The Prion Protein Has RNA Binding and Chaperoning
Properties Characteristic of Nucleocapsid Protein NCp7 of
HIV-1*
Caroline
Gabus
,
Edmund
Derrington
,
Pascal
Leblanc
,
Jonas
Chnaiderman
,
Dominique
Dormont§,
Wieslaw
Swietnicki¶,
Manuel
Morillas¶,
Witold K.
Surewicz¶,
Daniel
Marc
,
Pradip
Nandi
, and
Jean-Luc
Darlix
**
From the
LaboRetro, Unité de Virologie Humaine
INSERM-Ecole Normale Superieure de Lyon (ENS) 412, ENS de Lyon,
46 Allée d'Italie, Lyon 69364, France, the
§ Département de Recherche Médicale,
Commissariat à l'Energie Anatomique, BP6, Fontenay-aux-Roses
92265, France,
Institut National de la Recherche Agronomique,
Centre de Recherches de Tours, Nouzilly 37380, France, and the
¶ Department of Pathology, Case Western Reserve University,
Cleveland, Ohio 44106
Received for publication, October 25, 2000, and in revised form, February 2, 2001
 |
ABSTRACT |
Transmissible spongiform encephalopathies are
fatal neurodegenerative diseases associated with the accumulation of a
protease-resistant form of the prion protein (PrP). Although PrP is
conserved in vertebrates, its function remains to be identified.
In vitro PrP binds large nucleic acids causing the
formation of nucleoprotein complexes resembling human immunodeficiency
virus type 1 (HIV-1) nucleocapsid-RNA complexes and in vivo
MuLV replication accelerates the scrapie infectious process, suggesting
possible interactions between retroviruses and PrP. Retroviruses,
including HIV-1 encode a major nucleic acid binding protein (NC
protein) found within the virus where 2000 NC protein molecules coat
the dimeric genome. NC is required in virus assembly and infection to
chaperone RNA dimerization and packaging and in proviral DNA synthesis
by reverse transcriptase (RT). In HIV-1, 5'-leader RNA/NC
interactions appear to control these viral processes. This prompted us
to compare and contrast the interactions of human and ovine PrP and
HIV-1 NCp7 with HIV-1 5'-leader RNA. Results show that PrP has
properties characteristic of NCp7 with respect to viral RNA
dimerization and proviral DNA synthesis by RT. The NC-like properties
of huPrP map to the N-terminal region of huPrP. Interestingly, PrP
localizes in the membrane and cytoplasm of PrP-expressing cells. These
findings suggest that PrP is a multifunctional protein possibly
participating in nucleic acid metabolism.
 |
INTRODUCTION |
Transmissible spongiform encephalopathies are fatal
neurodegenerative diseases that include Creutzfeld-Jakob disease, kuru, fatal familial insomnia, and Gerstmann-Sträussler-Scheinker
(GSS)1 syndrome in humans, scrapie in sheep and
goats, and bovine spongiform encephalopathy in cattle (1). A major characteristic of transmissible spongiform encephalopathy diseases is the accumulation of an abnormal partially protease-resistant prion protein (PrPSC), which
is derived from the normal protease-sensitive prion protein (PrPC). Although accumulation of PrPSC may
damage cells of the central nervous system, it is clear that the
generation of spongiform encephalopathy requires the presence of both
PrPSC and PrPC (2). The abnormal
PrPSC is thought to recruit the normal PrPC
through a proposed trans-conformation process causing the accumulation of PrPSC in the form of plaques and fibrils (3-5).
The cellular prion protein (PrP) is encoded by a single gene
corresponding to about 250 amino acids and is highly conserved in
vertebrates (4-6). PrP is widely expressed, but it appears to
accumulate in the central nervous system and the lymphoreticular system. PrP may be produced via several pathways, because it can be
found in the extracellular fluid, on the outer surface of the plasma
membrane and Golgi, and as a transmembrane form, designated ctmPrP,
where the N-terminal domain is oriented within the cytoplasm (7, 8). In
addition, a truncated version of PrP, resulting from a stop codon at
position 145 in human PrP (huPrP), is associated with a variant of the
GSS syndrome and is found in the cytoplasm (9).
Although PrP is a highly conserved protein in vertebrates, its role
remains to be identified. Interestingly, PrP null mice develop normally
and appear to be healthy (2). Nonetheless, a number of functions have
been proposed for PrP such as superoxide dismutase activity,
involvement in copper metabolism (reviewed in Ref. 10), and, very
recently, participation in signal transduction during neuronal
differentiation (11). In addition, PrP was shown to interact with
sulfated glycans (12), RNA aptamers (13), and large nucleic acids (14,
15), causing the formation of nucleoprotein complexes similar to HIV-1
nucleocapsid-RNA complexes formed in vitro (16). A recent
report shows that MuLV replication accelerates the scrapie infectious
process (17), suggesting possible in vivo interactions
between retroviruses and PrP.
These findings prompted us to compare and contrast the interactions of
human PrP and the HIV-1 Gag-encoded nucleocapsid protein with
viral nucleic acids. HIV-1, like all retroviruses examined to date
(except for the Spumaretroviruses), encodes a major nucleic acid
binding protein (NC protein) found in the virion core where about 2000 NC protein molecules completely coat the dimeric RNA genome. The
biological roles of NC include the chaperoning of RNA dimerization and
packaging during virus assembly and proviral DNA synthesis by reverse
transcriptase (RT) in the course of viral infection. These functions of
NC are thought to be largely governed by interactions with the HIV-1
5'-leader RNA (16, 18-20).
