Effects of insertional and point mutations on the functions of the duck hepatitis B virus polymerase.

Effects of

Insertional and Point

Mutations

on

the Functions of the

Duck

Hepatitis

B

Virus Polymerase

LUNG-JI CHANG,1t RUSSELL C. HIRSCH,2 DON GANEM,"3 ANDHAROLD E. VARMUSl2* Departments of Microbiology andImmunology,1 Medicine,3 and Biochemistry and Biophysics,2

University of California, SanFrancisco, California 94143-0502 Received 4 June1990/Accepted 16 August1990

The polymerase (P)geneof hepadnaviruses encodesalargepolypeptide thatappearstoparticipate in several

stepsinthevirallifecycle: packaging of viral RNA, providing the primer for synthesis of minus-strand DNA,

synthesizing minus-strand DNA from an RNA template and plus-strand DNA from a DNA template, and

degrading viral RNA in RNA-DNA hybrids. To assist in the assignment of these functionstodomains of the duckhepatitisBvirus polymerase protein,wehave constructed aseriesof substitution mutations andalarge insertion mutation, based in parton amino acidsequence comparisonswith other proteins known toexhibit

reversetranscriptase (RT) and RNase H activities. We foundthatchanges in highly conserved sequences in

putativeRTand RNase Hdomainsin the carboxy-terminal half of the protein dramatically reduced synthesis of both strands of viral DNA without major effects on RNA packaging into subviral cores. Thus we can uncoupleRNApackagingand DNAsynthesisbutcannot separateRT and RNase H activitiesashas beendone

with human hepatitis B virus. The viability ofamutantwithalargeinsertion (123 amino acids)upstreamof

theRT and RNase H domain indicates that a hinge region may separate parts of the polymerase protein

implicatedinprimingandpolymerization.

Replication ofhepadnaviruses involves synthesis of the viral DNA genome from an RNA pregenome packaged in cytoplasmic core particles (16). Several of the functions required forreverse transcription of viral RNA-packaging

of viral RNA (8), priming of DNA synthesis (1), DNA

polymerization on RNA and DNAtemplates (reverse tran-scriptase [RT]), anddigestion of RNAin RNA-DNAhybrids (RNase H)-are believed to be encoded in a long open reading frame calledPthatoverlaps the 3' end of thecore(C) geneandall of thesurfaceantigengene(7). Unlike retroviral

polgenes, which areexpressed by synthesis ofcore

(gag)-pol fusion proteins, the hepadnavirus pol genes are ex-pressed by translational initiationatastartcodonnearthe5'

end of the P reading frame that is preceded by several upstreamAUGs in PmRNA(5, 14).

Comparison ofproteins with RT or RNase H activities

encoded by hepadnaviruses, retroviruses, and othergenetic

elements (see Fig. 1) indicates that a domainof the

hepad-navirus P protein likely to be essential for RT function is

positioned in thecarboxy-terminal halfof the duckhepatitis

B virus (DHBV) Pgene product (i.e., residues 477 to 515) upstream ofa region resembling known RNase H domains

(i.e., residues 665 to 755). Experiments with antipeptide

antibodies have shown that protein linked to the 5' end of

minus-strand DNA, believed to prime synthesis of that

strand,is derived from the Pgeneproduct (1).Theregionof

the P protein that facilitates packaging of viral RNA into

cytoplasmic core particleshas notbeenidentified, although

several P frameshift mutants are deficient in this recently

described function(8).

