Avian retroviral RNA encapsidation: reexamination of functional 5' RNA sequences and the role of nucleocapsid Cys-His motifs.

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JOURNALOFVIROLOGY, Jan. 1993,p.178-188 Vol. 67,No. 1 0022-538X/93/010178-11$02.00/0

Avian

Retroviral

RNA

Encapsidation: Reexamination of

Functional 5'

RNA

Sequences and the Role

of

Nucleocapsid Cys-His

Motifs

RACHELARONOFF,1'2tADELINE M. HAJJAR,1'2'3ANDMAXINE L. LINIAL12* Divisionof Basic Sciences, Fred Hutchinson CancerResearch Center, 1124 ColumbiaStreet,

Seattle, Washington 98104,1* andDepartments

of Microbiology2

and Comparative

Medicine,3 Universityof Washington, Seattle, Washington 98195

Received 14 August 1992/Accepted 20 October 1992

RNApackaging signals

(*)

from the5' ends of murine and avian retroviralgenomeshave

previously

been shown to directencapsidationofheterologousmRNA into the retroviralvirion.The avian5'packagingregionhas

now beenfurther characterized, and we have defined a 270-nucleotide sequence,

A*,

which is sufficienttodirect packaging of heterologous RNA. Identification ofthe

A*

sequence suggests that several retroviral cis-acting sequences containedin

*+

(theprimer binding site, the putativedimerlinkage sequence, and thesplicedonor

site)aredispensable forspecific RNA encapsidation. SubgenomicenvmRNA isnotefficientlyencapsidatedinto particles, even thoughtheA1 sequenceis presentinthis RNA.In contrast,spliced heterologous

*-containing

RNA is packaged into virions as

efficiently

asunspliced species; thus splicing perse is notresponsibleforthe failure ofenv mRNA to be encapsidated. We also found that an avian retroviral mutant deleted for both

nucleocapsid Cys-His boxes retains thecapacityto encapsidate RNA containing

*

sequences,althoughthis RNA

is unstable andis thusdifficult to detectinmature particles. Electron microscopy reveals that virions produced bythis mutant lack a condensedcore,whichmayallow the RNA to be accessible to nucleases.

Packaging signals are the cis-acting retroviral sequences required forspecific encapsidation of genomic RNA into the virion. These signals were first identified by analysis of a

quailcell linecontainingaspontaneous Roussarcomavirus

(RSV) mutant provirus, SE21Q1b (29), which has 179 bp deleted from the 5' leader of the recombinant proviral genome(3, 42). Analysesof thismutant and of other avian and mammalian retroviral mutants have localized RNA

packaging signals

(*)

to the 5' ends of retroviral genomes

(25, 26, 32, 48). Assayshave also been developed todefine sequences which are required for efficient packaging of nonviral RNAs. Such assays have shown that about 800 nucleotides (nt)of5' sequences containing part of the gag

gene(called

*+)

candirectpackagingofheterologousRNA,

equivalent to that of genomic RNA, into murine leukemia

virus (MLV) particles (1, 5). Sequences from the 5' end of the avian retroviral genome (avian leukosis virus

[ALV])

have also been tested for the ability to induce specific

packagingof nonviral RNA(4).A683-base

*

+ sequencehas

been foundtodirectefficient encapsidation ofaheterologous mRNA into avian retroviral virions. However, unlike the

case of MLV, this packaging was reduced about 10-fold

relative to that ofRNAencoded bya retroviral genome or complete retroviralvectors (4).

Subgenomicenv mRNAs are notefficiently encapsidated

bywild-type avianormurine retroviruses. The 'I sequences in MLV and spleen necrosis virus are located between the splice donorand acceptor sites; thus,

*

is not contained in the subgenomic env mRNA. In contrast, the defined 5'

*

signal in avian viruses is upstream of the splice donor and thus isincluded in both the genomic and the splicedmRNAs.

Nevertheless, env RNA is inefficiently encapsidated into

* Correspondingauthor.

tPresent address: Laboratory of Molecular Biology, Medical

ResearchCouncil, HillsRoad, Cambridge CB2 2QH, England.

ALVvirions. Anothercis-actingpackagingsignal, the 115-nt direct repeat(DR)sequence,originallyfoundflankingthesrc

gene of RSV, has been implicated in the encapsidation of avian retroviral RNA into virions (43). However, because the DR is present in bothsplicedsubgenomicandunspliced

genomicRNAs, itcannotexplainthe difference inpackaging

between these RNAs.

The retroviral genome encodes threepolyprotein precur-sors, Gag, Pol, and Env. In thecaseof avianretroviruses, Gag is proteolytically cleaved to yield five polypeptides: matrix (MA), a protein of unknown function (plO), capsid

(CA), nucleocapsid (NC), and protease (PR). Only the gag

geneproductisrequiredforproductionof retroviralparticles

containing RNA. Viruses with mutations in the PR active

site in which no processing of Gag occurs form particles which containwild-type amounts of RNA (13, 44). These and other data(38)supportthe idea that thefull-length Gag precursoris bothrequiredand sufficient for RNA packaging. AfterproteolysisofGag, the NCpolypeptidebinds RNA in vitrowithhigh affinitybut withno apparentspecificity(17).

Proteolytic cleavage also coincides with the formation of

mature C-type virions containing ahighly condensed elec-tron-densecore(24).

Analyses of virions produced by mutated proviruses have indicated that a 14-amino-acid Cys-His motif, CX2CX3

(G)HX4C (whereXrepresents anyaminoacid)found within

the NCdomain ofGag, is required forproduction of particles

containing genomic RNA (34, 35). Cys-His boxes have

generallybeenthoughttoform metalbinding domains

some-what similar to zinc finger metal binding domains. It was reported that retroviruses donotcontain zinc(21), but more

recent work has detected zinc in association with Gag proteins (for example, see reference 7). Although zinc fin-gers are generally associated with DNA binding activity,

transcriptionfactor IIIA bindstoRNAaswellasDNAand

containsmultiplezincfingers (6).

