Hsp70 Protein Positively Regulates Rabies

Virus Infection
Xavier Lahaye, Aurore Vidy, Baptiste Fouquet and Danielle
Blondel
J. Virol. 2012, 86(9):4743. DOI: 10.1128/JVI.06501-11.
Published Ahead of Print 15 February 2012.

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Hsp70 Protein Positively Regulates Rabies Virus Infection
Xavier Lahaye,* Aurore Vidy,* Baptiste Fouquet, and Danielle Blondel
CNRS, UPR 3296 Virologie Moléculaire et Structurale, Gif sur Yvette, France

The Hsp70 chaperone plays a central role in multiple processes within cells, including protein translation, folding, intracellular
trafficking, and degradation. This protein is implicated in the replication of numerous viruses. We have shown that rabies virus
infection induced the cellular expression of Hsp70, which accumulated in Negri body-like structures, where viral transcription
and replication take place. In addition, Hsp70 is present in both nucleocapsids purified from infected cells and in purified viri-
ons. Hsp70 has been shown to interact with the nucleoprotein N. The downregulation of Hsp70, using specific chaperone inhibi-

Downloaded from http://jvi.asm.org/ on May 11, 2014 by UCSF Library & CKM
tors, such as quercetin or RNA interference, resulted in a significant decrease of the amount of viral mRNAs, viral proteins, and
virus particles. These results indicate that Hsp70 has a proviral function during rabies virus infection and suggest that Hsp70 is
involved in at least one stage(s) of the viral life cycle, such as viral transcription, translation, and/or production. The mechanism
by which Hsp70 controls viral infection will be discussed.

I n eukaryotic cells, the posttranslational folding of proteins and

quality control are performed by molecular chaperones and heat
shock proteins (HSPs). The ubiquitous chaperone family of the
70-kDa HSPs plays a central role in protein homeostasis and pro-
tection against proteotoxic stresses by preventing protein misfold-
ing and aggregation by directing damaged protein to the ubiqui-
tin-proteasome system for degradation (4, 15, 34, 40). Hsp70
chaperones not only survey the folding status of proteins as part of
the quality control function, which is very important under stress
conditions, but also are involved in the regulation of fundamental
cellular processes, such as signal transduction, cell cycle regula-
tion, apoptosis, and innate immunity (23, 25).
Viral infection depends on the successful recruitment of host
cellular factors at different steps of the viral life cycle: genome
replication, viral protein synthesis, viral assembly, and counterde-
fense against cell apoptosis and innate immunity. During viral
infection, large amounts of viral proteins are synthesized and pro-
tein folding can become a limiting step. Therefore, on the one
hand viruses need cellular chaperones for their own protein fold-
ing processes; on the other hand, as chaperones are involved in the
regulation of fundamental cellular processes, viruses have to in-
teract with them. Hsp70 chaperone, the major inducible heat
shock protein, is frequently recruited by viruses. Research during
the past 30 years has revealed the involvement of Hsp70 in all steps
of viral life cycle replication and for viruses from numerous fam-
ilies of diverse orders (24, 28). Several DNA viruses, such as her-
pesvirus (HSV1) (10, 38), vaccinia virus (18), adenovirus type 5
(24), and simian virus 40 (SV40) (27, 31), induce the specific
expression of Hsp70 (33). In most cases this induction has a pro-
viral effect. Evidence is growing that the Hsp70 induction is also
important for the replication of many positive-strand RNA and
negative-strand RNA viruses (28). In the case of measles virus,
Hsp70 interacts with nucleocapsids (42) and induces the in-
creased expression of viral genes (5). Nevertheless, in some cases
Hsp70 was found to inhibit viral infection. An increase of its ex-
pression confers to cells a protection against rotavirus (2), vesic-
ular stomatitis virus (9), respiratory syncytial virus (41), and in-
fluenza virus (16) infections. In the latter case, it has been reported
that Hsp70, which interacts with the ribonucleoprotein complex,
interferes with the polymerase activity and negatively regulates
viral transcription and replication (16, 22). Interestingly, Hsp70
was found to have both positive and negative regulatory effects on
the RNA replication of flock house virus, a nodavirus (39), high-
lighting the complexity of this particular virus-chaperone interac-
tion.
Rabies virus, the prototype of the Lyssavirus genus that belongs
to the Rhabdoviridae family (Mononegavirales order), causes a fa-
tal disease that is associated with intense viral replication in the
central nervous system. Its single-stranded negative-sense RNA
genome (⬃12 kb), encoding five viral proteins, is encapsidated by
the nucleoprotein (N; 40 kDa) to form the nucleocapsid that is
associated with the RNA-dependent RNA polymerase L (220 kDa)
and its cofactor, phosphoprotein (P; 33 kDa). Inside the viral par-
ticle, the nucleocapsid has a tightly coiled helical structure that is
associated with the matrix protein (M; 22 kDa) and is surrounded
by a membrane containing a unique glycoprotein (G; 62 kDa).
The virus enters the host cell through the endosomal transport
pathway via a low-pH-induced membrane fusion process cata-
lyzed by G (14). The nucleocapsid released into the cytoplasm
serves as a template for transcription and replication processes
that are catalyzed by the L-P polymerase complex (for a review see
reference 1). It has been shown recently that rabies virus transcrip-
tion and replication take place within Negri body-like (NBL)
structures, which are inclusion bodies formed during viral infec-
tion (20). During transcription, a positive-stranded leader RNA
and five capped and polyadenylated mRNAs are synthesized. The
replication process yields nucleocapsids containing full-length
antigenome-sense RNA, which in turn serve as templates for the
synthesis of genome-sense RNA. During their synthesis, both the
nascent antigenome and the genome are encapsidated by N pro-
Received 6 October 2011 Accepted 9 February 2012
Published ahead of print 15 February 2012
Address correspondence to Danielle Blondel, blondel@vms.cnrs-gif.fr.
* Present address: X. Lahaye, Institut Curie, Centre de Recherche, Human Innate
Immunity, Paris, France; A. Vidy, Max von Pettenkofer Institute für Virologie,
Ludwig Maximilians University, Munich, Germany.
X.L. and A.V. contributed equally to this article.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.06501-11

