detected in Laemmli-type DDDP activity gels(unpublished observations). Although the strongestMgCl2 ; and finally 8 hr with two changes of buffer 2without glycerol. DDDP activities were concordant with peak amountsof viral markers (DHBV DNA and EPR) in fractions 25Fractions 22 through 28 from the first CsCl gradientcontained peak amounts of EPR and were pooled for to 29, significant DDDP activity was also detected infractions containing less abundant amounts of theserebanding in the second CsCl gradient. These fractionswere from the middle of the gradient and ranged in viral markers (fractions 23, 24, and 30 – 35). However,DDDP activity was not detected in the absence of ei-density from 1.2 to 1.3 g/ml, approximately halfway be-tween the density of virions (1.16 g/ml) and core parti- ther viral DNA or EPR. The minor discordance betweenpeak amounts of viral markers with peak DDDP activi-cles (1.34 g/ml). Western blots of these fractions indi-cated that these particles contained core protein, a ties detected in the activity gel assay probably reflectsdifferences in the sensitivity of detection among thesemuch reduced level of the viral pre-S protein (p36),none of the S envelope protein (p17), and a significant three assays. In a separate experiment, crude virionswere banded once in a CsCl gradient and selectedamount of a species (p28) presumed to be derived fromp36 (data not shown). Fractions 20 to 38 from the final fractions were tested in a partially denaturing RT activ-ity gel assay. The profile of DHBV DNA and EPR forCsCl gradient were tested using the endogenousDNAp reaction and the products were analyzed by fractions 20 to 38 was nearly identical to those shownin Figs. 1A and 1B and a very faint RT activity with anagarose gel electrophoresis (Fig. 1A). EPR was de-tected in fractions 24 to 38, but was strongest in frac- approximate molecular mass of 80 kDa (group II) wasdetected in fractions 25 to 36 (data not shown).tions 26 to 28. The same fractions were also assayedfor the presence of DHBV DNA by Southern blot analy- These results show that DHBV isolated from viremicserum is associated with DDDP activities nearly identicalsis. Viral DNA was detected in all fractions tested, butwas most abundant in fractions 24 to 27 (Fig. 1B). The to two major size groups of DDDP activities detectedwith liver-derived DHBV core particles, suggesting thatdensity of fractions 24 to 28 was between 1.26 and1.28 g/ml, nearly identical to the range observed for they are specifically packaged into nucleocapsids whichare later secreted into the circulation as virions. A studyfractions containing most of the EPR in the first CsClgradient. Fractions 21 to 36 were tested, without heat- measuring the specific infectivity of DHBV finds that it isessentially one, indicating that defective virions are rareing prior to electrophoresis, in a Laemmli-type DDDPactivity gel assay. DDDP activities were detected in in viremic serum (Jilbert and Burrell, personal communi-cation). Since the DDDPs we detected are not likely tofractions 22 to 36, but were greater in number andintensities in fractions 25 to 31 (Fig. 2). Five DDDP be derived from defective virions, it follows that they arenot derived from defective or aberrant core particles, butactive bands were discernible in these fractions withapproximate molecular masses of 155, 98, 78, 70, and rather from replication-competent core particles whicheventually mature into infectious virions. The pol gene68 kDa. These DDDP activities were divided into twosize groups, I and II, based on similar sizes and migra- origin of at least three of the DNAp activities is supportedby previous studies with human hepatitis B virus (HBV).tion patterns seen with DHBV core particle-associatedAID VY 8242 / 6a21$$$622 11-01-96 11:29:59 vira AP: Virology134 SHORT COMMUNICATIONFIG. 2. Fractions from the second CsCl gradient in which serum-derived virions were banded and tested for DDDP activities in Laemmli-type,8% PA DDDP activity gel assays (the same fractions described in the legend of Fig. 1). Samples were not heated prior to electrophoresis. DDDPactivities were designated I and II based on their estimated molecular masses (in kDa) shown on the right side of the gels. E. coli DNAp I (p109)and Moloney murine leukemia virus (MMLV) RT (p80) are represented by the bars on the left side of the gels and were included as molecularmass markers and positive controls for the assay as described previously (8). The two separate gels shown here were run and assayed in parallel.They were aligned using the E. coli DNAp I and MMLV RT molecular mass markers.HBV particles from patient sera and from a cell line stably detected are host proteins incorporated into viral nucleo-capsids; however, their consistent detection in virus par-transfected with HBV genomes contained, respectively,100-kDa (11) and 70-kDa (12) proteins with DDDP and ticle preparations suggests that their association is notadventitious, but functionally relevant. Our findings sup-RT activities which were identified as pol gene productsport the contention that multiple pol gene products areby