178

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A

'TI SI SV40PA

Sa=

Length Packaging

S.D.

683 ....

S.D.

AS.D.

5'ST

3S T

B

C(rTA) (A)n

ACGAACCACTGAATTCCGCATCGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAAT

AAACGCCATTTTACCATTCACCACATTGGTGTGCACCTGGGTTGATGGCCGGACCGTCGA

PBS

TTCCCTAACGATTGCGAACACCTGAATGAAGCAGAAGGCTTCATTTGGTGACCCCGACGT

LIP A'T 5'Sv

GATAGTTAGGGAATAGTGGTCGGCCACAGACGGCGTGGCGATCCTGCCCTCATCCGTCTC

GCTTATTCGGGGAGCGGACGATGACCCTAGTAGAGGGGGCTGCGGCTTAGGAGGGCAGAA

3'5JS

GCTGAGTGGCGTCGGAG§GAGTACTGCAGGGAGCCCAGATACCCTACCGAGAACTCA

GAGAGTCGTTGGAAGACGGGAAGGAAGCCCGACGACTGAGCAGTCCACCCCAGGCGTGAT

Gag S.D.

TCTGGTCGCCCGGTGGATCAAGCATGGAAGCCGTCATAAAGGTGATTTCGTCCGCGTGTA

4

GCT-AT

AAACCTATTGCGGGAAAACCTCTCCTTCTAAGAAGGAAATAGGGGCCATGTTGTCCCTCT

(dLs)

TACAAAAGGAAGGGTTGCTTATGTCTCCCTCAGACTTATATTCCCCGGGGTCCTGGGATC

4-L¶' B'S'!

CCATTACCGCGGCGCTATCCCAGCGGGCTATGATACTTGGGAAATCGGGAGAGTTAAAAA

CCTGGGGATTGTTTTGGGGGCATTGAAGGCGGCTCGAGAGGAACA I+

In thisreport,we demonstrate thatasequenceof 270nt,

A*,

can direct heterologous neo RNA into ALV. We also

show that incontrast tosplicedenvmRNA inthe retroviral

context, both spliced and unspliced heterologous mRNAs

containing

*

are efficiently packaged. Inaddition,

reexami-nation of the role of the ALV Cys-His motifs in RNA

packaging suggests that these are dispensable for initial

interactionsofGagwith 4.AnRSVCys-Hisdeletionmutant

produces particles containing RNA, but this RNA is not

intact and thusis more difficult to detect in mutant than in

wild-typevirion RNApreparations.

MATERIALS ANDMETHODS

Cell culture and transfections. ThequailcelllinesSE21Q1b (29), Q2bn-4D (46),andQT35 (36) andQT35 containingthe

398 ....

270 ....

135 +

263

FIG. 1. (A) Constructs usedtoassay5'regions of thegenomefor

4ifunction. Portionsof the 5' endof the RSVgenometobe tested

werecloned into the plasmidpCMVneo asdescribed in Materials and Methods.S.D., splice donor site ingag; AS.D.,splice donor site mutated inconstruct;S.A., spliceacceptorsite inSV40sequences. (B) Sequences of*+,L+,A*,and

S*.

Arrowsdenote the limits of the 683-base *+, 398-baseL+, 270-base A+, 135-base 5'S*, and 270-base 3'S4 sequences.The PBSis denoted byaline above the sequence. The promoter element (TATA), the polyadenylation [(A)n] signal, thestartof Gagsynthesis, the major 5' splice donor site (S.D.), and the putative dimer linkage sequence (dls) are indicated above thesequence. Thelocation of theSacl restriction endonuclease site(GAGCI7C) is indicated byabox.The splice site is mutated inA*toaHindIII restriction siteasindicated by GCTat

the arrowhead.

dell,2 provirus (35)weregrowninGM+D+CK(Ham'sF10

medium containing 10% tryptose phosphate broth [Difco], 5% calf serum, 1% heat-inactivated chick serum, and 1% dimethyl sulfoxide). The dell,2 producer cell line was

de-rivedby transfecting QT35cellswith theRSV dell,2 proviral plasmid (see below) andthepCMVneoL4, plasmidataratio

of 10:1. Cellsweregrownat37°Cina6% CO2 atmosphere.

Transfections were performed by the modified calcium

phosphatemethod ofChenandOkayama (10). All cellswere

seeded in Dulbecco modified Eagle medium with 10% calf

serum prior to the addition of plasmid DNA. Cells were

selected for drug resistance in CM (Ham's F10 medium

containing 10% tryptose phosphate broth, 12% calf serum,

4% chick serum, and 1% nonessential vitamins [GIBCO]). Hygromycin (Sigma) was used at 0.1 mg/ml and G418 (Sigma) was used at 0.25 mg/ml. Mass cultures of

drug-resistant cellswereobtainedafter 1to 3 weeksofselection. Individualdrug-resistantcell cloneswerepickedwithfinely

pulledoutPasteurpipetsintofresh selectivemedia. Plasmids. Plasmids were constructed by standard

tech-niques (31). pCMVneo and

pCMVhph4,'

have been previ-ouslydescribed(4, 27). testsequences(A4,

L*4,

5'S+, and

3'S+)wereinserted into the SmaI site ofpCMVneoin either

thesense orantisense

(QI)

orientation. Figure 1Acompares

the

Ali,

L+, and

S*

sequences relative to the previously

described 4+ sequence (4). To construct Lx4, the 5'-most

BstEII-XhoI fragment of pATV8R was cloned into vector pSL1180 (9), creating pSL1180. This was the template

for amplification with the following oligonucleotides: 5' primer (5'--3'), CGAGTCGACGGGAATAGTGGTC, and 3'primer, CGATCATGAGCCTTCAATGCC.Theamplified productwas isolated, digestedwithHincIIandBamHI,and

CMVIF

zx %.PIVI V IL- VW I

I A

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180 ARONOFF ET AL.

