0022-538X/90/041803-05$02.00/0
Copyright C) 1990,American Society for Microbiology
NOTES
Effects of
a
Highly Basic Region of
Human
Immunodeficiency Virus
Tat Protein
on
Nucleolar Localization
HARUHIKO SIOMI, HISATOSHI SHIDA, MASATOSHI MAKI, AND MASAKAZU HATANAKA* Institute for Virus Research, Kyoto University, Kyoto 606, Japan
Received 28 September 1989/Accepted 22 December 1989
Human immunodeficiency virus type 1 encodes a positive trans-activator protein, Tat, which is located predominantly in the cell nucleolus. To study the role of the basic region of Tat in nucleolar localization,we constructed fusion genesencoding serially deleted segments of Tat joined to theamino-terminal end ofthe Escherichia coli
0-galactosidase
molecule. We show that the basic region of Tat was sufficient for nuclearlocalizationbut notfornucleolarlocalization. Addition of three amino acids (59, 60, and 61) of the Tatsequence
atthe C-terminal end ofthe basic region was neccesary for the chimeric I-galactosidase to localize in the
nucleusaswellasin the nucleolus. We demonstrate thatashort amino acidsequence(G-48 RKKRRQRRRA
HQN-61), when fusedtothe amino terminus of
0-galactosidase,
canactas a nucleolar localization signal.Human immunodeficiency virus type 1(HIV-I), the
caus-ative agent of acquired immunodeficiency syndrome, is a
highly regulated retrovirus; geneexpression ofthe virus is
controlledby several trans-actinggenes aswellascis-acting
regulatory sequences located withinthe viral long terminal
repeat(LTR) and elsewhere in the viralgenome(28). Of the
trans-acting proteins, both Tat and Rev are essential for
virus growth, but their precise functions remain to be
defined. It has beenproposed that the Tat protein
acceler-ates the rate of virus production at one or more levels of
control, such as RNAtransport, RNAstability, and
trans-lation efficiency, as well as having effects on transcription
(28). Hauber et al. (11) demonstrated that Tat is
predomi-nantly located in the nucleolus. Other trans-regulatory
pro-teins of human retroviruses, Rex of human T-cell leukemia
virus type I (HTLV-I) (26) and Rev ofHIV-I (7, 20), have
recentlybeenshowntobe located predominantly in nucleoli.
Rex and Rev augment the production of viral structural
polypeptides by increasing the cytoplasmic concentration of
theintron-containing envandgag-pol mRNAs(6, 7, 13, 16,
24, 27).Thesefactssuggestthepossibilitythat thisnucleolar
eventmaybeinvolved in the regulation of human retrovirus
geneexpression.Atpresent, weknowverylittle about how
certain proteins accumulate in the nucleolus, although
evi-dencefor activetransportof proteinstothenucleus and for
thefunctioning of nucleartransportsignals (3) in relationto
cellulartransportmachinery (5, 12, 18, 19)hasaccumulated.
Identification ofanucleolar localization signal of Tat would
contributetoourunderstandingof the molecular mechanism ofnucleolartargetingand further Tat-mediated trans-activa-tion.
Tatpossesses astretchof basicamino acidresidues which
is highly conserved among various HIV-I isolates, and a
highly basic amino-terminal sequenceofRex of HTLV-I has
been shown to constitute a nucleolar accumulating signal
(26). Toexamine whetherthe basic region ofTat is crucial
fornuclear and nucleolarmigration, weconstructed
recom-binantplasmids encodingforhybrid proteinswith Tatatthe
* Corresponding author.
aminoterminusand 3-galactosidaseatthe carboxyterminus.
We also usedavacciniavirus(VV) expressionvectorsystem to achieve a high level oftransient gene expression in the
VV-infected cells (2, 17, 26).Inthis VVsystem,Tatwasalso
localized predominantlyin thenucleolus (4). Toexpressthe
chimericgeneswhich harbor 5'portionsof thetatgenefused
in frame to lacZ, the Sall fragments from pFtat (4) were
inserted into the BamHI-SmaI site present on plasmid
p7.5C40X (26).ResultantplasmidpV7.5Ftat,which contains
the VV 7.5 promoter (2) followed by the entire tat-coding sequenceand the HTLV-Ilongterminalrepeat,wasdigested
at its unique SmaI site, and then exonuclease III and Si
nuclease digestion (9)wereperformedto createdeletionsin
the 3' portion ofthe tat-coding region. Todetermine exact
nucleotideendpoints, clonesweresequenced bythedideoxy
method ofSangeretal. (22). After ligationofaSall linker
(5'-GGTCGACC-3'), the Hindlll-Sall fragments containing
the 3'-deletedtat-coding regions and VV 7.5promoterwere
ligated into pMC-LTR (26), which contains the Escherichia
coli lacZgenefollowedbythe HTLV-Ilongterminal repeat. This resulted insevenplasmidswhichproducesevenhybrid
proteins having 41, 49, 50, 52, 55, 58, or61 amino-terminal
residues fused to ,B-galactosidase (Tat-p-galactosidase
fu-sions) (Fig. 1).
