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JOURNALOFVIROLOGY, June1993,p. 3246-3253 0022-538X/93/063246-08$02.00/0

Copyright X 1993, AmericanSocietyforMicrobiology

Identification

of tRNAs

Incorporated into Wild-Type and

Mutant

Human

Immunodeficiency Virus

Type 1

MIN JIANG,'JOHNSON MAK,1AZIMLADHA,2 ERIC COHEN,2 MICHELKLEIN,3 BENJAMIN ROVINSKI,3ANDLAWRENCE

KLEIMAN"*

Lady Davis Institute for Medical Research oftheSirMortimer B. Davis-Jewish GeneralHospital,' Departmentof Microbiology, Universite6deMontreal,2 Montreal, Quebec H3T 1E2, and

TheConnaught Centre for Biotechnology Research, Willowdale, Ontario,3 Canada Received 10December1992/Accepted9March 1993

Wehave identified the tRNAs whichare incorporatedintobothwild-type human immunodeficiencyvirus type 1 strain HIB

(HIV-111,B)

produced in COS-7 cells transfected with HlV-1 proviral DNA and mutant,

noninfectious

HIV-1L,

particlesproducedinageneticallyengineeredVero cell line. The mutantproviralDNA containsnucleotides678 to8944; i.e.,bothlongterminalrepeats and theprimer binding site areabsent. As analyzed bytwo-dimensional polyacrylamide gel electrophoresis, bothmutantandwild-type HIV-1 contain fourmajor-abundancetRNAspecies,whichincludetRNALYS,

tRNAI"Y

(theputative primer forHIV-1reverse transcriptase) and tRNA". Identificationwasaccomplished by comparingtheelectrophoretic mobilities and RNase

T,

digestswith those of

tRNA3LY'

and

tRNAj',

purifiedfromhumanplacentaandcomparingthepartial nucleotidesequenceatthe 3' end of each viral tRNAspecieswithpublishedtRNAsequences.Thus,the absence of theprimer bindingsite in the mutant virus does not affect tRNALYSincorporationintoHIV-1. However,only thewild-typeviruscontains

tRNALY"

tightlyassociated with the viral RNAgenome.Theidentification of the tightlyassociatedtRNAas

tRNA3LY'

isbaseduponanelectrophoretic mobilityidenticaltothat of

tRNALYS

and theabilityof this RNA tohybridizewitha

tRNALys-specific

DNAprobe.Inadditionto thefourwild-typetRNA species,the mutantHIV-1-like particlecontains two

tRNAH'S

speciesand threetRNA-sizedspeciesthatwehave beenunable to identify. Their absence inwild-typevirus makes itunlikelythat they are requiredforviral infectivity.

Inthe earlystagesof viralinfection, theconversionof the retroviral RNAgenomeinto DNA whichcan beintegrated

into the infected hostcell genome is catalyzed by a virus-specific protein, reverse transcriptase (RT). This enzyme

requires a primer for activity, which in retroviruses is a

tRNA(9,10,12, 15,20, 21, 29, 33, 36, 37). The3'-terminal 18 nucleotides of primer tRNA bind to a complementary region just 3'of the5'-U5region, which is termed the primer binding site. Inaddition,recentevidence hasindicated that sixto seven nucleotides within the 5'-U5 region may also bind toa corresponding number of nucleotides in the TIC

loop(2)andthat genomic RNAsequences3' of the primer

binding site may play a role in facilitating primer tRNA bindingtotheRNAgenome(21). Thesequenceof the primer

binding site in human immunodeficiency virus type 1 (HIV-1)DNAsuggests that the primer tRNA in this virus is

tRNA3

ys(30),oneof the three major tRNALYS isoacceptors

inmammaliancells (29). Purified bovine

tRNA3YS

has also beenshowntohavethe abilitytointeract with HIV-1 RT and primereversetranscription in vitro (3, 34). In thisreport,we

showthat this tRNAisoneofafew selecttRNAs

incorpo-rated intoHIV-1during viral assembly.

The primer tRNA is selected for incorporation into the virus fromover 100 different host cell tRNA species. The

mechanismby whichthisoccursis largelyunknown, and it isnotclear whether othertRNAs found in retroviruses are

also incorporated by thesame mechanism. The number of abundanttRNA-sized RNA species reportedtobe found in retrovirusesisquite variable, ranging frommorethan 20 (9,

11, 14, 26, 33) toonly 2 (27). Even fora single-typevirus,

* Correspondingauthor.

