Structural and functional analysis of the human immunodeficiency virus type 2 Rev protein.

Vol. 65, No. 1 JOURNALOFVIROLOGY, Jan. 1991,p. 445-449

0022-538X/91/010445-05$02.00/0

Copyright © 1991,American Society for Microbiology

Structural

and Functional Analysis of the

Human

Immunodeficiency

Virus

Type

2

Rev

Protein

PATRICK J. DILLON, PETER NELBOCK, ANN PERKINS, ANDCRAIGA. ROSEN* Department of Molecular Oncology and Virology, Roche Institute of Molecular Biology,

340Kingsland Street, Nutley, New Jersey07110-1199

Received 17July 1990/Accepted 28 September 1990

The Rev proteins of the human immunodeficiency viruses (HIV) are necessary for expression of viral structural geneproducts. Site-directedmutationsweremade within theHIV-2revgenetoidentify functional domains.We observed that similartoHIV-1 Rev, the HIV-2 Rev proteinwasphosphorylated, albeittoamuch lesserextentthanwasHIV-1Rev. We alsofoundthat like HIV-1 Rev, HIV-2 Revlocalizedtothe nucleus,with amarked accumulationin the nucleolus. Mutations withinastretchof basic residues prevented both nuclear and nucleolar localization. Furthermore, mutant Rev proteins able tolocalize in the nucleus but unableto localize in the nucleolus werenonfunctional.

The human immunodeficiency viruses HIV-1 and HIV-2 encode several trans-acting regulatory proteins that are necessary for viral gene expression. The most-studied of these regulatory proteins are Tat, which activates gene

expression directed fromthe long terminal repeat (2, 36), and Rev,whichis necessary for the expression of viral structural proteins (13, 35). The Tat protein interacts with the TAR element (15, 31) present at the 5' end of all viral mRNAs. The HIV-1 and HIV-2 Rev proteins interact with an RNA sequence element present in the env gene (4, 9, 21, 27, 28,

37),originally referredto as CAR (8, 32) and now called the Rev-responsive element (RRE) (26). While the function of Tat is to stimulate an increase in expression of all viral genes, thefunction ofRevinvolves enhancement of

expres-sion from unspliced and singly spliced viral mRNAs (12, 17-19, 23, 26). In the absence of Rev, the viral proteins encoded by these mRNAs are not expressed because of entrapment of their respective mRNAs in the nucleus.

Interactionof Rev with the RRE RNA, either alone or with

additional cellularfactors, presumably mediates the export of these RNAs from the nucleus to the cytoplasm (12, 14, 18, 26).

The HIV-1 and HIV-2 Tat proteins can be functionally

substituted for each other, as demonstrated by their ability toutilize each other's TAR elements (11). However, the Rev proteins of HIV-1 and HIV-2 do not share this reciprocal

relationship(9, 23, 24). While HIV-1 Rev can function with both the HIV-1 and HIV-2 RREs, HIV-2 Rev functions only with its own RRE and that of the more closely related simian

immunodeficiency virus (9, 23, 24). This nonreciprocity is due in part to theinability of the HIV-2 Rev protein to form a stable interaction with HIV-1 RRE RNA (9). To gain insight into how HIV-2 Rev may be similar to or different from HIV-1 Rev, in vitro mutagenesis of HIV-2 Rev was used toidentify thedomains importantfor function.

We haverecently described astrategy forthe expression andpurification ofHIV-2RODRev protein from Escherichia

coli(9, 10). We have shown that this HIV-2 H6Revprotein

binds to the HIV-2 RRE RNA and is functional when

expressed in eucaryotic cells (9). A polyclonal antiserum

against purifiedHIV-2 H6Rev proteinwas raised in rabbits

* Correspondingauthor.

andwasusedtofurther characterize the HIV-2 Revprotein

expressed ineucaryotic cells.