Our results show that huPrP largely mimics the chaperone properties of
NCp7 with respect to the annealing of complementary nucleic acid
strands, viral RNA dimerization, the hybridization of replication
primer tRNA
to the HIV-1 5'-primer binding site sequence and the initiation of reverse transcription by RT. These NC-like activities of huPrP appear to map to
the N-terminal region of huPrP. Such properties are not unique to
huPrP, because the ovine PrP was also found to harbor nucleic
acid-chaperoning properties similar to those of HIV-1 NCp7 and
retroviral nucleocapsid proteins (reviewed in Ref. 16).
 |
EXPERIMENTAL PROCEDURES |
Recombinant Proteins, Enzymes, RNA, and Plasmid DNA--
The
full-length human PrP (huPrP), and the fragments
huPrP-(23-144) (N-terminal region) and huPrP-(122-231)
(C-terminal region) were produced in Escherichia coli and
purified as described previously (21). The ovine PrP (ovPrP) (residues
25-234) was prepared using a similar protocol (22). The purity of the
proteins was higher than 95% as judged by polyacrylamide gel
electrophoresis. HIV-1 nucleocapsid protein NCp7, NCp7-(12-53), and
Vpr were synthesized by the Fmoc
(N-(9-fluorenyl)methoxycarbonyl)/opfp chemical method and purified by high pressure liquid chromatography (23). Proteins were
dissolved at 1 mg/ml in buffer containing 30 mM Hepes, pH 6.5, 30 mM NaCl, and 0.1 mM ZnCl2.
DNA oligonucleotide corresponding to the Tar(
) 57 nt of HIV-1
sequences (pNL4.3 molecular clone) was from Eurogentec (Belgium). HIV-1
5'-leader RNA corresponding to nucleotides 1-415 was generated
in vitro as previously described (18) and phenol and
chloroform extracted and purified by gel filtration. HIV-1
3'-RNA of 650 nt (positions 8583-9208 of the HIV-1 genome) with a
poly(A) tail was prepared by transcription in vitro
(27).
Natural bovine tRNA
from beef liver
was a kind gift of Gérard Keith (Strasbourg, France). Synthetic tRNA
was generated in
vitro using T7 RNA polymerase. HIV-1 reverse transcriptase (RT
p66/p51), purified from E. coli, was provided by S. Le Grice
(Frederick, MD). HIV-1 integrase (INp32), purified from E. coli, was provided by J.-F. Mouscadet (Villejuif, France).
All plasmid DNAs were amplified in E. coli 1035 (RecA
) and
purified by affinity chromatography (Qiagen protocol).
Nucleoprotein Complex Formation--
Reactions with HIV-1
32P-labeled 5'-RNA, and either PrP or HIV-1 NCp7 were
performed for 10 min at 37 °C in 10 µl of a buffer containing 20 mM Tris·HCl, pH 7.5, 30 mM NaCl, 0.2 mM MgCl2, 5 mM dithiothreitol, 0.01 mM ZnCl2, and 1.5 pmol of RNA. PrP or NCp7 was
at the indicated molar protein to nucleotide ratios. Reactions were
stopped by 5 mM EDTA, and samples were electrophoresed on a
1.3% agarose gel in 50 mM Tris·borate, pH 8.3. Gels were visualized by ethidium bromide staining followed by gel fixation in 5%
trichloroacetic acid, drying, and autoradiography. To pellet nucleoprotein complexes, reaction mixtures were centrifuged at 4 °C
for 5 min at 10,000 × g.
Nucleic Acid Annealing Assays--
Reactions with HIV-1 RNA,
32P-labeled tRNA, or 32P-labeled minus strand
Tar DNA (Tar
) and either PrP or HIV-1 NCp7 were performed for 10 min
at 37 °C in 10 µl of a buffer containing 20 mM
Tris·HCl, pH 7.5, 30 mM NaCl, 0.2 mM
MgCl2, 5 mM dithiothreitol, 0.01 mM ZnCl2, 5 units of RNasin (Promega), 1.5 pmol of RNA, 3 pmol
of tRNA; or Tar(
) and PrP or NCp7 at the indicated molar
protein to nt ratios. Reactions were stopped by SDS/EDTA (0.5%/5
mM), and samples were treated with proteinase K (2 µg)
for 10 min at room temperature, phenol-chloroform was extracted, and
RNA was analyzed by electrophoresis on 1.3% agarose in 50 mM Tris·borate, pH 8.3. Gels were visualized by ethidium
bromide staining followed by gel fixation in 5% trichloroacetic acid,
drying, and autoradiography. A 0.16- to 1.77-kb RNA ladder was used for
size determination. The percentage of primer annealed to the HIV-1 RNA
was determined by densitometric scanning of the autoradiograph.