specific antisera. These two DNAp species are nearlyexpressed and packaged within core particles which areidentical in size to the Group I (98 kDa) and II (68 to 78the precursors to infectious virions.kDa) DDDPs we detected in preparations of virions andsimilar to two of the RTs (p109 and p80) detected withACKNOWLEDGMENTShighly purified core particles from DHBV-infected ducks(8). Two other studies identified a 65-kDa pol gene prod- S.M.O. is currently supported by NIH Molecular Biology–VirologyTraining Grant NIH-T32 NS0731 and a fellowship grant from the Ameri-uct solubilized from preparations of HBV from patientcan Cancer Society and the University of Colorado Cancer Center.serum (13) and liver-derived woodchuck hepatitis virus(WHV) core particles (14), in Western blots using Pol-REFERENCESspecific antisera. We have also detected a similarly sized1. Summers, J., and Mason, W. S., Cell 29, 403–415 (1982).protein (p63) with DDDP and RT activities in preparations2. Khudyakov, Y. E., and Makhov, A. M., FEBS Lett. 243, 115–118of DHBV core particles (8) and virions (p68 in Fig. 2).(1989).Since the predicted full-length protein encoded by the3. Toh, H., Hayashida, H., and Miyata, T., Nature (London) 305, 827–pol gene is 94 kDa for HBV and WHV and 85 kDa for 829 (1983).DHBV, the 65- to 70-kDa proteins likely represent less- 4. Chang, L-J., Hirsch, R. C., Ganem, D., and Varmus, H. E., J. Virol.64, 5553–5558 (1990).than-full-length proteins. The reactive antisera in the5. Faruqi, A. F., Roychoudhury, S., Greenberg, R., Israel, J., and Shih,WHV study were directed at the carboxy-terminal se-C., Virology 64, 764–768 (1991).quences of the WHV polymerase and also detected a6. Li, J-S., Cova, L., Buckland, R., Lambert, V., Deléage, G., and Trépo,31-kDa protein in WHV core particles which is nearly C., J. Virol. 63, 4965–4968 (1989).identical in size to the RNase H activity (p35) associated 7. Radziwill, G., Tucker, W., and Schaller, H., J. Virol. 64, 613–620(1990).with both DHBV and WHV viremic sera and purified8. Oberhaus, S. M., and Newbold, J. E., J. Virol. 67, 6558–6566 (1993).DHBV liver cores (9). The similarities in sizes and activi-9. Oberhaus, S. M., and Newbold, J. E., J. Virol. 69, 5697–5704 (1995).ties of the HBV and WHV Pol proteins identified by immu-10. Starnes, M. C., and Cheng, Y-C., J. Biol. Chem. 264, 7073–7077nostaining with many of the DNAp and RNase H activities (1989).associated with DHBV core particles and virions suggest 11. Shin, H. J., and Rho, H. M., J. Biol. Chem. 270, 11047–11050 (1995).12. Bavand, M., Feitelson, M., and Laub, O., J. Virol. 63, 1019–1021that these activities represent products of the viral pol(1989).gene present within virus particles and, although not co-13. Mack, D. H., Bloch, W., Nath, N., and Sninsky, J. J., J. Virol. 62, 4786–valently linked to the viral genome, were nonetheless 4790 (1988).involved in its replication. It remains possible that the 14. Feitelson, M. A., Clayton, M. M., and Duan, L-X., Virology 180, 430–433 (1991).DHBV-associated DNAp and RNase H activities we haveAID VY 8242 / 6a21$$$622 11-01-96 11:29:59 vira AP: Virology

Preparations of Duck Hepatitis B Virions Contain Multiple DNA Polymerase Activities

AbstractThe hepadnaviral DNA genome is synthesized by a viral-encoded reverse transcriptase, but the nature of this protein(s)in vivoremains obscure. We have previously described studies in which activity gel assays identified multiple DNA polymerase (DNAp) activities associated with highly purified duck hepatitis B virus (DHBV) core particles. We now report that virions isolated from viremic sera are associated with DNA-dependent DNAp activities which are nearly identical to major DNAp activities detected with highly purified DHBV core particles. These results suggest that the virion-associated polymerases are the same as those which are detected with core particles and are likely to represent DHBVpolgene products involved in replication of the genome

Elsevier - Publisher Connector 

VIROLOGY 226, 132–134 (1996)
ARTICLE NO. 0636
SHORT COMMUNICATION
Preparations of Duck Hepatitis B Virions Contain Multiple DNA Polymerase Activities
STEPHANIE M. OBERHAUS1 and JOHN E. NEWBOLD2
Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599-7290
Received June 11, 1996; revised September 13, 1996; accepted September 18, 1996
The hepadnaviral DNA genome is synthesized by a viral-encoded reverse transcriptase, but the nature of this protein(s)
in vivo remains obscure. We have previously described studies in which activity gel assays identified multiple DNA polymer-
ase (DNAp) activities associated with highly purified duck hepatitis B virus (DHBV) core particles. We now report that virions
isolated from viremic sera are associated with DNA-dependent DNAp activities which are nearly identical to major DNAp
activities detected with highly purified DHBV core particles. These results suggest that the virion-associated polymerases
are the same as those which are detected with core particles and are likely to represent DHBV pol gene products involved
in replication of the genome. q 1996 Academic Press, Inc.