Totest some of these predictions about Pgene function, we have constructed a set of substitution mutations in conserved regions of the P gene of DHBV and one large

* Correspondingauthor.

tPresent address: Laboratory of Molecular Microbiology,

Na-tional Institute ofAllergy and InfectiousDiseases, Bethesda, MD 20205.

insertion mutation inaputatively functionlessspacerregion. The mutant DNAs have been tested for their abilities to

directassembly ofparticles containingviral RNA(the pack-agingfunction) andtosynthesizetheminus andplusstrands ofviralDNA(theRT andRNase Hfunctions).Incontrast to

arecentreportthat the RT and RNase H domains of human

hepatitisBvirus(HBV)canbedissociated by mutation (12), we have found that DHBV mutations in two conserved

RNase H motifs aswell as anRTmotif impairsynthesis of

both DNA strands. The packaging function, however, is unaffected by these changes. Finally, the insertion mutant

appears abletoperformallknownfunctions of the

polymer-ase, implyingthat the insertion is situated in a dispensable

and thusmanipulable regionof thegene. MATERIALSANDMETHODS

Plasmidconstructionandmutagenesis. A369-bp proteinA

fragment(HindIII-SalI) was isolatedfromplasmid pRIT-2T (Pharmacia) and end-filled with Klenow fragment of DNA

polymerase I; thefragmentthenwas ligatedtotheDHBV3

genomic vector (15) blunted with Asp718 (nucleotide [nt] 1290)and Klenow. Site-specificmutagenesis wasperformed as described previously except that a subclone of the

pTZ18U vector (Bio-Rad) containing the DHBV3 genomic

sequence was used (5). For thesynthesis of the YXDD (nt

1703)mutant, asynthetic30-mer(TATATGCATGCCTTCC TCCTCTGCCACCCA) was used. For thesynthesis of

mu-tants ataminoacid 666(nt2165)and755(nt 2432),a20-mer [GTAGCTACG(C/G)A(T/A)GCTACCCCI and a 24-mer

[TATAATCCTGCT(I/A/C)A(AII/_i)GGCCCATCC]

were

used(themismatchednucleotidesareunderlined).The clon-ing junctions and mutation sites of the final plasmids were

verified byDNA sequencing.

Transfection ofHepG2cells.HepG2cellsweremaintained

in Dulbecco modifiedEaglemediumsupplementedwith10%

heat-inactivated fetal calf serum (GIBCO Laboratories). Calcium-phosphatetransfectionwasperformedasdescribed previously (5). The protein A-polymerase mutant was

con-5553

0022-538X/90/115553-06$02.00/0

Copyright C) 1990,American Society for Microbiology

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structedas adimerand used fortransfection

directly.

Other mutant

plasmids

contained DHBV monomer and therefore were

digested

and

religated

so as to present circularized monomers or

oligomers

for transfection.

Preparation

of viral nucleic acids. Viral cores were har-vested from transfected

HepG2

cells

by

immunoprecipita-tion with anticore antisera as

previously

described (5). To

exclude

input

plasmid

DNA as a contaminant in these assays, the cores were treated with DNase I and RNase A

beforethenucleic acidswereextracted. To harvest

particles

fromculture

media,

mediawerecollectedon

day

3or6after

transfection,

spuninaclinical

centrifuge (800

x g, 5min)to

removedetached

cells,

furtherclarified

by centrifugation

in

aSorvallSS-34rotor

(10,000

rpm, 10

min),

and

layered

onto

2 ml of15% sucrose. The viral

particles

were

pelleted

in a

SW41rotor at

39,000

rpm for4h. After

centrifugation,

the supernatantwas

carefully

removed. Beforefurther

analysis,

the

pellet

was

suspended

in 30,ul ofHBV-RTbuffer

(50

mM

Tris

[pH

7.6],

10 mM

MgCl2,

0.1%

2-mercaptoethanol,

1% Triton

X-100)

at

4°C overnight.

Southern blot

analysis, primer extension,

and RNase

map-ping.

Southern transfer was

performed

as

previously

de-scribed

(5).

Preparation

of radiolabeledRNAwas

performed

as

previously

described

(4).