Reverse Transcription Assays--
First the nucleic acid
annealing assay was carried out in 10 µl for 5 min at 37 °C as
above, and next the reaction volume was increased to 25 µl by
addition of 2 pmol of HIV-1 RT, 0.25 mM each of
dNTPs, 60 mM NaCl, and 2.5 mM
MgCl2. Samples were incubated for 20 min at 37 °C. The
reactions were stopped and processed as for the analysis of nucleic
acid annealing, except that after phenol extraction, nucleic acids were
ethanol-precipitated, recovered by centrifugation, dissolved in
formamide, denatured at 95 °C for 2 min, and analyzed on 8%
polyacrylamide gel electrophoresis in 7 M urea and 0.5×
TBE, pH 8.3. 5'-32P-Labeled FX174 DNA
Hinf markers (Promega) were used for size determination (not shown). The levels of cDNA synthesized by RT were quantified by densitometric scanning.
Cell Culture, DNA Transfection, and
Immunohistochemistry--
293T cells, derived from the HEK cell line
(human embryonic kidney cells), were cultured at 37 °C in an
atmosphere of 5% CO2 and in Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) complemented with 10% fetal calf
serum, penicillin (100 IU/ml), streptomycin (100 µg/ml), and
L-glutamine (2 mM). Transfection of 293T cells
were performed using the calcium phosphate method with 3 µg of
pDNA-huPrP for 106 cells. After 10 days under
puromycin selection (0.7 µg/ml), resistant cell colonies were
expanded and found to express huPrP.
For immunohistochemistry, cells were grown in eight-well Labtek
chamber slides and fixed in ice-cold 2% paraformaldehyde for 30 min at
4 °C. They were then washed twice in PBS and incubated for 30 min in
a blocking solution of PBS containing 5% bovine serum albumin, 1%
normal goat serum, and 0.2% Tween 20 prior to staining with
antibodies. This same solution served to dilute the anti-PrP SAF 37 antibody (IgG2a recognizing amino acids 79-92 of huPrP) used at 1/200
dilution, and the rhodamine-conjugated Helix pomatia
lectin, which was included with the primary antibody in some
experiments at a concentration of 5 µg/ml (kindly provided by Dr. F. Barde, ENS, Lyon, France). Primary incubation was for 2 h at room
temperature. After washing sections 5 × 10 min in PBS, bound
antibodies were revealed with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin at a final dilution of 1/400 in blocking
buffer containing bis-benzimide (1 µg/ml) to stain DNA. Controls included no primary antibody and non-transfected cells. Slides
were washed three times in PBS, mounted with moviol and analyzed
with a Zeiss axioplan fluorescence microscope, and a Zeiss axiovert
inverting microscope equipped with 488- and 514-nm lasers and LSM 510 software for confocal analysis.
 |
RESULTS |
The Human and Ovine PrP Bind HIV-1 RNA in Vitro--
HIV-1
nucleocapsid protein NCp7 is a small Gag-encoded protein. During the
infectious process NCp7 is required to chaperone proviral DNA synthesis
by reverse transcriptase (RT) (Fig.
1A) (16, 18, 19). In addition,
NCp7 is pivotal in virus assembly, because it recruits the genomic RNA
and chaperones its dimerization and packaging into assembling particles
(Fig. 1A). Interactions between the HIV-1 5'-leader RNA and
NCp7 appear to govern these viral processes (Fig. 1A). NCp7
with the two zinc fingers and an NCp7 mutant, designated NCp7-(12-53),
without the basic N- and C-terminal domains necessary for RNA
dimerization in vitro and virus assembly in vivo
(16, 24, 25), were generated by peptide synthesis (Fig. 1B
and data not shown) (23). Recombinant huPrP-(23-231) and the N
(23)- and C (122)-terminal regions were produced in E. coli and purified to homogeneity (21). To compare the nucleic acid
binding abilities of PrP and HIV-1 NCp7, we used the multifunctional
5'-leader of HIV-1 (Fig. 1), because it is formed of contiguous
functional domains that are required for viral DNA transcription (TAR
and poly(A) stem-loops), minus strand DNA synthesis by RT (5'-primer
binding site for initiation and Tar for strand transfer), viral
RNA dimerization and packaging (DIS and DLS), and translation (the AUG
of Gag) (Fig. 1). NCp7 interacts with the DIS-DLS domains to chaperone
viral RNA dimerization and packaging during virus assembly and with the
5'-primer binding site and TAR sequences during minus strand DNA
synthesis by RT in the course of infection (16, 19).

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Fig. 1.
HIV-1 RNA sequences and functions of NCp7 in
virus replication. A, the 5'- and 3'-regions of the
HIV-1 genomic RNA and the TAR oligonucleotides are shown. The 5'-leader
RNA is made up of several functional domains: TAR and poly(A) stem-loop
structures are required for provirus transcription and during reverse
transcription for minus strand DNA transfer. Note that the TAR and
poly(A) domains form R (Repeat). U5 is the untranslated 5'-sequence.
PBS is the primer tRNA
binding site and is 18-nt long. PBS is also required for
plus strand DNA transfer. The DIS (dimer initiation
sequence) and E/DLS (packaging/dimer linkage element) are
necessary for viral RNA dimerization and packaging in assembling
particles. AUG is the initiator codon for Gag. The 3'-RNA is formed of
the PPT (polypurine track), which is the start site for plus
strand DNA synthesis, U3 (3'-untranslated sequences), the
TAR and poly(A) stem-loop structures, and An tail (broken
line). The TAR and poly(A) are required for minus strand transfer
during proviral DNA synthesis. Nucleotide positions are indicated
above the lines representing the 5'- and 3'-RNAs.
