Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity.

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JOURNAL OF VIROLOGY, Nov. 1993,P. 6365-6378 0022-538X/93/116365-14$02.00/0

Alternative Splicing of

Human

Immunodeficiency

Virus

Type 1

mRNA

Modulates Viral Protein Expression,

Replication, and Infectivity

DAMIAN F. J. PURCELL* AND MALCOLM A. MARTIN Laboratory ofMoleclularMicrobiology, NationalInstituteof Allergy and

Infectious Diseases, Bethesda, Maryland 20892 Received 27April 1993/Accepted21 July 1993

Multiple RNA splicing sites exist within human immunodeficiencyvirus type 1 (HIV-1) genomic RNA, and

these sites enable the synthesis ofmanymRNAsfor each ofseveralviral proteins. We evaluated the biological significance of the alternatively spliced mRNA species during productive HIV-1 infectionsof peripheral blood lymphocytes and human T-celllinestodetermine the potential role of alternative RNA splicing inthe regulation of HIV-1 replication and infection. First,weusedasemiquantitative polymerase chain reaction of cDNAs that were radiolabeled for gel analysis todetermine the relative abundance of the diverse arrayofalternatively

spliced HIV-1 mRNAs. The predominant rev, tat, vpr, and env RNAs contained a minimum of noncoding

sequence,but thepredominant nef mRNAswereincompletely spliced and invariably included noncodingexons.

Second, the effect of altered RNA processingwasmeasuredfollowingmutagenesis of the major5' splice donor

and severalcryptic, constitutive, and competing 3' spliceacceptormotifs of

HIV-1NL4-3.

Mutations that ablated constitutive splice sites led tothe activation ofnewcryptic sites; some of these preserved biological function.

Mutations that ablated competing spliceacceptorsitescaused marked alterations inthe pool ofvirus-derived mRNAs and, in some instances, in virus infectivity and/or the profile of virus proteins.The redundant RNA splicing signals in the HIV-1genomeand alternativelyspliced mRNAs providesamechanism forregulating the

relative proportions of HIV-1 proteins and, insome cases, viral infectivity.

Eucaryotic cells control their metabolic activities by regulat-ing gene promoter activity and the processing of RNA and protein. In thesame waythat the study of viralpromoters has servedasaparadigm for the promotersof their host

mamma-lian cells, the investigation of viral RNA processing in

mam-malian cells has provided an insight into various mechanisms

usedtoregulate the steady-state level ofaspecific mRNA. The

complexnature of theprocessing of human immunodeficiency virus type 1 (HIV-1) RNAs provides an important model for

human RNA processing pathways. All retroviruses require RNA splicing to remove upstream gag and pol coding

se-quencesfrom theenvmRNA. Inaddition, HIV-1 generatesa

distinctly complex pattern of spliced RNA to encode the essential regulatory proteins, Tat and Rev, as well asseveral other proteins (Vif, Vpr, and Nef) needed for successful replication in vivo (3, 15, 18, 26, 32, 35, 36, 43, 47, 52). The HIV-1 Rev protein binds viral RNAspecies that contain the Rev-responsive element (RRE), located in the env gene,

thereby promoting the export, and possibly the stability and translation, of partially spliced and unspliced RNAs from the nucleus into thecytoplasmfor its translationand/or packaging

intoprogenyvirions(2, 6, 7, 9, 12-14, 19, 20, 22, 23, 28, 33, 36,

38). The Rev-RRE system alleviates theparadoxical require-mentfor bothspliced and unsplicedHIV-1 RNA for successful virus replication. Rev protein also regulates the temporal changefrommultiply spliced HIV-1 RNAstopartially spliced

orunspliced RNAsduring productive virus infection (27, 29). Thesplicingof HIV-1 RNA isextremely complexbecause of the presence of both constitutive and alternatively used 5' RNA splice donor (SD) and 3' splice acceptor (SA) motifs. Numerous weak SA motifs, located toward the center of the

*Corresponding author.

genomic RNA, are competing points of ligation for splicing,

and their alternate selection usually determines the protein encodedby themature RNA (3, 15, 18, 35, 43, 47). However,

someof these mRNAsare multicistronic, encodingmorethan oneprotein (15, 48, 49). Increased diversity of spliced mRNAs for several HIV-1 proteins results from the alternative casset-ting of two noncoding exons into a proportion of transcripts

(15, 43, 47). In addition, the useofseveral cryptic SA and SD

sitesmayleadtothe synthesis of novel chimeric viral proteins (5, 15, 46, 47). The varieduse of these diverse splicing signals

results in thesynthesis of several setsofstructurally different

RNAsthatserveasalternative templates for the translation of

thesame protein, includingthe viralenvelope, regulatory,and

accessory proteins. Because this complex pattern of RNA

expression is maintained among many HIV-1 isolates of

di-verse origins (42, 43, 50), it is likely that this complexity is

critical for the successfulcompletion of the HIV-1 infectious cycle and not simply an inherent redundancy in viral RNA processing.

The major advantage of examining regulated splicing of a

self-replicating entitysuchasHIV-1 is that suchinvestigations

can usewholecells, rather than in vitro splicing extracts, and the biological consequences can be readily correlated with RNA and protein expression as well aswith virus infectivity. Whilemixedgroups of mRNAs encodemost HIV-1 proteins, it isunclear whether different cell typesusealternativesplicing to regulate HIV-1 RNA expression. The principal cell types thatareinfectedby HIV-1, CD4+ lymphocytesand monocyte-derived macrophages, are known to alternatively splice RNA from some cellular genes, depending on the maturation or

activation statusof the cell (e.g., CD45[54, 55])oron the cell type (e.g., CD46 [44]). Given the wide sequence diversity of HIV-l strains (37), it is likely that sequence differences will

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6366 PURCELL AND MARTIN

affect the

splicing

motifs of different virus isolates in view of what is known in other systems

(17,

34).

We

exploited

the

self-replicating capacity

of HIV-1 to

examine the role of alternative RNA

splicing

in theregulation

ofvirus

replication

and

infectivity

andto evaluate the relative

importance

of the different RNAs

encoding

HIV-1

proteins.

First,

we used a

semiquantitative

polymerase

chain reaction

(PCR) protocol

that

preserved

the relative

proportions

of the

HIV-1 RNA

species

in the 1.8- or 4.0-kb class ofpoly(A)+

RNAtoevaluatethe

steady-state

levelsof viralmRNAsduring

productive

viral infection.

Second,

we introduced mutations into several SD and SA motifs of the HIV-1

proviral

clone

pNL4-3

to assess their effectsonthe

composition

and relative abundance of

alternatively

spliced

mRNAs

during

virus

repli-cation and infection. HIV-1

splice

site mutants

permitted

an

examination of the

biological significance

of the

large

redun-dant

pool

of

spliced

mRNAs and the

potential

roleof alterna-tive RNA

splicing

in the

regulation

of HIV-1

during

tissue culture infections.

MATERIALSANDMETHODS

Construction of

proviral

mutants. TheHIV-1

proviral

mo-lecularclone

pNL4-3

wasconstructed from the NY5 andLAV

(LAI)

HIV-1 isolates

(1).

A PCR-based

mutagenesis protocol

that used a

mutagenic oligonucleotide

and a second

primer

positioned

nearaconvenient restrictionendonuclease sitewas

usedtogenerateaPCR

product containing

the mutation. This

product

was

gel purified

and usedas a

megaprimer

withathird

oligonucleotide positioned

near a second convenient restric-tion site togenerate DNA

containing

the mutated

splice

site and thetworestriction sites

(31).

These

products

werecloned into the

pCR1000

vector

(Invitrogen,

San

Diego,

Calif.),

screened

by

restriction

mapping,

andthen cloned back into the HIV-1

provirus by using

the selected restriction sites. The

oligonucleotides

used,

with mutated nucleotides underlined, were as follows: for

SD1,

Odp.008

(5'-TGGCGTACTCTGC

AGTCGCCGCC-3')

with

Odp.002

(5'-CTCTGGTAACTAG

AGATCCCTCAG-3')

and then

Odp.007

(5'-CTCATCTGGC

CTGGTGCAATAAGG-3');

for

SA4b, Odp.023 (5'-AGGAG

ATGCTCAAGGC7lITYlGTCATG-3'),

and for SA5, Odp.025

(5'-GTCTCCGCTTlCTTCGAGCCATAGG-3'),

each with

Odp.021 (5'-GAATTGGGTGTCGACATAGCAG-3')

and

then

Odp.030

(5'-TTGFLT7AYTATTATlTTCCAAATTGTTC

TC-3');

for

SA6,

Odp.028

(5'-GTGTTAGYTTATCTTG

CACTGATTTGAAG-3')

with

Odp.030

and then Odp.021;

and for

SA7a,

Odp.033 (5'-CTATAGTGAATTCAGTTAG

GCAGGGAT-3'),

for

SA7a+7b, Odp.035

(5'-CTATAGT

GAATTCAGTlTlTCGCAGGGATATT-3'),

and for SA7,7a,

7b,

Odp.037

(5'-CTATAGTGAATTCAGFT[TCGCAGGGA

TATTCACCATTATCGT'Tl7CGTACCCACCTCCCCTATA

GTGAA T AGAGTTAG G

CAGGGATAT-3'),

each with

Odp.032 (5'-CCGCAGATCGTCCCAGATAAG-3')

andthen

Odp.031

(5'-AGTAGAGCAAAATGGAATGCCAC-3').