We examined the subcellular localization of the fusion
proteinsdescribed abovebyindirect immunofluorescenceby
using rabbit
anti-p-galactosidase
antibody (Fig. 2). Thefu-sion protein expressed from pV7.5tat(1-61)lacZ32 was
ob-served predominantly in the nucleus, as well as in the nucleolus. The fusion proteins expressed from both
pV7.5tat(1-58)lacZ30 and
pV7.5tat(1-52)lacZ25
werelocal-izedmainlyin nuclei butwereabsent fromnucleoli, although
somecytoplasmic stainingwas also observed. These results
strongly suggest that the basic region may be involved in
nuclear transport of the Tat protein. In contrast, cells transfected with pV7.5tat(1-55)lacZ18, pV7.5tat(1-50)lacZ34,
and pV7.5tat(1-41)lacZ1 lacked any localized staining and
exhibited fluorescence throughout the cell, as did cells
transfected with pV7.5tat(1-49)lacZ33 (data not shown).
Although Tat(1-55)LacZ contains the basic stretch of
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1804 NOTES
m
E
in0
tat
a
.' E
86
---ISYGRKKRRQRRRAHQN
---45 61
VV 7.5pro pV 7.5 tat(1-61)lacZ32 l 1
pVT.5tat(1-58)1acZ30
i-pV 7.5 tat(1-55) IacZi E1
ZJ-pV7.5tat(1-52)lacZ25 J1. tat
50 pV7.5tat(1-50)IBCZ34 = 1 4
49 pV7.5tat( -49)lacZa3
41 pV7.5 tat(1-41)lacZl 1i ii1
FIG. 1. TheTat-,3-galactosidasefusionproteins.The fusionproteinscontainaconstantportionof,B-galactosidase (thin line)at thecarboxy terminus and variousamountsof Tat protein (boldline)atthe aminoterminus. The numberattherightend ofabold lineindicates the number
of Tatamino acidsintheprotein.Thetwoproteinmoietiesarenotdrawntoscale;the,B-galactosidase moietyconsistsof1,012residues. The
Tat-1-galactosidasegenefusionsareunder the control of theVVp7.5promoter.No andNprefertonucleolarornucleoplasmiclocalization.
Cells expressingfusion proteins exhibited fluorescenceat similar intensitiesinboth thecytoplasmand the nucleus(N/C).
RKKRRQR, its nuclear accumulation was inefficient. The
onepossible explanation may be that the Tatportion of the
fusion protein was prevented from folding properly by its
association with 3-galactosidase, leading to the inability to
be associated with some element of the nuclear transport system.Thebehavior ofTat(1-58)LacZ, which contains the entirelysine-arginine-rich regionofTat,wasabsent from the
nucleolus oftransfected cells.However,afusedproteinwith
onlythreeadditionalamino acids ofTat,Tat(1-61)LacZ, was
targeted tothenucleolus aswellasthenucleoplasm.
There-fore,theseresults show that this basicregionisnotsufficient
for nucleolar migration and additional amino acids are
re-quired for efficient nucleolar localization, when fused to
3-galactosidase.
Next,wetested whether the shortpeptides, includingthe
basicregion ofTat, can direct
P-galactosidase
to the nucle-olus. A duplex DNA linker containing the translationalinitiationcodon followed bythe codons for Tatwas
chemi-cally synthesized with 5' SphI and 3' Sall overhangs and
ligated between the SphI and Sall sites of the plasmid
pGOM3 (26) containing the VV 7.5 promoter, the gene
encoding ,-galactosidase, and the HTLV-I long terminal
repeat, to give rise to the plasmid pV7.5tatBAAlacZ (Fig.
3A). pV7.5tatBAAlacZ or the control plasmid pV7.5NlacZ
wastransfectedinto CV-1cells, and thesubcellular
localiza-tionof thepolypeptides wasexamined byindirect
immuno-fluorescence. Figure 3B shows that the polypeptide
ex-pressedfrompV7.5NlacZ haspredominantly acytoplasmic
distribution. In sharp contrast, the fusion protein
TatBAA-lacZ is seen to accumulate in the nucleolus within the
nucleus, although the fusion protein isalsopartlyseeninthe
cytoplasm. These data showed that the putative Tat
nucle-olarlocalization signalsequence GRKKRRQRRRAHQN is
functional intargeting acytoplasmically localized
heterolo-gouspolypeptide tothenucleolus.