1

2

3

-dii lZ

P+

LT'

aLTP

FIG. 2. Primer extension ofanalysisof virion RNA todetermine

*+-versusLq,-directed encapsidation. Q2bn-4D cellswere

trans-fected with pCMVneo*+ (q+), pCMVneoL* (sense) (L+), or

pCMVneoLqi (antisense) (aLqi), andpoolsof G418-resistant clones

were selected. Equivalent amounts of viral supernatants were pelleted, and virions were extracted. RNA was hybridized to a

22-baseneo-specificantisenseoligonucleotide,andprimerextension assayswereperformed.

inserted into vectorpSL1180DNApreviously digestedwith BamHIand StuI.Theresultant subclone

(pSL1180L*)

was

thendigested withPvuII and SmaI and ligated into SmaI-linearizedpCMVneo. The A4 fragmentwas amplifiedfrom

pSL1180L4

by polymerase chain reaction with the

5'AqI

oligonucleotide primer, CGAGTCGACGGGAATAGTGG TC, and the 3'A primer, ATCAAQllATGACGGCT TCC. The 3'A primerintroducesamutation inthe retrovi-ralsplice donor site thatcreates aHindlIl restriction endo-nuclease site (underlined). The amplified fragmentwas iso-lated anddigestedwithHindIII andHincIl; the endswere

repairedwithKlenowpolymeraseandligatedintopCMVneo linearized with SmaI.

pCMVneo5'S*

and pCMVneo3'S4i plasmidswereconstructedby digestingthe

L*

fragmentwith Ec1136II (anisoschizomer ofSacI) andligatingthe 5' or 3' fragment into the SmaI site of pCMVneo. pVZLti was

constructed for generation of RNase protection probes by insertingtheNsiI-BamHIfragmentfrom

pSL1180L*4

into the

PstI-BamiHI-digested pVZ1 (pBluescribe [Stratagene, La

Jolla, Calif.] containingadditionalrestrictionenzymesitesin the polylinker [20]). When pVZLqi is digested withEcoRI and transcribed by T3 polymerase, a sense

L*

RNA is

generated;when

pVZL*

is digestedwithHindlIl and tran-scribedbyT7polymerase, an antisense RNAisgenerated. The RSV PRactivesite mutant(pgag.neo.D37N) and dele-tion(pgag.neo.APR)constructswerekindly provided byL. Stewart and V. Vogt (45). D37N also contains a second

mutation in PR (D--Y at amino acid 41 [47a]). These plasmids express only the appropriately mutated retroviral gag gene (not pol or env) and a neo gene (that is expressed from a spliced mRNA). The RSV NC mutant proviral construct, pAPrCdell,2, deleted for both Cys-His boxes, was kindly provided by P.-F. Spahr (35; the detailed con-struction is given in thisreference).

RNApreparations. Viral supematants were collected from

virus-producingcell line cultures every 12 h and clarified of

cell debris by low-speed centrifugation (2,500 rpm for 10 min) in an American Scientific Products Omnifuge. Virions

werepelleted througha2-ml20%sucrosecushion in isotonic buffer(SB) (0.1MNaCl, 1 mM EDTA, 10 mMTris-HCl,pH

7.4) by centrifugation at 23,000 rpm for 2 h in a Beckman

SW28rotor.Viralpelletswereresuspended in 100 ,ul of SB. The protein concentration was determined by Bradford analysis of resuspended pellets (Bio-Rad, Richmond, Calif.). Virions were lysed in 0.5% sodium dodecyl sulfate (SDS), and RNA was extracted by using two or three phenol-chloroform extractions followedbyachloroform extraction and ethanolprecipitation. In someexperiments, virion prep-arationswereincubated in the presence of0.5%SDS and 200

,ug of proteinase K (VWR Scientific) perml for 30 min at

37°Cpriortothephenol-chloroformextractions. Viral RNA

pelletswereresuspendedsothat each microliter of RNAwas

equivalentto1ml oforiginalviral supernatant. Total cellular

RNA was extracted by the guanidinium isothiocyanate method ofChomczynski and Sacchi (11) in 0.5 ml of solution D per10-cm2plate oftissue culture cells. RNAintegritywas

monitoredby ensuring the presence of distinct rRNA bands after ethidium bromide stainingof total cellular RNA elec-trophoresed in0.8% agaroseminigels.

RNAanalysis. Methods forprimer extension and RNase

protection have been previously described (4, 28). The

sequence of the neo-specific oligonucleotide (neo 22-mer) primer is (5'-3') GCAGCCCTlTGCGCCCTGAGTGC.

Sam-ples were resuspended inloading dye and electrophoresed

through 8% (primer extensions)or 6% (RNaseprotections)

polyacrylamide gels containing 8M urea.

Protein analysis. SDS-polyacrylamide gel electrophoresis

and techniques for cell labelling and immunoprecipitation

have beenpreviouslydescribed(41).Aftergel

electrophore-sis, proteinswere electroblotted ontonitrocellulose or

Im-mobilon P (Millipore) with a Semi-phor apparatus (Hoefer Scientific Instruments). Western immunoblots were per-formed withone ofthefollowing primaryantibodies (Abs); rabbit otRSV Pr-C (used at adilution of 1:500) and

mono-clonal Abs aCA and aMA (used at a dilution of 1:1,000) obtained from V. Vogt (Cornell University). To prevent

nonspecific Ab binding, filters were incubated for 2 h in

phosphate-bufferedsaline-0.5% Tween-5% driedmilk. The

filterswere then incubated with the primary Ab diluted in

phosphate-buffered saline-0.5% Tween-5%dried milkfor 1

hatroomtemperaturewithshaking and then washed for 10 minthreetimes with thesamebuffer. Secondary Abs

(Am-ersham, Arlington Heights, Ill.), either horseradish

peroxi-dase-conjugated donkey a-rabbit Abs (1:6,000) for

rabbit-derived antisera or horseradish peroxidase-conjugated a-mouse Abs (1:2,000) for reaction with monoclonal Abs,

wereincubated with the filter in phosphate-buffered

saline-0.5% Tween-5% dried milk for 1 h at room temperature.