B, HIV-1 NCp7 and mutant NCp7-(12-53) with the two CCHC
zinc fingers (Zn) are shown. The complete huPrP as well as the N- and C-terminal
regions are shown with the octarepeats and the H1 to H4 helices. Amino
acid positions are indicated. Functions of NC in HIV-1 replication are
mediated by nucleic acid interactions as indicated by
arrows. In the course of viral DNA synthesis NC chaperones
primer tRNA annealing to the primer binding site, initiation of reverse
transcription, and completion of viral DNA synthesis by RT. TAR and
poly(A) are required for NC to chaperone minus strand DNA transfer, and
primer binding site for plus strand DNA transfer. Also NC chaperones
viral RNA dimerization and packaging during virus assembly.
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As observed previously (18), NCp7 binds in a cooperative manner to the
multifunctional 5'-leader of HIV-1 RNA and forms nucleoprotein
complexes (Fig. 2, lanes
2-4), whereas the NCp7 mutant (12-53) does not form stable
complexes (lanes 6-8). huPrP binds to the 5'-leader RNA as
evidenced by the formation of nucleoprotein complexes (lanes
14-16). The N-terminal fragment (23) of huPrP was also able
to bind to the 5'-leader RNA (lanes 18-20) in a manner
similar to NCp7 (lanes 2-4), whereas the C-terminal
fragment (122) did not show any RNA binding affinity (lanes
22-24). The ovine PrP was also expressed in E. coli as
a recombinant protein and purified to homogeneity (not shown).
The ovine PrP also formed nucleoprotein complexes in a
dose-dependent manner similar to NCp7 (lanes
10-12). These NC- and PrP-nucleoprotein complexes are of high
molecular mass, because they can be recovered by 5-min centrifugation
at 10,000 × g (data not shown) (26).

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Fig. 2.
Formation of nucleoprotein complexes after
binding of PrP to HIV-1 RNA. 32P-Labeled HIV-1
5'-leader RNA of 415 nt in length (described in Fig. 1) was incubated
at 37 °C for 10 min with or without NCp7 or PrP. Formation of
nucleoprotein complexes was analyzed by electrophoresis on a 3%
polyacrylamide gel in 50 mM Tris·borate, pH 8.3. One
picomole of RNA was used, and the molar ratios of protein to nt are
indicated at the top. Lanes 1, 5,
9, 13, 17, and 21, CT
without protein; lanes 2-4, NCp7; lanes 6-8,
NCp7-(12-53); lanes 10-12, ovine PrP (ovPrP);
lanes 14-16, human PrP (huPrP(23-231));
lanes 18-20, huPrP-(23-144); lanes 22-24,
huPrP-(122-231). Vertical arrow is direction of
electrophoresis. Horizontal arrows are free RNA and
nucleoprotein complexes, respectively. Results shown indicate that
binding of PrP or NCp7 to HIV-1 RNA is probably cooperative.
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Human and Ovine PrP Have Nucleic Acid Chaperoning
Properties--
The interactions of PrP with nucleic acids suggested
that this cellular protein might have RNA and DNA
annealing activities similar to those of HIV-1 NCp7 or the human p53
protein (27). This was examined using HIV-1 sequences corresponding to
TAR and the 3'-region of the HIV-1 RNA genome corresponding to U3, TAR, and poly(A) sequences (Fig. 1A). The minus strand TAR
sequence (Tar
) represents the 3'-end of the so-called strong stop
cDNA (ss-cDNA(
)), the initial product of reverse
transcription. Under physiological conditions, NCp7 directed annealing
of Tar
to Tar+ present within the 3'-RNA (Fig.
3, lanes 2-4). Interestingly, huPrP was also able to hybridize Tar
to the 3'-RNA in a
dose-dependent manner (lanes 13-15). A similar
level of hybridization was obtained with huPrP-(23-144) (lanes
17-19). On the other hand, NCp7-(12-53) and huPrP-(122-231) did
not direct annealing of Tar
to HIV-1 3'-RNA (lanes 5-7
and 21-23, respectively). The ovine PrP (ovPrP) was found
to be as effective as huPrP and HIV-1 NCp7 in directing the specific
annealing reaction (lanes 9-11). These data demonstrate that PrP of human or ovine origin has nucleic acid annealing activity characteristic of HIV-1 NCp7 and retroviral NC proteins in general (16,
18, 19, 27-30).

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Fig. 3.
Nucleic acid annealing activity of PrP.
Assays were performed as indicated under "Experimental Procedures"
with HIV-1 3'-RNA and 32P-labeled Tar( ) DNA (Fig. 1). At
the end of the reaction, protein was removed by proteinase K digestion,
followed by phenol extraction. Analysis of 32P-Tar( )
annealed to HIV-1 3'-RNA was by electrophoresis on 1.3% agarose gels.
Gel was fixed with 5% trichloroacetic acid, dried, and
autoradiographed. The molar ratios of protein to nt are indicated at
the top. The vertical arrow shows direction of
electrophoresis and horizontal arrows correspond to Tar( )
DNA (bottom) and HIV-1 3'-RNA:Tar( ) DNA
(middle). Lanes 1, 8, 12,
16, 20, control incubation without NCp7 or PrP;
lanes 2-4, NCp7; lanes 5-7, NCp7-(12-53);
lanes 9-11, ovine PrP (ovPrP); lanes
13-15, huPrP-(23-231); lanes 17-19, huPrP-(23-144);
lanes 21-23, huPrP-(122-231).