Splice

site mutant

proviruses

were sequenced to verify the presence of the desired

change

aswell as additional changes that

might

have been introduced during the PCR procedure. Some mutations

(ASA4b

Tat G->S,

ASA5

Tat R->S, ASA6

Env

K--S,

ASA7a Env R->S,

ASA7b

Env R->S, and ASA7

Env

Q->R)

changed

the codon at that splice site. Other

changes

were asfollows:

pNLASD1,

756A-4T(asilent change in the

packaging

signal);

pNLASA4b,

6002

C-*T

(Tat A-V, Rev

L->F);

pNLASA5,

6143 A--G (Env E->G); pNLASA6,

6695T->C

(Env

F->L);

and

pNLASA7a+7b,

7361 T->C (Env

F--L),

8069 C->A

(Env

L--M),

8107

G->T

(EnvW->C), and 8321 T->C

(Env

S--P).

Several other proviral splice site

mutants were sought; the resultant plasmids proved to be unstable, however, precluding their functional

analysis.

Cellculture,transfections,andinfections. HeLa

cells,

main-tained in Dulbecco's modified Eagle's medium

supplemented

with 10% fetal calf serum

(FCS),

were obtained from the American Type Culture Collection. CEM

(12D7)

cells,

main-tained in RPMI 1640 medium with 10% FCS, were obtained fromMicrobiological Associates

(Gaithersburg,

Md.),

aswere

peripheral blood mononuclear cells (PBMC), which were

stimulated with phytohemagglutinin

(0.25

,ug/ml;

Wellcome Diagnostics, Dartford, UnitedKingdom)for 96 h and grown in RPMI 1640with 10% FCS and 10% interleukin-2

(Pharmacia

Diagnostics, Fairfield,N.J.).HeLacells(5 x

105)

in T25flasks

were cotransfected bythe calcium phosphate

coprecipitation

technique(57)with20

,ug

of

proviral plasmid

DNAand 0.5

,ug

ofahuman growth hormone reporter plasmid, pXGH5

(10);

transfection efficiencywasdeterminedbya

radioimmunoassay

for human growth hormone

(Nichols

Institute, San Juan Capistrano, Calif.). Virus production was monitored with an

assay for reverse transcriptase (RT) activity, using

[32P]TTP

incorporation withanoligo(dT)*poly(A) template (59). Cells (2 x 105) were infected with 105 cpm of RT activity of an

HIV-1 inoculum(approximately equivalent to a multiplicity of infection of0.002) in1ml ofRPMI 1640for2hat37°Cbefore addition of 4 ml ofRPMI 1640containing 10%FCS. The cells were fed with RPMI 1640 containing 10% FCS at 2-day

intervals, and aliquots of the medium were assayed for RT

activity.

Isolationof HIV-1mRNA,preparationofcDNA,and

semi-quantitative PCR for spliced HIV-1 mRNA. Total cell RNA was harvested by extraction with RNAzol (TelTest Inc., Friendswood,Tex.) 16or36haftertransfection of HeLa cells or immediatelyprior tothe peak ofRTproductionfollowing

infection of approximately 5 x 106 infected human PBMC; poly(A)+ RNA was selected by the Micro-Fast track

oli-go(dT)-cellulose method (Invitrogen). Forfirst-strand cDNA synthesis, either poly(A)+ RNA or in vitro-transcribed RNA

(seebelow) wasdenatured in 15 mM MeHgOH in the

pres-ence of 130 mM f-mercaptoethanol and 0.5 ,ug of random hexamers at 94°C for 2 min before reverse transcription (two

times) for 1 h with murineleukemia virus RT, using a cDNA cycle kit (Invitrogen). Semiquantitative PCR analysis of cDNA from the 1.8-kbclass of RNA was performed with oligonucle-otide primers Odp.045 (5'-CTGAGCCTGGGAGCTCTCTG

GC-3'; positions 477 to 499) and Odp.032 (5'-CCGCAG ATCGTCCCAGATAAG-3'; positions 8477 to 8498); for the 4.0-kb class of RNA, primers Odp.045 and Odp.070

(5'-ACTATTGCTATTATTATTGCTACTAC-3'; positions 6094

to 6115) were used. Twenty cycles of PCR were performed with 1 Uof Amplitaq (Perkin Elmer Cetus, Norwalk, Conn.), 2 ,lI of first-strand cDNA, 0.2 mM each dATP, dCTP, dGTP, and dTTP, and 1 ,uM each primer in 10 mM Tris-HCI (pH 8.3)-50 mM KCl-1.5 mM MgCl2-O.001% (wt/vol) gelatin by first incubating the mixtures for 5 min at 94°C and then

subjectingthem to thermocycling(94°C for 1.5

min,

55°Cfor 1

min,72°C for 2.5 min) before finally incubating them at

72°C

for7min. Ifthe DNA concentration in a reaction mixture was less than 10ng/,l, as assessed byagarose gel electrophoresis,

aliquotswere sequentially reamplified in steps of five cycles after dilution 1:4 with fresh reaction mix to maintain linear amplification. Amplification products (100 ng) were radiola-beledby performing a single round of PCR as before but with addition of 10 ,uCi of[32P]dCTPandsubsequently analyzed by electrophoresis on a 6% polyacrylamide-urea gel at sufficient

current tomaintain a temperature of

65°C;

bands were visu-alizedby autoradiography or quantified with a Fujix BAS 2000

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RNA SPLICING MUTANTS OF HIV-1 6367

Bio-image analyzer. An MspI digest of pBR322 was end labeled and used as size markers. Controls used in PCR experiments included cDNA from mock-transfected and

-in-fectedcells and poly(A)+orin vitro-transcribedRNA(below)

that was not incubated with RT during the cDNA synthesis

reaction.

Analysis of PCR products and cloning of HIV-1 cDNAs. Bands were excised from the PCR gel, eluted in 0.5 M

ammoniumacetate-10 mMmagnesium acetate-I mM

EDTA-0.1% sodium dodecyl sulfate (SDS) for 16 h at4°C, and then

precipitated with ethanolbefore rcamplificationwith thesame

primers. Afterit wasconfirmed that the PCRproductmigrated

as a singleband, residual primerswere removed with a Magic

PCR Prep column (Promega, Madison,Wis.), and theproduct

was directly sequenced by using end-labeled nested

oligonu-cleotideprimers in thedsDNACycleSequencing System(Life

Technologies, Inc., Gaithersburg, Md.). Purified PCR bands werealsoclonedintothepCRIIvector(Invitrogen)sothatthe

sense strand was downstream of the T7 promoter sequence, and the identities of clones were confirmed by sequencing. HIV-l cDNA cloneswere linearizedwith SpeI, extracted with phenol and chloroform (50%, vol/vol), and precipitated with

ethanol, and RNA was transcribed in vitro by usingT7 RNA

polymerase (Promega). The DNA template was removed

by

digestion (twice) with RNase-free DNase (Promega),and the RNA produced wasdirectly quantified by spectrophotometry

or density scanning of bands following agarose gel electro-phoresis. Selected in vitro-transcribedRNAswere diluted and then mixed in known proportion as controls for subsequent

cDNA synthesis and PCR amplification.

Analysis of HIV-1 proteins. Lysates were prepared from

transfected HeLacells(5 x 107cells perml) in0.5% Nonidet

P-40 in 10 mM Tris-HCI (pH 7.4)-150 mM NaCI-I mM EDTA-1 mMphenylmethylsulfonylfluoride.Differentamounts of the cell lysate, standardized on the basis of human

growth

hormone activity,wereseparated on SDS-5 to 20%

polyacryl-amide gradient gels, electroblotted onto Immobilon-P mem-branes (Millipore, Bedford, Mass.), and blocked with 5%

powdered skim milk in

phosphate-buffered

saline. HIV-1 proteins were detected by using serum

(1:1,000)

from an HIV-1-seropositiveindividual, orrabbitserum to a Nef

N-ter-minal peptide (1:100) (21) or to

purified

NL4-3

gpl60/120

(1:100) (58), and visualized with

'251-labeled

protein

A

by

autoradiography or

phosphoimaging

analysis.