0022-538X/12/$12.00 Journal of Virology p. 4743– 4751 jvi.asm.org 4743

Lahaye et al.
tein. This encapsidation is regulated by the P protein, which plays
the role of chaperone, preventing the N protein from binding to
cellular RNA and from aggregating (32). The neosynthesized
genomic RNA either serves as a template for secondary transcrip-
tion or is transported to the cell membrane and assembles with the
M and G proteins for the budding of neosynthesized virions.
We have previously shown that Hsp70 protein accumulates in
the NBL structures, which are sites of rabies virus transcription
and replication (20). In addition, Hsp70 has been previously
shown to be incorporated in purified rabies virus (37). Therefore,
we investigated the role of this chaperone during viral infection.
We report here that rabies virus infection induces the expression
of Hsp70. We also show that Hsp70 interacts with the nucleopro-
tein N, the major component of NBL structures, and that Hsp70
downregulation results in the inhibition of different steps of the
viral cycle. The inhibition of viral protein synthesis and of viral
production can reflect the inhibition of viral RNA synthesis, but it
also could be due to a specific role of Hsp70 in these further steps.
MATERIALS AND METHODS
Virus stock and cell infection. BSR cells, cloned from BHK 21 (baby
hamster kidney) cells, were grown in Dulbecco’s modified Eagle medium
(DMEM) supplemented with 10% fetal bovine serum (FBS). Human neu-
roblastoma SK-N-BE cell lines were grown in RPMI 1640 plus L-glu-
tamine (Gibco) supplemented with 15% fetal calf serum (FCS).
The CVS strain of rabies virus was grown in BSR cells. Virus titers were
determined by standard plaque assays of BSR cells.
Antibodies and drugs. The mouse polyclonal anti-P antibody has
been described previously, and the anti-P monoclonal antibodies (MAb)
25C2 and 30F2 have been previously described (36). The monoclonal
anti-N MAb 62B5 was produced in mice immunized with the virus. Rab-
bit polyclonal anti-P and anti-M antibodies were obtained by the repeated
injection of purified recombinant protein produced in Escherichia coli.
The mouse anti-Hsp70 MAb (SPA-810) and rabbit polyclonal anti-Hsp70
(SPA-812) antibody were obtained from Stressgen, and the anti-␣-tubu-
lin MAb (N356) was from Amersham. Quercetin (Qct; Q4951) and the
rabbit polyclonal anti-actin (20–33) antibody were obtained from Sigma.
The mouse anti-green fluorescent protein (GFP) MAb (11814460001)
was purchased from Roche.
Plasmids and siRNA. The plasmids pCDM8-P, encoding the P pro-
tein (CVS strain), and pCDM8-N, encoding the N protein (CVS strain),
have been described previously (6). The plasmid pRL-TK was described
elsewhere (11, 13) and was a generous gift of D. Garcin (University of
Geneva School of Medicine). The plasmids pTit-N, pTit-P, and pTit-L
were described previously (7) and were obtained from K. K. Conzelmann
(Ludwig-Maximilians University of Munich). The plasmid pDImut (21)
was provided by N. Tordo (Institut Pasteur). The plasmid pCMV-Hsp70,
encoding the Hsp70 protein, and pCMV-Neo were kindly provided by G.
Trugnan (2). The siRNA-Hsp70 duplex and scrambled siRNA were pur-
chased from Eurogentec.
Cell infection and transfection. BSR cells were grown on glass cover-
slips in 6-well plates and were infected with rabies virus at various multi-
plicities of infection (MOI) and at various times postinfection (p.i.). BSR
cells grown to 90% confluence in 60-mm-diameter dishes also were trans-
fected with 2 ␮g of plasmid using Lipofectamine 2000 as described by the
manufacturer (Invitrogen).
Purification of virus and nucleocapsids. Purified virus was obtained
as follows. Virions were pelleted through a cushion of 25% glycerol TNE
(10 mM Tris, pH 7.5, 1 mM EDTA, 50 mM NaCl) in TD (0.8 mM Tris, pH
7.4, 150 mM NaCl, 5 mM KCl, 0.7 mm Na2HPO4, 10 mM EDTA) and
further purified by centrifugation in a sucrose gradient (10 to 40%, wt/
vol) in TD.
Rabies virus nucleocapsids were isolated from infected BSR cells as
described previously (17).
4744 jvi.asm.org
Treatment of cells with drugs. Cells were kept in DMEM containing
25 to 200 ␮M quercetin for 1 h before virus inoculation and during infec-