Filters were washed as described above, and signal was detectedby usingthe enhanced chemiluminescence system (Amersham).

Electron microscopy. Culture supernatantswere clarified ofcell debrisbylow-speedcentrifugation, and viruses were J. VIROL.

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A.

B.

C.

D.

1 2 3 4 5 6 7 8 16 17 18 19 20 21 22

9 10 11 12 13

-112 nt

A

Al

5'S5'SI 3'S

virus

14 15

-112 nt- _ _4 w~

_L-A AI

cell

v none L A AL 5'S S'S 3'S 1Js

primer

neo L CH- A AIS'S 5'S 3'S

FIG. 3. Primerextension analysis ofviralRNAs containing

neo*

sequences. (A) RNAs were extracted from virion RNAs, and primer

extensionanalysiswith the neo 22-meroligonucleotidewasperformedasdescribedinMaterials and Methods. Equivalentamountsofviral supernatantswereloaded in each lane.Q2bn-4Dcells weretransfected with pCMVneo with the indicated test*sequence. Lanes: 1, none

(neo); 2,L+; 3, NC mutantdell,2;4, A+; 5,AI, (antisense or inverted); 6,5'S4;7,5'SI; 8,

3'S*.

Thegel wasexposed for 18 h with two

intensifyingscreens. (B)Exposureof thegelcontaining lanes 4 to 8 (panel A) for 3 days withintensifying screens; samples were as indicated

forpanelA. (C) CellularRNAextractedfrompCMVneoAe4iconstructswithAu,in the sense(lane14)orantisense (lane15)orientation. (D)

Westernblot analysis ofvirionsproduced by G418-resistant mass cultures. Test j sequencestransfected into each Q2bn-4D line arelisted

below each lane. The bandscorrespondingtothe Gag proteins CA (27 kDa) and MA (19kDa) areindicated. Lane 21 containsone-fifththe amountofsamplethatlane 20 contains.

pelleted through a 20% sucrose cushion. The viral pellets

were fixed in a 1:1 dilution of SB and half-strength Kar-novsky's fixative (22) for4 to6 h and thendehydrated in a graded series of ethanols. The pellet was then infiltrated with

amixture ofpropyleneoxide andLuft's Epon 812(30),with

gradual increasing of the proportion of Epon 812 to 100%.

Afterbeing infiltrated overnight, the pellet was embedded in Epon 812 andpolymerized ina60°Covenfor 48 h. Sections

were cut at athickness of 70to 80 nm, stained withuranyl

acetateandlead tartrate, and examined withaJEOL100SX transmission electronmicroscope at80 kV.

RESULTS

Packaging of heterologousRNAcontainingavianretroviral 4 sequences.Wepreviouslyfound thata683-base sequence called 4+ can directspecific encapsidation of hygromycin

(hph) RNA into the ALVvirion when inserted 3' of the

codingsequences(4).Inordertofurtherdefinefunctional

*

domains, sequences containedwithin

*+

were tested in a

similar heterologous system. These

*

test fragments were

inserted atthe 3' end ofpCMVneo in sense and antisense

(inverted)orientations (Fig. 1A). The sequencesof theA+,

L+,,

5'S+, and

3'S*

test fragments relative to that of the

previouslydefined

4i+

are shown in Fig. 1B.

Lls

lackstwo

retroviralcis-actingelements, theprimer binding site(PBS) anda sequence postulatedtobe involved ingenomic RNA

dimerization, the dimer linkage sequence (dls) (8, 12). The

A4,

sequence also lacks the PBS but ends at the major 5'

splice site,whichwasmutatedduringthecloningprocedure

sothat it isnolonger functional (Fig. 1B). Deletion analysis hadpreviously shown the regionsurroundingthe SacI site in theavian leader regiontobeimportant for RNApackaging

(25). Cleavage of

L*

atthe SacI site creates the

5'S*

and

3'S*

test fragments (Fig. 1B). ThepCMVneo vectors

con-tainingeach of these 4 testsequencesweretransfected into

theRous-associated virus type 1-derivedpackagingcell line Q2bn-4D (46). Virionsproducedby the cell lineswerethen

assayed for the presence ofheterologousneoRNA.

Wefirst directlycomparedthepackagingmediatedby

uP+

tothatdirected by thelongesttestfragment, L+. As shown

in Fig. 2,packagingdirectedby

L*

in thesenseorientation

(lane2)isequivalenttothat directedby

4.+

(lane 1),whereas in the antisense orientation

(aL4.;

lane 3),this packaging is decreased by more than 100-fold. Because there is no

difference between

L+.-

and

*+-directed

packaging, we

conclude that neither the PBS nor the dls contributes to

specificencapsidation.

Wenextexaminedtheabilityof all of thetestfragmentsto

direct RNA packaging by using primerextension analyses

(Fig. 3A). Both

L*

and

A*4

sequences direct equivalent

levels ofpackagingof theneoRNA(comparelanes 2and4). The

5'Sf

retains some abilityto induce

packaging (lane

6).

Thisismoreclearlyseeninalongerexposureof thisgel

(Fig.

3B, lane 11). The

3'SI

sequence does not direct

specific

encapsidation of the heterologous mRNA

(Fig.

3A, lanes 8

and13),nordo any of theinverted

*4

sequences

([denoted by

subscriptI]lanes5, 7,10,and

12)

relative

toA*

(lane

9).

neo

mRNAis present in all cell culturesatsimilar levels

(Fig. 3C,

V- fo,tw

AL. mmdo -cA

,x7ft",

kw MA

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182 ARONOFF ET AL.

Sac

S.D.