ExpressionofHIV-2 Rev was accomplished by

transfec-tion of Cos-7 cells with the plasmid BLpSVHIV-2Rev, which encodes anunmodifiedHIV-2Revprotein (9). Prior to

transfection, Cos-7 cellswereplatedat adensity of 106 cells per100-mmdish andtransfected with 5

jig

of plasmidDNA in a transfection cocktail containing Dulbecco modified Eagle medium supplemented with 500 ,ug of DEAE-dextran per ml, 50 ,ug ofchloroquine per ml, and 10% Nutridoma (Boehringer Mannheim). The transfection cocktail was placed oncells for 2.5 h, and the cellswereincubatedat37°C

in a5%CO2incubator. Following the incubation, the

trans-fection cocktailwasremoved and the cellsweretreated with

mediumcontaining10% dimethyl sulfoxide for 2.5 min. The medium was then removed, fresh, complete medium was addedtothecells, andincubation continued for48 hbefore

radiolabeling. While the predicted molecular mass of the 100-amino-acid

HIV-2ROD

Rev protein would be

approxi-mately 12 kDa, immunoprecipitation of the radiolabeled

lysate from transfected cells with the HIV-2 Rev-specific

antiserum showed that the HIV-2Revprotein migratedas a 16-kDa protein by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis analysis (Fig. 1A). A similaranomalous

gel migration was seenwith HIV-1 Rev.

Todetermine whether theHIV-2 Revproteinis

phosphor-ylated, as has been observed forHIV-1 Rev (3, 20), Cos-7 cells were transfected with either BLpSVRev or

BLpS

VHIV-2Revandradiolabeled with carrier-free

32p.

Whereas

immunoprecipitations of the radiolabeled lysates showed that the HIV-1 Rev proteinwas highly

phosphorylated,

the HIV-2 Rev protein displayedalower level of

phosphoryla-tion that could be seenonly afterlong exposures ofthe

gel

(Fig. 1B). However, the functional consequence ofthis is unclear since the extent ofphosphorylation ofHIV-1 Rev doesnotcorrelate with function (5).

Recentstudies have shownthat HIV-1 Revlocalizestothe nucleus and shows amarkedaccumulation in the nucleolus (6, 7, 30). To determine whether HIV-2 Rev

displayed

a similar nuclear localization pattern, Cos-7 cells were

trans-fected with BLpSVHIV-2Rev and

analyzed

by

indirect immunofluorescence(33),usingthe HIV-2

Rev-specific

rab-bitantiserum. SimilartoHIV-1Rev,HIV-2Revlocalizedto the nucleus andaccumulated in the nucleolus

(Fig.

2).

445

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446 NOTES

B c1

,; I

....W

"Ill__

69k- .

46k-_

30k-VP

21.5kI'*0

14.3k- IO

FIG. 1. Immunoprecipitation ofRevproteins from Cos-7 cells. (A)Cellsweretransfected with plasmid pSV2neo (-), BLpSVHIV-2Rev(HIV-2), BLpSVA73-74 (A73-74), BLpSVA75-76 (A75-76), or BLpSVRev (HIV-1)andradiolabeled at48 hposttransfectionwith

[35S]methionine. Cell lysates were immunoprecipitated with an HIV-2Rev-specific rabbit antiserumexceptfor theBLpSVRev cell lysate,whichwasimmunoprecipitated withanHIV-1 Rev-specific rabbit antiserum.Immunoprecipitated proteinswereanalyzedon a 15% sodium dodecyl sulfate-polyacrylamide gel. (B) To determine whether HIV-2 Rev was phosphorylated, Cos-7 cells were trans-fected withpSV2neo(-),BLpSVHIV-2Rev (HIV-2),orBLpSVRev (HIV-1) and radiolabeled with carrier-free 32p. Cell lysates from

pSV2neo andBLpSVHIV-2Revtransfectionswere immunoprecipi-tated with an HIV-2 Rev-specific rabbit antiserum, and the cell lysate from the BLpSVRev transfection was immunoprecipitated withanHIV-1 Rev-specificrabbit antiserum.

Todetermine whether the basic stretch ofamino acids in HIV-2 Revis involvedin nuclearlocalization, site-directed mutagenesis (22)wasusedtogeneratemutations thatdeleted or changed various residues within and outside this area

(Fig. 3). ThemutantHIV-2Rev proteinswereexpressedin Cos-7 cells, and theirsubcellular localizationwas analyzed

by indirect immunofluorescence (Fig. 2). Mutants A35-37, A39-43, and A43-49no longer accumulated in the nucleolus but gave a diffuse staining pattern throughout the nucleus and cytoplasm (Table 1). Mutations made outside the basic region retained the wild-type stainingpattern,with

accumu-lation inthenucleolus. These results indicate that the basic stretch of amino acids of HIV-2 Rev is important for nucleolar localization, aswould be predictedonthebasis of

previous studieswithHIV-1 Rev (6, 30).