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PrP Chaperones HIV-1 RNA Dimerization and Annealing of Primer tRNA
to the Primer Binding Site--
In the viral particle, the retroviral
genome is dimeric and has replication primer tRNA annealed to the
primer binding site, which constitutes the start site for reverse
transcription (see Fig. 1A). As demonstrated before, genomic
RNA dimerization and tRNA annealing to the primer binding site are
promoted by NC protein. In control experiments we used well
characterized nucleic acid binding proteins of viral origin such as
retroviral reverse transcriptases (RT), HIV-1 Vpr and integrase, T4
gp32 or nucleic acid binding proteins of cellular origin like E. coli RecA and human p53. All these proteins, some of which have
nucleic acid chaperone properties (40), were found to be inactive in
these viral processes (see Refss 18 and 23 for RTs and E. coli RecA; Ref. 27 for human p53 or RT). Using the HIV-1 system
with the multifunctional 5'-leader RNA and primer
tRNA
(Fig. 1B) NCp7 was indeed critical for HIV-1 RNA dimerization and primer
tRNA
annealing to the primer binding
site (Fig. 4, A and
B, lanes 2-4) (18, 23). Interestingly, we found
for the first time that a cellular protein, namely PrP, was capable of
promoting HIV-1 RNA dimer formation and
tRNA
annealing to the primer binding
site in a dose-dependent manner (lanes 14-16 for huPrP). Very similar data were obtained with the N terminus (23) of huPrP (lanes 18-20). NCp7-(12-53) retained
little activity (lanes 6-8) (16) and the C-terminal
fragment (122) of huPrP was completely inactive (lanes
22-24). The ovine PrP (ovPrP) was also found to show an NC-like
activity, because it directed HIV-1 RNA dimerization and primer
tRNA
annealing to the primer binding
site (lanes 10-12).

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Fig. 4.
PrP directs HIV-1 RNA dimerization and
annealing of primer tRNA to
the primer binding site. A, ethidium bromide staining of the
1.3% agarose gel. B, autoradiography to show primer
32P-tRNA annealing to
HIV-1 primer binding site. The HIV-1 5'-leader RNA and
32P-labeled tRNA were
incubated at 37 °C with or without NCp7 or PrP and reactions were
processed. The protein-to-nt molar ratios are indicated at the
top. The arrow shows direction of electrophoresis
and markers (in nt) are indicated on the left. HIV-1
monomer, dimer and multimer RNAs, and
tRNA are indicated on the
right. Lanes 1 and 7, CT without
protein. Lanes 2-4, with NCp7; lanes 5 and
6, with NCp7-(12-53); lanes 8-10, with huPrP;
lanes 11-13, with huPrP-(23-144); lanes 14 and
15, with huPrP-(122-231). Note that huPrP-(122-231) did
not promote HIV RNA dimerization or tRNA annealing to the primer
binding site (lanes 14 and 15), whereas huPrP and
huPrP-(23-144) were very active.
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Reverse transcription of the viral RNA is initiated by RT-mediated
extension of the primer tRNA hybridized to the primer binding site by
NCp7 (Figs. 1A and
5A). NCp7 was indeed critical
for the initiation of minus strand cDNA synthesis (Fig.
5B, compare lanes 1 and 2-4) (1-3,
16). Once again, huPrP was found to duplicate the properties of NCp7 by
actively promoting initiation of reverse transcription in a
dose-dependent fashion (Fig. 5B, lanes
8-10). The N-terminal fragment (23) of huPrP exhibited part
of this activity (lanes 11-13). NCp7-(12-53) retained some
activity, but only at a high protein to nt ratio (lanes 5 and 6, protein to nt molar ratio of 1:3) (16). The
C-terminal fragment (122) of huPrP was completely inactive
(lanes 14 and 15). Similarly to huPrP, the full-length ovine PrP was also very active in chaperoning the initiation of reverse transcription (lanes 16 and
17).

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Fig. 5.
PrP chaperones initiation of reverse
transcription of HIV-1 RNA. A, a scheme to show reverse
transcription initiation of HIV-1 RNA and synthesis of minus strand
strong stop cDNA, ss-cDNA( ), where PBS stands for
primer binding site. B, HIV-1 5'-RNA and
32P-labeled tRNA were
incubated with or without NCp7 or PrP. HIV-1 RTp66-p51 was added
together with the dNTPs to allow reverse transcription initiation.
Reactions were processed, and ss-cDNA( ) was analyzed.
The protein-to-nt molar ratios are indicated above the figure. The
arrow shows the direction of electrophoresis, and markers
(in nt) are indicated on the left. The ss-cDNA-tRNA and
primer tRNA are indicated on the
right. Lanes 1 and 7, CT with RT but
without NCp7 or huPrP. Lanes 2-4, with NCp7; lanes
5 and 6, with NCp7-(12-53); lanes 8-10,
with huPrP; lanes 11-13, with huPrP-(23-144); lanes
14 and 15, with huPrP-(122-231); lanes 16 and 17, with ovine PrP (ovPrP). Note that even at
a high molar ratio of huPrP-(122-231) ss-cDNA was not synthesized
(lane 15).