HIV-1,wehave found that the number ofmajor-abundance

tRNAsincorporatedrangesfrom 4to25,dependinguponthe cell type producing the virus (18). HIV-1 produced from transfected COS cells containsonlyfour majortRNAs (18) and hasbeen showntobeinfectious (1, 31). Theinfectivity of the virusproduced bytheparticularHIV-1proviralDNA vector and COS-7 cell line used in this work has been documented previously (13, 42). Since this virus contains onlyfourmajor-abundancetRNAspecies, thegreater

com-plexityof thetRNApopulation seen in HIV-1 producedin other cell types may not reflect the actual tRNA require-ments for the viral life cycle. Are the tRNAs in COS cell-produced virus selected because of unique functions thattheyperformin the virus lifecycle,or arethesetRNAs incorporated into the virus because of common structural properties recognized bytheselectionmechanism, irrespec-tive of having a viral function? The results in this report indicate that the tRNAs incorporated probablyshare

com-monstructuralproperties,since in theCOS-7cell-produced virions, the most abundant tRNAs incorporated are the

major tRNALYS isoacceptors. One of these,

tRNALYS,

be-haves as a primer tRNA; i.e., it is the major viral tRNA

speciesfound tightly associatedwith theviralgenome.

Is primer tRNA first placed onto the primer binding site and thencarried intothe viruswith thegenomic RNA, oris

it packaged independently of genomic RNA? It has been observed that in defective murine leukemia virus (23) and avian sarcomavirus (SE21Q1 [28]) lackinggenomic RNA, the patterns of tRNA incorporated match those of the wild-typevirus.In thiswork,we haveexamined the ability

ofanHIV-1 mutantlackingtheprimer binding site (as well

as both long terminal repeats) to incorporate the normal 3246

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pattern of tRNAs, including primer

tRNA"

Ys, and show that select tRNA incorporation in HIV-1 does not depend upon the presence of the primer binding site.

MATERIALSAND METHODS

Virus. HIV-1 produced in COS-7 cells was obtained by transfecting these cells with HIV-1IIIB proviral DNA as previously described (18). Virus was isolated from the su-pernatant 63 h posttransfection. The supernatant was first centrifuged inaSorvall SS-34 rotor at 3,000 rpm for 30 min, andthe virus wasthen pelleted from the resulting

superna-tantby centrifugation in a Beckman Ti45 rotor at 35,000 rpm for 1 h. Theviralpellet was then purified by centrifugation at 26,500 rpm for 1 h through 15% sucrose onto a 65% sucrose

cushion, using a Beckman SW41 rotor.

Theconstruction ofplasmid pMT-HIV and the establish-ment and characterizationofastable Vero monkey cell line

expressing the mutant virus have been described elsewhere (16). Briefly, a proviral fragment from

HIV-1_.i

containing only protein-coding information (nucleotides 678 to 8944)

was expressed in monkey kidney Vero cells by using the inducible human metallothionein promoter. Noninfectious HIV-like particles were secreted into the culture

superna-tant. Thesemutantvirions contained all of the HIV-1

struc-turalproteins andwerepurified bysucrose cushion centrif-ugation.

Viral RNAisolation andfractionation. Total viral RNAwas extracted from viral pellets by the guanidinium

isothiocy-anate procedure (5). Total viral RNAcontaining both high-molecular-weight genomic RNA and low-molecular-weight

tRNA was fractionated by using commercial Nucleobond AX-20 columns (Nest Group, Southboro, Mass.). This

col-umn bindstotal viral RNA ina low-saltbuffer

(0.2

M KCl, 15% ethanol[EtOH], 100mM

Tris-P04

[pH 6.3]). Low- and

high-molecular-weight RNAs are eluted

sequentially

with

higher-salt buffers (18). tRNA is eluted with a 0.8 M KCl

buffer(0.8 MKCl, 15% EtOH, 100 mM

Tris-P04 [pH

6.3]), while genomic RNA and any tRNA associated with it are

eluted next,

using

a 1.3 M KCl buffer

(1.3

M

KCl,

15% EtOH, 100mMTris-PO4

[pH

6.3]).

Thefractionationof free and total associated tRNAwas done asfollows. An AX-20 column was

equilibrated

with 1 ml of 0.2 M KCl buffer.

Solutions were

passed

through

the column

by

gravity.

Twentymicrogramsof totalviral RNAwasdissolved in 500 ,ul of 0.2 M KCI buffer and loaded on the column. The columnwas then

sequentially

washed,

first with 2 ml of 0.8 MKCIbuffer,which elutes free viral

tRNA,

and then with 2 ml of 1.3 MKCIbuffer,which elutes viral

genomic

RNAand the tRNAassociated with

it,

termed totalassociatedtRNA. Each 2-ml washwas collected as 4- to

500-,ul

fractions in

1.5-ml

microcentrifuge

tubes.After the additionof 10 ,ug of carrier DNA

(pBR322),

400 ,ul of

isopropanol

was addedto eachtube,and the tubeswere

immediately

centrifuged

attop speed in an

Eppendorf

microcentrifuge

for 30 min.