tion. As all drugs were reconstituted in dimethyl sulfoxide (DMSO), un-
treated cells were maintained in medium containing the same concentra-
tion of DMSO. The expression of Hsp70 was induced by a heat shock (HS)
at 45°C for 20 min, and then cells were reincubated at 37°C.
siRNA experiments. The siRNA-Hsp70 sequence of 21 nucleotides
(nt) was chosen based on the rules proposed by Elbashir et al. (12) and was
designed to interfere with the two mRNAs encoding Hsp70 (GenBank
accession number NM_005345 for HSPA1A and BC057397 for HSPA1B)
but not with the two Hsc70 mRNA variants. The sequences were the
following: siRNA-Hsp70 sense, 5=-CACGGCAACCUGGAGAUCA-de-
oxythymidine (dTdT)-3=; siRNA-Hsp70 antisense, 5=-UGAUCUCCACC
UUGCCGUG-dTdT-3=; siRNA-scrambled sense, 5=-ACAAUUCCGGGG
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GAGAGAG-dTdT-3=; and siRNA-scrambled antisense, 5=-CUCUCUCC
CCCGGAAUUGA-dTdT-3=. Transfections were obtained using an
Invitrogen Lipofectamine kit. BSR cells (in serum-free DMEM) were
transfected with 400 pmol of scrambled siRNA or 200 to 400 pmol of
siRNA-Hsp70. After 6 h at 37°C, the medium was completed with 5%
FCS. After 48 h at 37°C, it was possible to induce a heat shock and to infect
cells. After incubation, the culture supernatant was collected to quantify
viral production. The cell layer was recovered to obtain total extract for
Western blot analysis.
Detection of viral mRNA and Hsp70 mRNA by RT-PCR. The pres-
ence of Hsp70 mRNA or rabies N and P mRNA was determined by RT-
PCR. Total cellular RNA was isolated from cell culture using an RNA kit
according to the manufacturer’s instructions (RNA NOW). Oligo(dT)
primer was used for the synthesis of the first strand of cDNA. The
expression of Hsp70 and rabies genes was carried out by the PCR
amplification of the Hsp70 or rabies N and P genes using the following
primers: Hsp70 sense (Hsp70-A), 5=-GCCGGATCCATATGGCCAAAG
CC-3=; Hsp70 antisense (XhoI-B), 5=-GCCCTCGAGCTAATCTACCTC
CT-3=; CVS-N sense (N-A), 5=-GCCGGATCCATGGATGCCGACAAG
A-3=; CVS-N antisense (N-B), 5=-GCCGGATCCTTATGAGTCACTCG
A-3=; CVS-P sense (P-A), 5=-GCCGCTAGCATGAGCAAGATCTTTGT
T-3=; and CVS-P antisense (P-B), 5=-CTCGAGTTAGCAGGATGTATAG
CG-3=. The PCRs were performed using three different volumes of RT
mixture (cDNA; 1, 3, and 5 ␮l). The PCR products were electrophoresed
in a 1.2% agarose gel and visualized with ethidium bromide.
Northern blot analysis. RNA was isolated from cells with the RNA
NOW kit (Ozyme). Total RNA was separated on a 1.5% agarose gel under
denaturing conditions and blotted onto nylon membranes (Roche Mo-
lecular Biochemicals). Hybridizations were performed with digoxigenin
(DIG)-labeled oligonucleotides recognizing the rabies virus (RAV) N or P
gene sequence and by incubation with anti-DIG antibody conjugated to
alkaline phosphatase, followed by CDP Star analysis.
Minireplicon system assay and luciferase assay. The minireplicon
system assay was slightly modified from Le Mercier et al. (21). BSR cells
were grown in a 12-well plate (3 ⫻ 105 cells per well) in DMEM supple-
mented with 5% FCS and incubated for 24 h at 37°C (in 5% CO2).
The cells were transfected with pTit-N (1.2 ␮g), pTit-P (1.2 ␮g),
pTit-L (0.3 ␮g), pT7 (1 ␮g; to allow the cytoplasmic expression of the T7
RNA polymerase), pRL-TK (0.3 ␮g), and pDImut (0.75 ␮g) using the
FuGENE 6 reagent as described by the manufacturer (number
11814443001 from Roche). The N, P, and L proteins and the RNA mini-
genome formed a functional RNP template resulting in luciferase gene
transcription, thus the amount of luciferase was related to the transcrip-
tional activity of the reconstituted RNP. The inhibition of Hsp70 expres-
sion was obtained by siRNA-Hsp70 transfection 48 h before minireplicon
transfection, and the overexpression of Hsp70 was induced by transfec-
tion with pCMV-Hsp70 (1.2 ␮g). In the absence of pCMV-Hsp70, all cells
were transfected with pCMV-Neo. Thirty-six hours after the transfection
of the minireplicon system, the measurement of Firefly and Renilla lucif-
erase activities were performed according to the manufacturer’s protocol
(dual-luciferase assay system from Promega). Relative expression levels
Journal of Virology
 Role of Hsp70 in Rabies Infection