Alternatively,

cells were

biosynthetically

labeled with

[35S]methionine-cys-teine (Tran

35S-label; ICN, Irvine,

Calif.)

as described

previ-ously (59) and

immunoprecipitated

with rabbit serum to Rev

produced inEscherichiacoli

(1:100)

(gift

from D. M.

D'Agosti-noand G. N. Pavlakis). Levels of Revfunctional

activity

were

measuredasdescribed

previously

(24) by

cotransfecting

0.5

[Jg

of the pDM128 Rev reporter

plasmid

with 20 jig of

proviral

plasmid and 0.5

p.g

of

pXGH5

human

growth

hormone reporterplasmidandthen

assaying

for

chloramphenicol

acetyl-transferase

(CAT)

activity

on cell extractsthatwere

standard-ized for growth hormone expression.

RESULTS

The selection of different

competing

SA sites in

primary

HIV-1 transcriptsleads tothe alternativeexon

composition

of

fully

processed

mRNA

(Fig.

1).

For

example,

functional Tat

protein, a transactivatorofHIV-I

transcription

(8,

35,

40,

51),

isexpressedfromthetwotypesof mRNA

using

SA4

(Fig.

IA):

RNAs containing exon

4E,

which is continuous from SA4 to

thepoly(A) addition site

(one-exon

tat),

or

transcripts

joining

exons 4 and 7 (two-exon

tat)

(Fig.

I

C)

(45).

Two other

competing

SAsites,SA4a and SA4b

(Fig. IA),

mapping

fewer than200bases downstream from

SA4,

give

risetoexons4aand

4b,

which are

spliced

to exon 7 to generate mRNA for Rev

(Fig.

IC).

Another

competing splice

site,

SA5

(Fig.

lA),

is used for the

expression

ofexons 5and SE

(Fig.

IC);

RNA

species

that contain exon 5E encode

envelope

gp16O,

and those

splicing

exon 5 to exon 7 encode Nef

protein (15, 47).

The

translation initiation sites ofmRNAs

using

SA4a, SA4b, and

SA5have poor ribosome

binding

capacities

andthereforehave the

potential

to be multicistronic: mRNAs

containing

exon

SE,

4aE, or 4bE encode

Vpu

and

gpl6O

envelope

proteins,

whereas

transcripts joining

exon 4aor4bwith exon 7 encode both Rev and Nef

(15,

47, 48,

53).

This increases the

coding

potential

of several HIV-1 mRNAs and the

complexity

of

HIV-1 mRNA

pool.

Further

complexity

results from the

interchangeable

incorporation

oftwo

noncoding

exons, exons 2and 3

(Fig.

IC),

into the

spliced

RNAspecies

utilizing

anyof the downstream SA motifs.

PCR

protocols

using primers

that promote

amplification

of

limited

subgroups

of RNA have been usedin

conjunction

with selective

hybridization probes

to map SA6,

SD5,

SA7a,

and

SA7b

(Fig. lA)

withinenv,thus

generating

frameshifting

exons

6,

7a,

and 7b

(Fig.

IC).

These exons may

generate

novel

chimeric Tat-Env-Rev

(Tev

or

Tnv),

Rev-X-Tat, and Tat-Env

fusion

proteins

following

transfection-infection

by

some

deriv-atives ofthe HIV-I1Alstrain

(5,

15, 46,

47).

These are

merely

cryptic

sites in the

HIV-1NL4-3

genome

(see

below),

andtheir role

during

HIV-1

replication

is notclear

(16).

Semiquantitative

PCR

analysis

of

spliced

HIV-1 mRNA.

Since Northern

(RNA)

blot

analysis

ofHIV-1 RNA does not

distinguish

thefull array of

alternatively

spliced

RNAs

encod-ing

thesameviral

protein,

weusedasensitive

semiquantitative

PCR assay and

urea-acrylamide gel

analysis

thatdiscriminated

between RNA species

differing

in size

by

a

single

nucleotide.

Twosuchassayswerecarriedout.Thefirst

selectively

detected the smaller

multiply

spliced

1.8-kb RNAs that use

SD4

and

SA7, SA7a,

orSA7b

(nef,

rev',

tat,or

vpr) by

using the

Odp.045

and

Odp.032

primers

(Fig. lD);

the second

specifically

ana-lyzed

the

larger

4.0-kb RNA

species,

which containexonsthat

extend

beyond SD4

into the env

reading

frame

(vpu/env,

one-exontat, vpr, and

viJf),

by

using

the

Odp.045

and

Odp.070

primers

(Fig. IE).

Representative gels,

depicting

the PCR

products

that resulted from random hexamer

priming

ofthe

multiple

species

of HIV-1 RNA

present

in infected PBMC priorto the

peak

ofRT

production,

areshown in

Fig.

1D

and

E. Each of these

cDNA

bands was excised from the

gel,

reamplified,

and

directly

sequenced.

Several

representative

cDNA clones of each band were also introduced into the

pCRII

vector, which contains a T7

promoter

adjacent

to the

cloning

site,

and also

sequenced.

To ascertain whether the relative

quantity

ofthe

PCR-amplified

cDNAbands visualized

on the

gel

faithfully

represented

the relative levels of the

various RNA

species

isolated from infected

cells,

plus-sense

HIV-1 RNAwas

synthesized

from

eight

differentcDNAclones invitro

by

using

T7RNA

polymerase, directly

quantified,

and

then mixed in known

proportions.

Included among these RNAs was one,

designated cryptic,

from a cDNA clone

containing

exons

1,

2x, 5,

and

7,

where exon 2xreads

through

SD2

toa

cryptic

SDat

position

5059 in

vif.

The RNAmixtures

were then used as

templates

for cDNA

synthesis

and PCR

amplification,

and the -P-labeled PCR

products generated

were

analyzed

by

phosphoimaging (Fig.

2).

Wefoundthat the

proportion

of

radioactivity

measured for each cDNA band

closely

matched the

proportion

ofRNAaddedto themixture

prior

to cDNA

synthesis

(Fig. 2A).

Thus, each cDNA reverse VOL.67, 1993

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http://jvi.asm.org/

(4)

A. Splice junctions

- Splice

fL HJ donors

-SI): I

B. Open reading frames

C'DC7O'

Ln i"in 4' mG, C;i)

Spike CIDO1%IIt-I~Oil IILn 1101,

>acptnjrs tiy t

-S: 2 3 4-ih: 6:

2 3 4

I'c L'Iev ' -'

7 -L

tE=

,-L

LIn.-L

_NT'_P

PU'

CO CO

CS} Cc 0

..i.J

CiD

(D C

CD G

_I c

iAAA,^.i.

71)

-8

7 ..

tat21tcv.3

n

,

_rvXr

C.

Exons 02 *3 -5 06

M41) M4a - 4

-4

-- 3a

M.

ml

-

7

_ _ 7a

I 2 J7b

- S~~E

4hE 4aE

4cE 4E

3E 2E

D. PCR for

1.8 kb class of RNA

P('GRGelI RNAname Exoncontent lBand size

- Vpr 2 1/2J3a/ 7 1101 [it

VprI 11/3a/7 1051 nt

Tat4

Tat 3

I'aat2

_doo..It Tat I

Rev 12

Rev 11

Rev 11)

Rev 8

Revr7 Ref 6 Nef4

Re

Reesr4

Nef3 Rev3 Rev 2 Rev I N-ef 2

Nsef I

2/ /4/-7

113/ 7

I1,4/7 4/

1 / 3/ 4t 7

/2/3/4i /7

/2/3/4 /7

3 4c7

13 4a 7

3/4b 7 / /4a 7

I_/12i4b 7_/_'7

/ /7

44il7

/4 /7

/ 5/

788rit 73>8Tlt

714 rIt

664 iit 629 nI 61 ntri

605lit

589Tit 5-79rii

93rin

555 nit 537n nt

5 15ior

487nt

480rit 464jit

-t -- Vif 2

_Vpr

4

Vpr3 Tat 8 Tat 7

Tfat6

TI'at5 Env 16 Env 15 - / Env 14 Ern 13

/{nv 12

Env 11 Env 1(I _lp - .Env 8

W-f HlEiv7

lEnv6

. Envrh

En\ 4

Env I

/7 396Iot

E. PCR for

4.0 kb

class

of

RNA

PCURCGel RNAname Exoncontent Band size

I/ 2E

I1/2 / 3E I/3E

I/2/3/4E 3/4E /2/4E /4E /2/3 /4cE I/2 /3 4aE

I/2/3 / 4bE

I/ 3/SE

/3!4cE

I/3!4aE I/3/4bE I1214cR /3/5E I/2/4aE I12/4bE

I/2/5E

/4cE

I/4aF

I/4bE I/5E

1474nt

1047nt

997ot

734 nt 684 nt 660 nt

610nt

575nt 557 nt 551 nt 535 nt

525nt 507nt 501 nt 501 nt 485 nt 483nt

477 nt 461 nt

451 nt 433nt

426nt 410nt

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transcribed from the in vitro RNAwas amplified with equal

efficiency withuseof the Odp.045-Odp.032 primerpair.