In some cases, the blot filters

containing

viral DNA were first

hybridized

to the DHBV

probe

or a

plus-stranded

RNA

probe (nt

1658 to

14)

in the

buffer described

previously by

Church and Gilbert (6). To

strip

off the

probe,

the filter was incubated in a solution

containing

80%

formamide,

1%sodium

dodecyl sulfate,

12.5

mM

EDTA,

and 10mMTris

(pH 7.5)

at

65°C

for30 minto1 h. After

prehybridization,

thefilterwas

rehybridized

with a

minus-stranded RNA

probe (DHBV

nt 14 to

1658).

For

primer

extension,

viralcoreRNAwas

hybridized

to a5'-end

32P-labeled

oligonucleotide primer

(DHBV

nt2785 to

2765)

ina solution

containing

80%

formamide,

0.4M

NaCl,

1mM

EDTA,

and 40 mM PIPES

[piperazine-N,N'-bis(2-ethane-sulfonic

acid)]

(pH 6.5),

at

95°C

for3 min and then

placed

on

ice for10 min. After

precipitation

with ethanol and

suspen-sionin 10 ,ul of avian

myeloblastosis

virus-RT buffer

(50

mM

Tris

[pH 8.3],

6 mM

MgCl2,

40 mM

KCl,

1 ,ug ofbovine

serum

albumin,

0.5 mM

deoxynucleoside triphosphate),

the

RNA-primer

hybrid

was incubatedwith avian

myeloblasto-sis virus reverse

transcriptase

at

37°C

for 45 min. The

products

were

precipitated

once withethanol and

analyzed

on an 8%

sequencing

gel

next to a

sequencing

ladder

generated

with thesame

phosphorylated primer.

For RNase

mapping,

the viralcoreswereharvested andthenucleic acid

contents were

digested

with DNase I before

hybridization

with a

specific

antisenseRNA

probe,

which could annealto the 5' end of the viral pregenomes. The RNA

probe

was

made

by

T7 RNA

polymerase

(Promega)

and incubatedwith thecore pregenomesat

85°C

for5 min, whichwasfollowed

by

incubation at

30°C overnight

in the 80% formamide

hybridization

buffer described above. The RNA-RNA

hy-bridswere

digested

with RNaseA

(40

,ug/ml)

and RNaseT1

(2

,ug/ml)

at

30°C

for1h.This reactionwasterminatedbythe

additionof sodium

dodecyl

sulfate

(to

1%)

and

proteinase

K

(50

pug)

followed

by

an additionalincubation at 37°C for 15 min. After

phenol

extraction and ethanol precipitation,the

duplex

RNAs

together

with unlabeled DNA markers were

analyzed

on a5%

polyacrylamide gel.

Endogenous polymerase

assay. For the assayof viral

poly-merase

activity,

viral materialspelletedfrom 10 ml of culture

mediawere

suspended

in 30 ,ulof HBV-RT buffer. To the mixture

dATP, TTP,

dGTP(0.5 mMeach), dCTP(12

puM),

and 1 ,ul of

[a-32PIdCTP

(3,000

Ci/mmol;

Amersham) were

added. The reaction was performedat 37°C for 3 h. At the end, cold dCTPwasaddedto0.2 mM and further incubated for 30 min. The reaction was stopped by the addition of sodiumdodecyl sulfate(to1%),10,ug of yeasttRNA,and 20 ,ug of proteinase K inafinal volume of100

pd

and incubated at 37°C for 30 min. The 32P-labeled viral DNA was then isolated by phenol-chloroform extraction and ethanol pre-cipitation. The reaction product wasanalyzedby

electropho-resis on a 0.7% agarose gel, dried onto 3MM paper, and

subjected toautoradiography. RESULTS

Experimental strategy. Site-directed mutagenesis tech-niques were used togenerate several substitution mutations at three highly conserved positions in putative RT- and RNase H-coding domains of cloned DHBV DNA (Fig. 1).