were calculated by dividing the Firefly luciferase values by those of the

Renilla luciferase.
Pulse-chase experiments and immunoprecipitation. At 16 h postin-
fection, the culture medium was replaced with methionine- and cysteine-
free medium, and the culture was incubated for 1 h. The cells then were
labeled with 2 ml of prewarmed medium containing 20 ␮Ci of [35S]
methionine and [35S]cysteine (0.8 Mbq) (express protein labeling mix;
Perkin Elmer) for 5 min. Prewarmed medium containing 5 mM cold
methionine and 1 mM cold cysteine then was added for the chase period.
The cells were washed in cold PBS and lysed on ice in 500 ␮l of buffer
containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.5%
NP-40, and an antiprotease cocktail (2 ␮g of leupeptin per ml, 2 ␮g of
antipain per ml, 2 ␮g pepstatin per ml, 2 ␮g of chymostatin per ml, and 16
␮g of aprotinin per ml). Nuclei were eliminated from the lysate by cen-

trifugation at 12,000 ⫻ g for 2 min at 4°C. The cytoplasmic fractions were
incubated for 2 h at 4°C with specific antibodies (anti-P, anti-N, or anti-
M). Immune complexes were precipitated by incubation with protein
A-Sepharose for 1 h at 4°C, washed three times, denatured in Laemmli
buffer, and analyzed by SDS-PAGE. The quantification of radioactivity
was performed by a phosphorimager.
Coimmunoprecipitation. Cells were harvested by being scraped into
cold phosphate-buffered saline (PBS), and cell extracts were prepared as
described above. The cytoplasmic fraction was incubated overnight at 4°C
with specific antibodies (anti-Hsp70 or anti-N). Immune complexes were
precipitated by incubation with protein A-Sepharose for 1 h at 4°C,
washed three times, and denatured in Laemmli buffer. Immunoprecipi-
tated proteins were analyzed by Western immunoblotting using different
antibodies.
Western blot analysis. Cells were washed and resuspended in PBS,
lysed in hot Laemmli sample buffer, and boiled for 5 min. About 20 ␮g of
protein was analyzed on a 10% SDS-PAGE gel and transferred onto a
nitrocellulose membrane. The proteins were blocked on the membranes
with 10% skimmed milk in PTBS for 2 h and incubated overnight at 4°C
with rabbit polyclonal anti-Hsp70, anti-P, anti-N, anti-actin, or anti-tu-
bulin antibody. The blots then were washed extensively in PBS-Tween and
incubated for 1 h with the appropriate peroxidase-coupled secondary
antibodies (Amersham). All of the blots then were subjected to chemilu-
minescence (ECL; Amersham). Protein spot levels were determined by
using ImageJ quantification software.
Immunoaffinity columns and proteomic analysis. (i) Immunoaffin-
ity columns. Monoclonal anti-N antibodies (62B5 and 64B6), which were
previously described (36), were first purified by binding to protein A
Sepharose, eluted with 100 mM glycine (pH 3), and immediately read-
justed to neutral pH by adding Tris (pH 8).
Second, the purified antibodies were applied on a protein A-Sepharose
column equilibrated in Tris M, pH 8. Protein A was then washed and
resuspended in 0.2 M borate buffer, pH 9.
The cross-linking reaction takes place in the presence of 20 mM di-
methyl pimelimidate during 30 min at room temperature under gentle
rotation. Protein A then is washed and resuspended with 0.2 M ethanol-
amine (pH 8) for 2 h. After the removal of ethanolamine, the free anti-
bodies (not cross-linked to protein A) were eluted with 100 mM glycine
buffer (pH 3).
(ii) Purification of viral and cellular proteins associated with N. Cy-
toplasmic fractions of uninfected or infected SK-N-BE cells (1 ⫻ 108 cells
at an MOI of 10) were loaded separately on the anti-N immunoaffinity
columns. The columns then were washed extensively with PBS, and the
proteins bound to the column were eluted using 100 mM glycine buffer
(pH 3).
(iii) Analysis of proteins bound to columns of anti-N. The eluates
were resolved on SDS-PAGE, and the proteins were revealed using silver
nitrate (silver stain plus kit; Bio-Rad) or colloidal Coomassie blue. Unique
protein bands that had been compared to proper controls were
trypsinized for proteomic analysis by matrix-assisted laser desorption
ionization–time of flight (MALDI-TOF; Platform of Proteomics in the
Downloaded from http://jvi.asm.org/ on May 11, 2014 by UCSF Library & CKM
FIG 1 Hsp70 interacts with the rabies virus N protein. (A) Hsp70 is detected
in purified virus and in purified nucleocapsid. Purified virus (5 ␮g) and puri-
fied nucleocapsid (5 ␮g), prepared as described in Materials and Methods,
were analyzed by SDS-PAGE followed by Coomassie blue staining (lanes 1 and
4) and Western blotting using a mixture of anti-N, anti-P, and anti-M anti-
bodies (lanes 2 and 5) and a polyclonal anti-Hsp70 (lanes 3 and 6). (B) BSR
cells were cotransfected with plasmids expressing Hsp70 and rabies N protein.
Two days after transfection, cells were harvested and lysed. Cell lysate was
incubated with the irrelevant polyclonal anti-GFP antibody (lanes 2 and 6),
with the mouse MAb anti-N antibody (lane 3), or with the polyclonal anti-
Hsp70 antibody (lane 5). Immune complexes were analyzed by Western im-
munoblotting using anti-Hsp 70 (lanes 2 and 3) and anti-N (lanes 5 and 6)
antibodies. Direct cellular extracts of transfected cells (input) also were ana-
lyzed by WB with anti-Hsp70 (lane 1) and anti-N (lane 4). Bands correspond-
ing to Hsp70, N, and heavy-chain IgG are indicated.
Research Unit of Biochemistry and Structure Protein, INRA, Jouy en Jo-
sas, France).
RESULTS
Hsp70 interacts with the nucleoprotein. Hsp70 has been shown
to be present in rabies virions (37) and within NBL structures
containing the viral L, N, and P proteins and viral RNAs (20, 26).
We confirmed here that Hsp70 was also incorporated in purified
virions (Fig. 1A, lanes 1 to 3). NBL structures are sites of viral
transcription and replication (20). To identify the viral partner(s)
of Hsp70, we first investigated whether Hsp70 protein was present
in the nucleocapsids composed of the viral RNA tightly associated
with the N protein. Rabies nucleocapsids were purified from in-
fected cells by cesium chloride gradient and analyzed by Western
blotting with anti-N and anti-Hsp70 antibodies. Hsp70 was found
to be associated with the nucleocapsids containing exclusively the
N protein (Fig. 1A, lanes 4 to 6). This result suggests that Hsp70
binds to the N protein.
To examine whether Hsp70 interacts with the N protein in
rabies-infected cells, two types of experiments were conducted. In
the first one, an immunoaffinity column immobilized with anti-N
MAbs was used to identify cellular factors interacting with the N
protein. The cytoplasmic fraction of human neuroblastoma SK-

May 2012 Volume 86 Number 9 jvi.asm.org 4745

Lahaye et al.