For hepadnaviruses, synthesis of the virion DNA occurs ml of serum taken from DHBV congenitally infected Pekin
ducks. Viremic serum contains two kinds of viral parti-primarily in the liver within cytoplasmic core particles con-
cles, infectious virions, and surface antigen particlestaining the pregenomic RNA and viral polymerase mole-
which are present in excess of virions. Surface antigencules. The RNA pregenome templates synthesis of the
particles are composed of viral S and pre-S proteins, asvirion DNA by reverse transcription (1). This process in-
well as host-derived lipids, but do not contain viral nu-cludes polymerization of minus-strand DNA (via RNA-de-
cleic acids, core, or polymerase proteins. Virions andpendent DNA synthesis), degradation of the pregenome
surface antigen particles have different densities, but arefrom an RNA:DNA heteroduplex (by a putative RNase H
difficult to separate completely by banding in buoyantactivity) as minus-strand DNA synthesis proceeds, and
CsCl density gradients. A 40-ml sample from the crudesynthesis of plus-strand DNA from the minus-strand DNA
virion preparation (900 ml) was mixed with CsCl in coretemplate (via DNA-dependent DNA synthesis). Enzymatic
buffer (CB) containing Triton X-100 and 2-mercaptoetha-activities responsible for these steps are encoded by the
nol as has been described for banding DHBV core parti-viral polymerase gene. Sequence analyses (2, 3) and mu-
cles (8). By using this procedure we hoped to isolatetational studies have indicated four domains within the
core particles derived from virions stripped of envelopepolymerase open reading frame: an amino terminal
(S and pre-S) proteins and to solubilize surface antigenprimer, a spacer region, a reverse transcriptase (RT), and a
particles. Fifty-one (100-ml) fractions were collected fromcarboxy terminal RNase H (4–7). However, little is known
the gradient and tested for the presence of virus particlesregarding expression of this viral gene and the nature of
using the endogenous DNAp assay and scintillationthe protein(s) in vivo which carry out the enzymatic func-
counting (8). Fractions exhibiting endogenous DNAp re-tions required during reverse transcription. Our previous
activity (EPR) were pooled and banded again in a secondstudies using activity gel assays identified multiple DNA-
CsCl density gradient. Fractions (100-ml) were collectedand RNA-dependent DNA polymerase (DNAp) activities
and analyzed for the presence of EPR and viral DNA byassociated with highly purified duck hepatitis B virus
Southern blotting and hybridization with a radiolabeled(DHBV) core particles (8). In this report we show that multi-
plus-stranded RNA probe (9) and then diluted with CBple DNA-dependent DNAp (DDDP) activities are also de-
and centrifuged. Pellets were dissolved in sample buffertected in preparations of virions.
and tested in the DDDP activity gel assay. The DDDPA crude preparation of virions was made (9) from 100
activity gel assay was done as described previously (8)
with a modified washing protocol (10). After electrophore-
1 Current address: Department of Neurology, University of Colorado sis, each gel was incubated at 47 on a shaker with 1-
Health Sciences Center, Campus Box B182, 4200 East Ninth Avenue,
liter buffer changes as follows: 1 hr with two changes ofDenver, CO 80262.
buffer 1:50 mM Tris–HCl (pH 8.0), 2 mM dithiothreitol2 To whom correspondence and reprint requests should be ad-
dressed. and 20% glycerol; then 16 hr with two changes of buffer
1320042-6822/96 $18.00
Copyright q 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
AID VY 8242 / 6a21$$$621 11-01-96 11:29:59 vira AP: Virology
133SHORT COMMUNICATION
FIG. 1. Fractions from the second CsCl gradient in which serum-derived virions were banded and tested for the presence of nucleocapsids using
the endogenous DNAp reaction (EPR) and Southern blotting (S. blot). Products of the EPR were analyzed by agarose gel electrophoresis (A). DHBV
DNA was detected using a radiolabeled plus-stranded RNA probe (/ probe) (B). Fractions were numbered from the bottom of the gradient to the
top. Open circular (OC) and single-stranded linear (SSL) molecules are designated by arrows.
