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the presence ofXMRV.
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In the present study, we analyzed 105 RNA samples from the
prostate tissue of 87 sporadic prcstate cancer patients and also
biopsy samples from 70 healthy men without prostate cancer for
,
factors.l4-16
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't386-6532/t- re. front @s.rO20(ts Elrdier B.V.All rights reseiled.
doiil0,l0l6Djd.20OE.e.0lE
revealed that prostatic stroma cells were infected at low frequency
(0.s-1.2%).
an endoribonuclease of the antivlrel defense
RNase
p:thway, was one of the first prostate cancer suJceptibiF
ity genes recognized. The missense mutation R462Q has been
linked to hereditary prostate cancer 11-t3 and has been implicated in up to 13X of all prostate cancer cases in some
studies.rl Not all studies have confirmed this finding, perhaps
because of differences in populadon genetics or etrvircnmental
8 9
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virus-related gammaretrovirus (XMRV), was dlscovered in prostatic tissse ftom patients with familial prostate canceliro
specifically in patients homozygous for a missense mutation itr
the RNase L gene, R462q Huorescence in situ hybridization
,
Abb@iationt: PCR polymerisa ch.in rcaction; PCA, grcstate qncer; SNP. singla nucleotida polyhorphirm; XMRV. x.notrcpic mu.inc lcukemia virus-related
virusi PSA, prost:ta sp.cifrcrntigen; PlA, polif.r.tiv. inllamrutory.trcphyi HPC,
hcradihry proshre on@i
CgRsgonding.uthoi T€1.: +49 42$3 8184; f.r: {g 42803 3250
E-moil oddrAsi nfi$h€roulc.dc (l{ Fisch.rl
nant stages ofthe disease.r viral infections may be trigg€tr for the
inRammatory prccess. However, epidemiological studies deiigned
to detect links between specificviral infections and prosQte cancer
have been inconclusive.2-e
Recently, a new gammaretrovirus, xenotropic murine leukemia
5““ 5∞4 3
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5
66
1
,,
Conclurio6j XMRVwas rarely detected in non-famllial prostate cancer$Dples from Northcrn Europe.n
patients. The homozygous mut.tion R462Q(qO wa5 signinc.ntly undc(ep.escntld (<64) in this cohort
when comp'red to other studies ( 1 - l7x)
@ 200g Ersryier B.v. Arr right5 rcserved.
cef,
Recent work empbasizes thet prostate cancer is frequently
associated with chronic prostatic inflammation. A lesion called
prclifentive inflammat6y atrophy is often found in the premalig-
56
55
prostate cancer patients heterozJEous for R462Qor carrying the wild typc allelc.
Objectivesj To detcrmine the Drrsrnce ofXMSJ in non-familial pD$atc c.ncer samples.
Study design: RNA from prostate tis5u€ was an.lyzed for XMRV usingnested RT-PCR In.ll samples, RNase
L (R462O genotyplng was perfomcd using an allele-specific PCR
.|05
R6ulfs: XMRv-specific sequenccs werc detectcd In on. of
tissue samples frqm non-familial prostate
canc€r patients atrd from one of 70 tissue emplcs frcm ms without prostrtc rancer. Thc t{o y\MRV.
positive patients wcrc wild typc or heterozJ€ous for thr R462Q mut tion and thus caried rt lcst one
fully functional RNasc L allele.
most common form of prcstate cancer (80-909) and its incidance incrcrses with age. Femilial prostate cancer, which accounts
for lO-202 of all prcState canccr cases, occun much earlier in
life and is defined as prostate cancer occuffing in individuals
with three or morc first degree relatives who had prostate can-
,
mut tioninRN.seL(R462q)wercpositivcforXMRV.whilcthevirusw.srarcly(l/56)detectedinfamilial
Prcstate cancer is the most frequent cancer of men in North
America and Europe. well krown factors contributing to the
risk of prostate canccr arc age, androgens, environmental and
genetic factoF.l Sporadic (non'familial) prostate cancer is the
6
“5
Sqcttroundr We previously identified a novel ercgenous gammaretrovlrus (xenotppic murlne leukemia
virus-related Sammaretrovirus{XMnV)) usinga pan-viral micro..ray. XMRVi5 the firstMlv-rclatedvirus
tound in human infection. Forty pcrc.nt (8/20) of famili.l prostate canccr p.tients homoz),gous for a
1- lntroduction
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Nicole Fischerl,° ,OlaFH11lwinkelb,Claudia Schulza,Felix K.H.Chunb,
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Clinicrl and prthologicrl
Contents lists available at Sci€nc€Diroct
Prevalence of human gammaretrovirus Xヽ 4RV in sporadic prostate cancer
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4. Dlsssslon
XMRV, a novel gammaretrovlrus, was rcccntly identified in
familial prGtate cancer samples using a pan-viral mlcrcarray.lo
Our eerlier studies suggested that functional mutationi in RNase L
might be important forthe acquisidon ofXMRV, Almost all XMRVpositive prostate cancer cases described so far carry a mutation
within RNase L (R462Q), resulting In reduced RNas€ Lactivlty,lo
However, thc currcnt study found only a low prevalence of
XMRV in non-famillal prostate $ssue of men In Norlhdn.Europe.