I~~~~~~~~~I

le:thnI(nt)

420 Pro'ected species

328 270

410

B

V1i-ion RNA

MI 1 2 3 4 2' 5

527

--404 4--4 -U

309 -

-242 W -s

2429

spliced L 'V

[--C

Cell RNA 1 2 3 4 5

527 -404-

:w.5

YIP

309--s Q

242 - _

275 260

FIG. 4. RNase protection analysistodeterminepackaging of spliced and unspliced+-containingRNAs.(A)Location and size ofexpected protection products forthetest*sequences.The antisenseL++ probeis shownonthetopline,with theexpected protected specieslisted below. The small boxes indicateplasmid-derived polylinkersequencesshared withsomeof the RNAs. (B)RNaseprotection analyseswere performedasdescribed in Materials and Methods. Virion RNAswereisolated fromG418-resistantmasscultures ofQ2bn-4Dcellstransfected with plasmids as follows (per lane): 1, pCMVneo; 2and 2', pCMVneoqi+ (virusobtained after transfections withtwo differentplasmid preparations); 3,pCMVneoL*;4,pCMVneoL*(antisense).Lane5containsQT35cellsinfected withreplication-competentALV butcontains

noneoplasmid. Unspliced and splicedRNAsaredenoted U and S. M indicates molecularweightmarkers of the indicated nucleotidelengths. The faint band above the unsplicedprotected product,bestseenin lane 1,is causedbyresidualundigested full-lengthaLI probe. (C)Total guanidinium isothiocyanate-extracted cellular RNAwasexamined by usingRNaseprotectionassays asdescribed forpanelB. Each lane contains RNA extracted from the cellsusedtoproducethe virions inpanelB. Lanes:1,pCMVneo; 2,pCMVneoqi+;3,pCMVneoL*;4,

pCMVneoL*(antisense); 5, QT35cellsinfected with ALV. The location of theQ2bn-4D packagingvectorband is denoted(Q).There isno

sequencemismatch betweentheprobeandthisvector.

lanes 14 and 15 for A4, and

A*,,

and data not shown), regardless of the orientation of the test insert. As a measure of viral particle production, the level of viral

proteins was examined by Western blot analysis. Similar

levels of particles (within threefold) are produced byeach

Q2bn-4D culture (Fig. 3D). When the primer extension

experimentshown in Fig. 3was quantitated by

densitome-try, after correction for theamount of RNAloaded, itwas

found that A4, directs encapsidation that is greater than

100-fold-more efficient than the level seen with no

A*

sequence orwithA4I,. Although5'S4 directssome

encapsi-dation ofneoRNA,the level isincreasedonly byabout5-to

10-fold abovebackground (eitherneo aloneorneo

contain-inginverted sequences).

Spliced RNA is efficiently packaged in the heterologous vector system. Although both genomic and spliced

subge-nomic avian retroviral RNAs contain sequences, only genomic RNA is efficiently encapsidated. Because the

Lq4

and 4+ sequencescontain the retroviral 5'splicesite and the simian virus 40 (SV40) sequences included in the

heterolo-gous vector contain a 3' splice site (Fig. 1A), the neo plasmidscould be usedtodeterminewhethersplicingaffects

packaging ofheterologous RNAs. We previously detected several different 4,+ RNA bands by Northern (RNA) blot

analysis, indicating that the splice sites are used in the

pCMVneo4+ construct(datanotshown). In orderto deter-mine whether spliced heterologous RNA is packaged into virions, an antisenseL4 probewasused for RNase protec-tion analysiswith cell and virion RNA from Q2bn-4D cell

linestransfected withneo4, vectors.

Figure4AdepictstheRNase-protected products expected

for each 4-containing RNA species. The results of the RNase protection assays with virion RNAs (Fig. 4B) and

cellular RNAs (Fig. 4C) are shown. Control cells were

transfected with plasmid encoding ALV. As expected for

wild-type virus, both unspliced and spliced ALV RNA is detected in cell RNA(Fig. 4C, lane 5) but only unspliced

RNAcanbereadilydetected in virion RNA(Fig. 4B,lane5).

We found that the retroviralsplicesite is usedin thecaseof

the

pCMVneo*+

construct butnot the

Lq,

construct (Fig.

4C, lanes 2 and 3). Both the spliced and unspliced

*+-containing

pCMVneoq,+

RNAspeciesareefficiently

encap-sidated into virions (Fig. 4B, lanes 2 and 2'). Since the identicalsplice sites arenotused in transcriptsencodedby

the

pCMVneoL*

construct,nospliced productis detected in

virions (Fig. 4B, lane 3). In the case of the

L*

protected species,theunsplicedRNAwasdifficulttoseparate fromthe freeprobe on this gel,but these could be distinguished by

using gradient gels (datanotshown). The RNA encodedby

theQ2bn-4Dpackagingcellvectorcanclearlybeseeninall of theQ2bncell RNAs(band QinFig. 4C,lanes1 to4),but this +-deleted RNA ispackagedveryinefficiently (Fig. 4B,

lanes 1 to4).

Acomparison of the ratio ofunspliced to spliced (U/S)

RNAs in thecell and virion RNAsprovides an estimateof

theefficiency ofencapsidationof each RNA. In thecase of

pCMVneo4,+,the total intracellularU/Sratio is 8.3(Fig. 4C,

lane2),while intwodifferent virion RNApreparations, the

U/S ratios are2.9 and 1.4 (Fig. 4B, lanes 2 and 2').These numbersarenotcorrected for the difference in the numberof radioactive residues protected by each RNA species and thus overrepresent unspliced RNA. We conclude that

splicedneo RNAs arenotexcluded from thevirion, unlike thecasefor thesplicedALVenvmRNA(whichisthesame

sizeasthesplicedvectorproducts; Fig.4B andC,lanes5).

The ratio ofU/SALV RNAs inthe cell is9.8; however, in the virus it ismorethan65(atthe limit ofquantitationofthis

gel). As the 270-base

A*

lacking a splice donor site is

packaged, theseresultssuggestthatsequenceswhichare3'

A

Probe: {x L-v+

fIulllenq,h k'' spliced 41

fulllength L4'

full lencrih A 'v

02bn-4Dgenome

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virus: as

PL

- - 410

-- 270

-, *- 260

--u

1%

4.