2:buffer 1 with the addition of 70 mM KCl and 10 mM DDDPs detected in Laemmli-type DDDP activity gels
(unpublished observations). Although the strongestMgCl2 ; and finally 8 hr with two changes of buffer 2
without glycerol. DDDP activities were concordant with peak amounts
of viral markers (DHBV DNA and EPR) in fractions 25Fractions 22 through 28 from the first CsCl gradient
contained peak amounts of EPR and were pooled for to 29, significant DDDP activity was also detected in
fractions containing less abundant amounts of theserebanding in the second CsCl gradient. These fractions
were from the middle of the gradient and ranged in viral markers (fractions 23, 24, and 30 – 35). However,
DDDP activity was not detected in the absence of ei-density from 1.2 to 1.3 g/ml, approximately halfway be-
tween the density of virions (1.16 g/ml) and core parti- ther viral DNA or EPR. The minor discordance between
peak amounts of viral markers with peak DDDP activi-cles (1.34 g/ml). Western blots of these fractions indi-
cated that these particles contained core protein, a ties detected in the activity gel assay probably reflects
differences in the sensitivity of detection among thesemuch reduced level of the viral pre-S protein (p36),
none of the S envelope protein (p17), and a significant three assays. In a separate experiment, crude virions
were banded once in a CsCl gradient and selectedamount of a species (p28) presumed to be derived from
p36 (data not shown). Fractions 20 to 38 from the final fractions were tested in a partially denaturing RT activ-
ity gel assay. The profile of DHBV DNA and EPR forCsCl gradient were tested using the endogenous
DNAp reaction and the products were analyzed by fractions 20 to 38 was nearly identical to those shown
in Figs. 1A and 1B and a very faint RT activity with anagarose gel electrophoresis (Fig. 1A). EPR was de-
tected in fractions 24 to 38, but was strongest in frac- approximate molecular mass of 80 kDa (group II) was
detected in fractions 25 to 36 (data not shown).tions 26 to 28. The same fractions were also assayed
for the presence of DHBV DNA by Southern blot analy- These results show that DHBV isolated from viremic
serum is associated with DDDP activities nearly identicalsis. Viral DNA was detected in all fractions tested, but
was most abundant in fractions 24 to 27 (Fig. 1B). The to two major size groups of DDDP activities detected
with liver-derived DHBV core particles, suggesting thatdensity of fractions 24 to 28 was between 1.26 and
1.28 g/ml, nearly identical to the range observed for they are specifically packaged into nucleocapsids which
are later secreted into the circulation as virions. A studyfractions containing most of the EPR in the first CsCl
gradient. Fractions 21 to 36 were tested, without heat- measuring the specific infectivity of DHBV finds that it is
essentially one, indicating that defective virions are rareing prior to electrophoresis, in a Laemmli-type DDDP
activity gel assay. DDDP activities were detected in in viremic serum (Jilbert and Burrell, personal communi-
cation). Since the DDDPs we detected are not likely tofractions 22 to 36, but were greater in number and
intensities in fractions 25 to 31 (Fig. 2). Five DDDP be derived from defective virions, it follows that they are
not derived from defective or aberrant core particles, butactive bands were discernible in these fractions with
approximate molecular masses of 155, 98, 78, 70, and rather from replication-competent core particles which
eventually mature into infectious virions. The pol gene68 kDa. These DDDP activities were divided into two
size groups, I and II, based on similar sizes and migra- origin of at least three of the DNAp activities is supported
by previous studies with human hepatitis B virus (HBV).tion patterns seen with DHBV core particle-associated
AID VY 8242 / 6a21$$$622 11-01-96 11:29:59 vira AP: Virology
134 SHORT COMMUNICATION
FIG. 2. Fractions from the second CsCl gradient in which serum-derived virions were banded and tested for DDDP activities in Laemmli-type,
8% PA DDDP activity gel assays (the same fractions described in the legend of Fig. 1). Samples were not heated prior to electrophoresis. DDDP
activities were designated I and II based on their estimated molecular masses (in kDa) shown on the right side of the gels. E. coli DNAp I (p109)
and Moloney murine leukemia virus (MMLV) RT (p80) are represented by the bars on the left side of the gels and were included as molecular
mass markers and positive controls for the assay as described previously (8). The two separate gels shown here were run and assayed in parallel.