(The

addition ofcarrierDNAtothe

sample

prior

to

loading

iton the AX-20 column is unnecessary, sinceit doesnot

improve

recoveryof thesample from the

column.)

Pelleted

samples

werewashedoncewith70%EtOH andoncewith95%EtOH and then dried invacuo.Withthis

procedure,

>80%oftotal viral RNA isrecovered.Toisolate

tightly

associated

tRNA,

total viral RNAwasheated for 3 minat

65°C

indissociation

buffer

(20

mMTris

[pH

7.5],

200 mM

NaCl,

2 mMdisodium

EDTA),

which will cause the release of

loosely

associated

tRNA from the

genomic

RNA. After

quick

cooling

on

ice,

the RNA

sample

was fractionated with an AX-20

column,

isopropanol precipitated in the presence of 10 ,ug of carrier

DNA, washed,anddried as described above. The 0.8 MKCl buffer will elute the freed loosely associated tRNA along with theinitiallyfreeRNA,while the 1.3 MKClbuffer will

elute the genomic RNA along with the tRNA still tightly associated withit.

Purification of human

tRNAI

8and

tRNAL'3.

Ten to 20 mg

of total tRNAcanbeisolated from200 g of humanplacenta

by using

the method of Roe (32), which uses standard

phenol-chloroform

extractions to isolate total RNA,

fol-lowed by DEAE-cellulose chromatography to isolate total tRNA. The tRNAwas aminoacylated with

[3H]lysine,

and

tRNALYS fractions were isolated by using standard

chro-matographic

methods for tRNA purification (sequentially,

DEAE-Sephadex

A-50,reverse-phase chromatography

[RPC-5],

and PorexC4

chromatography [8, 29]).

Afinal

purifica-tion step used two-dimensional

(2D)

polyacrylamide gel

electrophoresis (PAGE)

as described below. The Porex C4

tRNA'Y'

fractionwasresolvedintotwo

lysine

tRNAs, while

the Porex C4 tRNALYS fraction remained a

single species

during electrophoresis.

The tRNALYs

isoacceptor

spotswere

eluted from

gel

slices

by soaking

inwater

overnight

andwere

concentrated

by

ethanol

precipitation.

Their

identity

as

lysine tRNAswasconfirmed

through partial

RNA

sequenc-ing

(see below)

and

hybridization

with DNA

probes

specific

for tRNALys.

Inthisreport,wereferto twotRNA

species

representing

tRNALYs. The term tRNALYs refers to a

population

oftwo tRNALYS

species

which differ

by

one base

pair

in the

anti-codon stem

(29).

The first

major peak eluting

from RPC-5 containsthesetwotRNA

species,

and thesearenotresolved

by

PorexC4

chromatography.

We

do, however,

resolvethis

population

intotwo

tRNALYS

species by

the final step of 2D

PAGE. Sincewehavenotdetermined which spot is

tRNA~1Ys

and which istRNALYS,wecall both spots

tRNA'S.

RNA

labeling.

The fractionated RNA

samples

were

la-beled

by

the

[32P]pCp

3'-end-labeling technique

(4).

[32P]pCp

was made as follows. Five microcuries of

[y-32P]ATP

(spe-cific

activity, 3,000

Ci/mmol; Dupont

Canada)

was dried down in a

microcentrifuge tube, using N2.

Then 100

,lI

of

reactionsolution

(50

mMTris-HCl

[pH 9.2],

5mM

MgCl2,

3

mM

dithiothreitol,

5% bovineserum

albumin,

1

FiM

3'-CMP,

10 U of T4

kinase)

was added. The reaction mixture was incubated at

37°C

for 3

h,

and the conversion of 3'-CMP

to

[32P]pCp

wasmonitored

by

polyethyleneimine thin-layer

chromatography

in 0.8 M

NH2SO4,

which separates

[32P]

pCp

from [3

P]ATP.

Labeling

of RNA with [32P]pCp was done as

previously

described

(4,

19).

Free viral tRNA was labeled without furthertreatment.Total associatedtRNAwasfirstheatedto

65°C

for 3

min,

quickly cooled,

and then labeled. Labeled

tightly

associated tRNA was obtained

by

fractionating

a

similar amount of

labeled,

total associated tRNA on an

AX-20 column as described above. After

labeling

of the various RNA

fractions,

free

[32P]pCp

wasremovedfrom the

labeled macromolecules either

by

using

Sephadex

G-50

(Pharmacia)

homemade

spin

columns,

equilibrated

with TE buffer

(10

mM Tris

[pH 7.5],

1 mM

EDTA),

or

during

the

electrophoresis

run. Before

analysis by

PAGE,

the

samples

wereheatedat

90°C

for2 min.