-7 8

0p

*:

1 2 3456

FIG. 5. Effect of proteinase K treatment onvirion RNAs de-tected by using RNase protection. (A) RNA was extracted as described in Materials and Methods from cellscontainingthedell,2 provirus and transfected with pCMVneoL4i (lane 1) or from Q2bn-4D cells transfected with pCMVneoA* (lane 2). Virus was isolated from these cultures, and RNAwasextracted either after digestion (lanes4and6)with 200 pLgofproteinaseKpermlorwith noaddition ofproteinaseK(lanes3 and5).Viruswasfromdell,2 cells (lanes 3 and 4) and Q2bn-4D cells (lanes 5 and 6). RNA

extracted fromequivalentvolumes of viralsupernatantwereloaded

ineach lane.(B)RNAwasextracted from cellscontainingthe APR provirus (lane 7)orthe PR active sitepointmutantprovirusD37N (lane 8). Virion RNAwas extracted after proteinaseKtreatment

(lanes9 and 10).

of the splice site and the splicingprocessitselfare

dispens-able forspecific RNAencapsidation.

Because the antisense

L*

probe is alsoprotected by the

+-deleted RNA derived from the Q2bn-4D packaging line

vector, it was possible to directly compare packaging of RNAscontaining and lacking

*

in the same virion popula-tion. The ratio of the Q2bn-4D vector-derived RNA to unspliced

neo*+

RNA in the cell is 6.4 (Fig. 4C, lane 2), while this ratio is0.007in thevirion(Fig. 4B,lanes2and2'; theQ2bn-4D-protected productof260ntisbarelydetectable

atthisexposureof thegel). Thus, packagingof the heterol-ogous +-containing RNA is about 1,000-fold more efficient than that of thevector RNAlacking Ji.

NCCys-Hismotifsarenotrequiredfor RNApackaging.An intactGag polyprotein precursor appearstoberequiredfor avian retroviral genomic RNA encapsidation (38), although

processing of the precursor is not required (44). It has been suggested that the NC Cys-His motifs are an essential component of the packaging machinery, as deletion of both of the Cys-His motifs from the NC protein of RSV (dell,2) leads to production of virions in which RNA cannot be detected by Northern blot analysis (35). We confirmed this resultby using denaturing Northern blots (data not shown). However, Northern blot analysis will detect only intact RNA molecules and is thus not a very sensitive assay. During the course of these studies, we obtained evidence that RNA was present in dell,2 viral particles, albeit in a degraded form. Primer extension analysis with virion RNA obtained from cells containing both a dell,2 provirus and pCMVneoL4 sometimes showed a smear of neo extension products, possibly indicative of degraded RNA (for exam-ple,seeFig. 3A, lane3). In this experiment, only the dell,2

RNA yielded this result, although all virion RNAs were prepared at the same time and with the same reagents. In other experiments, we could sometimes detect a faint band ofthe expected size (data not shown).

This prompted us to reexamine whether

*-containing

RNAis packaged into the NC dell,2 virions by using RNase protection assays and a variation of our standard virion RNA extraction protocol. Intact virion RNA is readily obtained from wild-type virions by using phenol-chloroform extrac-tioninthe presence of0.5%SDS. RNA can also be extracted fromvirion suspensions after digestion with proteinase K in the presence of0.5% SDS (38, 44), although we have not found this treatment to affect RNA integrity when using wild-type virus. We reasoned, however, that if RNA in the

dell,2 particleswas moresusceptibletoRNasedegradation,

aproteinase Ktreatment might inhibit such nuclease activ-ity. To examine whether proteinase K treatment would allow detection of dell,2 virion RNA, RNase protection assays wereperformed with the

aLLI

probe and RNA obtained from virions which had been extracted either by our usual method

or after proteinase K treatment (Fig. 5). We compared cellularand virion RNAs from dell,2 cells transfected with

pCMVneoL*

and Q2bn-4D cells transfected with pCMV

neoA,4. When total cellular RNA from dell,2 cells

trans-fected with

pCMVneoLfi

was analyzed, the expected

Lqi

band at410nt(Fig. 4A) is readily detectable (Fig. 5A, lane

1). Aprotected species of 270 ntis expected from hybrid-ization of theodL14probe

toA*

RNA(Fig. 4A),and both the 270-baseband and the Q2bn-4D +-deleted provirusbandat

260 nt are evident in the Q2bn-4D cellular RNA (Fig. 5A, lane 2).

Aqi

RNA, as expected, is enriched in virion RNA relative to the +-deleted packaging vector RNA, although the apparent ratio islessenedbythelongexposureof thegel

tofilm. Furthermore, proteinaseK treatment hasnoeffect

onthe amount of A or Q2bn-4D RNA detectable (lanes 5 and 6). In contrast, proteinase Ktreatment hasa profound effectontheamountof RNA detectable in thedell,2virions

(lanes3and4).Inadditiontothemajor protected species at

410bases, there are many shorterprotected species which

are notfound in the cellular RNA(comparelanes 1and4).In contrast, most of the shorter species found in Q2bn-4D virionsareattributabletosimilarspeciesin the cellularRNA

(lanes 2, 5, and 6). These results are consistent with the

presenceofpartially degradedRNAinthedell,2virions.In

thisexperiment,RNAs extracted fromequalvolumes of cell

supernatant were loaded in each lane. The dell,2 QT35

culture produces about 40% of the number of

particles

produced by the Q2bn-4D cultures as detected byWestern

blot analysis(data notshown).

We also examined the PRmutantAPR and D37N virions

A. B.

cell: b C; neoI

neo RNA L A

,irus: dell,2 Q2bn

RENA. L L A A

PK - + - +

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184 ARONOFF ET AL.