The mutant DNAs were introduced into the human hepa-tomacell lineHepG2 by calcium-phosphate-mediated

trans-fection to observe effects on P gene functions. Tojudge RNApackaging efficiency, core particles were isolated from cytoplasmic fractions by immunoprecipitation, and prege-nomic RNA was measured either by primer extension assay

or by RNase mapping (8). Synthesis of viral DNA was

assessed by gel electrophoresis and Southern blotting of DNA extracted from thecytoplasmiccores;when appropri-ate, probes that distinguish minus and plus strands of viral DNA were also used (data not shown). In some cases, we

also tested the culture medium for released virus particles that could incorporate radioactive nucleotides into DNA, a measureofendogenous DNA polymerase activity. Since the RTsofhepadnaviruseshave notbeendissociatedfrom their native templates without loss of enzymatic activity (13) and since we have been unsuccessful in assaying hepadnaviral RTactivityasdescribedbyBavand and Laub (2), itwasnot possible to test our mutants for polymerizing activity on

heterologous templates or to test directly for RNase H activity. Instead we sought indirect evidence for impaired RNase H in selectedcasesby looking for synthesis of minus strands butnotplus strands, the phenotype which would be

expected if RNase H were unable to remove pregenomic RNAfrom RNA-DNAhybrids. Nospecific tests for primer functionwereperformed.

A mutation in the conserved RTmotif, YXDD, inactivates

DHBV DNA polymerase. As an initial test of our ability to

perturb P gene functions with substitution mutations, we

changed the most highly conserved motif among RTs

(YXDD)by alteringthe YMDD sequence encodedby wild-type (wt) DHBV at amino acid positions 511 to 514 to

YMHA(Fig. 1). Aswehavepreviouslyshown(8), synthesis and packaging ofpregenomic RNA occurred normally in

cells transfected with the mutant genome (Fig. 2b, lane 4,

analyzed by primer extension). However, core particles from cells that received the mutant genome contained no

viral DNA (Fig. 2a, lane 4), which is consistent with the

prediction that the RT function was inactivated, thereby

preventingthesynthesisof either minus or plus strands. This conclusion was confirmed by the absence of endogenous DNApolymerase activityinextracellular particles (Fig. 2c, lane 3). This YMDD to YMHAmutation also changes two amino acidsatthecarboxy terminus ofthesurface antigen;

however, interruption of translation of the surface antigen frame in other mutants did not affect polymerase function

(see below) and cannot account for the results with this

mutant.

Mutations in twoconserved RNase H motifs inactivate RT

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Conserved RT: Conserved RNaseH: ...LLPQGA--SP...YADDL...TDGS...MELMAAIVAL. ...APPPHL--ND ...FVDDM...

...ALPQGI--NS... YVDDV.

...VLPQGM--SP

...YMDDL...TDAS... LEARAVAMAL.

...RLPQGF--SP... YVDDL...TDGS... AELIALTQAL.

...VLPQGF--SP

...YMDDI...SDGA ...GELAGLLAGL.

...VLPQGW--SP... YMDDL...VDGA ...TELQAIYLAL.

...VVPFGL--AP ...YVDDI...TDAS... KETLAVINTI.

...KIPMGV--SP ...YMDDV...ADAT ...AELLAACFAR.

...KAPMGV--SP .

YMDDF...T.DAT..

QELIMSCLAK.

nutations 'HA H "

".

E "

)Sition 513/514 666 ' "

..TDSQYV.. .NERCD...

..TDSAFV ...NDVAD...

..TDSRYA.. .NRMAD... ..VDSKYL...NNYVD....

..TDSQYA...NEQVD....

..TDNTHF...NHFAD...

..TDNSVV...NP AD...

..SDSTFV...NP AD...

Q H K

K

I 755

DHBV

I

I

insertion

Tetner

LI

PreS I

Core S

RT

I

65

a.a.

FIG. 1. (Top) Sequence alignments of the conserved RT and RNase H domains of known polymerases. Alignmentsweregeneratedwith

the program Genalign from the package of sequence analysis programs ofHugo Martinez, Department of Biochemistry, University of

California, San Francisco. Alignmentswere made withcomplete sequencesandpiecewise by aligning 50-amino-acid stretchesatatime.