We have previously described a modified Rev-dependent heterologousgeneexpressionassaytoassessthe functional activity of HIV-2 Rev (9). The Rev-responsive reporter plasmid pIIIH2 containsthe HIV-1envgenesequencesthat mediatethenuclear retentionof viralmRNAandtheHIV-2 RRE substituted for the HIV-1 RRE. These sequences are

positioned3'tothetermination codon andincorporated into the 3' untranslated portion ofthebacterialchloramphenicol acetyltransferase (CAT) reporter gene transcript (Fig. 4A). The result of these modifications is that CAT gene expres-sion becomes subjecttoregulation by HIV-2 Rev.

Cotransfection of

CHOZiptauIII

cells (9, 31) with the re-porter plasmid pIIIH2 and with BLpSVHIV-2Rev, which

expresses the wild-type HIV-2 Rev protein, fully restored CATgene expression (Fig. 4). Mutant HIV-2 Rev proteins

which did not localize to the nucleolus did not function, indicating that nucleolarlocalization may be necessary for

E

F

FIG. 2. Indirect immunofluorescencestainingof Cos-7 cells ex-pressing wild-typeandmutantHIV-2 Revproteins.Cos-7 cellswere transfected with plasmid DNA and stained 48 h posttransfection, using anHIV-2Rev-specific antiserum. (A) Phase-contrast

micro-graph of wild-typeHIV-2Rev;(BtoF)fluorescencemicrographsof

wild-type HIV-2 Rev (B), mutant A35-37 (C), mutantA39-43 (D), mutantA43-49(E), andmutantA46-49(F).

HIV-2 Rev activity. Mutations were made in the carboxy half of HIV-2 Rev to further assess its role in function. A mutation involving residues 61 through 64 (A61-64) func-tionedtoasimilarextentaswild-type HIV-2Rev.However, mutations affecting residues 73 through 76 (A73-74 and A75-76)producednonfunctionalproteins, eventhoughthese proteins localizedto the nucleolus. The loss of functionfor these mutantsmay reflect a change inconformation,

espe-ciallysince thesetwoproteins migratedmoreslowlythan the wild-type protein in gel electrophoresis (Fig. 1A). While mutantA73-74 doesnotfunctiononits own,itbehavesas a transdominant suppressorof HIV-2 Rev function (datanot shown),furtherillustratingthefunctionalimportanceof the carboxyregion.Inaddition,thetwo mutantscA80andcA90, which contain carboxy deletions of 20 and 10 amino acids respectively, were also nonfunctional, although they local-izedtothe nucleus.

In this report, we have characterized the HIV-2 Rev protein in an effort to identify possible similarities and

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NOTES 447

M N E R A D E E G L Q R K L R L I R A35-37

L L H Q T N P Y P Q G P G T

I

A S

QI

R

A39-43 A43-49 A46-49

R N

JR

R R R W K Q RW R Q I L A L A

A61-6

D S I Y T F P D PP A N S P L D Q T

A73-74 A75-76

I Q H L Q G L T I Q E L P D P P T H

HIV-2

_IIIH2 CAT I HIVe 1RRE

pill

H2 - -

---HVn

mRNA+1

AAAAAA

hQJ

L P E S Q R L A E T

FIG. 3. Amino acidsequenceofHIV-2RODRevprotein encoded by BLpSVHIV-2Rev. The mutagenized areas are indicated by

boxes withmutantdesignations above.

differences between the HIV-2 and HIV-1 Rev proteins. Using amonospecific anti-HIV-2 Rev antiserum, we show thatHIV-2RoD Rev isa16-kDa protein which localizestothe nucleus and accumulates within the nucleolus. The nucleolar accumulation of HIV-2 Rev is similartothat observed for HIV-2 Rev (6, 7, 30). Previous studies have shownthat the arginine-rich basic stretch of amino acids in HIV-1 Rev is involved in nucleolar localization (6). Deletions and alter-ations ofresidues found in this basic stretch of amino acids resulted in the loss of nucleolarlocalization.The HIV-2 Rev protein contains a similar arginine-rich region, and

site-directedmutagenesiswasusedto study its role in nucleolar localization. Three mutant HIV-2 Rev proteins (A35-37, A39-43,andA43-49), which contain amino acid substitutions

[image:3.612.60.298.74.194.2]

ordeletions betweenresidues35 and 49, failedtoaccumulate in the nucleolus. In addition, these mutants displayed a substantialcytoplasmic stainingpattern,indicating that mu-tations in thisregionnotonlyaffected nucleolar localization but also impaired the ability to reach the nucleus. While deletion ofamino acids 43 to49 (mutant A43-49) abolished nucleolar localization,asmaller deletion of residues 46to49 retainedthe ability of HIV-2 Revtolocalizetothe nucleolus. Interestingly, mutantsof HIV-2 Rev that didnotaccumulate inthenucleoluswerenonfunctional. This result is consistent with that observed for HIV-1 Rev and further supports a

