BBRC
Biochemical and Biophysical Research Communications 334 (2005) 631–637
www.elsevier.com/locate/ybbrc
Inhibition of lytic infection of pseudorabies virus by arginine depletion
Hsien-Chi Wang, Yung-Ching Kao, Tien-Jye Chang, Min-Liang Wong *
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung 402, Taiwan
Received 15 June 2005
Available online 5 July 2005
Abstract
Pseudorabies virus (PRV) is a member of Alphahepesviruses; it is an enveloped virus with a double-stranded DNA genome. Polyamines (such as spermine and spermidine) are ubiquitous in animal cells and participate in cellular proliferation and differentiation.
Previous results of our laboratory showed that the PRV can accomplish lytic infection either in the presence of exogenous spermine
(or spermidine) or depletion of cellular polyamines. The amino acid arginine is a precursor of polyamine biosynthesis. In this work,
we investigated the role of arginine in PRV infection. It was found that the plaque formation of PRV was inhibited by arginase
(enzyme catalyzing the conversion of arginine into ornithine and urea) treatment whereas this inhibition can be reversed by exogenous arginine, suggesting that arginine is essential for PRV proliferation. Western blotting was conducted to study the effect of
arginine depletion on the levels of structural proteins of PRV in virus-infected cells. Four PRV structural proteins (gB, gE,
UL47, and UL48) were chosen for examination, and results revealed that the levels of viral proteins were obviously reduced in long
time arginase treatment. However, the overall protein synthesis machinery was apparently not influenced by arginase treatment
either in mock or PRV-infected cells. Analyzing with native gel, we found that arginase treatment affected the mobility of PRV structural proteins, suggesting the conformational change of viral proteins by arginine depletion. Heat shock proteins, acting as molecular chaperons, participate in protein folding and translocation. Our results demonstrated that long time arginase treatment could
reduce the expression of cellular heat shock proteins 70 (hsc70 and hsp70), and transcriptional suppression of heat shock protein 70
gene promoter was one of the mechanisms involved in this reduced expression.
2005 Elsevier Inc. All rights reserved.
Keywords: Pseudorabies virus; Arginine; Arginase
Pseudorabies virus (PRV; suid herpesvirus 1) belongs
to the Alphaherpesvirus family and can cause AujeszkyÕs
disease in pigs. The capsid of PRV virion contains a
double-stranded DNA genome of about 143 kbp in
length, and the capsid is surrounded by a protein layer
named the tegument and a lipid membrane containing
virus-encoded proteins called envelop [1,2]. From early
time of infection to late stage of viral morphogenesis,
complicated interactions between PRV and the host cell
occur [3].
We previously reported that PRV achieved productive infection with either exogenous spermine (or
*
Corresponding author. Fax: +886 4 22862073.
E-mail address: [email protected] (M.-L. Wong).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2005.06.139
spermidine) treatment or depletion of endogenous polyamines [4]. The amino acid arginine is a precursor in the
biosynthetic pathway of polyamines [5]. Arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is a hydrolytic enzyme responsible for catalyzing the conversion of
arginine to ornithine and urea [6,7]. In this work, we
studied the effect of exogenous arginase on the productive infection of PRV; and the possible function of arginine in mediating the correct conformation of PRV
proteins was investigated. Although the 20 amino acids
are essential nutrients for culturing animal cells in vitro,
arginine is specially required for virion production of
nuclear DNA viruses [8]. Most studies on the role of
arginine for virion production were conducted in adenovirus-infected cells [8–10], whereas the relationship
632
H.-C. Wang et al. / Biochemical and Biophysical Research Communications 334 (2005) 631–637
between arginine and herpesviruses was relatively poorly
understood. In this report, we found that the plaque
formation of PRV was blocked by arginine-depletion
treatment. Results showed that the amounts of viral
structural proteins were reduced in long time arginase
treatment and that the conformation of PRV proteins
was altered by arginine depletion.