They were aligned using the E. coli DNAp I and MMLV RT molecular mass markers.
HBV particles from patient sera and from a cell line stably detected are host proteins incorporated into viral nucleo-
capsids; however, their consistent detection in virus par-transfected with HBV genomes contained, respectively,
100-kDa (11) and 70-kDa (12) proteins with DDDP and ticle preparations suggests that their association is not
adventitious, but functionally relevant. Our findings sup-RT activities which were identified as pol gene products
port the contention that multiple pol gene products areby specific antisera. These two DNAp species are nearly
expressed and packaged within core particles which areidentical in size to the Group I (98 kDa) and II (68 to 78
the precursors to infectious virions.kDa) DDDPs we detected in preparations of virions and
similar to two of the RTs (p109 and p80) detected with
ACKNOWLEDGMENTShighly purified core particles from DHBV-infected ducks
(8). Two other studies identified a 65-kDa pol gene prod- S.M.O. is currently supported by NIH Molecular Biology–Virology
Training Grant NIH-T32 NS0731 and a fellowship grant from the Ameri-uct solubilized from preparations of HBV from patient
can Cancer Society and the University of Colorado Cancer Center.serum (13) and liver-derived woodchuck hepatitis virus
(WHV) core particles (14), in Western blots using Pol-
REFERENCESspecific antisera. We have also detected a similarly sized
1. Summers, J., and Mason, W. S., Cell 29, 403–415 (1982).protein (p63) with DDDP and RT activities in preparations
2. Khudyakov, Y. E., and Makhov, A. M., FEBS Lett. 243, 115–118of DHBV core particles (8) and virions (p68 in Fig. 2).
(1989).Since the predicted full-length protein encoded by the
3. Toh, H., Hayashida, H., and Miyata, T., Nature (London) 305, 827–
pol gene is 94 kDa for HBV and WHV and 85 kDa for 829 (1983).
DHBV, the 65- to 70-kDa proteins likely represent less- 4. Chang, L-J., Hirsch, R. C., Ganem, D., and Varmus, H. E., J. Virol.
64, 5553–5558 (1990).than-full-length proteins. The reactive antisera in the
5. Faruqi, A. F., Roychoudhury, S., Greenberg, R., Israel, J., and Shih,WHV study were directed at the carboxy-terminal se-
C., Virology 64, 764–768 (1991).quences of the WHV polymerase and also detected a
6. Li, J-S., Cova, L., Buckland, R., Lambert, V., Dele´age, G., and Tre´po,
31-kDa protein in WHV core particles which is nearly C., J. Virol. 63, 4965–4968 (1989).
identical in size to the RNase H activity (p35) associated 7. Radziwill, G., Tucker, W., and Schaller, H., J. Virol. 64, 613–620
(1990).with both DHBV and WHV viremic sera and purified
8. Oberhaus, S. M., and Newbold, J. E., J. Virol. 67, 6558–6566 (1993).DHBV liver cores (9). The similarities in sizes and activi-
9. Oberhaus, S. M., and Newbold, J. E., J. Virol. 69, 5697–5704 (1995).ties of the HBV and WHV Pol proteins identified by immu-
10. Starnes, M. C., and Cheng, Y-C., J. Biol. Chem. 264, 7073–7077
nostaining with many of the DNAp and RNase H activities (1989).
associated with DHBV core particles and virions suggest 11. Shin, H. J., and Rho, H. M., J. Biol. Chem. 270, 11047–11050 (1995).
12. Bavand, M., Feitelson, M., and Laub, O., J. Virol. 63, 1019–1021that these activities represent products of the viral pol
(1989).gene present within virus particles and, although not co-
13. Mack, D. H., Bloch, W., Nath, N., and Sninsky, J. J., J. Virol. 62, 4786–
valently linked to the viral genome, were nonetheless 4790 (1988).
involved in its replication. It remains possible that the 14. Feitelson, M. A., Clayton, M. M., and Duan, L-X., Virology 180, 430–
433 (1991).DHBV-associated DNAp and RNase H activities we have
AID VY 8242 / 6a21$$$622 11-01-96 11:29:59 vira AP: Virology


Preparations of Duck Hepatitis B Virions Contain Multiple DNA Polymerase Activities 

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Preparations of Duck Hepatitis B Virions Contain Multiple DNA Polymerase Activities

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