Alignmentswereoptimized visually by givinggreaterweighttoregions of high conservation.The most-conserved amino acid stretches in both

the RT and RNase Hgenes are listed; hyphens indicate spaceofasingle, nonconserved amino acid. Conserved amino acids of DHBV P

protein chosen formutagenesisareshowninboldface; the substituted aminoacids and their residue numbersareshown below. RSV,Rous sarcoma virus; MoMLV, Moloney murine leukemia virus; BLV, bovine leukemia virus; HIV, humanimmunodeficiency virus; CaMV, cauliflowermosaic virus. (Bottom) Coding organization ofthe DHBVgenome.Open boxes denoteopenreading frames,with the Pgeneand

itsputative domains indicated atthetop.pA, ProteinAcoding region inserted into the indicated siteinopenreadingframe P in theprotein

Amutant shown inFig. 4;a.a.,amino acid.

function. We next asked whether changes in highly

con-servedresidueswithinmotifs foundinseveral known RNase H domains would affect the synthesis of viral DNA. In

particular, we wanted to verify the importance ofthe

con-served sequences and to ask whether we could generate mutants specifically deficient inRNase H function without

loss of the abilitytosynthesize minus-strand DNA.

Two amino acidsuniversally conservedamongknownand

putativeRNase Hswereselectedastargetsformutagenesis (Fig. 1). TheAspresidueatposition666waschangedtoHis

(D666H)orGlu(D666E),and theAspresidueatposition755

waschangedtoGln (D755Q), His (D755H),orLys (D755K). Surprisingly, all five of these mutants were severely or completely deficient in DNA-synthetic capacity, as

mea-sured both by Southern blotting of nucleic acids from

cytoplasmic cores(Fig. 3a andb) andby endogenous

poly-merase assay with particles pelleted from culture medium

(Fig. 3c). The only viral DNA observed was a markedly

reducedamountof minus-strand DNA incoreparticlesfrom

cellstransfected withmutant D666E, indicating slight resid-ual RT activity (Fig. 3a, lane 3; data not shown). These

resultscannotbe ascribedtoafailuretopackage pregenomic RNA, since primer extension assays showed no difference between mutant and wt packaging efficiencies (data not shown). We conclude that the conserved residues in the putativeRNase H domain areessential forviralreplication

and that mutations at positions 666 and 755 affect RT

function, thereby preventingadissociation of RT and RNase

Hactivities (see Discussion).

Alarge insertionupstreamof the RT domain iscompatible

with Pprotein functions.Thesequencesbetween the RT and protein primer regionsofhepadnavirusPgeneproductsare poorly conserved, have no known functions, and might be dispensable for virus replication. To begin to assess these possibilities,we constructed aninsertionmutantby placing a 369-bp sequence encoding bacterial protein A into the

DHBV Pgene4bpdownstream of the translationinitiation

codon for the major surface antigen gene (Fig. 1). (This mutant had the additional potentialvirtue ofdirecting

syn-thesis ofaprotein A-P fusionproductthatcould bereadily purified by binding to immunoglobulin G-Sepharose; al-though this strategy has been successfully appliedto prod-uctsof in vitrosynthesis,wehave been unabletodetectthe predicted fusion protein in lysates ofHepG2 or Cos7 cells

transfected with the mutant gene under the control of a cytomegalovirus promoter, presumably because of low

lev-elsofexpression [L.-J. Chang, unpublished results].)

When expressed in HepG2 cells in the context of the

complete DHBV genome, the protein A insertion mutant

appeared to retain most or all viral functions. Pregenomic

and subgenomic RNAs (Fig. 4a) and core protein (not shown)werepresentatlevels similartothosefound in cells

transfected with wild-type DHBV DNA. The pregenomic

RNAwasalso detectedin thecytoplasmic-core particlesof

E.coli Tyl

Copia

RSV MoMLV BLV HIV

CaMV

HBV DHBV

point n

a.a. pc

RNaSeH I

T..