RNase T1

fingerprinting

and RNA

sequencing.

Partial RNase T1

digests

are

analyzed by

using

1D

PAGE to separate the RNA

fragments

(25).

To sequence the nucle-otides at the 3' end of the tRNA

molecule,

the

enzymatic

sequencing

methodology

wasused. This

method,

first

devel-oped by

Donis-Kelleretal.

(7)

andreviewedinreference

22,

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3248 JIANG ET AL.

uses enzymes which cut 3'- or 5'-end-labeled RNA at specific bases. The radioactive fragments areseparated by 1DPAGE, and the sequence is read from theelectrophoretic

band pattern in a manner analogous to reading DNA

se-quencinggels. Specific enzymatic cleavageis altered in the presence of modified bases, and other methodologies are required to identify them. However, modified bases are normally missing within the 18-nucleotide region at the 3' end of tRNAs, and this method can result in the rapid

identificationof tRNAs.

1D and 2D PAGE. Electrophoresis of viral RNA was carried outat4°Cinathe HoeferSE620gel electrophoresis

apparatus.Gelsizewas14by32cm.The first dimensionwas runina10%polyacrylamide-7Mureagelforapproximately

16h at800 V,until thebromophenolbluedyewasbeginning

toelutefrom the bottom of thegel.After autoradiography,

thepiece of gelcontainingRNAwas cutout,embedded ina second gel (20% polyacrylamide-7 M

urea),

run for 46 h (25-Wlimiting), and thensubjected to autoradiography. All

electrophoreticruns werecarriedoutin 0.5xTBE

(1

xTBE is 5 mM Tris, 5 mM boricacid,and 1 mM disodiumEDTA).

The electrophoretic gel patterns presented in this report show onlylow-molecular-weight RNA, since the high-mo-lecular-weight viral genomic RNA cannot enter the poly-acrylamidegels. Furthermore,these patterns representonly

the most abundant tRNA species present, since the high specific activities of the labeled tRNAs used will reveal

manymoreminor-abundance species with longerfilm expo-sures.

The amount of32P radioactivity in each major-abundance

tRNArelative to othertRNA-sized speciesseen in the 2D PAGEwasdetermined eitherby excisingeach spot fromthe gel and directly countingitsradioactivityinaliquid scintil-lation counter,usingCerenkovcounting,orbyanalyzingthe 2D PAGE tRNA pattern via phosphor imaging (Bio-Rad,

Toronto, Ontario, Canada). The two methods gave similar

results.

Detection oftRNALYSisoacceptors by RNA-DNA hybridiza-tion. To detect the presence oftRNAY'S and

tRNA3'YS

in viralRNA, we have synthesized 18-mer DNA

oligonucleo-tides complementary to the 3' 18 nucleotides of

tRNA'S

(5'-TGGCGCCCAACGTGGGGC-3')

or

tRNA'YS

(5'-TGGC

GCCCGAACAGGGAC-3'). These DNA probes hybridize

specifically with tRNAY's or

tRNA3YS

(see Fig. 7A) and werehybridizedtodotblotsof RNA samples on Hybond N

(Amersham). The DNAoligomers were 5' end labeled with T4 polynucleotide kinase and

[-y-32P]ATP

(3,000 Ci/mmol;

DupontCanada),andspecificactivities of108to 109cpm/,Lg

were generally reached. Approximately 107 cpm per oli-gomerwasgenerallyused perblot in hybridization reactions.

RESULTS

Figure1Ashows the 2D PAGE pattern of tRNA incorpo-rated into HIV-1 transiently produced in transfected COS-7

cells,andFig.2Ashows the tRNA species incorporated into the mutantvirions produced in Vero cells established with pMT-HIV-1. COS-7 cell-produced virus contain four major-abundance tRNA species, spots 1, 2, and 4 being most

abundant. Figure 1B shows that in 1D PAGE, species 1 and 2have electrophoreticmobilities similar to those of the two

lysine tRNAs that we have isolated from the tRNALys isoacceptor family, while species 3 and 4 have mobilities

similar tothatofpurified

tRNALYS.