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PrP Promotes Specific Proviral DNA Synthesis--
A succession of
specific events is required for the conversion of the genomic RNA into
a complete proviral DNA with the two LTRs. Nevertheless, nonspecific
replicative events can take place, such as self-initiation of reverse
transcription, which is enhanced by template interactions, nicks in the
genome or folding back of the template (31-33). Retroviral NC proteins
behave as nucleic acid chaperones, which can destabilize RNA secondary
structures and thus inhibit self priming of reverse transcription (see
scheme in Fig. 6A)
(31-33).

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Fig. 6.
PrP inhibits self-priming of reverse
transcription. A, self-priming of reverse transcription
is shown in Ref. 1. Coating of the free RNA by NCp7 or PrP, which
results in the inhibition of self-priming is schematized in Ref. 2.
B, self-priming of reverse transcription. Assays with HIV-1
3'-RNA were performed as described under "Experimental Procedures"
except that [32P]dCTP was used. The protein-to-nt molar
ratios are indicated at the top. The vertical
arrow on the right shows direction of electrophoresis
and DNA markers (in nt) are indicated. Lanes 1,
4, 7, 10, 13, and
16 are CT without added protein; lanes 2 and
3, NCp7; lanes 5 and 6, NCp7-(12-53);
lanes 8 and 9, huPrP-(23-231); lanes
11 and 12, huPrP-(23-144); lanes 14 and
15, huPrP-(122-231); lanes 17 and 18,
ovine PrP (ovPrP).
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A 650-nt RNA, with a 25-nt poly(A) tail, corresponding to the 3'-region
of the HIV-1 genomic RNA was used to mimic resumption of minus strand
cDNA synthesis after minus strand transfer (Fig. 1B)
(31-34). However, a possibility exists that RT transcribes the genomic
RNA by virtue of self-priming at the 3'-end and thus in the absence of
minus strand transfer after ss-cDNA synthesis (Fig. 6A).
As shown in Fig. 6B and in agreement with published data
(31, 32) NCp7 strongly inhibited self-primed cDNA synthesis in a
dose-dependent manner (lanes 2 and
3), whereas the zinc finger domain (NCp7-(12-53)) was
completely inactive in this process (lanes 5 and
6). Interestingly, PrP of both human and ovine origins strongly inhibited self-priming of cDNA synthesis (lanes
8-9 and 17-18, respectively), and this activity
mapped to the N terminus (positions 23-144) and not the C terminus
(positions 122-231) (lanes 11-12 and 14-15, respectively).
To examine the continuation of cDNA synthesis by reverse
transcription of HIV-1 3'-RNA after minus strand transfer, the Tar
DNA representing the 3'-region of ss-cDNA(
) was included in the assays (Fig. 7A). Under these physiological conditions (see
"Experimental Procedures") both NCp7 and PrP were able to hybridize
Tar
to HIV-1 3'-RNA (Fig. 3). Extension of Tar
by reverse
transcription of the 3'-RNA was clearly specific in the presence of
HIV-1 NCp7 (Fig. 7B, compare lane 1 CT with
lane 3 where the NCp7:nt ratio was 1:6; see also Fig.
6B) in agreement with published data (30, 31). PrP of either
ovine or human origin was found to completely replace NCp7, because in
both cases specific cDNA(
) synthesis was observed (Fig.
7B; compare CT lane
1, with lane 7 (ovPrP-to-nt ratio of 1:6) and
lane 9 (huPrP-to-nt ratio of 1:6). In addition, the N
terminus of huPrP was also functional in these assays (see lane
10 in which the huPrP-(23-144)-to-nt ratio was 1:6). Mutant NCp7-(12-53) started to be partially effective at a protein-to-nt molar ratio of 1:3 (lane 5). However, huPrP-(122-231) was
completely inactive in promoting specific cDNA synthesis
(lanes 12 and 13).

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Fig. 7.
PrP chaperones-specific cDNA synthesis by
RT. A, schemes showing reverse transcription by
self-priming (lane 1 in B) and primer-directed
cDNA synthesis (lanes 3, 7, 9, and
11 in B). B, primer-directed reverse
transcription of HIV-1 RNA. Assays were performed in the presence of
HIV-1 3'-RNA and primer Tar( ) DNA (see Fig. 2) and in
conditions described under "Experimental Procedures" except
that [32P]dCTP was used (31). The protein-to-nt molar
ratios are indicated at the top. The vertical
arrow shows the direction of electrophoresis and DNA markers (in
nt) are indicated. The horizontal arrow shows the
full-length cDNA. Lanes 1, CT without added protein;
lanes 2 and 3, NCp7; lanes 4 and
5, NCp7-(12-53); lanes 6 and 7, ovine
PrP; lanes 8 and 9, huPrP-(23-231); lanes
10 and 11, huPrP-(23-144); lanes 12 and
13, huPrP-(122-231).
|
|
Subcellular Localization of PrP--
The above results clearly
show that PrP has the ability not only to interact with nucleic acids
(Fig. 2) but also to modify their conformation (Fig. 5) and chaperone
the hybridization of nucleic acids with complementary sequences (Fig.