TP

I

-..O...

_I

_pA

_I

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FIG. 2. Analysis of the YMDDtoYMHAmutant.(a)Southern blot analysis ofviral DNA in cytoplasmic-core particles. HepG2 cellsweretransfected withnoDNA(lane 2),wtDHBVDNA(lane

3), or mutant DHBV DNA (lane 4). Cytoplasmic cores were

prepared 4 days posttransfection, and total nucleic acids were

extracted as described in Materials and Methods; samples were

electrophoresed through 1.2% agarose gels, transferred to nylon filters, and hybridized to 32P-labeled DHBV DNA. Lane 1, 3-kbp DHBV linear DNA marker. (b) Analysis of encapsidated RNA. Cellsweretransfected withnoDNA(lane 1),wtDHBV DNA(lanes 2 and3),orYMHAmutantDNA(lane 4) and either totalpoly(A)+ RNA(lanes 1 and2)orRNA fromcytoplasmiccores(lanes 3 and 4) was prepared. These RNAs were annealed to a 5' end-labeled

oligonucleotide homologous tont 2658to 2641 nearthe 5' end of

pregenomic RNA; following extension with avian myeloblastosis virus polymerase, the labeled products were analyzed on an 8%

sequencing gelalongsideaDNA-sequencing ladder generated with

thesameprimer, shown in the four lanesonthe left. Arrowdenotes theposition of the 5' end of pregenomic RNA (3). (c) Endogenous polymerase assay. Particles were pelleted by ultracentrifugation

from themedium of HepG2 cells transfected with wtDHBV DNA (lane 1), noDNA(lane 2),orYMHAmutantDNA(lane 3). Pellets

were incubated with [32P]dCTP and unlabeled dATP, dGTP, and dTTP; labeled DNA productswerethen extracted and analyzedby

agarose gel electrophoresis and autoradiography. OC, Open

(re-laxed) circular DNA; SS, single-stranded DNA.

the protein A Pmutant (Fig. 4b), suggesting that the

inser-tion didnotpreventpackaging. Totestits effecton polymer-ase function, we analyzed the DNA in the mutant cores. Interestingly, the proteinAinsertionmutantretained

DNA-synthetic capacity, since cytoplasmic cores contained both open circular DNA and partially single-stranded

intermedi-ates (data not shown); in the culture media, the released mutantparticlescontainedopencircular DNA(Fig. 4c, lane 2). (The reduced mobility of the mutant DNA, compared

withDNAfound inwtcores,is attributabletothe size ofthe insertion mutation.) The retention ofDNA polymerase ac-tivity by the insertion mutant wasconfirmed byan

endoge-nous polymerase assay on particles harvestedfrom culture medium (Fig. 4d). Thus the region between the protein

primer and RT domains appears dispensable for P gene

functionandsubjecttoextensive manipulation;moreover, a

10% enlargement of the viral genome is compatible with packagingand complete DNAsynthesis.

Thepresence of viral DNA in themedium of cells

trans-fected with amutantthatdisrupts surface antigen synthesis

was a surprise, particularly in view ofour recent findings that Sproteinsarerequired for HBVDaneparticle assembly

and export (V. Bruss and D. Ganem, unpublished data). However, wehave also found thatHepG2 cells transfected with DHBV DNArelease significant quantities of viralcores

(V. Bruss, unpublished data); presumably the active parti-clesdetected in thisexperimentweresuchreleasedcores.