Heat shock proteins (hsps) mediated protein folding
and translocation [11]. Eukaryotic cells can express several hsp families, including hsp100, hsp90, hsp70, hsp60,
and hsp40 [12]. Among these hsp families, the hsp70
family plays a major role in assisting the folding of nascent polypeptides in the cytosol [11]. The roles of heat
shock protein 70 family in the assembly of polyomavirus, papillomavirus, and vaccinia virus were reported
[13–15]. The constitutively expressed hsc70 and the
stress-induced hsp70 are two members of cellular heat
shock protein 70 family. We found that expression of
the stress-induced hsp70 was severely suppressed with
long time arginase treatment; these data together with
the observations from arginine-depletion experiments
implied a vital function of arginine and of heat shock
protein 70 in the assembly of PRV.
Materials and methods
Chemicals and antibodies. The L-arginase (cat. no. A-8013) purified
from bovine liver was purchased from Sigma (St. Louis, MO, USA)
and was prepared as 1 u/ll. L-Arginine (cat. no. A-8094) and D-arginine (cat. no. A-2646) were purchased from Sigma and were prepared
as 1 M (1.74 g in 10 ml water) aqueous stock solution. Both the mouse
monoclonal antibodies against gB and gE of PRV and rabbit polyclonal antibodies against UL47 and UL48 proteins of PRV were
provided by T.C. Mettenleiter, Insel Riems, Germany [16]. The rat
monoclonal antibody (SPA-815) against the constitutively expressed
hsc70, mouse monoclonal antibody (SPA-810) against stress-induced
hsp70, and mouse monoclonal antibody (SPA-820) against both heat
shock proteins (hsc70 and hsp70) were purchased from StressGen
(Victoria, BC, Canada).
Cell and virus. Monolayer LM (tk ) cells (thymidine kinase negative) were maintained in DulbeccoÕs modified EagleÕs medium
(DMEM) with 10% fetal bovine serum (Hyclone), penicillin, and
streptomycin sulfate. This cell line was used for PRV (TNL strain)
infection, metabolic labeling, and drug treatment in the following
experiments.
Plaque assay and arginase treatment. Confluent LM (tk ) monolayer cells in 2-cm diameter multiwell plates were infected with PRV
(108 PFU/ml). Arginase was added 2 h prior to PRV infection and
incubated for another 2 h postinfection (hpi). Then cells were washed
with DMEM and overlaid with medium containing 1% methylcellulose
(2 ml per well). After 5 days of incubation, the plaque formation was
visualized by staining with 0.5% crystal blue.
Metabolic labeling of cells and gel electrophoresis. For metabolic
labeling of protein synthesis, mock or PRV-infected cells (multiplicity
of infection: 2 PFU/cell) were labeled with [35S]methionine (10 lCi/ml;
PerkinElmer Life Sciences) in methionine-free DMEM [17]. Following
labeling, cells were washed with ice-cold PBS and lysed with 2· disruption buffer (62.5 mM Tris [pH 6.8], 27.5% glycerol, 2% SDS, 1.4 M
b-mercaptoethanol, and 0.02% bromophenol blue), and samples were
boiled and separated by SDS–10% polyacrylamide gel electrophoresis
(SDS–PAGE). The radioactively labeled proteins were visualized by
autoradiography.
Western blotting. Following SDS–PAGE, the separated proteins
were transferred onto a nitrocellulose membrane, incubated with
blocking solution (5% skimmed milk), and probed with various antibodies, respectively, as indicated in the figure legends. Dilution of
antibodies was done according to the instruction from the manufacturer or from T.C. Mettenleiter laboratory. The procedures for
detecting primary antibodies were performed with a chemiluminescence system (SuperSignal West Pico Chemiluminescent Substrate,
Pierce).
Native gels. The cell lysis PBS buffer contained 0.5% Triton X-100,
0.1% deoxycholate, 1 mM PMSF; the 5· loading buffer was 0.326 M
Tris, pH 8.8, and 52.6% glycerol. Samples were not boiled prior to
loading. The procedures for electrophoresis under nondenaturing
condition were followed as described with slight modifications [18]. In
our experiments, the percentages of acrylamide (acrylamide/bis-acrylamide 37.5:1; Amresco) for resolving gel and stacking gel were 5.5%
and 4%, respectively. The pH of resolving gel and stacking gel were 8.8
and 6.8, respectively. The electrophoresis condition of power supply
was 70 V for 10 h.