3) that are critical for HIV-1 replication (Figs. 3-6). PrP is thought
to localize mainly at the cell surface of PrP-expressing cells (4, 6).
This would render interactions between PrP and cellular or viral
nucleic acids unlikely. However, PrP has also been reported to localize to the Golgi and the nucleus (8, 35). To examine the subcellular localization of PrP, we used the 293T cells derived from a human embryonic kidney cell line. Endogenous expression of PrP in these cells
is below the threshold of detection by immunocytochemistry, and no
staining was observed in control 293T cells (Fig.
8, A and B). This
allowed us to be confident that staining of transfected PrP in 293T
cells was selective, giving an accurate picture of its subcellular
distribution other than being nonspecific reactivity against other
cellular proteins. 293T cells were transfected with a huPrP
plasmid construct, and a 293T cell population constitutively expressing
PrP was derived upon puromycin selection. Both 293T and 293T-huPrP
cells were examined by immunocytochemistry using an antibody
recognizing amino acids 79-92 of huPrP (SAF 37, J. Grassi,
Commissariat à l'Energie Anatomique Saclay, France). As reported
in Fig. 8, intense punctate staining of huPrP occurred in the plasma
membrane of many cells, as was expected, but also in a cytoplasmic
compartment and more rarely in what appeared to be the nucleus (Fig. 8,
C and D). In cells undergoing mitosis, as
determined by the organization of chromatin revealed by DNA staining, a
quite different pattern of expression was seen. In such cells there was
an intense staining throughout the cytoplasm. This was clearly not an
artifact, because metaphase control cells showed no staining (compare
A with C). Confocal analysis with double labeling
of PrP and a Golgi marker (rhodamine-conjugated H. pomatia
lectin) showed that, in cells not undergoing mitosis, PrP was
selectively localized in the plasma membrane and an intracellular compartment, including the Golgi, although cytoplasmic PrP staining did
not co-localize very well with the Golgi marker (Fig. 8, E, G, and I, from the plasma membrane to the
nucleus). When nuclear staining of PrP appeared to occur, the Golgi
marker was also present, indicating that in such cells the Golgi
compartment was superimposed on the nucleus. In cells undergoing
mitosis, the cytoplasmic localization of PrP was confirmed. Indeed the
Golgi is dispersed during mitosis, and it was apparent that PrP was not
so uniformly localized as the Golgi marker, indicating that PrP is
probably present in both the Golgi and a distinct cytoplasmic
compartment (vertical arrows in E-J). In
conclusion, these data strongly suggest that huPrP can accumulate in
several cell compartments where it might interact with nucleic
acids.

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Fig. 8.
Localization of PRP in 293T cells.
293T cells expressing human PrPC (C-J)
were analyzed by immunocytochemistry for the expression of PrP. In
control 293T cells (A and B) no staining was
observed and background was very low. In 293T cells stably expressing
huPrP, we observed strong staining of PrP in the membrane and cell body
(see arrowheads in C and D).
Cytoplasmic staining was intense throughout the cytoplasm of cells
determined to be in metaphase by DNA staining with
bis-benzimide (B and D). This was not
simply a cell cycle-related artifact, because no staining occurred in
metaphase cells that were not transfected with the huPrP plasmid
(compare cells indicated by arrowheads in A and
B with those in C and D). Confocal
microscopy was performed to analyze the subcellular distribution of PrP
in stained cells (E-J). The same cells are shown at three
different depths in the Z axis (going progressively deeper
from the cell membrane to the nucleus). Green fluorescence
shows anti-PRP staining, and red fluorescence shows binding
of the H. pomatia lectin to show the Golgi (E,
G, and I), close co-localization appears as
yellow labeling. Phase contrast in F,
G, and H corresponds to the immunofluorescent
staining in E, G, and I, respectively.
The vertical downward pointing arrow indicates a cell that
was established to be in metaphase by DNA labeling (not shown). Note
the round morphology and absence of a defined nuclear compartment under
phase contrast. The cell indicated by the horizontal arrow
was apparently not in metaphase, because it contains a well defined
nucleus and typical fusiform morphology with a short process. In cells
that were not undergoing mitosis, huPrP was localized primarily in the
membrane and also in a cellular compartment depicted by the Golgi label
(red fluorescence). Co-localization, indicated by
yellow staining was not perfect, and often
patches and points of green anti-PrP
staining occurred in the midst of a patch of red
Golgi staining (blue arrows in E and
G). In the metaphase cell the Golgi stain was dispersed
quite evenly throughout the majority of the cell body, although
it was excluded from the region occupied by the chromatin (unstained
region of the metaphase cell in G and I). PrP
staining was also diffuse and cytoplasmic; however, intense
points and patches of labeling occurred in the
cytoplasm with a distribution distinct from that of the more evenly
distributed Golgi label. Occasionally, PrP staining appeared to occur
in the nucleus, as indicated by the blue arrows in
G and H, however, the Golgi label also stained
and co-localized in the same region suggesting that the apparent
nuclear labeling may well be in a distinct overlying compartment.
|
|
 |
DISCUSSION |
The findings that PrP of human or ovine origin binds RNA confirm
and extend previous data showing that the human prion protein exhibits
nucleic acid binding properties (13-15). Furthermore, we have found
that binding of PrP to HIV-1 RNA causes the formation of condensed
nucleoprotein structures similar to those obtained with retroviral NC
proteins. Interestingly, the interactions between PrP and the viral RNA
in these nucleoprotein structures appear to share many of the specific
functional characteristics of HIV-1 NCp7 in the viral context (see
below) (16, 20). PrP differs from other nucleic acid binding proteins
and nucleic acid chaperone proteins, because it mimics the functions of
NCp7 in HIV-1 replication such as HIV-1 RNA dimerization and primer
tRNA
annealing to the 5'-primer
binding site during virus assembly and the chaperoning of proviral DNA
synthesis by RT during virus infection (see Figs. 1, 4, and 5)
(reviewed in Ref. 16). The latter properties are not shared by nucleic
acid binding proteins such as HIV-1 Vpr and RT, or human p53 (see Figs.