PCRanalysisof the 1.8-kb classof mRNAwasperformedon

samples from HeLa cells transfected with HIV-1 proviral

clones or from human PBMC infected with cell-free HIV-1

(Fig. 2B). RNA directed bythe pNL4-3 cloned provirusDNA

intransfected HeLa cellsorby

HIV-lNL4-3

ininfectedhuman

T lymphocytes yielded thesamepatternof bands innumerous

independent experiments irrespective of whether RNA was

synthesized followingtransfectionorinfection.Asimilar

band-ingpatternwasalso observed withRNApreparedfrom PBMC

infected with HIV-1LAI (Fig. 2B, lane 10). The results

pre-sented in Fig. 2 demonstrate that HIV-1 RNA is spliced

equivalently in transfectedHeLacellsandinfected PBMC and thatthesplicingpatternissimilar for closely related strainsof

HIV-1 suchas NL4-3and LAI.

These control PCR amplifications also illustrate two other

important points: (i) for each RNA mixture, the relative

proportion of the different amplified RNA species, as

deter-mined experimentally, closely approximated theoriginal

pro-portion added, and (ii) only PCR productsderiving from input RNAwere detected on the gel, indicating that only RNAs

spliced by genuine processing pathways gave bands in this

analysis. AbnormalcDNA species,potentially arising by

tem-plate jumping during reverse transcription of RNA, did not

appear asbands on the final autoradiogram. Thus, the PCR

protocol used here accurately reflects the relative quantityof

oneHIV-1 RNAspeciescompared with the others amplified in

thesamereaction.

Identification ofa newSA site forrevandvpu/envRNAs. Our

PCR analyses ofviral RNA synthesized in transfected HeLa cellsand infected PBMCindicatedthepresenceofapreviously

unreported competing SA site, designated SA4c, thatwasused

togeneratethreenovelrevmRNAs,rev3, rev6, and rev9(Fig.

1D), andthreenovelvpulenvmRNAs, env4, env9, and envl6 (Fig. 1E). TheSA4csite is 18 nucleotides (nt)upstreamfrom SA4a and isconservedamongmost,butnotall,HIV-1 isolates

(Fig. 3).TheSA4csplice site exists inHIV-lHXB2,

HIV-1LAI,

HIV-lJR-CsF,

andHIV-lBA-L,thestrains used in earlier studies

mapping thesplicing motifsofHIV-1, althoughitsuse wasnot noted(3, 15, 18, 35, 43, 47).Sinceno newtranslation initiation sites are introduced into mRNAs using SA4c, transcripts containingexons4c and 4cE would stillencodetypicalHIV-1 Revor Env protein. However, structural changes introduced into these mRNAs may affect their translatability. An ATG codonexists in theHIV-1NL43betweenSA4c and SA4a butis present in acontext unfavorable for efficient translation(30).

Relative abundance of the alternatively spliced HIV-1 mRNAs. Since PCR amplificationofHIV-1 cDNA preserved the relative proportion of the various alternatively spliced forms of HIV-1 RNA, we used phosphoimaging analysis to

directly determine the relative abundance of each of these

RNAs in a spreading

HIV-1NL4-3

infection of PBMC (Fig. 2

and4).Within the1.8-kb class of HIV-1mRNAs(Fig. 4A), nef,

rev,tat,andvprspeciesexisted inaratio of 56:34:9:1;within the

4.0-kb class (Fig. 4B),env,tat, vpr,and vif species existedina

ratio of 92:5:2:1. Of the nef mRNAs, nef2, which includes

noncodingexon 5flanked bySA5 and SD4,wasthe

predom-inant type, comprising 49% of all nefRNA and 28% of all

1.8-kbRNAs.nefRNAscontaining noncodingexon3(nef4)or

2 (nef3) or both 2 and 3 (nef5) were present in decreasing amounts. Nefl RNA, which lacks a noncoding exon, was the

leastabundant nefmRNA. Itisunclear whether the

nontrans-lated RNAfromexon5 contributestonef function,asthenef gene has not been evaluated as an RNA element, only as

protein.

Incontrast, thepredominant spliced mRNAsfor the other

HIV-1 proteins lacked noncoding exons. Only 20% of rev

mRNA includes anoncodingexon, and theuse ofnoncoding exon 3 (rev7, rev8, and rev9) or both exons 2 and 3 (revl0,

revll, andrevl2) predominatesover useofexon2alone(rev4,

revS, and rev6). In addition, revl and rev2 mRNAs, utilizing

SA4a and SA4b, respectively, occur approximately fivefold morefrequently than rev3 mRNA, which usesthe previously

unreportedSA4c. Both theone-andtwo-coding-exonforms of tat mRNA (1.8- and 4.0-kb class RNAs, respectively) infre-quentlyusenoncodingexons2and 3(tatlandtat5). However,

when a tat mRNA contained a noncoding exon, exon 2 was

predominantly used (tat2andtat6).Therelativeproportion of

theone-andtwo-exonformsoftatorvprmRNA couldnotbe

determined here, since the assays for the 1.8- and 4.0-kb mRNAsaremerelysemiquantitativeamongthespecies

repre-sented in each PCR assay.

EightypercentofenvmRNAsuseSA5 (exon SE);however,

12% ofenvRNAs(env2, env3,andenv4)utilize the upstream

SA4a, SA4b,orSA4c(exon4aE, 4bE,or4cE)whennoncoding

exons 2 and 3wereexcluded. Noncoding exons 2and 3were

usedonlyatlow(<5%) frequencyinenvmRNA andmostlyin

conjunction with SA5 (envS, env8, and envl3). This study clearly identifies mRNA species containing both noncoding

exons2and3 in thesametranscript.Thisfindingcontrastswith

previous analyses of RNA directed by the HIV-lHXB2 strain that identified these as mutually exclusive exons (15, 18, 47)

but is in agreement witharecentreportthatcharacterizedviral

FIG. 1. Identification by RT-PCR of alternatively spliced HIV-1 mRNAscontaining a variety ofexons that arise from the existence of numeroussplicejunctionsencodedintheHIV-1 genome.(A) Mapshowingthelocations of the SD and SA sites in thepNL4-3proviral molecular cloneofHIV-1,witheachpositionshown innucleotidesfromthestartof the 5'longterminal repeat(LTR).The SD and SA sitesarenumbered asdoneby Schwartzetal.(47). (B)Organizationofthe HIV-1 genome.Openboxes show locations of theopenreadingframes that encode the HIVproteins. (C)ThedifferentHIV-1 exonsgeneratedfrom the useof different combinations of SA and SD motifs during RNA splicingare shownasbarsandnumberedasdonebyMuesingetal.(35).Exonsrepresented bygraybarswerenot found inanyof the RNAspecies examined inthesestudies.Exons1,2,3,and 5 donotencodeHIV-1protein,andthose numbered withanEreadthroughSD4 into theenvgene. (D) The various HIV-1 mRNA species falling into the 1.8-kb size range in Northern blot analyses were distinguished by acrylamide gel analysis of

PCR-amplifiedcDNA,usingprimersOdp.045(positions477to499)andOdp.032 (positions8477to8498).Arepresentativegel from PCRanalysis

of RNA frompNL4-3-infectedPBMC is shown. Thedistinct HIV-1RNAspecieswereidentifiedbydirect PCRsequencingof excised bands and bycloning,and theidentity,exoncontent,and size ofthe PCRproductareshown.The mRNAspecieshave been namedaccording to theprincipal

protein that they encode andbytheirsize,with thesmallestRNAas1. Faint bandswerevisibleonlongerexposuresof the autoradiogram.rev mRNAsarebicistronicand alsoencodeNef(15, 48,49).(E) Representativeacrylamide gelfrom PCRanalysis of HIV-1 mRNA species falling into the4.0-kb size range frompNL4-3-infectedPBMC,usingprimers Odp.045andOdp.070 (positions6094 to6115). The identity of each RNA species, determinedbydirectsequenceanalysisofexcisedbands,is shown with theexoncontent and size of the PCR product. RNA species not matched to abandonthegelweredifficulttodetectexceptonverylong gelexposuresandwerenotquantified above the background level in

phosphoimaginganalysis.AllenvmRNAsaremulticistronic and also encodeVpuand Nef(48,49).