Ourexperiments showed thatthe first 52 ofthe 86amino
acidsofTatweresufficienttolocalize the fused
3-galactosi-dase to the nucleus. This result is in accord with a recent
findingthata,3-galactosidasefusionprotein thatcontaineda
5-amino-acid sequence (GRKKR) derived from the basic
region ofTat at the amino-terminal end of ,3-galactosidase
accumulatedwithin thenucleoplasmandwasexcludedfrom
the nucleolus (21). The RKKR sequence is homologous to
the nuclear localization signal of simian virus 40 large T
antigen (14). The requirement of nonconserved Tat
se-quences at thecarboxyl end of the basicregion in orderto generate sequences thatareableto localize ,-galactosidase
tothe cell nucleolusprovidesanimportant hintfor postulat-ing models for the interaction of this basic stretch with a
nucleolar constituent. It is reasonable to assume that the
basic stretch will be functional onlyif itisexposed properly
onthe surface ofaproteinand that itsfunction issensitively
affected by the structural environment within which it is presentin anygiven protein.
The amino acid residues 48 to 61 of Tat contain the
sequence RKKRRQRRR. A similar sequence of two stretchesof basic amino acidsflankingaglutamineresidueis presentinanucleolar localization signal of the Rex protein
of HTLV-1 (26). The HIV Rev protein contains a similar
highlybasicregionwithapredominanceofarginineresidues
(27). Glutamines also play a role in the sequence of Rev
protein. The amino acid sequence (R-35 QARRNRRRRW
RERQR-50) of Rev could actually replacethe basicregion
of Tat (4), and when glutamines were fused to the amino terminusof 3-galactosidase, they also acted as anucleolar
localization signal (15). Although the characteristic feature of the nucleolar localization signals in the three proteins is apparent, it may not require exact primary sequences, as
shown by the comparison of the three sequences and the mutational analysis of Rex and Tat (10, 26). Protein se-quences that bear little sequence homology have been
re-ported to manifest identical biological functions in other
61 lacZ
58
55
52
Location No/Np
Np
N/C
Np
N/C
N/C
N/C
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[image:2.612.152.466.69.300.2]CDt
=r
N:
0CD CD
_
-vo G 3
CDO ,
0 c
O 7
-0)
_+tcn
CD q (jC
G~ _. ea
ONCD
_. z
- 5 -0*P
'eo5 .
--S
O m
sA 0 o
*,.< 0
P: 0
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1806 NOTES
A
VV 7.5 pro
LI
}
lacZ
pV 7.5
tatBAAlacZ
MGRKKRRQRRRAHQNGRPGDPVV
i .
48
tat
61
9
B-galactosidase
pV7.5 NIacZ
MGAQNGRPGDPVV
---9
B-galactosidase
[image:4.612.69.553.70.673.2]B
FIG. 3. Subcellularlocationof the fusion protein consisting of the putative nucleolar localization signal of Tat andp-galactosidase. (A) pV7.5tat BAAlacZ contains the only short coding sequence for Tat fused to lacZ under the control of the VV 7.5 promoter. (B) Indirect immunofluorescenceof cells transfected with pV7.5tatBAAlacZ. Theplasmidstransfected werepV7.5tatBAAlacZ (a) and pV7.5NlacZ (c). Phase-contrast micrographs (b and d) of the same field are shown.
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cases. For example, signal sequences that direct the
inser-tion ofproteins into the endoplasmic reticulum bear little,if
any, sequencehomology (1, 29); the same istruefor signal
sequences ofmitochondrialproteins (23) and also for signal sequences of nuclear proteins (3). Therefore, it is possible
thatthe three sequences share an underlying structural trait
and may participate in the same mechanism for nucleolar
localization. Althoughtheexperiments reported heredo not
directly probe the mechanism of nucleolar accumulation, a
catalog ofnucleolar targeting sequences from
trans-regula-toryproteins of human retroviruses may provide a clue for
furtherinvestigation.
We thank Koreaki Ito for anti-3-galactosidase serum, Hiromu TakematsuandSatoshi Kubota for technical assistance, and Hifumi Maedafor excellent secretarial assistance.
Thiswork was supported by grants from the Ministry of Educa-tion, Science,andCulture of Japan.
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