Sucrose gradient. LM (tk ) cells in 10-cm diameter dish were
infected with PRV at 10 MOI per cell and incubated at 37 C. Cells
were treated with arginase (10 U/ml) at PRV infection. After 12 h,
cells were harvested by cell scraper and disrupted with a needle of
28G. The lysate containing virus particles were loaded onto 20–60%
sucrose gradient and were subjected to centrifugation (26,000 rpm
for 90 min, Beckman SW41Ti rotor). After centrifugation, the gradient was fractionated into 11 fractions (750 ll each) from top to
bottom of the tube. Each fraction was separated by SDS–10%
PAGE. And viral structural proteins were detected by Western
blotting.
Plasmid, transfection, and CAT assay. DNA used in transfection
was purified by QIAprep Spin miniprep kit (Qiagen). LM (tk ) cells
were transfected using Lipofectamine 2000 reagent (Invitrogen). Cells
were seeded in six-well plates in a volume of 2.5 ml of complete
medium on the day prior to transfection. Plasmid pHBCAT for
transfection was a gift from R.I. Morimoto (Northwestern University,
Evanston, Illinois) and pCAT-Basic was from Promega. Plasmid DNA
(1 lg) was mixed with DMEM to 50 ll, Lipofectamine 2000 (3 ll) was
mixed with DMEM to 50 ll, and both were incubated separately for
10 min at room temperature. Following mixing and 20 min incubation,
the mixture was added dropwise to the cells. After 46–63 h of transfection, arginase was added into the medium as indicated in the figure
legend.
At 65 h of transfection, cells were washed three times with phosphate-buffered saline, harvested by scraping into TEN solution
(40 mM Tris–HCl (pH 7.5), 10 mM EDTA, and 150 mM NaCl), and
collected by centrifugation. Cell pellets were resuspended in 250 mM
Tris–HCl (pH 8.0) and then lysed by three-cycle of freeze–thaws.
Lysates were heated for 10 min at 60 C and centrifuged, and the
supernatants were collected for following reactions. The total protein
concentration from each transfection was measured by the Bradford
method (Bio-Rad). To each reaction, 50 lg protein, 2 ll [14C]chloramphenicol (CM) (0.1 lCi, 50 mCi/mmol, NEN), and 2 ll acetyl
coenzyme A (35 mg/ml, Sigma) were added and sample volumes were
adjusted to 150 ll by addition of 250 mM Tris–HCl (pH 8.0). After
incubation at 37 C for 1 h, chloramphenicol and its acetylated
products were extracted with ethyl acetate and then the ethyl acetate
was evaporated. Samples were resuspended in 20 ll ethyl acetate and
spotted onto thin layer chromatography (TLC) sheets (MachereyNagel). The products were separated with a solvent system of chloroform–methanol (19:1, vol/vol) on a silica gel. TLC sheets were
autoradiographed at 70 C. Intensity of radioactivity of [14C]CM
and acetylated [14C]CM was analyzed by computerized imaging
system.
H.-C. Wang et al. / Biochemical and Biophysical Research Communications 334 (2005) 631–637
Results and discussion
633
Effects of arginase on levels of intracellular viral proteins,
translational machinery, and protein conformation
Inhibition of PRV plaque formation by arginase
In this work, we showed the inhibition of PRV plaque
formation by arginase treatment, whereas this inhibition
can be reversed by exogenous L-arginine, suggesting that
arginine is essential for PRV proliferation (Fig. 1). The
result also demonstrated that exogenous D-arginine
could also counteract the inhibitory effect of arginase,
indicating that the function of L-arginine could be replaced by D-arginine or D-arginine was converted into
L-arginine by cellular enzymes. Our data were consistent
with earlier studies on adenovirus, herpesvirus, MarekÕs
disease virus, and pseudorabies virus, which showed that
the yield of infectious virus was reduced by using
arginine-deprived medium [8,9,19–21].