TABLE 1. Structural andfunctional characterization of HIV-2 Revproteinmutants

Plasmid Mutationa Nucleolar Functionb

localization

pSV2neo None NTC

BLpSVHIV-2Rev None + + +

BLpSVA35-37 QRRtoAHG -

-BLpSVA39-43 RRRRWtoS -

-BLpSVA43-49 DeletesWKQRWRQ

BLpSVA46-49 DeletesRWRQ + +

BLpSVA61-64 PDPPtoGS + + +

BLpSVA73-74 IQtoGS +

-BLpSVA75-76 HLtoGS +

-BLpSVcA80 Deletes 81-100 +

BLpSVcA90 Deletes 91-100 +

aDeletions and substitutions of amino acids affected by mutations are

indicated; the positions of the mutations are reflected in mutant plasmid

names.

bMeasured in the HIV-2 Rev-dependent heterologous gene expression

assay(Fig. 4). Resultsaveragesfrom severalexperiments. ++,Fullactivity; +, intermediateactivity;-,noactivity.

cNT, Not tested.

Pr- cP eD en a qw m

U, 40 V- ,

+ + I* V.* +.* O

[image:3.612.318.560.76.283.2]

I

~~~~~~~~~~~~~~~~~~~~~~~~~~.

!

... .. ... .. ... ... ....

FIG. 4. Functional analysis ofmutant HIV-2 Rev proteins. (A) Schematicillustration of the indicator plasmid pIIIH2, which con-tains the HIV-2 RRE.(B)Cotransfection of

CHOZip,a,tij

cells with pIIIH2 and pSV2neo (-) or with the indicated wild-type (W.T.) HIV-2 Rev or mutant HIV-2 Revexpressionvector. pU3R-III isa control plasmid that contains the CAT gene under controlof the HIV-1long terminalrepeat andisnotdependenton Rev (36).The CATassayresults werefromcelllysates prepared48h posttrans-fection;assays were performedaspreviously described(16).

possible role of the nucleolus in Rev function (6, 7, 30). It should be noted that the functionally similar Rex protein from human T-cell leukemia virus type1alsoaccumulatesin thenucleolus (34). While theprecise role that the nucleolus plays in Rev and Rex function remains unknown, it is possible that thenucleolus providesan accessory hostcell factor which assists Rev in the productive export of un-spliced and partially spliced viral transcripts from the nu-cleus.

Although the HIV-1 and HIV-2 Rev proteins contain similar basic stretches ofaminoacids,thecarboxyhalves of these twoproteins are quite dissimilar. The

HIV-2ROD

Rev

protein is 100 amino acids long, compared with the

116-amino-acid-longHIV-1 Revprotein. A previous studyshows that deletion of 25 amino acidsfromthecarboxyterminus of HIV-1 Revgives rise toatruncatedproteinthat retainsfull function (30). However,weobserved thatdeletion ofas little as 10 amino acids (cA90) from HIV-2 Rev resulted in a nonfunctionalprotein.Thisfindingsuggeststhat most,if not all, of the carboxy terminus of HIV-2RoD Rev is necessary for functionorthe properfoldingof theprotein. Inaddition, while there exists little homology between the carboxy termini of the HIV-1 and HIV-2 Rev and HTLV-1 Rex

proteins, some mutations in thecarboxy regionsof allthree of theseproteinscangiverise toatransdominant suppressor phenotype (1, 25, 29). IfRevfunction requiresthe presence ofahost cell cofactorthat interacts with this domain of Rev or Rex, the diversity of residues present in this domain suggests that a

family

of cellular factors with similar func-tions may recognize the individual Rev and Rex proteins.

Alternatively, if a single cofactor is involved, the lack of

homology between HIV-1 and HIV-2 Rev and Rex would suggest that a property other than amino acid sequence VOL.65, 1991

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448 NOTES

might be responsible for association of these proteins with cellularfactors.

Further studies on these proteins should enable elucida-tion of the mechanismof Rev function and the basis for the inability of HIV-2 Rev to function with the HIV-1 RRE.

This work was supported in part with funds from a National Cooperative Drug Discoverygrant.

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