5-7 and Refs. 18, 26, 27, 32, 33, and data not shown). Many cellular proteins with nucleic acid chaperoning properties are essential to
nucleic acid maintenance and metabolism (36), such as DNA repair for
p53 (37), pre-mRNA processing for hnRNP proteins (38), and
telomere elongation for hnRNP A1 (39). This suggests that PrP
may exert one of its functions at the level of nucleic acid metabolism
(36, 40).
The length and precise location of the functional domain responsible
for the nucleic acid binding and chaperoning activities of PrP is at
present unknown. However, our data clearly show that this domain is
localized within the N-terminal region encompassing residues 23-144
(Figs. 3-7) (41) analogous to a mutant PrP associated with a variant
of the GSS syndrome (9).
The capacity of PrP to interact with nucleic acids (RNA, DNA, and
oligonucleotides) and to modify their conformation and degree of
compactness as well as to chaperone nucleic acids interactions in a
manner characteristic of retroviral NC proteins (Figs. 5-7) (16, 42,
43) raises a number of questions. PrP is found in several cell
compartments. A major fraction is usually attached to the plasma
membrane via the GPI anchor. However, a transmembrane form with
the N terminus in the cytosol, designated ctmPrP, has also been
described (44). In addition to accumulation in the plasma membrane,
huPrP was also found in the cytosol and probably in the nucleus, where
it might interact with cellular and/or viral nucleic acids (Fig. 8).
PrP may thus belong to a growing class of proteins exhibiting numerous
activities and functions, for example the 37-kDa laminin receptor
precursor (45). The latter protein is present on the cell surface as
well as in a form associated with ribosomes as a component of the
mRNA translational machinery (46, 47). Interestingly, PrP interacts
with this 37-kDa laminin receptor precursor (45). Another example
of a protein with both membrane- and nucleic acid-associated functions
is C17. This protein is either membrane-associated, functioning in
G-protein-mediated signal transduction related to the calcitonin
gene-related receptor (48) or associated with RNA polymerase III
for tRNA gene transcription (49). The possibility that PrP is
associated with ribosomes or other cellular nucleoprotein
components is presently under investigation.
In cells infected by HIV-1, the retroviral Gag polyprotein encoding NC
is membrane-anchored via its N terminus during virion formation and
budding at the cell surface (16, 25, 43, 50). Therefore, it is tempting
to speculate that Gag and PrP interact at the plasma membrane in
infected cells. This might well influence virus replication and
accelerate the scrapie infectious process as suggested by Carp et
al. (17) in the MuLV/mouse system. In that respect, the impact of
huPrP expression on HIV-1 formation and replication is currently being
investigated, and preliminary results indicate that PrP interactions
with NC and the viral RNA can strongly influence HIV-1 virion formation
and replication.
 |
ACKNOWLEDGEMENTS |
Thanks are due to Stuart Le Grice (Frederick,
MD) for HIV-1 RT, Bernard Roques (Paris, France), and Damien
Ficheux (Lyon, France) for HIV-1 NCp7, NCp7-(12-53), and Vpr; to
Jean-François Mouscadet (Villejuif, France) for HIV-1 IN; to
Gérard Keith (Strasbourg) for
tRNA
; to Jacques Grassi and Yvelyne Frobert (CEA Saclay, France) for the SAF 37 anti-PrP antibody; and to
J. Julien (ENS Lyon) for assistance in confocal microscopy.
 |
FOOTNOTES |
*
This work was supported by Agence Nationale de Recherche sur
le SIDA, INSERM, and Mutrielle Gènèrale de l' Education
Nationale (to J.-L. D.) and by National Institutes of Health
Grant NS38604 (to W. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Tel.: 33-47-27-28-169;
Fax: 33-47-27-28-777; E-mail: Jean-Luc.Darlix@ens-lyon.fr.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M009754200
 |
ABBREVIATIONS |
The abbreviations used are:
GSS, Gerstmann-Sträussler-Scheinker syndrome;
PrP, prion protein;
PrPSC, abnormal partially protease-resistant PrP;
PrPC, normal protease-sensitive PrP;
ovPrP, ovine PrP;
huPrP, human PrP;
HIV-1, human immunodeficiency virus type 1;
MuLV, murine leukemia virus;
NC protein, nucleic acid binding protein;
RT, reverse transcriptase;
nt, nucleotide(s);
PBS, phosphate-buffered
saline;
DIS, dimer initiation sequence;
DLS, dimer linkage element;
ss-cDNA, strong stop cDNA;
TAR, trans-activation response
element;
CT, control.
 |
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