Species 1 to 4 isolated fromthe mutant virus present a pattern similar to that of the

wild-type viraltRNAs in 2D PAGE (Fig. 2A) and migrate in

A

20%L

10%

3 2

B

201

A B C 1 2 3 4

FIG. 1. tRNAincorporated into HIV-1produced in transfected COS-7 cells.(A) 2D PAGE(performedasdescribed in thetext) of free viral tRNA; (B) comparison of electrophoretic mobilities of pure human

tRNAjL3s

and

tRNA3LYS

with that ofHIV-1tRNAspecies found in the free tRNApopulation.Lanes AtoCrepresentpurifiedhuman placental

tRNA'3YS

and thetwocomponentsoftRNAlL, respectively; lanes1 to 4correspondtothe like-numberedspotsinpanel A.

1D PAGE with mobilities similar to those of their

like-numbered counterparts in wild-type virus (Fig. 2B). How-ever, the mutant virus contains an additional five RNA

speciesnot seenin theCOS-7cell-producedHIV-1.

A

20%t

10%

9

5 .2

B

__

20%1 g *

A n C I 2 3 4 5 6 7 8 9

FIG. 2. tRNAincorporated into the mutant HIV-1 produced in chronically infected Vero cells. (A) 2D PAGE (performed as de-scribedin thetext) of freeviral tRNA; (B) comparison of electro-phoretic mobilities of pure human tRNALY' and tRNA3YS with HIV-1 tRNAspecies foundinthe freetRNA population. Lanes A to andC represent purified human placental

tRNA3LYS

and the two componentsoftRNA,S,respectively;lanes 1 to 9 correspond to the like-numbered spotsinpanelA.

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tRNAs IN HIV-1 3249

A B C 1 2 3 4

.-

o o

-,-,W .n.o

I

5w

4 %

'lap

es

*47@

ob #A

I

OH A II A B C 2 3 4 5 6 7 8 9

At

-_

4~~_w~ ~.s~ ~

lif b ow xIt

_ l _ _~~~~~~~~m _. _ _

_ am_~

_. t_ _o d

4w__

_s_ _ atia*5_

_m a w _0d

0 * *

[image:4.612.131.235.78.410.2]

4k'b.0

FIG. 3. 1DPAGE ofpartial RNaseT1digestsofhuman placental

tRNALYS

and free tRNAs incorporated into HIV-1 produced in

transfected COS-7 cells. Lanes: A to C, human

tRNA3LYS

andthe two human tRNA species resolved from the tRNALys fraction, respectively; 1 to4, the freeviral tRNAspecies, numbered accord-ingtothesystem used tolabel theindividualtRNA spotsshown in Fig. 1A.

The fingerprints of partial T1 digests ofpurified human tRNALYSisoacceptorsandviral tRNAsisolatedfromCOS-7 cell-producedHIV-1werecompared (Fig. 3). Itisclear that spots 1 and 2produce thesame fingerprint pattern asdoes

tRNAiLYs, while spot 4 (but not spot 3) produces the same

fingerprint patternas does tRNA yS.

Figure4 showssimilarT1 digest fingerprintsfor the RNA

species present in the mutant virus. The T1 digest finger-prints ofspecies 1 to4 of themutant virus areidentical to those oftRNAs 1 to 4 found in the wild-type virus. The

identity of these mutant viral RNAspecies is further con-firmed in Fig. 5, which shows 1D PAGE RNA

sequencing

gels of spots 1, 2, and 4. The sequences of the last 18 nucleotides of the 3' ends of these viral tRNAs againindicate that spots 1 and 2aretRNALYS and spot4 is

tRNA3LYS

(29).

The partial sequences of the 3' termini of all nine RNA

speciesfoundin themutantvirusarelisted in Table 1. Spot

3, found in bothwild-typeandmutantviruses,appearstobe a

tRNAI'le

isoacceptor, while the sequences of spots 8 and 9 match thatof mammalian tRNAHIS. We havenotbeen able to match the partial sequences of spots 5 to 7 with any known RNAlisted in thecurrent data bases.

The tRNA incorporated into wild-type HIV-1 contains

FIG. 4. 1D PAGEofpartialRNaseT1digests ofhumanplacental tRNALYS and free tRNAs incorporatedinto mutant HIV-1 produced inchronically infected Vero cells. Lanes:OH-, alkalinehydrolysis of

tRNAlL,s

withbase;AtoC, human

tRNA3LYS

andthetwo human tRNAspecies resolved from the

tRNAL3S

fraction, respectively;1 to 9, the freeviral tRNAspecies, numbered accordingtothesystem usedtolabel theindividualtRNA spotsshown inFig. 2A. Because of the weak signal of placental

tRNA3LYS

in panel II, another fingerprint ofthis tRNA isshowninpanelI.

three,andpossibly four,major-abundance species(withthe threetRNALYs isoacceptors beingthemostabundant).