DISCUSSION

The generation of duplex DNA from a single-stranded

RNA template requiresthattheresponsible machinerycarry outmultiple reactions (7). These include minus-strand prim-ing and elongation onanRNAtemplate, the removal of the RNA from the RNA-DNA hybrid during DNA synthesis, and the priming and elongation of plus-strand DNA on a DNAtemplate. Inaddition, in viruses and retrotransposons

all ofthese reactionsareprecededby the selective encapsi-dation of the RNA template in a subviral nucleoprotein

complexorcapsid.For hepatitis B viruses, all of thesesteps

require the participation of product(s) of the Pgene. In this study, with the homologies that exist between known and presumed reverse transcriptasesas aguide, we

have attempted agenetic dissection of the functional

orga-nization of the DHBV P gene product. This approach is conceptually straightforward for those activities (RT and RNase H) that have been mapped to specific conserved regions of well-characterizedenzymes.Forexample,

exper-iments with murineleukemia virus polymerase expressed in

bacteria (17) have indicated that the RT and RNase H activities of this enzyme can be cleanly dissociated by mutationand that the two activitieslikelyreside in separate

protein domains separated byafunctionless spacerregion. Our study indicates thatsomebut notall of the activities of the DHBVenzyme can be dissociatedbymutation. First, the fact that multiple mutations affecting viral DNA synthe-sis remain competent for RNA packaging indicates that thesefunctionsareseparable.However,wedo notyetknow

whether the encapsidation function of the protein mapsto a discreteregionor(ifso)where thatregion maybe. Wehave recently examinedaseries offrameshift mutations

through-out the gene and found all of them to be defective for

encapsidation (8); this might implythatmultiple regions of

the chain arerequired or thattheactivity might mapto the

extreme C terminus. However, since mutant proteins are often unstable and since we have no way at present to measurethelevels ofPproteins, this result may simplybe a trivial consequence ofdegradation of the mutant

polypep-tides.

Ourattemptstoseparatethe RTand RNaseHfunctionsof

theDHBVenzyme, onthe otherhand, were to noavail. In

these studies we used as an indirect indicator of these activities the structure of theproductDNAs in mutant core

particles. Mutants with lesions affectingRTalone shouldbe

unable toproduce either strand of DNA, while pureRNase H mutants should accumulate RNA-DNA hybrids

contain-ing only minus-strand DNA. As expected, mutation of the YXDDmotif conserved in all RTsinactivated the DHBVRT

altogether. However, lesions in motifs implicated in RNase Hactivity also resultedin severedefects in minus-aswellas

plus-strand DNA synthesis. Again, this result could mean

either that these activities might not reside in independent domains or that the mutations we have chosen may have drastic effectsonprotein structure orstability.

Interestingly,recentstudiesindicate thatRTandRNase H

functionscanbe separated by mutation in human HBV (12). By using similar assays, Radziwill et al. have found that almost two-thirds of deletion mutations in the RNase H

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a.

1 2 3

b.

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FIG. 3. Analysis of the RNaseH homologyregion mutants. (a) Southern blotanalysis of viralDNA incytoplasmic cores.Cells were

transfected with wt DHBVDNA(lane 1), D666Hmutant DNA(lane2),orD666Emutant DNA(lane 3);3days laterDNAextractedfrom cytoplasmic cores was analyzed by agarose gel electrophoresis and Southern hybridization with a 32P-DHBV DNA probe. (b) Primer extensionanalysis of viralRNApackaged intocytoplasmiccoresaftertransfection with D666H andD666E mutantDNAs. Two lanes from

asequencing ladderareshowntotheleft,asdescribed in the legendtoFig. 2b. Thearrowindicates thepositionofproductsexpectedfrom full-length RNA. (c)Southernblotanalysis of additionalmutants.Cellsweretransfected withwtDHBV DNA(lane1)ortheindicatedmutant

DNAs(lanes 2 through 4) and analyzedasdescribed forpanelA.Hybridizingmaterialnearthebottom of thegelrepresentsDpnI-digested

plasmidDNAusedfor transfection. (d)Endogenous polymerase assay. Cellsweretransfected withwtDHBV DNA(lane 1)ortheindicated mutant DNA(lanes2through 6);4days later virusparticleswerepelleted from

tfie

medium of each culture andanendogenouspolymerase

reaction wascarried out. Labeled productDNAwasanalyzedas described in thelegendtoFig. 2c.OC,Open circular DHBVDNA;SS,

single-stranded unit-lengthDHBV DNA.