To investigate the molecular mechanism of the blocking of plaque formation by arginase, we first examined
Fig. 2. Western blots of PRV structural proteins gE, gB, UL47, and
UL48 with arginase treatment. Lane 1: PRV infection without
arginase. Lane 2: arginase treatment 2 h before PRV infection and
2 hpi. Lane 3: arginase treatment 2 h before harvesting at 17 hpi. Lane
4: arginase treatment 10 h before harvesting at 17 hpi. Lane 5: arginase
treatment in the entire infection process. The levels of viral proteins
decreased in long time arginase treatment (lanes 4 and 5).
Fig. 1. The plaque formation of PRV was inhibited by arginase
treatment. Plaque formation was visualized by staining with 0.5%
crystal violet. (A) Row 1: PRV infection without arginase treatment.
Row 2: arginase (10 u/ml) pre-treatment 2 h and co-incubation with
PRV for another 2 h; then cells were overlaid with medium containing
1% methylcellulose (2 ml per well). Rows 3 and 4: the arginase
treatment was same as row 2, however the subsequent methycellulose
medium contained 5 mM L-arginine, and 5 mM D-arginine, respectively. (B) Plaque numbers are the means of three independent infections,
and error bars indicate standard errors of the means.
Fig. 3. The incorporation of [35S]methionine into newly synthesized
proteins in either mock or PRV-infected cells was generally not affected
by arginase. Cells were pulsed-labeled with [35S]methionine (10 lCi/ml)
for 2 h and then harvested for SDS–10% PAGE and autoradiography.
Lanes 1–5: mock infection with same arginase treatment as PRV
infection (lanes 6–10). Lane 6: PRV infection without arginase. Lane 7:
arginase treatment 2 h before PRV infection and 2 h postinfection
(hpi). Lane 8: arginase treatment 2 h before harvesting at 17 hpi. Lane
9: arginase treatment 10 h before harvesting at 17 hpi. Lane 10:
arginase treatment in the entire infection process.
634
H.-C. Wang et al. / Biochemical and Biophysical Research Communications 334 (2005) 631–637
the influence of arginase on the total levels of four viral
structural proteins (gB, gE, UL47, and UL48) in PRV-infected cells. Results of Western blotting revealed that
these viral proteins were decreased with long time (10 h
and more) arginase treatment (Fig. 2). We also studied
the effect of arginase on the protein synthesis machinery
by labeling mock or PRV-infected cells with radioactive
[35S]methionine. The results indicated that the global profile of protein synthesis process was not influenced by arginase (Fig. 3); these data were consistent with a study on
adenovirus by using arginine-deprived medium [8]. As it
can be seen in Fig. 2, the amount of gE was dramatically
reduced in long time arginase treatment. Relative to other
viral proteins, it seemed that gE was preferentially
degraded with arginase treatment, and this was not due
to intrinsic instability of gE because examination on the
half-lives of two PRV proteins gB and gE revealed no significant difference (data not shown). Taken the protein results described together, we proposed that arginine played
a role in folding or conformational maintenance of viral
proteins, and arginine depletion would eventually promote the degradation of viral proteins. These results of
PRV were generally similar with an earlier study of adenovirus, which showed that adenoviral DNA and proteins
were produced in arginine-negative medium, and viral
DNA could be packaged into virions after arginine restoration [8,10].
It is known that arginine may suppress aggregation of
proteins during refolding in vitro [22,23]; therefore, it is
very likely that intracellular arginine exhibits a similar
function in protein folding. We hypothesized that intracellular arginine might influence the protein conformation. To explore this, cells were lysed with milder buffer
and lysates were not boiled prior to loading into a native
gel. After gel electrophoresis and Western blotting, we
examined the effect of arginase on the mobility shift of
gE and gB in native gel; and our results demonstrated that
arginase treatment altered the mobility of gE and gB, suggesting that a conformation change caused by arginine
depletion (Fig. 4). We proposed that arginine was also
essential for proper folding of intracellular proteins and
that proteins were susceptible to degradation in arginine-depletion milieu. Thus, the reduction of total viral
proteins with long time arginase treatment in Western
blots of SDS–PAGE (Fig. 2) as well as of native gel
(Fig. 4) was mainly a consequence of protein degradation,
not biosynthesis of polypeptides.