Sim-pletRNApatternsofincorporationhave also been found for mousemammarytumorvirus(27)andin avian myeloblasto-sis virusand Roussarcomavirusaswell(20).Inall retrovi-rusesexamined,includingHIV-1, therearealsomore minor-abundancespecieswhose appearance isdependentuponfilm exposure. In HIV-1, their amount relative to the tRNALYS

isoacceptors also varies from one viral preparation to an-other.

Analysis

ofthe

electrophoretic

patterns offree tRNA inwild-typeHIV-1 revealsthat,onaverage,the

radioactiv-ity in the four major-abundance speciesrepresents 80% of the total radioactivity on the gel. Among the four major

tRNAspecies,therelative abundancesare asfollows: spot1

(tRNALys),

46%; spot2

(tRNALYs), 16%;

spot3

(tRNAI1e),

8%;and spot 4(tRNA

Ys),

30%.The 2:1 ratio oftRNALysto

tRNALYsis similartotheconcentration ratio of these tRNAs in the cell.

The primer tRNA for reverse

transcription

in HIV-1 is believedtobe

tRNA3LYS.

Inotherretroviruses

examined,

the

primer tRNA is

distinguished

from other viral tRNAs

by

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3250 JIANG ET AL.

OH T1 U2 PhYMB.C. B C

OH T1 U 2 PhyM B.c

D

OH T1 U2 PhyM B.c.

tw

:w

F6

a

W a ,NM,

'a

;a

at

.a

t

d_

9

a0

608:

C-

U-*? 65G

U-

C-Op

G-a

a

Op~ ~~4C.-*

A- 4f

C- As

65

G- *

U-u- .

,oG *

C-G- *

-G

70

c--.

G- w

0- Y!

C- 0

G-

c-

G-c

-

c-FIG. 5. RNAsequencegels showingthepartialnucleotidesequencesatthe3' termini of humanplacental

tRNA3LYS

(A),mutantviralspot 4(B),humanplacental

tRNAlL,s

(C),andmutantviral spot 1(D).Lanes:OH-,alkalinehydrolysis; Ti, U2, PhyM,andB.c.,RNasesused forenzymatic sequencing (T1, U2, PhyM, andBacilluscereus,respectively).

beingfoundtightlyassociated with the viral RNAgenome.

Figure 6 shows 1D PAGE of tightly associated tRNA iso-lated fromCOS-7cell-producedHIV-1. AsingletRNA-sized

species which moves with the electrophoretic mobility of

tRNA3

y'isfound, supportingthepremisethatthis tRNAis

the primer. This tRNA also hybridizes with a

tRNA"-Ys-specific probe. We have developed 18-mer DNA probes

which specifically hybridize with either

tRNA,

Ys or

tRNA3YS.

These probes are complementary to the last 18

nucleotides at the 3' ends of these tRNAs. The tRNA dot

blots in Fig. 7A demonstrate that these probes hybridize

only to their respective tRNALYS isoacceptors and to the tRNALYS-rich fraction obtained during the purification of humanplacentaltRNALYSbyDEAE-cellulose chromatogra-phy. These oligomer probes also do not hybridize to the tRNALYs-poorfraction from the DEAE-cellulose columnor

toEscherichiacolitRNAPhe, which showsa67%homology

to

tRNALys

inthe last 3' 18 nucleotides.Usingtheseprobes,

we find (Fig. 7B) that the free tRNA

populations

of both wild-typeandmutantHIV-1containtRNA1,2sand

tRNALYS.

A OH T1 U2 PhyMB.c.

4' vo w 9

- x~

4w X, .e

:w

W

"I

* _-'Aw2

...

4w

.1

Ob

.'i

do

a a

OS

*0 .7.z

I _m

A _0 _ to

ea

*

-"l

ap

a0

G-60U

-

c-

u-r6sG

-

u-

c-*il .0

a

*p

G-60§=

C- A-65C

- G-

u-*?.'

U-0- *4#

.

70G

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[image:6.612.422.482.77.267.2]

TABLE 1. Sequencesof 3' termini oflow-molecular-weight viralRNAs

Spot Sequence(3'-5') Identity

1 ACCGCGGGUUGCACCCCG

tRNA1,2

2 ACCGCGGGUUGCACCCCG tRNA1s

3 ACCACCGGGCAUGCCCCU

tRNA'G

4 ACCGCGGGCUUGUCCCUG

tRNA3LYs

5 ACCGCGGGGUGGUGACUG ?

6 ACCACGGAUACAGGUCUU ?

7 ACCGCAAGGGACUAAACG ?

8 ACCACGGCACUGAGCCUA tRNAHiS

9 ACCACGGCACUGAGCCUA tRNAHiS

The tightly associated tRNA from COS-7 cell-produced HIV-1 hybridizes only with the

tRNALY'-specific

probe, while neither tRNALYS isoacceptor isdetected in the tightly associated fraction isolatedfrom similar amountsofmutant virus.