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B.

100 100 100 100 102 106 102 100 0.4 5 10 50

0.2 2 8 58

100 100 100 100 95 98 97 95

50 10 5 0.4 58 8 0.2 0.1 100 100 100 100

71 82 84 79 0.4 5 10 50 0.1 2 10 58 100 100 100 100 117 123 113 115

Tat2

Tat1 cyptic

Nef4

Nef3 Rev2 Nef2

4~

RNAmixtures r g, *

ABCD4tt4*~t

q9¼~

A B C D + ?q #h

Vpr2

Vpr1

Tat4 Tat3

P-'-

- Tat2

Tati

Rev12

-nquie

....

Rev1O,11

''Nef5

RevB,9

Net3 Rev3

_,n,. NRev2

_ _ = = ~~~~~~~~~RevI

-~~ ~~~~~Nef~~~~~2

Nef1 50 10 5 0.4

71 7 5 0.8 Nef 1 *r

N

A..- Netl

1 234567

Invitro RNA HeLa

89

10

[image:6.612.144.457.77.408.2]

PBMC

FIG. 2. The relativeproportionsofalternativelyspliced HIV-1 mRNAswereaccuratelydeterminedbyRT-PCR assay. Thesemiquantitative

capacityof the RT-PCR assay for the 1.8-kb class ofspliced HIV-1 mRNAwasevaluatedby mixing predetermined concentrations of RNA transcribed in vitro fromHIV-1cDNAclones with T7 RNApolymerase(A)andthenperformingreversetranscription,PCRamplification,labeling

with[32P]dCTP,andgelanalysis (B).Proportionsof each RNA added intofourmixtures,Athrough D,areshowninpanelAwith theproportion

of cDNAexperimentallyamplifiedbyPCR(panelB, lanes1to4)and determinedbyphosphoimaging analysis (showninitalics inpanelA). (B)

Lane 5showsapoolof RNA mixtures AthroughDthatweretreatedinparallelbutwithout theadditionof RTduringcDNAsynthesis.RNA from HeLa cellstransfected withreporterhumangrowthhormoneonly(lane6)andwithpNL4-3(lane7)orwith RNAfromPBMC(lane8)and PBMC infected with

HIV-lNL,43

(lane9) orHIV-lLAI (lane 10)wasexamined in thesamesetofreactionsasthe control. Theidentityof each amplified speciesis shownontheright (seeFig. 1D).

S

A

ACCAATTGCTATTGTAAAAAGTGTTGCTTTCATTGCCA

----C---A---T

----C--- --_-_

4-SA 4a PTTGTTTCATGACAAPACF

---CA

---

__---A---G---CA--- -

---CA---CA ----

--SA 4b CCTI AC

G----__

"I__

__-

4-Rev SA

start 5 GCATCTCCTT}{GCAC

_________ _____

_________ ____ _

---_-_

-AT- -F---C----CA-G----__

G---C--- ---C---

-A---__G---A--F---________________ _---A---G-- __---

Si

-A----G---C--- ----

-C--T-A-AC---__G---AT-G ---C--- -G---A-A--G--__G---J_ _ _ _ __

FIG. 3. LocationofSA4c,a newSAsite forrevandvpu/envmRNAs.Shown is alignment of nucleotidesequencesof severalHIV-1 isolates surroundingthe newSA site forrevandvpu/envmRNAs, SA4c(adaptedfrom Myersetal. [37]).

A.

In vitro RNA mixtures

A B C D

Tat2 Tatl

cryptic

Net4

Nef3 Rev2

Nef2

NL4-3

LAI HXB2 SF2

MN

NY5

ADA BAL

SF162

ELI

MAL

GAAGA

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A.

forwardprimer

Odp.045:

II

- splice 2

,* acceptors

I M_IU k}

-111illor"Giv.splc.e- ! N.-4a

_~-splice

1 donors

RNA species -Nef

1-=mL.- Nef 2 mmm-- Nef3 mom- Nef4 miii- Nef 5 Rev 1

Rev2 i.

Rev 3 ' Rev 4

-,--Rev5

-Rev6 -Rev 7 Rev 8 Rev 9 Rev 10

-,_i-Rev 11 Rev 12

-1.8kb class of RNA

reverseprimer :Odp.032.

I II

3a 4ca7

I

n7.,

1I

"O*2

2

Vlli

3

2 3

- Tat1 - Tat 2 - Tat3 - Tat4

:L I. L.!.,

I.... 4

:: Relative %

;;;i5 -j~

....- Af

49

11 24 7

-,-,-- 32 -j

.... 40 8

1

1-_Li~

44

_ -33

.-.. 2

-:_..

2

_ -1

48

28

16 9

- Vpr1

- Vpr2 -.n- 7228 -.

B.

4.0 kb class of RNA

forwardprimer reverse primer

Odp.1145 Odp.070.'

2 3. 4cab -splice

acceptors 1 , '2 . pl3 vpr Irevi4 I to do s

~~L Ll w~~~~~it 1 Lrz| splice

I

|2|~~~~~~

3 4 donors

RNA species o. Env1

-.-Env 2

-Env 3

Env 4 Env -Env6

in-Env7

-. Env8 Env 9 - Env 10 - Env 11 - Env 12.-- Env

13.-in- Env 14 - Env 15 - Env 16

- Tat5 - Tat6 - Tat7 - Tat 8

Relative % 80

5 _6

mo-_ 1

_o 5

_ 1

_io 1 _ _ _NO O

85 9 6

0

- Vpr 3 - Vpr4 - Vif2

98

2

[image:7.612.72.562.86.376.2]

p 100 FIG. 4. Structure and relative abundance of each alternatively spliced HIV-1 mRNA. (A) Splice site usageofthe 1.8-kbclassofHIV-1 mRNA amplified by PCR of randomly primed cDNA, using primers Odp.045 and Odp.032. (B) Splice site usage of the 4.0-kb class of HIV-1 mRNA amplifiedby PCR of randomly primed cDNA, using primers Odp.045 and Odp.070. The solid boxes raised above the line represent the regions ofretained RNA. Shown at the right of each panel is the relative proportion of each mRNA species quantified by phosphoimaging from pNL4-3 virus infections of PBMC, using semiquantitative PCR analysis for the 1.8- and 4.0-kb classes of RNA. In general, values less than 1 were not measured at levels significantly above background and were recorded as zero; however, the existence of these cDNA species was evident on long gelexposures.

RNAininfected MT-2cells (50).Noevidencewasfound for a

vif

RNA among the 1.8-kb species that spliced SD4 to SA7

(vifl).

Splicesite mutants ofpNL4-3.During HIV-1 mRNA splic-ing, the cellular spliceosome cleaves at GT and AG dinucle-otides within the SD and SA motifs, respectively. Several of these highly conserved dinucleotide motifs present in the pNL4-3 proviral DNA clone of HIV-1 were replaced with different dinucleotides to block RNA splicing, using a PCR-based strategy (Fig. 5). Seven site-directed provirus mutants weregenerated by inactivating (i) the constitutivelyusedmajor splice donor,pNLASD1; (ii)thecompeting splice acceptorfor

thefirstcodingexonofrev,pNLASA4b; (iii)thecompetitively

selectedmajorspliceacceptorforenvandvpu,pNLASA5; (iv)

the cryptic splice acceptor within env purportedly used to generate theTevprotein (5, 46, 47), pNLASA6; (v) SA7a,the

most5'of twoconservedcrypticSA sequencesmapping34 and 28bases upstreamfrom thesecondcodingexon oftatandrev,

designated pNLASA7a; (vi) both the SA7a and SA7b cryptic sites, pNLASA7a+7b; or (vii) both of these in combination withSA7, the constitutive SA forthesecondcodingexonoftat

and rev, pNLASA7+7a+7b. Some of these mutations also

altered an amino acid codon(s) overlapping the

splice

site

(Materials and Methods). The integrityofallmutant

proviral

clones was confirmed by nucleotide sequence

analysis

of the reconstructed regions.