N-BE-infected cells was loaded on an anti-N immunoaffinity col-

umn. In parallel, uninfected cell extracts or infected cell extracts
incubated without antibodies were used as controls. The proteins
retained on the column were eluted, analyzed by SDS-PAGE, and
identified by MALDI-TOF proteomic analysis after excision from
the gels. Among the cellular proteins, the chaperone Hsp70 was
identified as a cellular partner of the N protein (data not shown).
As expected, the viral phosphoprotein P was also detected as a viral
partner of N. In the second experiment, BSR cells were cotrans-
fected by two plasmids individually expressing Hsp70 and N pro-
tein. Cell extracts were immunoprecipitated with a mouse mono-
clonal anti-N antibody. The proteins present in the immune
complexes then were analyzed by Western blotting with anti-

Hsp70 antibodies. Hsp70 was detected in the immunoprecipitates
obtained with the anti-N antibody but not with the unrelated
anti-GFP antibody, indicating the specificity of the interaction
(Fig. 1B, lanes 2 and 3). The reverse experiment showed that the N
protein also could be coimmunoprecipitated with Hsp70 (Fig. 1B,
lane 5). Thus, all of these data indicated that Hsp70 interacts with
the viral N protein in the absence of other viral proteins, and this
supports the idea that this interaction results in the localization of
Hsp70 in NBL structures (20).
Rabies infection induces Hsp70 expression. We then investi-
gated the effect of viral infection on the expression of Hsp70. In-
fected cells were analyzed at different times postinfection (p.i.) by
Western blotting. Hsp70 was detected at early times postinfection
(4 h p.i.), whereas no detectable trace was observed in uninfected
cells (Fig. 2A). The Hsp70 level was enhanced during viral infec-
tion (Fig. 2A). This was confirmed by the confocal immunofluo-
rescence analysis of infected cells, which showed that Hsp70 levels
increased in the NBL structures concomitantly with those of viral
N and P proteins (Fig. 2B, 16 h p.i.) before spreading throughout
the cytoplasm (Fig. 2B, 24 h p.i.). These results indicated that
Hsp70 is induced by rabies infection.
Modulation of cellular Hsp70 and effect on rabies virus in-
fection. It is not known whether the induction of Hsp70 expres-
sion is the result of a cellular stress response due to viral infection
or if the protein is induced specifically by some viral mechanism
because it would play a role during the viral life cycle. To analyze
the function of this Hsp70 induction during rabies infection, dif-
ferent experiments were performed to modulate the intracellular
level of Hsp70.
First, we determined the conditions to modulate the Hsp70
expression in noninfected cells. To induce the expression of
Hsp70 protein, BSR cells were subjected to heat shock (HS) for 20
min at 45°C, and then the cells were reincubated at 37°C for dif-
ferent times. The Western blotting indicated that the induction of
the expression of Hsp70 was maximal at 6 h postshock and was
maintained thereafter (Fig. 3A). In contrast, treatment with an
inhibitor of Hsp synthesis, quercetin (Qct), before HS inhibited
the synthesis of Hsp70 proteins. The application of 100 ␮M Qct
resulted in a drastic inhibition of Hsp70 expression (Fig. 3B) with-
out causing visible cell damage to the cell (data not shown). Thus,
the following experiments were performed at 6 h after HS or with
100 ␮M Qct to induce or inhibit Hsp70 expression, respectively.
We first analyzed the effect of the modulation of Hsp70 on the
synthesis of viral proteins during viral infection (Fig. 4A and B).
When Hsp70 was overexpressed (HS), we observed an increase of
P protein synthesis (factor of 4 at 12 h p.i.) compared to control
cells (Fig. 4A), although this increase became less pronounced
Downloaded from http://jvi.asm.org/ on May 11, 2014 by UCSF Library & CKM
FIG 2 Expression of Hsp70 during rabies virus infection. (A) BSR cells were
uninfected (Ni) or infected at an MOI of 1. At different times p.i. as indicated,
cell extracts were analyzed by Western blotting using anti-Hsp70, anti-N, an-
ti-P, and anti-actin antibodies. (B) BSR cells were infected as described for
panel A. Double-immunofluorescence staining was performed with anti-
Hsp70 and anti-P antibodies. The scale bars correspond to 20 ␮m.
with the progression of infection (Fig. 4A, 16 to 20 h p.i.). In
contrast, the inhibition of Hsp70 (Qct) resulted in a drastic reduc-
tion of the level of P protein at 12 and 16 h p.i. (factors of 5 and 20,
respectively). The accumulation of the other viral proteins was
also inhibited (data not shown). This inhibition was not due to
drug toxicity, as the number of infected BSR cells obtained by
counting the fluorescent cells was unchanged in the absence of
Hsp70 (data not shown). This drastic effect on the viral protein
level was attenuated at later stages of infection concomitantly with
the induced Hsp70 expression (Fig. 4A). To investigate whether
the inhibition of viral protein accumulation was due to the inhi-
bition of viral protein translation or to an increase of viral protein
degradation, we analyzed the effect of Qct on viral translation by
pulse-chase experiments with [35S]methionine/cysteine. Infected
cells treated with Qct were labeled for 5 min and chased for peri-
ods of up to 60 min. Cells then were lysed and immunoprecipi-
tated with anti-N and anti-P or anti-M antibodies. The immuno-
precipitates were analyzed by SDS-PAGE. Treatment with Qct
dramatically affected viral protein synthesis (Fig. 4B). To analyze
the effect of Qct on cellular protein synthesis, mock-infected cells
left untreated or treated with Qct were labeled for 5 min and an-
alyzed directly by SDS-PAGE. In contrast to viral proteins, cellular
protein synthesis did not seem to be affected (Fig. 4B, lower, lanes

4746 jvi.asm.org Journal of Virology

 Role of Hsp70 in Rabies Infection

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FIG 3 Modulation of Hsp70 expression in the absence of infection. (A) Uninfected BSR cells were incubated for 20 min at 45°C and then at 37°C for various times
after heat shock (HS) as indicated. Cell extracts were analyzed by Western blotting using anti-Hsp70 and anti-tubulin antibodies (used as a loading control). The
level of Hsp70 was quantified by immunoblot scanning and normalized with respect to the amount of tubulin (lower panel). (B) Uninfected BSR cells were
untreated (0) or were treated with different concentrations of quercetin (Qct) (as indicated) for 1 h before HS at 45°C for 20 min in the presence of quercetin.
After HS, the cells were incubated at 37°C for 6 h. Cell extracts were analyzed by Western blotting using anti-Hsp70 and anti-tubulin antibodies. The level of
Hsp70 was quantified by immunoblot scanning and normalized with respect to the amount of tubulin (lower panel).