Figure 6 shows that inadditionto

tRNALYS,

alarger RNA

species is found tightlyassociated with the viralgenome.We

have previously reported the presenceof this RNA in the

tightly associated fraction of RNAs isolated from HIV-1 produced from chronically infected H9 cells. It has an

approximate size of 185 nucleotides (18). We also find this RNA species inmouse mammarytumorvirus (19)butdonot

find it inthemutantvirus RNAreported here. Theidentity of this RNA is unknown. In addition to this 7S-sized RNA, HIV-1 low-molecular-weight RNA containsadiscrete RNA

migrating as a 5S RNA, i.e., approximately 124 bp in size

(18). Both5S and7S RNAs have also been found in avian retrovirus (35, 39).

DISCUSSION

Inretroviruses,tRNAsarefoundbothinafreepopulation

and in a subfraction associated with the genomic RNA,

which can be further fractionated into loosely and tightly associated tRNA(39). Inthisreport,wehave characterized the free and tightly associated tRNApopulations found in infectious, wild-type HIV-1 and in a noninfectious mutant HIV-1 particlewhich lacks both long terminal repeats and

10% *

i

[image:6.612.155.230.520.698.2]

1 2

FIG. 6. 1D PAGE of tightly associated tRNA found in HIV-1

producedinCOS-7 cells(lane1)andpurifiedhumantRNA'YS(lane 2).

*8

vp

9

FIG. 7. Hybridization of RNA dot blots with DNA probes

spe-cificfortRNALYS(A) and

tRNA3LYS

(B). Rows1to5 contain 100ng

ofpurifiedorpartially purified tRNAs andareusedtodemonstrate the specificity of thetwo DNAprobes; rows6 to 9 contain viral RNAsamples. Rows: 1,tRNALYs; 2,tRNA3Ys;3 and 4,

tRNALYs-poor and tRNALYs-rich fractions, respectively, from the DEAE-Sephadex A-SO chromatography used in the purifications of the placental tRNALYSisoacceptors; 5, E. coli tRNAPhC; 6 and 7, free andtightly associated tRNA, respectively, from HIV-1 produced in COS-7cells;8 and9,freeandtightly associatedtRNA, respectively, frommutantHIV-1-like particles produced in Vero cells.

the primer binding site in theproviral DNAgenome. (The

loosely associated fraction was not characterized in this work.) Primer tRNA is found in the freefraction and has also been found to be more tightly associated with the RNA

genomethan areother viral tRNAs (11, 26, 27, 38, 40). In thesestudies,wehave foundthat the free tRNApopulations of bothwild-type andmutantHIV-1 containtwospecies of

tRNALys,

tRNALYS,

andtRNAIIC.

tRNALYS

is the putative

primerforreverse transcription; insupportof this hypothe-sis, we have found that the COS-7 cell-produced virus contains a tRNA tightly associated with the viral genome

which migrates with the same electrophoretic mobility as

does

tRNALYS

and hybridizes to a

tRNALYS-specific

DNA

probe. Themutantvirus doesnotcontain this tightly

asso-ciated tRNA. Our data show that neitheradifferent cell type northe absence of the primer bindingsite alters the select incorporation of the tRNALYs isoacceptors. We conclude that the primer binding site is not required for tRNALYS incorporation,unless the mechanism forprimertRNA incor-poration in Vero cells is different from thatin COS-7 cells. Thisseemsunlikely,sinceourresult supportspreviouswork which indicates that the RNAgenome is not required for primertRNAincorporationin either murine leukemiavirus (23)oraviansarcomavirus(28).

Theincorporationof thetRNALysisoacceptorsinto HIV-1 need notreflect aviral function thatthey perform butmay occur because the recognition signal for selecting primer

tRNA3LYS

is shared with theothertRNALYSisoacceptors.Of

course,one cannoteliminate the possibilityofaviral func-tion for tRNAs presentinmoreminor amounts inwild-type virus.However,wefeel that the variation thatweobservein the abundance, or even detectability, of sometRNA-sized speciespresentin HIV-1producedin different celltypes (18)

arguesagainsttheirplayinganessential role in the viral life

A B

1

2 e

3 54 e

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http://jvi.asm.org/

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3252 JIANG ET AL.