Consensus motif

Mutantname pNLASD1

pNLASA4b

pNLASA5 pNLASA6 pNLASA7a pNLASA7a+7b pNLASA7+7a+7b

CAG(1I AAGT

A IG

3' Spilice Acceptor

TTTTTTTTTTTTNC,,2 CCCCCCCCCCCCNTC G

CTGCAGAGTACGCCA

'1

TGACAAAAGCCTTGAG

41

CATCTCCTATGGClPCG

4i TGTGTTAGTTTAIC11T TTCTATAGTGAATI(CA

4,i "I TTCTATAGTGAAT1PCAGTT1PCG

'1 'I I

AAT1CAGTTTCGCAGGGATATTCACCATTATCGTTTCGPA FIG. 5. Basesubstitutions introduced into SD and SA motifs of the pNL4-3proviralclone ofHIV-1.Theconsensusmotifs of mammalian SD and SA sites are shown at the top, with the essential two

dinucleotides of the motif shown in outlined font. These dinucleotides were changed in the pNL4-3 proviral clone to the bases shown in outlined font so as to inactivate several SD and SA sites. Arrows indicate thepointofcleavageand ligationof RNAduringsplicing.

t 'W'st-7- I

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A.

9 eB.

gpl60/120o Z gp160/120

p55

...,>.. ~~~~~~~~~~~~~~~..~ ~ p55

p24 ..IE...-...

p17 _ _

36 hrs

p24 p17

72hrs

1 2 3

4 5

6

7

8 9

0 1 2

FIG. 6. Some HIV-1 splicingmutants direct the synthesis ofan altered profile of HIV-1 protein. Western blot analysis of HIV-1 protein detected with patient serum from HeLa cell lysates was performed 48 h(A) and 72 h(B) after cotransfection of 0.5 pLgofa growth hormone reporter plasmid with 20 pLg ofwild-type pNL4-3 (lane 1), with RNA splice mutant provirus pNLtASD1 (lane 2), pNLASA4b (lane 3), pNLASA5 (lane 4), pNLASA6 (lane 5), pNLASA7a (lane 6), pNLASA7a+7b (lane 7),orpNLASA7+7a+7b (lane8),orwith reporterplasmidalone(lanes9 and0).The volume of celllysatewasstandardizedfor transfectionefficiency accordingtothe determination of humangrowthhormone in the culturesupernatants.

Protein synthesis bysplicingmutantsofHIV-1. Inthe first groupofexperiments, theeffects of thesplicesite mutationson

viral protein production were assessed by Western blotting

(immunoblotting) lysates from transfectedHeLacells, usingan

AIDSpatient'sserum.AsshowninFig.6, each of themutants

except pNLASA7+7a+7b directed the synthesis of the same

complement ofHIV-1 proteinsasdidwild-type pNL4-3,with thefollowing exceptions. Mutation ofSD1 caused a marked decreasein thequantity of viral proteins accumulatingin HeLa

cellsduring the first 48 h despite transfection efficiency

equiv-alent tothatof the wild type,as measuredby coexpression of

ahumangrowth hormone expression plasmid (Fig. 6A, lanes1

and 2). By 72 h after transfection, however, HIV-1 protein accumulation directedbymutantplasmidpNLASD1was

com-parabletothewild-type level (Fig. 6B, lanes1and2). Mutation ofSA4b,used for theproductionofrevandenvmRNAs, ledto

consistent and substantial elevations ofgpl60/120 levels

com-pared with the wild type (Fig. 6A, lane 3). In contrast, the mutation of SA5 markedly reduced, but did not eliminate,

gpl60/120production (Fig. 6A, lane 4). Mutation of SA6 (for

tevmRNA)and the SA7aorSA7a+7bcryptic splice sites (for the secondcodingexonoftatandrev)had little if any effect on

HIV-1protein synthesis (Fig. 6A, lanes 5 to 7), indicating that these three SA sites (and exons 6, 7a, and 7b) play no significant role for HIV-1 protein synthesis in transfected HeLa cells. In contrast, mutant

pNLzASA7+7a+7b,

which contains a triple splice site mutation including SA7, the in-frame acceptor used constitutively for the second coding

exonoftatand rev, directs the synthesis of a markedly altered protein profile (Fig. 6A, lane 8), similar to that previously reported for Rev-deficient HIV-1 mutants: minimal levels of

p55,p24, or pl7gagprotein and no detectable gpl60/120 (12, 14, 20, 33) accompanied by a novel and abundant 20-kDa protein that reacted with Tat antiserum (data not shown). No increased accumulation of HIV-1structural proteins was noted

at later times (72 or 96 h after transfection) despite the

HeLa transfection ct

%99 99>D 9999>¢COrC5>rc>rtA 2;c,~~~~~-P7SstvX=sS7?<z,

622bpI 527bpi

404bp

1 2

3 4 5 6

7 8 910

FIG. 7. HIV-1provirusesmutatedatcryptic splicesitessynthesize awild-typeprofileof RNA.SemiquantitativeRT-PCRanalysisof the 1.8-kb class HIV-1 RNAfrom HeLacells transfectedwith 0.5pLgof reporterplasmid alone(lane 2)orcotransfectedwith 20,utgofpNL4-3

(lane 3) or HIV-1 RNA splicing mutant pNLASD1 (lane 4),

pNLASA4b (lane 5), pNLASA5 (lane 6), pNLASA6 (lane 7),

pNLASA7a (lane 8),pNLASA7a+7b (lane 9),orpNLASA7+7a+7b (lane 10).AnMspI digest of pBR322is shownontheleft(lane 1)as a sizemarker.

expression of levels of total cellular HIV-1 RNA similar to

wild-type levels by Northern blot analysis (not shown). These

results suggest that mutant pNLASA7+7a+7b failed to

ex-press functional Rev and resulted in reduced amounts of Rev-dependent cytoplasmic mRNAs encoding Gag and Env

proteins.

mRNAanalysis from cryptic splice site mutants of HIV-1.

When the 1.8-kb class of RNAfrom HeLa cells transfected with HIV-1 proviralDNAclonescontaining splice site

muta-tions was analyzed, it was clear that several expressedthesame

RNApattern as didwild-typepNL4-3 (Fig. 7). For example, mutants pNLASA6, pNLASA7a, and pNLASA7a+7b were indistinguishable from the wild type (Fig. 7, lanes 7 to 9), indicating that the SA6, SA7a, and SA7b sites are not used with significant frequency by HIV-1NL43, even though it is virtually identical to HIV-lHXB2, the strain in which these splice siteswereoriginally described. Furthermore, no cDNAs

usingthe SA6, SA7a, or SA7bsplice site were ever detected amongthePCR-amplifiedbands that were directly sequenced

fromcellstransfected or infected with derivatives ofHIVLAIor

HIV-lN"-3,

and no clone generated from these PCR-ampli-fiedcDNAscould beshown to utilize these splice sites. Thus, the SA6, SA7a, and SA7b cryptic sites are used extremely rarely, if at all, and may be active only in the

HIV-1LAI

derivativeHIV-lHxB2.

Wefound that careful selection and testing of PCR primers

was required, in conjunction with the use of poly(A)+ RNA andnonspecific cDNA priming, to avoid the amplification of aberrant cDNAs. Our semiquantitative PCR assays depended

onequal use of all the HIV-1 RNA species in either the 1.8- or 4.0-kb mRNA classes as competing templates for amplifica-tion. Otherprotocols may selectively amplify transcripts using SA6, SA7a, and SA7b (47): reports characterizing the

se-J. VIROL.

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A. e

B. Q

9~,'o

Vpr3-

-Tat4

Tat3.

Tat2-Tati- _ _ _

Nef5- Rev6-

Nel4- Revl,-Net2- F

Nett-__

Tat7-

Tat6-Tat5- __

Envg-Env8-..

Env5-

Env4- Env3-Env2- _

Envi- -__

1 2 3 4 5 1 2 3 4 5

FIG. 8. An altered profile of spliced RNA is synthesized byHIV-1

provirus mutated at constitutive orcompeting splice sites. Shown is

semiquantitativeRT-PCR analysis of the1.8-kb class HIV-1 RNA (A)

and the 4.4- to 5.5-kb class of HIV-1 RNA (B) from HeLa cells

transfected with 20 p.g of pNL4-3 (lane 1) or HIV-1 RNA splicing

mutantpNLASD1 (lane 2),pNLA&SA4b (lane 3),pNLA&SA5 (lane 4),

orpNLASA7+7a+7b(lane 5).

quence of cDNA clones around the SA7 splice site (15) or

using hybridization analyses of cDNAs (50) also failed to

detectusageof SA7aorSA7b.