17 and 18). This indicates that the drug led to the inhibition of
viral protein translation but did not affect protein stability.
We next studied the effect of Hsp70 on viral production. Sim-
ilar experiments were performed and supernatants were used for
viral titration (Fig. 5A and B). When Hsp70 was overexpressed
(HS), viral production at 16 h p.i. was slightly higher (factor 2)
than that in normal conditions (Fig. 5A and B). In contrast, the
inhibition of Hsp70 (with Qct) induced a dose-dependent inhibi-
tion of the viral production during the viral cycle (Fig. 5A). Viral
yield was 10- to 20-fold and up to 100-fold lower than that in
control cells in the presence of 100 and 200 mM Qct, respectively
(Fig. 5A and B). These data indicated that the reduction of Hsp70
significantly impairs viral protein synthesis and virus production,
suggesting that Hsp70 has a proviral effect during rabies infection.
However, the overexpression of Hsp70 in infected cells has at most
a small effect on viral life cycle. This could be explained by the fact
that Hsp70 is efficiently induced by rabies infection, and this in-
duction would be sufficient to support the viral life cycle.
Specific inhibition of Hsp70 expression by siRNA and effect
on viral infection. As quercetin is not a specific inhibitor of Hsp70
but inhibits the synthesis of other heat shock proteins, we per-
formed silencing experiments to investigate more specifically the
function of Hsp70 during rabies viral infection. BSR cells were
transfected with two different concentrations of Hsp70 siRNA
(200 and 400 pmol) for 48 h, and then cells were infected under
normal (control) or heat shock conditions. Hsp70 was efficiently
downregulated, as shown in HS-treated cells (Fig. 6A, lanes 9 and
10) compared to control and scrambled siRNA (Fig. 6A, lanes 6 to
8). In the absence of HS, the induction of Hsp70 by viral infection
(Fig. 6A, lanes 2 and 3) was also inhibited after Hsp70 silencing
(Fig. 6A, lanes 4 and 5). These observations were confirmed by the
quantification of the cellular Hsp70 protein level (Fig. 6B). Under
these conditions, the knockdown of Hsp70 expression was associ-
ated with a significant reduction of the amounts of viral N and P
proteins of 3- to 8-fold depending on the siRNA dose (Fig. 6A,
lanes 4 and 5 versus lane 3, and B). The decrease of the amount of
viral N and P proteins was maintained in HS conditions between a
factor of 2 to 4 depending on siRNA-Hsp70 quantities (Fig. 6A,
lanes 9 and 10 versus lane 8, and B). As expected, the N and P
protein levels were similar in the presence of scrambled siRNA
(Fig. 6A, lanes 2 and 3 versus 7 and 8, and B). These results con-
firmed that the specific inhibition of Hsp70 synthesis led to a re-
duction of viral protein levels and indicated that Hsp70 has a
proviral role.
The effect of siRNA-Hsp70 on viral production was also ana-
lyzed in the same conditions as those described above. The siRNA-
Hsp70 treatment resulted in an inhibition of the viral production
of 10- to 15-fold compared to control and scrambled siRNA,
whatever the dose of siRNA and conditions (Fig. 6C). These re-
sults confirmed that Hsp70 plays a positive role in rabies infection.
Effect of Hsp70 on viral RNA synthesis. We next tested
whether the inhibition of viral protein synthesis (observed in the
presence of Qct or in siRNA experiments) could reflect an inhibi-
tion of viral transcription. First, we analyzed the effect of Qct on
viral mRNA synthesis. Total RNAs were extracted from untreated
infected cells, cells treated with Qct, or cells submitted to HS and
were analyzed for the presence of the viral N mRNA by Northern
blot analysis. In the presence of Qct, the amount of N mRNA was
reduced by a factor 5 (Fig. 7A), indicating that Qct impaired viral
transcription. However, the induction of Hsp70 expression (HS)
did not result in an increase of the amount of rabies N mRNA
(Fig. 7A).
We then tried to confirm these effects on viral RNA synthesis in
the presence of a more specific Hsp70 depletion. As Northern
blotting is not the most sensitive method, we first used RT-PCR to
detect the viral mRNA of N and P proteins and the mRNA of
Hsp70 in infected cells after Hsp70 silencing experiments and in
cells infected under normal (control) or heat shock (HS) condi-
tions (Fig. 7B). Increasing amounts of RT reactions (1, 3, and 5 ␮l)
were used for PCR amplification. As expected, HS or/and rabies

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Lahaye et al.

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FIG 4 Effect of modulation of Hsp70 on viral protein synthesis. (A) BSR cells were left untreated (DMSO) or were treated with quercetin (Qct; 100 ␮M) for 1
h and infected (MOI of 1) in the presence of Qct, or cells were submitted to heat shock (HS) (after 1 h of DMSO treatment) and infected (MOI of 1) 6 h after HS.
Cells were lysed at different times p.i. as indicated, and cell extracts were analyzed by Western blotting using anti-Hsp70, anti-P, and anti-tubulin antibodies. The
levels of P and Hsp70 proteins present in different conditions (DMSO, Qct, and HS) were quantified by Western blot scanning and normalized with respect to
the amount of tubulin (expressed in logarithmic scale) (lower panel). An arbitrary level of 100 is applied to P and Hsp70 levels (*) in a classical infection (DMSO)
for each hour (p.i.) to allow comparisons. (B) BSR cells were left untreated (⫺) or were treated with 100 ␮M Qct (⫹) for 1 h and infected (MOI of 1) in the
presence of Qct. After 16 h p.i., cell were pulse labeled with [35S]methionine and [35S]cysteine for 5 min and then chased in cold medium for the times indicated
before lysis. The lysates were immunoprecipitated with anti-N and anti-P antibodies (upper panel) or anti-M antibodies (lower panel). The immunoprecipitates
were analyzed by SDS-PAGE followed by autoradiography. Mock-infected cells were labeled for 5 min (without chase), and cell extracts (CE) were directly
analyzed by SDS-PAGE followed by autoradiography (lower panel, lanes 17 and 18).