The in vitro role of arginine in maintaining the
stability of PRV virion was also examined. We incubated virions that were purified from sucrose gradient
with arginase, and Western blotting result revealed
that the virion protein was susceptible to partial degradation in arginine-depleted condition (data not
shown).
Fig. 4. Western blots of the native gel showing the effect of arginase on the mobility shift of PRV gE and gB. After gel electrophoresis and
transferring samples onto membrane, the membrane was cut into two pieces and probed with antibody against gE or gB, respectively. Lanes 1 and 7:
mock infection. Lanes 2 and 6: PRV infection without arginase. Lanes 3 and 9: arginase treatment 2 h before PRV infection and 2 hpi. Lanes 4 and
10: arginase treatment 2 h before harvesting at 17 hpi. Lanes 5 and 11: arginase treatment 10 h before harvesting at 17 hpi. Lanes 6 and 12: arginase
treatment in the entire infection process. The positions of gE and gB are indicated by arrowheads; the degree of band shifts is proportional to the time
of arginase treatment. A weak band resulted from background signal (at the very high molecular weight region) in the left panel (gE) does not show
the same direction of shift as the major band (between 75 and 120 kDa).
H.-C. Wang et al. / Biochemical and Biophysical Research Communications 334 (2005) 631–637
The assembly of PRV structural protein was affected by
arginase treatment
Using 20–60% sucrose gradient centrifugation, the effect of arginase on the sedimentation rate of PRV structural protein UL48 was studied. As shown in Fig. 5,
result revealed that arginase treatment caused the reduction of UL48 in the fractions of higher percentage sucrose, indicating that the association between UL48
and other proteins as well as the assembly of virions
were blocked in the arginine-depleted condition.
635
stress-induced hsp70 was reduced with long time arginase treatment in mock-infected cells (Fig. 6). However,
in PRV-infected cells, the stress-induced hsp70 was reduced with long time arginase treatment; the constitutively expressed hsc70 was not affected (Fig. 6).
Influence of heat shock protein 70 gene expression by
arginase
Heat shock proteins, acting as molecular chaperones,
can assist protein folding and translocation [12]. It was
shown that the heat shock protein 70 family can mediate
the assembly of polyomavirus, papillomavirus, and vaccinia virus [13–15]. To our knowledge, the effect of arginine depletion on the expression of heat shock proteins
has not been investigated. In the following, we studied
the effect of arginase on the expression of two members
of heat shock protein 70. Results revealed that the
expression of either constitutively expressed hsc70 or
Fig. 6. Western blot showed the reduced expression of stress-induced
hsp70 by arginase in long time treatment. The expression of constitutively expressed hsc70 was relatively less affected by arginase. Lanes
1–5: mock infection with same arginase treatment as in PRV infection
described in the following. Lane 6: PRV infection without arginase.
Lane 7: arginase treatment 2 h before PRV infection and 2 hpi. Lane 8:
arginase treatment 2 h before harvesting at 17 hpi. Lane 9: arginase
treatment 10 h before harvesting at 17 hpi. Lane 10: arginase treatment
in the entire infection process.
Fig. 5. Using sucrose gradient centrifugation, the distribution of PRV structural protein UL48 without (A) or with (B) arginase treatment was
examined. After centrifugation, the sucrose gradient was fractionated into 11 fractions (lanes 2–12); lane 1 was the portion of cellular lysate. These
fractions were subjected to SDS–PAGE and probed with anti-UL48 antibody. Arginase treatment decreased the UL48 structural protein in the
fractions of 4–12 (B).