Analysis of mRNA from HIV-1 with mutated constitutive and competitive splice sites. The cDNAs amplified from cells

transfected withmutantpNLASD1 had apatternverysimilar to that of the wild type but with a slightly slower

electro-phoretic mobility (compare lanes 3 and 4 in Fig. 7). Direct

sequence analysis of these PCR bands as well as several

individual cDNA clones indicated that spliced RNA from

pNLASD1 usedaGTdinucleotide4ntdownstream from the

mutated majorSD (constitutiveSD1) siteas acrypticSDsite

in this HIV-1 proviral DNA. The HIV-1 proteins translated

fromtheseslightly largermRNAswereindistinguishablefrom thewild type (Fig. 6), but the rate of splicing of these viral

transcripts may be slower, perhaps explaining the delay in

protein expression previouslyobserved. This cryptic SD

(incor-rectly annotated as the major SD in the alignment of this

region byMyers et al. [37]) is strongly conserved among all

HIV-1 isolates.BecausethecrypticSD1sitemaybeused only

whenthegenuine siteis inactivated, it is likely that thestrong

conservation of this alternative splice site results from addi-tional selective pressures. Nevertheless, its existence greatly

reduces thepossible loss of virus infectivity due to a

sponta-neous mutation affecting the major SD that would otherwise

blocktheproduction of functional splicedmRNAs.

ThecDNAsamplified from the remaining mutantsdiffered from the pNL4-3 wild-type pattern (Fig. 7 and 8). For the provirus mutant inactivating the constitutive SA7 for the

secondcoding exon oftat andrev, pNLASA7+7a+7b, every

cDNA bandclearly differedinsizecompared with the wildtype

followingamplificationofthe 1.8-kbspecies ofHIV-1 mRNA

(Fig. 8A, lane 4); the overall pattern was similar to the

wild-typepattern except that each band had a faster

electro-phoretic mobility. A direct sequence analysis of these PCR bands and sequencing of individual pNLASA7+7a+7b cDNA clones indicated that all used an AG dinucleotide situated 20 nt downstream from SA7 as the alternative SA site; this resulted in cDNAs that were 20 bases shorter than the wild type. The activation of this cryptic downstream SA site, however, precluded the generation of mRNA capable of encoding a functional Rev protein and resulted in an immu-noblot devoid of HIV-1 structural proteins (Fig. 6A, lane 8). It should be noted that all of the wild-type 1.8-kb spliced RNAs were transcribed as truncated species by mutant pNLA SA7+7a+7b except for the nefl RNA, which was not detected on the autoradiogram shown in Fig. 8A. The cDNA bands obtained for pNLASA7+7a+7b after PCR for the 4.0-kb mRNA were identical to those of wild-type virus, indicating that RNA splicing to SA and SD sites upstream of SA7 was not affected by the absence of functional two-exon Rev protein. This finding demonstrates that Rev is not required for splice site selection in HIV-1 RNAs; however, protein expression from RNAs containing an RRE clearly requires functional Rev protein.

Mutation of each of two competing SA sites, SA4b and SA5, caused a different usage frequency for neighboring SA sites but not the activation of any cryptic sites. The first competing SA mutant, pNLASA4b, failed to generate bands for revl (Fig. 8A, lane 2), rev4, rev7, andrevlO (evident after long gel exposures; not shown) following PCR with primers for the 1.8-kb mRNA species and for env2 (Fig. 8B, lane 2), env6, envlO, andenvl4

(evident after long gel exposures; not shown) after PCR with primers for the 4.0-kb mRNA. The failure to detect these bands was consistent with the absence of RNAs splicing to the mutagenized SA4b site. All other cDNAs amplified from pNLASA4b were identical to wild-type cDNAs. The cDNA pattern associated with the second competing SA mutant, pNLASA5, lacked several predominant nef species (nef2, nef3, nef4, andnef5)after PCR for the 1.8-kb mRNA (Fig. 8A, lane 3), as well as the major env species (envl, env5, env8, and envl3) after PCR for the 4.0-kb mRNA (Fig. 8B, lane 3), reflecting the absence of RNAs splicing to the mutagenized SA5 site. In addition, there was a compensatory increase in the use of SA4, SA4a, SA4b, and SA4c, resulting in increased levels ofrevl, rev2, rev3, tatl, tat2, tat3, tat4, tat5, tat6, tat7, env2, env3, and env4 mRNAs.

Translational consequences of altered RNA profile for com-petitive splice site mutants. Since the mRNAs using the competing SA4a, SA4b, SA4c, andSA5 splice sites are multi-cistronic, potentially coding several proteins from each mRNA (15, 48, 49), we examined the effect of altered proportions of HIV-1 mRNAs on protein expression (Fig. 9). Mutant

pNLAiSA4b

directed the synthesis of a significantly increased level of envelopegpl60/120and Nef but decreased amounts of Revcompared with wild-type provirus (Fig. 9A to C, lanes 3). Because Rev protein plays a prominent role in regulating the level of gpl60/120, we evaluated Rev functional activity by measuring theRev-dependent rescue of CAT activity following cotransfection ofpNLASA4b with the pDM128 Rev reporter plasmid (Fig. 9E and F) (24). Again, Rev activity directed by

pNLASA4b, as measured in this assay, was lower than that directed bywild-type pNL4-3. Thus, the observed elevation of

gpl60/120wasnot due to any increase in Rev activity. Signif-icantly, the expression of both Rev-dependent gpl60/120 and Rev-independent Nef proteins were increased in similar pro-portions. These proteins share SA5 as the predominant com-peting SA for theirmRNAs. Thus, the increased synthesis of

gpl60/120 and Nef from the pNLASA4b mutant most likely reflects the increased use of SA5 (and SA4c) and results in

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A. '.

* gpl60/120

1 2 3 4

B.

,15 ey.

as* t,.. ,...

__ Nef

1 2 3 4

C.

*.- Re

r)- 2 3

E. Mock pNL4-3

0 5 1

0.5 0.5 0.5

pNL.NSA4b

5

0.5 0.5

pNL.\SA5

5 1 Ltg provirus

0.5 0.5 LtgpDM128

*

_W. _... AcCM SA7

CM

0 14.0 13.7 3.1 0.4 26.2 11.6 %conversion

FIG. 9. Translational consequences ofalternativeusageofcompeting splicesitesbymutantHIV-1proviruses.Westernblotanalysiswithrabbit serum togpI60/120(A)orNef(B)andimmunoprecipitationwithrabbitserum toRevfrom HeLacelllysatesprepared48 h after cotransfection with 0.5 ,ug of humangrowthhormone reporterplasmidalone(lane 1)orwith 20 ,ug ofpNL4-3(lane 2),pNLASA4b (lane 3),orpNLA&SA5 (lane

4)(C). Lysatevolumeswerestandardizedaccordingtohumangrowthhormonedetermination. Functional Revactivitywasmeasured withuseof the pDM128 Rev-dependent reporter plasmid of Hopeet al. (24) (D) by measuringthe amountofCAT activity rescueddue to Rev-RRE interactionaftercotransfectingthe indicated amountsofplasmids pDM128andpNL4-3, pNLASA4b, pNLASA5with0.5jigof humangrowth

hormone reporterplasmid(E).The percentageofchloramphenicol(CM)convertedtotheacetylatedforms(AcCM)is shownatthe bottom.SV40,

simianvirus40 promoterandenhancer; LTR, long terminalrepeat.

increased synthesis of both proteins. This occurs despite the loss ofmRNA species using SA4b which encode Rev. Since increaseduseof SA4c does not preventthe reduction ofRev

expression, if islikelythat multicistronic mRNAs usingSA4c

arelessefficient for Revexpression than for Neforgpl60/120

expression.

HeLa cells transfected with pNLASA5 express very low levels ofgpl60/120and Nefproteinsbut elevated levels ofRev

compared with wild-type pNL4-3 (Fig. 9A to C, lanes 4).

Elevated Rev activity was also measured in the assay of Rev-dependent rescue of CATactivity, showing that the low level ofgpl60/120 expression didnotresult from anydeficiency

in Rev function. The low-level expression ofgpl60/120most

likely results from the inefficient translation of the multicis-tronic env2, env3, and env4 mRNAs, which are present at

relatively increased levels in cells transfected with pNLASA5 compared with the wild-type pNL4-3 (Fig.8B, lanes 1 and3). This result indicates that the multicistronic env mRNAs are markedly less competent forgpl60/120expression than is envl.

pNLASA5 alsofails to synthesize the major nef RNAs (nef2, through nef5; Fig. 4), although the levels of the nefl cDNA species, which results from splicing of SD1 directly to consti-tutive SA7,were equivalent in thepNLASA5- and wild-type-transfected cells (Fig. 8A). This low expression of Nef protein indicates that the increased amount of bicistronic

revinef

and

envinefmRNAs encoded by mutant pNLASA5 fails to com-pensate for the loss of the predominant monocistronic nef mRNAs.The elevated levels of Rev protein observed

demon-strate that the bicistronic

revinef

and

envinef

mRNAs encode

Revwith significantly higher efficiency than Nef.