RSV

SE

21Qlb

X

40K

Q2bn

del

1,2

[image:7.612.151.476.73.411.2]

X

50K

FIG. 6. Analysis of pelleted virions by electron microscopy. Preparationwas as described in Materialsand Methods. Magnifications: x40,000(top panels) andx50,000(bottom panels).

in the presence of proteinase K as controls. It has been shown by using assays other than RNase protection that APR, which has a Gag polyprotein deleted for the PR

domain,doesnotpackageRNA(38),while the PR active site

point mutant D37Nvirions do contain wild-type levels of

RNA(44).Theexpectedresultswereobtained in bothcases.

The active site mutant D37N packageswild-type levels of

unspliced genomicRNAbutnotsplicedRNA(Fig. 5B,lanes

8 and10).APRvirions haveaseverepackagingdefect(Fig.

5B,

lanes 7 and9).The fact thatnoRNAwasdetectableby

our techniques with the APR virions further validates the

fact that RNA is encapsidated into dell,2 particles by Gag

lacking Cys-His domains.IfthisRNA wasmerely associated

with theoutside of thevirions, it would have been detectable in all viralpreparations.

Weconclude from these studies that dell,2 virions retain theabilitytoencapsidateRNA.This doesnotcontradict the previous conclusion that dell,2 virions do not contain intact virion RNA (35). However, it demonstrates that other do-mains of the Gag precursor, not the Cys-His boxes of the NC domain, are required for specific

*

recognition and RNA encapsidation.

Morphology of dell,2 andpackaging cell line virions.

Viri-onsproducedby packaging cell lines generally contain RNA,

even in the absence of apackageable retroviral vector (4). Our studies withdell,2 ledustopostulate that encapsidated RNA in this mutant is susceptible to degradation because

matureNCbinding is disrupted by theCys-His box deletion. Wewishedto seewhether this differencemightbe reflected in an altered dell,2 virion ultrastructural morphology. In

addition, we wished to determine whether the presence of

+-containing RNAmightcontribute to theformation of the

well-characterizedmorphology of the mature type C virus (for example, see reference 39). Wechose three viruses to

analyzein orderto addressthissecondquestion. Wild-type

RSV encapsidates abundant genomic RNA containing 4

sequences. Q2bn-4D virions contain little or no +-deleted RNA, derived from the packaging vector, and a subset of cellular RNAs suchasglyceraldehyde3-phosphate dehydro-genase (4). SE21Q1bis apackaging mutantwhich encapsi-dates cellular mRNAs, including the 4,-deleted mutant ge-nome, randomly (3, 4, 42). Pelleted virions fromwild-type RSV,Q2bn-4D,SE21Q1b,anddell,2 cellswereanalyzed by electronmicroscopy (Fig. 6).We found that virionsreleased by the Q2bn-4Dpackagingcell line and cellscontaining the

SE21Q1bmutant areindistinguishable fromwild-type RSV

and containanelectron-densematureviralcore.Incontrast,

the dell,2 virions have a diffuse central core, with a

mor-phology resembling neither mature nor immature C-type

particles. The virion proteins are processed in this mutant

(35 and data not shown), so an immature morphologywas notexpected. However, the lack ofawild-type, hexagonal, and condensedmature coremostlikelyreflects theinability J. VIROL.

k'm -;.v,

'.C. ANMN-.

,.I.

ill

ip

I

Vi".ik.,f-v.

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A 3B

x14f+

A

,o

B

i_ C

LXf

A

C

FIG. 7. Predictedsecondarystructuresof*+,L+,and A i.The GeneticsComputerGroup (UniversityofWisconsin) (14)RNAfolding

program,FOLD,basedonthecomputeralgorithmof Zucker(49),and thegraphics outputprogramSQUIGGLESwereused.Folding energies obtained(intenths of kilocalories[1cal= 4.184J])were asfollows: for*+,-204.0; forL+, -134.3; and forA,-90.9.

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186 ARONOFF ET AL.

of

dell,2

mutantNC

protein

tobindwith

high affinity

tothe

packaged

RNA.

DISCUSSION

Inthis

study,

wehave examined thecis-and

trans-acting

determinants ofRNA

encapsidation

into the avian retroviral virion. We show that the functional ALV 5' 4 sequence is 270 nt or less in

length,

that

spliced

RNA can be encapsi-dated

efficiently

into retroviral

particles,

and that NC

Cys-His boxes are not

required

for the initial

Gag-qij

RNA interaction which

brings specific

4-linked RNA into the

particle.

Regions

of the

cis-acting packaging domain, 4,

from the 5'

end ofthe retroviral genomewere tested in a

heterologous

packaging

assay. A270-nt sequence,

A+,

was found to be sufficient to direct

encapsidation

of

heterologous

neo RNA into virions

produced by

an ALV-derived

packaging

cell line. Neither Ai nor any of the other avian retroviral sequences testedcan direct

packaging

that is asefficient as that ofa

complete

retroviralvector

RNA,

possibly

because of the lack of the 3' DRsequence.

However,

we have not been able to show that the addition of DR sequences can either induce

packaging

when

placed

3' of a

heterologous

mRNAoraugment

packaging

of

heterologous

+-containing

RNA

(4a).

These data thus show that the 4 sequence is

necessaryfor

packaging

in aretroviralcontextandsufficient

for

packaging

ina

heterologous

context.

However,

it should

be noted that 4 sequencesdefined assufficient ina heterol-ogous context may be different from those defined

by

deletions within

genomic

RNA,

perhaps

because of differ-ences in RNA

folding

constraints. For

example, although

MLV sequences in U5 are not

required

for

packaging

of

heterologous

RNA

(1),

deletions inU5canaffect

encapsida-tion of

genomic

RNA

(37).

TheA4, sequencedefined in the

heterologous

assaylacks the retroviral

splice

donor

site,

the

putative

dimer

linkage

sequence, and the PBS.