636
H.-C. Wang et al. / Biochemical and Biophysical Research Communications 334 (2005) 631–637
Fig. 7. (A) The influence of arginase on the transcription of human heat shock protein 70 gene promoter was examined by CAT assay. The
promoter activity was suppressed in long time arginase treatment (lanes 5 and 6) but was stimulated in short time treatment (lanes 3 and 4). One
microgram of plasmid (pHBCAT) was transfected into cells. After 2 days of transfection, arginase was added or not (time of treatment was
indicated as following. (a) Arginase was added during 46–50 h of transfection and cells were lysed at 65 h after transfection; (b) arginase treatment
was in 63–65 h of transfection and cells were harvested at 65 h of transfection; (c) arginase treatment was in 55–65 h of transfection and cells were
harvested at 65 h of transfection; (d) arginase treatment was in 48–65 h of transfection and cells were lysed at 65 h of transfection). Cell lyates
were prepared after 65 h of transfection and the CAT assay was done as described under Materials and methods. (B,C) Quantitation of CAT
assays at 37 and 42 C, respectively. The values are the means of three independent transfections and CAT reactions, and error bars indicate
standard errors of the means. The numbers (1–6) of lanes are corresponding to those of (A). The plasmid pCAT-Basic devoid of a promoter is a
negative control.
In contrast, our data showed that the expression of
stress-induced hsp70 was stimulated in short time arginase treatment (Fig. 6); this stimulation perhaps was a
cellular response to external stress.
To the blot of stress-induced hsp70, we striped the
anti-hsp70 antibody and re-probed with an antibody
that can recognize both hsp70 and hsc70; and the over
pattern of Western blot was similar to the hsc70 blot
(Fig. 6).
To further investigate the effect of arginine depletion
on the heat shock protein 70 gene expression, we examined the influence of arginase on the transcription of
heat shock protein gene promoter by CAT assay. Cells
were transfected with plasmid pHBCAT containing a
promoter of human heat shock protein gene [24,25].
Following 2 days of transfection, arginase was added
as indicated (Fig. 7). It was observed that arginase suppressed the promoter activity of heat shock protein gene
in long time treatment but stimulated the promoter in
short time treatment (Fig. 7). This downregulation of
the pHBCAT transcription was consistent with the Western blotting results described above.
In summary, given that proper folding and maintaining native state of viral proteins is a prerequisite for virion assembly, our study indicates that both arginine and
heat shock protein 70 are indispensable for the production of mature PRV virions in cultured cells. It deserves
further study to explore whether there is a connecting
pathway between arginine and heat shock proteins.
Acknowledgments
This study was supported by a grant from the
National Science Council (Taipei, Taiwan). We are
grateful to Professor T.C. Mettenleiter (Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, Insel Riems, Germany) for the
antibodies against structural proteins of PRV and to
Professor Richard I. Morimoto (Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois, USA) for the
plasmid containing the promoter of human heat shock
protein 70 gene.
H.-C. Wang et al. / Biochemical and Biophysical Research Communications 334 (2005) 631–637
References
[1] B.G. Klupp, C.J. Hengartner, T.C. Mettenleiter, L.W. Enquist,
Complete, annotated sequence of the pseudorabies virus genome,
J. Virol. 78 (2004) 424–440.
[2] T.C. Mettenleiter, AujeszkyÕs disease (pseudorabies) virus: the
virus and molecular pathogenesis—state of the art, June 1999,
Vet. Res. 31 (2000) 99–115.
[3] H. Granzow, B.G. Klupp, W. Fuchs, J. Veits, N. Osterrieder,
T.C. Mettenleiter, Egress of alphaherpesviruses: comparative
ultrastructural study, J. Virol. 75 (2001) 3675–3684.
[4] H.C. Wang, M.L. Wong, Lytic infection of pseudorabies virus in
the presence of spermine, spermidine, or DFMO, Virus Res. 94
(2003) 121–127.
[5] H.M. Wallace, A.V. Fraser, A. Hughes, A perspective of
polyamine metabolism, Biochem. J. 376 (2003) 1–14.
[6] C.P. Jenkinson, W.W. Grody, S.D. Cederbaum, Comparative
properties of arginases, Comp. Biochem. Physiol. B Biochem.
Mol. Biol. 114 (1996) 107–132.
[7] J. Perozich, J. Hempel, S.M. Morris Jr., Roles of conserved
residues in the arginase family, Biochim. Biophys. Acta 1382
(1998) 23–37.