virus infection induced the expression of Hsp70 mRNA (Fig. 7A,
lanes 1 to 3 and 7 to 9) and Hsp70 was efficiently downregulated in
the presence of siRNA-Hsp70 compared to control and scrambled
siRNA (Fig. 7B, lanes 4 to 6 and 10 to 12). Hsp70 induction re-
sulted in a slight increase of the amount of viral mRNA of N and P
(Fig. 7B, lanes 7, 8, and 9 versus lanes 1, 2, and 3); however, Hsp70
silencing did not result in a decrease of the amount of viral mRNA
by this method (Fig. 7B, lanes 10, 11, and 12 versus lanes 4, 5,
and 6).
We then used a more quantitative assay, the luciferase reporter
system in a minireplicon system, that allows the reconstitution of
a functional rabies virus RNP. The luciferase gene reporter is
framed by 3= leader and 5= trailer sequences and is under the con-
trol of rabies transcription signals (21). The production of lucif-
erase activity is the result of the encapsidation of the minigenome
followed by its transcription by the L-P complex and by its repli-
cation in the presence of N protein. The siRNA-Hsp70-treated
BSR cells were cotransfected with plasmids encoding L, N, and P
proteins and the minigenome. As expected, luciferase activity
background was obtained in the absence of the L protein (Fig. 7C).
The overexpression of Hsp70 induced a slight stimulation of the
luciferase activity (to about 20%) compared to that of the control
(Fig. 7C). Similarly, the Hsp70 silencing weakly reduced the lucif-
erase activity (to about 20 to 40%) in a dose-dependent manner.
We ensured that the amounts of viral N and P proteins expressed
from the plasmids were unchanged in the different conditions
(Fig. 7C). Taken together, these data suggested that Hsp70 plays a
minor role even in viral transcription. However, whether this role
is sufficient to explain further effects on viral protein synthesis and
viral replication should be further investigated.
DISCUSSION
In this study, we have investigated the role of Hsp70 during rabies
virus infection. Coimmunoprecipitation and proteomic analyses
indicated that Hsp70 interacts with N. However, we cannot ex-
clude that this interaction is indirect and mediated by one or more
other factors. Whatever the case, this interaction probably results
in the presence of Hsp70 in purified nucleocapsids, within NBL
structures, and in purified rabies particles. In addition, we have
shown that rabies infection induces an increase of cellular Hsp70
levels as soon as 4 h p.i. in BSR cells and in neuroblastoma cells
(data not shown). The increase in Hsp70 levels following viral
infection has been widely observed. This can be explained by dif-
ferent mechanisms. The induction may occur indirectly through
the production of a large number of unfolded proteins during
viral infection. Second, the viruses could interfere with stress sig-
nal transduction pathways independently of the folding status of
proteins. Indeed, Hsp70, which is involved in many cellular pro-
cesses, such as cell cycle, apoptosis, and cellular innate immunity
pathways, is frequently diverted by viruses for their benefit (23,
25). In the case of rabies virus, Hsp70 enrichment might simply be
correlated with excess amounts of viral proteins (particularly N