Infectivity of splicing mutants of HIV-1. The ability of the

HIV-1 splice site mutants to generate progeny virions was assessed by measuring the RT activity released into the

medium following cotransfection of HeLa cells with proviral

and human growth hormone DNAs (Fig. 10A). All of the

splice sitemutantsexceptpNLASA7a+7b generated less par-ticle-associated RT than did the wild-type plasmid pNL4-3;

pNLASA7+7a+7b andpNLASD1 produced only 2and 15% of viral progeny, respectively, comparedwith the wild type.

Theinfectivity of the splice site mutants was evaluated by

inoculating CEM (12D7) cells with equal amounts of virus harvested from transfected HeLa cell supernatants as deter-minedby RTassay (for wild-type pNL4-3, themultiplicity of infectionwas approximately 0.002) (Fig. lOB). Spreading in-fectionwasestablishedbyoneof thetwoproviruses containing

amutated constitutive site. Mutant pNLASD1 was infectious butexhibiteddelayedreplication kinetics compared with wild-typepNL4-3,reflectingthedelayed kinetics of protein synthe-sisbythismutant (Fig. lOB).LowerefficiencyofRNAsplicing from the cryptic donor activated in pNLASD1 is very likely responsible for the delayed replication and infectivity kinetics.

Mutant

pNLASA7+7a+7b,

which lacks the constitutive SA7

site,couldnot infect CEM(12D7) cells, reflecting its inability

toproduceRev and the HIV-1 structuralproteins.

Mutationsaffecting competingSAsitesin HIV-1 had

differ-enteffectsonviralinfectivity. MutantpNLASA4b,whichfails

togenerate severalHIV-1mRNAs(revl, rev4, rev7, and revlO andenv2, env6,envlO, and envl4)and consistently expressed elevated levels ofgpl60/120,exhibited growth kinetics similar

tothe wild-typevirus kinetics (Fig. lOB). In this case, altered

splicing of viral mRNA had no effect on viral infectivity. Mutant pNLASA5, which lacks the processing site for the

major envl mRNAspecies, was not infectious despite being abletosynthesize low levels ofgpl60/120in transfected HeLa cells by utilizing alternative SAs (Fig. 6A, lane 4; Fig. 9A). Thesecontrasting results demonstrate that alternative splicing

tocompetingSAsitescanaffectgpl60/120production, leading

tosynthesisof either fullyinfectious or defective virion.

D.

Revdependent reporterplasmid pDM128 SD4

SV40 CAT RRE

* *.

s ,

*

@ .

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A.

20000

- 15000

jk.

E

a,6

10000 5000

0

Bo

40000

'a

0. ie

30000

-200000

10000

DISCUSSION

0

* pNL4-3wildtype * pNLASDI O pNLASA4b

0 pNLASA5 O pNLASA6 13 pN1ASA7a a pNLASA7a+7b M pNLASA7+7a+7b

* Mock

pNL4-3wildtype pNLA pNLASA4b

pNLASAS

pNLA pNLA pNLASA7a+7b

pNLASA7+7a+7b

Mock 0

--+

-0---20

Days post-infection

30

FIG. 10. Efficiency ofvirion production and infectivity of HIV-1 splicing mutants. (A) Particle production by HIV-1 RNA splicing

mutantswasmeasuredby usingthe accumulation of RT activity40h aftercotransfectingHeLa cells with20 jig ofmutantprovirus plasmids and 0.5 ,ug of human growth hormone reporter plasmid. The RT activitywasstandardized fortransfectionefficiency by using the ratio of human growth hormonecomparedwithwild-type pNL4-3 virus. (B) Kinetics ofinfection of HIV-1 RNAsplicingmutantsfollowing trans-fer ofcell-free virionobtained from transfectionsupernatants(105cpm

fromtheRTassay)toCEM(12D7)cells.

The infectivity of mutants affecting the cryptic splice sites

was moresubtle andcouldnotbepredictedfrom theirability

to generate virus particles in the transfection experiments

shown in Fig. 1OA. Mutant pNLASA7a was infectious, but

peakvirus productionwasdelayed4 to5 days comparedwith the wild type. Mutant pNLASA7a+7b, which directed large

amountsofprogenyvirionproduction followingtransfection of HeLacells,was notinfectious. MutantpNLASA6wasalsonot

infectious, despite directing the synthesis of the wild-type complement and quantities of protein and spliced mRNAs

(Fig. 6 and 7). These latter results could indicate that the

crypticSA6 and SA7b sitesparticipateinsomeotheraspectof RNA processing (e.g., folding or branch point formation). Alternatively, amino acid changes introduced into the

enve-lope protein as a result of mutagenesis

'(see

Materials and

Methods) mayeliminate virus infectivity, possibly by affecting gpl60 processing as suggested by reduced amounts ofgpl20

observed inFig. 6A. Inaprevious study,asimilar mutation of SA6 in theHIV-lHXB2isolate resultedinthe loss ofinfectivity,

whereas SD5 mutants,also defectivefor Tevexpression,hada

wild-type phenotype (16).

Relativeproportions of alternatively spliced HIV-1mRNAs.

Various regulatory mechanisms control the expression and function of HIV-1 during a cycle of virus infection. The

regulation of RNAprocessing is one such prominent

mecha-nism,andthe balancedsplicing of genomic length RNA into a

complexset of alternative RNA transcripts is required for the

synthesis of several viral proteins essential for replication. Several transcripts are capable of expressing each of the regulatory and accessory HIV-1 proteins, and most of these

transcripts have the potential to encode two or more proteins with different efficiencies. To evaluate the importance of the

complex group of mRNAs synthesized during infection by

HIV-1, wefirstrigorouslydetermined the identities and rela-tivequantitiesof viralmRNAsresulting from transfection and infection experiments. Our analysis shows that some RNA

speciesaresynthesizedinpreference to others. Generally, the

most highly spliced forms of RNA that exclude noncoding

exons aremostcommonexceptinthe case of nef, in which case the inclusion of the 68-nt noncoding exon 5 is favored. A

previouslyunrecognized SA site forrev-and env/vpu-encoding

mRNAs (designated SA4c)wasidentified among a cluster of

competing SA sites in the tat coding sequence. This site was selected at fivefold-lower frequency than the SA4a or SA4b site for both rev and env mRNAs in PBMC infected with

HIV-lNL-3

or

HIV-lLAI.

The SA4csite is used by many strains of HIV-1 and is thepredominant SA used for rev mRNA by

someHIV-1 strains(42).Theaddition of thenewSA4c site to the centralcompeting SA sites(SA4,SA4a, SA4b, SA4c, and

SA5)

determined that 16 alternative mRNAs may encode

gpl60/120. However,mostofthese existatverylow levels, and the most common env mRNAs either used SA5 or excluded both noncoding exons 2 and 3. The shortest possible env

transcript (envl)accountsfor 80% of allenvRNAdirected by

HIV-'NL4-3.

Competing SA site usage determines gpl60/120 levels and

virus infectivity. Changing the balanced usage of competing

splicesites caused alterations in theproportionsof both RNA andprotein species and,in somecases, viralinfectivity.HIV-1 mutants containing changes affecting SA4b gave rise to an

increasedproportion of themRNAspecies using neighboring

SA4a, SA4c,

and SA5. This caused elevated expression of

envelope

gpl60/120but no loss of virus infectivity. Increased

synthesis

of HIV-1 env mRNA using SA5, which more

effi-ciently yields gpl60/120, is likely to explain the increase in

gpl60/120.Mutation of theclosely adjacent SA5,themajorSA forenvmRNA,resulted in increased useofSA4a, SA4b, and SA4c butwas

accompanied

byamarked reduction in boththe

expression of envelope gp160/120 and virus infectivity. The reduced levels ofgpl60/120werenotassociated with reduced

Rev activity, and Gag protein production was not altered.

These results demonstrate that the level of expression of

envelope proteins

can be

dramatically

altered

by

forcing

different

splicing

patternson

HIV-lNLA-3

through

the

dispro-portionate

usage of the

seemingly

redundant SA sites in this

region

of the viral genome.

Two determinants could control the selection of the

com-peting

SA sites if this alternative

splicing

mechanismwereto

operate invivo.

First,

the different sequence structure of the

competing

SA motifs

(Fig. 3)

orthe branch

point

structure(s)

in individual HIV-1 strains could alter thebalance of the SA usage. Thelocation of the branch

point(s)

for the

competing

HIV-1 SA sites is unknown. We have confirmed that HIV-1

strains with different sequences have different

splicing

patterns in a survey of various HIV-1 isolates

exhibiting

variable

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