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1 2 1 2

FIG. 4. Analysis of theproteinA(pA) insertionmutant.(A)Northern immunoblot of viralRNA. Poly(A)+RNAfrom cells transfected withwtDHBVDNA(lane 1)orproteinAmutant DNA(lane 2)wasseparatedon a1.6% agarose-2.2Mformaldehyde gel,transferredto a

nylon filter, andhybridized to

32P-DHBV

DNA. Positions of RNA size markersare shown at left. k, Kilobase. (b) RNase mappingof encapsidated viralRNA. Nucleic acidswere purifiedfromcytoplasmiccoresisolated from cells transfected withwtDHBV DNA(lane1), proteinAmutantDNA(lane 2),or noDNA(lane 3). Thesesampleswereannealedtoauniformlylabeled antisenseRNAprobespanningnt

3021to2352,generatedbyin vitrotranscription withT7 RNApolymerase. Hybridswere digestedwithRNase, and theprotected duplex

fragmentwasanalyzed by electrophoresison a5%polyacrylamidegel.Positions of molecularweightmarkersareindicatedatleft.(candd)

Viral DNA synthesis. Pelleted material from culture medium of cells transfected withwtDHBV DNA(lanes 1)orproteinAmutantDNA (lanes2)wasanalyzedbySouthernhybridizationto32P-DHBVDNAinpanelcorbyendogenouspolymeraseassaywith

[32P]dCTP

inpanel

d,asdescribed in thelegendtoFig.3d.

., zo

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A.. .o co -,. C.l

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domain of the HBV P gene selectively affect plus- rather than minus-strand synthesis (12). As in our experiment, mutations that changed the highly conserved YXDD motif in the RT domain impaired synthesis of both strands. A parallel difference exists among retroviral polymerases; unlike their murine leukemia virus counterparts (17), the RT and RNase Hfunctions of humanimmunodeficiencyvirushave not been separatedby mutation (9, 11).

Inspection of the sequence of hepadnavirus genomes reveals extensive divergence within the region of the P gene overlying the pre-S region of the overlapping S open reading frame. Thus this region ofthe P protein can tolerate many amino acid changes, which is compatible with the possibility that it encodes afunctionless spacer domain. Such a domain would separate the region of the protein linked to the 5' end ofminus-strand DNA (1), where it presumably functions as a primer (7), from the region of homology to other RT domains. The fact that our insertion of 369 nt in this region remained competent for all polymerase functions is consis-tent withthisnotion, as are several observations by others. Li et al. (10) recently noted that when DHBV genomes bearing frameshift mutations in the distal pre-S region were

inoculated intoducks, pseudorevertant viruses bearing com-pensatoryframeshift mutations several codons downstream wereregularly isolated. These mutants substitute a stretch of irrelevant amino acids for the authentic P sequences in this region yet grow and spread normally in vivo. Similarly, Schaller and co-workers (12) have shown that HBV genomes with deletions in this region are replication competent in

culturedhepatomacells,implyingthat thisregion is dispens-able.

These studies indicate both the power and the limitations of purely genetic analyses of protein function. Further progress in understanding the many activities of this com-plex enzyme will likely depend on either the purification of the native enzyme or the expression of large quantities of active protein by recombinant vectors and on the develop-ment ofbiochemical assays for the individual steps in the reverse transcription pathway.

ACKNOWLEDGMENTS

We thankRobin Colgrove forhelp with the sequence inspection andTitia DeLange, PeterPryciak, and Kuen-Teh Jeangfor helpful

discussions.

This workwas supported byPublic Health Service grants from

the National Institutes ofHealth. H.E.V. isan American Cancer

Society Professor of Molecular Virology.

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