[8] C.A. Heilman, H. Rouse, Adenovirus type 2 polypeptide synthesis
in arginine-deprived cells, Virology 105 (1980) 159–170.
[9] E. Everitt, B. Sundquist, L. Philipson, Mechanism of the arginine
requirement for adenovirus synthesis. I. Synthesis of structural
proteins, J. Virol. 8 (1971) 742–753.
[10] H.C. Rouse, R.W. Schlesinger, The effects of arginine starvation
on macromolecular synthesis in infection with type 2 adenovirus.
I. Synthesis and utilization of structural proteins, Virology 48
(1972) 463–471.
[11] F.U. Hartl, M. Hayer-Hartl, Molecular chaperones in the cytosol:
from nascent chain to folded protein, Science 295 (2002) 1852–1858.
[12] A.L. Fink, Chaperone-mediated protein folding, Physiol. Rev. 79
(1999) 425–449.
[13] L.R. Chromy, J.M. Pipas, R.L. Garcea, Chaperone-mediated in
vitro assembly of Polyomavirus capsids, Proc. Natl. Acad. Sci.
USA 100 (2003) 10477–10482.
[14] L. Florin, K.A. Becker, C. Sapp, C. Lambert, H. Sirma, M.
Muller, R.E. Streeck, M. Sapp, Nuclear translocation of papillomavirus minor capsid protein L2 requires Hsc70, J. Virol. 78
(2004) 5546–5553.
637
[15] S. Jindal, R.A. Young, Vaccinia virus infection induces a stress
response that leads to association of Hsp70 with viral proteins, J.
Virol. 66 (1992) 5357–5362.
[16] W. Fuchs, H. Granzow, T.C. Mettenleiter, A pseudorabies
virus recombinant simultaneously lacking the major tegument
proteins encoded by the UL46, UL47, UL48, and UL49
genes is viable in cultured cells, J. Virol. 77 (2003) 12891–
12900.
[17] M.L. Wong, Y.R. Yen, Protein synthesis in pseudorabies virusinfected cells: decreased expression of protein kinase PKR, and
effects of 2-aminopurine and adenine, Virus Res. 56 (1998) 199–
206.
[18] G.H. Cohen, V.J. Isola, J. Kuhns, P.W. Berman, R.J. Eisenberg,
Localization of discontinuous epitopes of herpes simplex virus
glycoprotein D: use of a nondenaturing (‘‘native’’ gel) system of
polyacrylamide gel electrophoresis coupled with Western blotting,
J. Virol. 60 (1986) 157–166.
[19] V.B. Inglis, Requirement of arginine for the replication of herpes
virus, J. Gen. Virol. 3 (1968) 9–17.
[20] G.E. Mark, A.S. Kaplan, Synthesis of proteins in cells infected
with herpesvirus. VII. Lack of migration of structural viral
proteins to the nucleus of arginine-deprived cells, Virology 45
(1971) 53–60.
[21] T. Mikami, M. Onuma, T.T. Hayashi, Requirement of arginine
for the replication of MarekÕs disease herpes virus, J. Gen. Virol.
22 (1974) 115–128.
[22] T. Arakawa, K. Tsumoto, The effects of arginine on refolding
of aggregated proteins: not facilitate refolding, but suppress
aggregation, Biochem. Biophys. Res. Commun. 304 (2003) 148–
152.
[23] K. Tsumoto, M. Umetsu, I. Kumagai, D. Ejima, T. Arakawa,
Solubilization of active green fluorescent protein from insoluble
particles by guanidine and arginine, Biochem. Biophys. Res.
Commun. 312 (2003) 1383–1386.
[24] W.D. Morgan, G.T. Williams, R.I. Morimoto, J. Greene, R.E.
Kingston, R. Tjian, Two transcriptional activators, CCAAT-boxbinding transcription factor and heat shock transcription factor,
interact with a human hsp70 gene promoter, Mol. Cell. Biol. 7
(1987) 1129–1138.
[25] B.J. Wu, R.E. Kingston, R.I. Morimoto, Human HSP70 promoter contains at least two distinct regulatory domains, Proc.
Natl. Acad. Sci. USA 83 (1986) 629–633.