cycle. Suchvariabilityis also demonstrated

by

the absence of mutant spots 8 and 9

(tRNAHiS)

and spots

5, 6,

and 7

(unidentified) in thewild-type virus,andwetherefore feel it

unlikely that the abundance of these

species

in the mutant

virusreflectsaviralfunction.

tRNA3'YS

isbelievedtobe theprimertRNA for HIV-1 RT. Wehave in fact found onlyonetRNAtightlyassociatedto the viral RNA genome inwild-typevirus. It has the

electro-phoretic mobility of

tRNALYS,

and it hybridizes with a

tRNALYs-specific

probe but not with a

tRNALYs-specific

probe.Thisfindingindicates that thetwo

tRNA1S

isoaccep-torsincorporatedinto the virusprobablydonotalsoserveas

primertRNA.Theirincorporation alongwith

tRNA'YS

may

thereforereflecta

recognition signal

for

incorporation

shared

by all three species. This phenomenon is also observed in mousemammarytumorvirus, avirus thatusestRNALYSas a primer but also incorporates tRNALys as the only other

majortRNAspecies (27).

Inconsideringwhat feature these tRNAs have incommon which allows for themtobe selected forincorporation,one must consider what process might be used to bring the tRNAs into the virus. This mechanism islargely unknown,

but a candidate viral protein which mayplay a role in this process is the

p1609a9-Po

precursor. This

protein

contains

the RT sequences, and the role of RT in select tRNA

incorporation has been reported. In mutant avian sarcoma virus(28)and murine leukemia virus(24) lackingRTprotein,

the incorporation of host cell tRNA becomes nonselective. Mature HIV-1 RT

(p66/pSi)

has also been showntointeract withtRNALYSin vitro

(3).

However,

p1609'9-J°I

rather than mature RT is implicated in tRNA selection, since the pre-cursoris notcleaved until after viral

budding (6, 41). (The

p1609ag-Pol

precursoris cleaved byaviral protease, andwe have recently observed that in a protease-negative mutant HIV-1which is unabletoprocessthese precursors,tRNALYS

is bothincorporated into thevirusandplacedontothe viral genome,

indicating

that theseprocessesdonotdependupon precursorprocessing

[17].)

It is unlikely that pl609&9-P°'would interact with naked

tRNA, since tRNA is found complexed in the cell with

proteins such as EF-lac and aminoacyl tRNAsynthetases. Therefore, a specific interaction between

p1609a&PO'

and

tRNALYS

mightinvolveaninitial interaction with theprotein

that the tRNAsare complexedwith. Ifthisproteinislysine tRNALYS synthetase, the specificityofbinding mightresult from an initial protein-protein, rather than protein-tRNA,

interaction. This interaction could occur during

p16099a-Po

synthesis, since all ofthese components would be in close

proximityaround thetranslational apparatus. Further exper-iments will be needed to determine whether

p1609a9-Po

demonstrates specific interaction with either deproteinized tRNALYSor

tRNALYS-protein

complexes.

Thepopularityof HIV-1 as anobject of study has resulted in the availability of a large number of well-characterized mutant proviral DNAs. Through transfection, these DNAs canbe usedtoproducemutantvirus in the COS cell system. The ability to now measure both the incorporation and

genomic placementofprimer

tRNA3LYs

in HIV-1 produced in transfected COS cells will facilitate the elucidation of the mechanism underlying these processes through a study of

the effectsofdifferentmutations on these processes.

ACKNOWLEDGMENTS

This work was supported in part by grants from the National Health ResearchDevelopmentProgram, Health and Welfare

Can-ada. B.R. was alsosupported inpart by the Ontario Technology Fund.

Thanks to M. A. Wainberg for the use of the tissue culture facilities at the McGill AIDS Center and to Sandy Fraiberg for assistance inpreparation of the manuscript.

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on November 9, 2019 by guest

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Figure

Figure 1Awild-typefrompMT-HIV-1.similarratedthecells,lysineisoacceptorabundant.2abundance have shows the 2D PAGE pattern of tRNA incorpo- into HIV-1 transiently produced in transfected COS-7 and Fig
FIG. 3.Fig.ingrespectively;transfectedtRNALYStwo 1D PAGE of partial RNase T1 digests of human placental and free tRNAs incorporated into HIV-1 produced in COS-7 cells
FIG. 5.4for (B), RNA sequence gels showing the partial nucleotide sequences at the 3' termini of human placental tRNA3LYS (A), mutant viral spot human placental tRNAlL,s (C), and mutant viral spot 1 (D)
FIG. 6.produced 1D PAGE of tightly associated tRNA found in HIV-1 in COS-7 cells (lane 1) and purified human tRNA'YS (lane2).

References

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