Thus,

none of these

cis-acting

retroviral elements is

required

for

specific

RNA

encapsida-tion. In

addition,

the sequences present in the 398-base

L+,

which lacks both the PBS and the

dls,

aresufficienttoallow detection of dimers and

higher-order

complexes

in

nondena-turing

gel

systems

(4a).

Thus,

the dls is

unlikely

to be the

sole determinant of RNA

multimerization,

an eventwhich

probably

occurs

only

after

proteolytic

maturation of

Gag

(40), subsequent

to the interactions

determining specific

RNA

encapsidation.

What is

required

for sequences to have

*4

function in a

heterologous

context? It is

generally

believed that RNA

folds into a

particular

structural

configuration

to allow

pro-tein

recognition (33).

Wehave shown that 4 sequencescan

function when

placed

next to both

hph

and neo, and it is reasonable to assume that the functional 4 folds

indepen-dently

of the linked

heterologous

sequences. In order to

analyze potential secondary

structuresthatareconserved in the functional 4 sequences identified in this work, the Genetics

Computer

Group (University

of

Wisconsin)

FOLD program

(14),

basedonthe Zuckeralgorithm (49),wasused to fold sense,

antisense,

and scrambled 4, sequences. A

variety

ofstructures

resulted,

and themoststablestructures

obtained forsense

4,+,

L+,

andAq4

areshown in

Fig.

7. Each contains three identicalstem

loops,

denotedA, B, C,which could constitute a core

packaging

domain. Inverse and scrambled

4,

sequences resulted in

quite

different predicted

secondary

structureswhich donotcontainA, B,orC(data

not

shown).

Similar computer

modeling

coupledwith direct

biochemical assays have definedpotential 4, structures for humanimmunodeficiency virus and MLV(2, 19).However, it hasnotbeen determined whether the human immunodefi-ciency virus and MLV sequencesanalyzedactually function biologically in a heterologous context.

We have found thatsplicing doesnotaffect theability of a heterologous

4,

RNA to be encapsidated. When the splice donor site is specifically mutated in A+, packaging is not

affected. Furthermore, when neo4,+ RNA is spliced to the

SV40spliceacceptor,the level ofpackaging of this species is

equivalenttothat of the unspliced RNA. Swain and Coffin have also demonstrated efficient packaging of spliced ALV readthrough RNA (47). The packaged spliced mRNAwas

generated by using the gag 5' splice site and the SV40 3'

splicesite so that 4 was at the5' end of the RNA (47). In

contrast, retroviral spliced subgenomic RNAs are not

effi-ciently encapsidated. Together, these results show that the

splice site is not required for packaging and that some

feature other than an interaction with thesplicing machinery

distinguishes unspliced and spliced retroviral RNAs. The

lack ofpackagingof viralenvmRNA could be causedby a

negative effect ofjuxtaposition of 4 andenv sequences in

spliced RNAs,or evenlong-distance interactions with DRor

U3 regions which prevent proper folding. An analysis of packaging directed by heterologousconstructsin which the

3' splice siteandenv sequences ofan avian retrovirus are

inserted inplace of the SV40 pA sequences of pCMVneo4+ could address this issue. Alternatively, sequences within gag orpol could interact positively with 5' 4 and 3' DR sequencesingenomic RNA so that, when these sequences

aresplicedfrom thetranscript,efficientpackagingnolonger

occurs. However, such positive elements have not been identified in avian viruses.

Suggestions have been made that Gag recognition of 4

involves the NC Cys-His domains (15, 17, 18, 35). Studies with chimeric viruses havereaffirmed the importance of the NC domain in

4,

recognition (16).Our results with the RSV

mutantlackingbothNCCys-Hisboxesstrongly suggest that

theprimary RNA-proteininteractiondeterminingpackaging

specificityis genetically separable from the ability to form

matureparticles containing intactgenomic RNA. By using RNase protectionassays,wefound that4,-containing RNA

is encapsidated into particles producedby cells containing

the dell,2 provirus but that it is extremely susceptible to

nucleolytic degradation. This is also reflected in the

mor-phologyof the dell,2 particles as seenby electron

micros-copy. The structure of the diffuse core seen in

dell,2

particles is consistent with the idea that packaged RNAis

readilyaccessibletonucleases and thus difficulttodetect in

virion RNApreparations.Nucleolytic degradation of encap-sidated RNA could

begin

immediatelyafter virionbudding. Nevertheless, in the absence of Cys-His boxes the Gag

polyproteiniscapable of specifically recognizing and

pack-aging

4-containing

RNA.TheCys-His boxes are required for

bindingofmatureNCto RNA,aninteractionimportant for

completevirionmaturation, protection ofthe encapsidated

RNA from degradation, and subsequent infectivity.

How-ever, it is not known whether the protein domain

encom-passingtheCys-HisboxesdirectlybindsRNA orwhether it

mediates another interaction, perhaps protein

multimeriza-tion,

which is indirectly responsible for RNA binding and

maturevirion corecondensation. Recent work with bovine leukosis virus suggests that theMAdomain may play a role

in specific RNA encapsidation (23). Combined with a

re-quirementforthePRdomain of ALV Gag, it is likely that the

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interaction of Gag and

*

is complex and involves more than oneprotein domain.

ACKNOWLEDGMENTS

This work was supported in part by grant CA18282 from the

National CancerInstitute to M.L.L. andbyComprehensive Cancer

Center core grant 5P30 CA15704. R.A. was supported by the UniversityofWashington Molecular and Cellular Biology training

grant GM07270, and A.M.H. was supported by University of Washington Comparative Medicine Training Program grant RR07019.

We thank L. Caldwell and J. Groombridge of the FHCRC

Electron Microscope Facility for invaluable assistance and the

Pittsburgh Supercomputing Center for help with some RNA fold-ings. We also thank V. Vogt and P.-F. Spahr for their generous contribution of reagents for these studies. We are grateful to M.

Emerman, J. Overbaugh, and L. Sharmeen for providing critical

assessment of the data.

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