4748 jvi.asm.org Journal of Virology


FIG 5 Effect of Qct treatment on viral production. BSR cells were infected at
an MOI of 1 without any treatment (DMSO) or in the presence of Qct as
indicated or were subjected to HS treatments as described in the text. Culture
media were collected at 16 h p.i. (A) or at different times pi (B), and viral titers
were determined as described in Materials and Methods. P values (**, P ⬍
0.001; ***, P ⬍ 0.0001) were calculated using the Student’s test.
and P); nevertheless, the recruitment of Hsp70 inside the NBLs,
which are sites of viral transcription and replication, in the puri-
fied nucleocapsids and in the purified virions suggests that Hsp70
plays an active role in the viral life cycle.
The downregulation of Hsp70, using specific chaperons inhib-
itors such as quercetin or RNA interference, significantly reduced
the intracellular level of viral proteins and viral production. The
reduction of viral protein accumulation appeared to be the result
of an inhibition of viral translation more than protein instability,
as shown by pulse-chase labeling (in the presence of Qct). Viral
transcription was also decreased. Interestingly, the inhibition at
different steps of the viral cycle (transcription, translation, and
viral production) was more drastic in the presence of Qct than
siRNA, suggesting that other chaperones are involved in the rabies
virus infection cycle. Unexpectedly, the overexpression of Hsp70
following heat shock treatment or pCMV-Hsp70 transfection re-
sulted in a slight increase of viral protein synthesis or viral pro-
duction. This is likely because infected cells already expressed a
sufficiently high level of Hsp70, and the overexpression before the
infection did not have much effect. The siRNA-induced reduction
of viral yield may be due to a decrease of the amount of viral
particles released, but it could well be related to the reduced infec-
tivity of the progeny virions lacking Hsp70.
All of these data indicate that Hsp70 has a proviral effect on
rabies virus replication by acting during at least one step of the
viral cycle. As these steps are connected, it is difficult to clearly
determine the mechanism by which Hsp 70 controls viral infec-
May 2012 Volume 86 Number 9
Role of Hsp70 in Rabies Infection
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FIG 6 Effect of specific inhibition of Hsp70 by siRNA interference on rabies
replication. (A) BSR cells were not transfected (Control) or were transfected
with scrambled siRNA (Scr) or siRNA-Hsp70 (Hsp) (two concentrations as
indicated). After 48 h, cells were submitted to HS (⫹HS) or not (⫺HS), and 6
h later cells were infected at an MOI of 3 for 16 h. Cell extracts were analyzed by
Western blotting using anti-Hsp70, anti-N, anti-P, and anti-tubulin antibod-
ies. (B) Quantitative analysis of cellular P, N, and Hsp70 protein levels in cells
treated as described for panel A. The resulting Western blots (three indepen-
dent experiments) were quantified using immunoblot scanning and normal-
ized with respect to the amount of tubulin. The amounts of N (black histo-
grams), P (gray histograms), and Hsp70 (white histograms) were measured,
and an arbitrary level of 100 is applied to N, P, and Hsp70 levels in a classical
infection (lane 2 of panel A) for comparisons. P values (***, P ⬍ 0.0001) were
calculated using the Student’s test. (C). Culture media of infected cells treated
as described for panel A were collected at 16 h p.i., and viral titers were deter-
mined as described in Materials and Methods. Viral titers represent averages
from three independent experiments. Error bars indicate the standard devia-
tions.
tion. Indeed, the resulting inhibition in viral protein synthesis and
in viral production could be a direct consequence of the viral
transcription inhibition, but it could also be amplified by an
Hsp70-specific role in these further steps of the viral infection.
Interestingly, the potential role of Hsp70 in viral RNA synthe-
sis is consistent with the interaction of Hsp70 with N and its asso-
ciation with the NBLs, which are sites of viral transcription and
replication, suggesting a possible interaction with the polymerase
jvi.asm.org 4749
Lahaye et al.
FIG 7 Effect of Hsp70 on viral RNA synthesis. (A) Effect of Quercetin on viral
RNA by Northern blotting. BSR cells were left untreated (C) or were treated with
quercetin (Qct; 100 ␮M) for 1 h and infected (MOI of 1) in the presence of Qct, or
cells were submitted to heat shock (HS) (after 1 h of DMSO treatment) and in-
fected (MOI of 1) 6 h after HS. Total RNA was extracted from cells, and samples
(10 ␮g of RNA per lane) were analyzed for the presence of rabies N mRNA and
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA, as described in
Materials and Methods. (B) Detection of viral RNA by RT-PCR. BSR cells were left
untreated (Control) or were treated with scrambled siRNA or siRNA-Hsp70 (two
concentrations as indicated). After 48 h, cells were submitted to HS (⫹) or not
(⫺), and 6 h later cells were infected by CVS at an MOI of 3 for 16 h and total RNA
was extracted. The presence of Hsp70 mRNA or rabies N and P mRNA was deter-
mined by RT-PCR as described in Materials and Methods. The PCRs were per-
formed using three different volumes of RT mixture (cDNA; 1, 3, and 5 ␮l) and
were analyzed on a 1.2% agarose gel with ethidium bromide. (C) Luciferase ex-
pression from a rabies minireplicon system. BSR cells were left untreated (control),
were treated with scrambled siRNA or siRNA-Hsp70 (different concentrations),
or were transfected by a plasmid encoding Hsp70 (pCMV-Hsp70). After 48 h, cells
were transfected with plasmids encoding L, N, and P proteins, T7 polymerase, and
the plasmid (pRL-TK) encoding Renilla luciferase in the presence of a rabies mini-
replicon system plasmid for the expression of Firefly luciferase. The L plasmid was
missing from one experiment, and this served as a negative control (without L).
After 36 h, cells extracts were analyzed for luciferase expression. Data represent the
separate assays for Firefly luciferase normalized to the expression of Renilla lucif-
erase and are expressed as percentages of the control (*). Error bars indicate stan-
dard deviations (from three independent experiments). In the bottom panel of B,
the expression of N, P, and tubulin (Tub) present in the cell extracts of control (C),
scramble (Sc), and siRNA Hsp was analyzed by Western blot analysis with anti-N,
anti-P, and anti-tubulin antibodies.
4750 jvi.asm.org
complex. Indeed, previous studies of three paramyxoviruses, mea-
sles virus, canine distemper virus, and respiratory syncytial virus,
have provided evidence for an interaction between the N protein
and Hsp70 (3, 29, 30). In these cases, Hsp70 has been shown to
stimulate viral polymerase in vitro activity, but it is not clear
whether Hsp70 acts directly as a cofactor of the enzymatic process
or whether it assists the folding of the N protein in an active form.
Interestingly, the positive effect of Hsp70 on viral infection is not
a common property of negative-strand RNA viruses belonging to
the same order, highlighting the complexity of the virus-Hsp con-
nections.
Other chaperones could play a role in rabies viral RNA synthe-
sis. For example, Hsp60 has been detected in the transcriptase
Downloaded from http://jvi.asm.org/ on May 11, 2014 by UCSF Library & CKM
complex of VSV, although its function has not been investigated
so far (35), and Hsp40 has been shown to enhance the binding of
Hsp70 with the N protein of measles virus (8). More recently, it
has been reported that Hsp70 is associated with complexes formed
between Hsp40 and the viral protein Nef in HIV-1-infected cells
and that the differential expression of Hsp40 and Hsp70 recipro-
cally regulates viral gene expression and replication (19).
In conclusion, these data support a positive regulatory role for
Hsp70 in the rabies virus infection cycle and suggest that Hsp70,
consistently with its multifunctional role, participates at the dif-
ferent stages of the viral life cycle, such as the transcriptional
and/or translational level and/or viral assembly and budding.
ACKNOWLEDGMENTS
We are grateful to Yves Gaudin for careful reading of the manuscript. We
acknowledge Noël Tordo (Unit Antiviral Strategies, Institut Pasteur,
France) for providing the rabies minireplicon system. We thank the Plat-
form of Proteomics (Unités de Recherches de Biochimie et Structures des
Protéines, Jouy en Josas, France). Confocal microscopy was performed at
the Plate-forme Imagerie et Biologie Cellulaire of the CNRS campus,
which is supported by the Institut Fédératif de Recherche 87 La Plante et
Son Environnement and the ASTRE program of the Conseil Général de
l’Essonne.
We acknowledge support from the CNRS.
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