RNase I- an endoribonuclese ofthe andviral defense pathway,
was one of the 6rst susceptlbility genes discowted in prostate
cancer The HPCI (hereditery prcstate amcei) locus was linked
to prostatc cancer in sevenl genetic linkagE studies performcd
in Nor h Amedca and Finland.ll'12:03 This inding was no! bc
connmed In a large case control study recently conducd in
Cemany,r6 RNase L ls implicated in the interfcron-medlated antiviral defcnce pathw:y and has been shown to play a rcle ln several
models of vinlinfection including influenzaA, WestNllcvtrus and
herpes simpls( viru s.21 2221'26
ln our previous study, XMRV was detectcd ln famllial prostate
cancer patients homozlrtous for thc R462Q yarlant (QQ) of RNate
Lro Overall, 40X.(8120) of patients hombzlryous for the SNp
R
(3)57(groupA):Genoけ pe QR
CCAAAATGTCCTGTCATC`
CCGAAATGTCCTGTCATCv
6{group B):Genotype RR
CCGAAATGTCCTGTCATC
CCCAAATGTCCTGTCATC
R462Q(qQ) has XMRV Infecrion, whereas only 1.51 (1/66) padents
hetemzygous (Q!.) or carrying the wild type allele (RR) were
XMRv-positive. Subsquent in vitro experlrDcn6 demonstrating
thatXMR\y' replication lncreases wlth reduced RNase Lactivity further comborated our prcvious rcsults.2?
So hr, th€re are no epidemiologlcal data rcgardlng the prwalence of XMRVin pmstate tissue,independenlofthe RNas€ Lstatus.
The prevalenc€ of XMRV in our cohoft wrs lou/ (l.l4t). 'Ihc two
XMRV-posidw patients w€rc heterozygous (HR).or.wild type (RR)
genorype and showed no deficiency in RNase L cxpresslon, These
results are in accord with our previous obsewation that XMRV
sequences are gredominantly fgund In prcstrte cancer patlents
with a deficiencyin RNase Land only rarely found in irrcstate cancer
patients with at least one fully funcdonal RNase Lalfte,
Genotyping for the sNP R462Q ln thc C-tlrminal domain of
RNase L revealcd that homozygoslty of M62e(ee) is a relativety
rare ev€nt (<6!). Th.se rcsults arc in conbast to pr€viou studies
tbat have reported homozygous R462e(@) mutadons In ll-t7x
of cases, indcpendent of the genedc backgound of thc population,
At present we do nothavean uplanadon for thls obser€tlon,slnce
all ofourpadents are ofCaucasian bac(grcundand livcin Northem
RNarc L mRNA rbundrncc
コ
,
聖 く3 嬌E ■E
Europe.
nRNA dpRrslon in prcitet! tissua r.drples. RMs! L mRNA axprcsquentitatiE rcal dm PCR s drsaibed in Sedon 2. St ndad
d€viatlons tod thrc? Ind.pand.nt cxlErlmcnts (6ur Epllors .&h) aE shom
PEfrre cmerell lin.s DUl45.nd INC:P(rhom.s bl*k tEE) rcE u$d.5contbb. nNrsr L hRM l.wls fmm he.lthycontFl prtl.ng(6roup B).r. shdn In ilght
gry nmplar trcm ItA p.ti.nts (Group A) are shM al whit. b.R. T lndicaias RM
fFm tumot Fglo[ N rbnds for RM froh noml tlssu?. Th.
XMtU-pGltlE
arc andic:tcd a5 sFcklcd brF. The RNr* L gEnotyp€ ofrll s.mplcs ir 5h@n
FtSl
3, RNsc
L
slon was mcasuEd br
I'
XMRV g.g saqurnc?s derived fDm s por.dic rnd fa mil ial prosbt? c. ncer semples. Phylogenctlc tr.e @mparint a 390-nt Ff-pCR gag fraSmen I . mplified fiom sporedic
tumr9mplc! (57 in CrouPAand 6 in GDUP B)wth eently published XMwsequen.es fmm f.milial pprt.iec.nc.r psd.nts.loThesequencc
aligned uringOuitalX
andthc
was 8!n.Eted uslngtha neighboFjoiningmthod, Sequences ara l:b.hd:5 xenotropic(x), polytropic(p), mdi6cd potytropic(pm). or ecoiropic(E).F1&
mc
tE
3.3. RNoie I expression
RelativeRNaseLmRNAexpression levels wereassayedin XMRVpositive sampler using quantitative real time RT-PCR. LNCap cells,
which have an inactivating deletion mutation in one ellele of
RNASE!.20 These cells had a zo-fold reduction in RNase L expres-
iI br4kcts,
cells.
T.bh
llz
3
RN$c Lgrnotyping ofSNP R462Q
Studygrcup
T.bl.2
XMRV detection u5ing n.stcd
g.g
PCA
XMRV ttl● )
Medan age(yr,)
(Croup A)
17105
is
p.r.ll.l s .n inlcrBl cosrFl.
RNASEL(SNP,162)
RQ Qo
Cont、1(Cmup 3)
Total RNA hol.tcd from prcshre tissue from pati.nB with prostarc oncer (pCA)
was:n.lyzed for(h€ pr.sence ofXIURV sequ.nccs usingan RT-nest€d pm. cApDH
us .hpliflcd
Numberscre.ned
RT-PCn
″“
sion levels compared to DUl45 cells (Fig.3). In contrast, the two
XMRv-positive samples (6 and 57) did not have reduced RNase
L expression when compared to DUl45 cells. Randomly selected
samples from our cohort representing Croup A and croup B did
not show major differences in RNase L spression. However, we
obseryed a 5o-fold difference in RNase L mRJ.,lA exDression when
comparing RNA frcm tumor cells (T) with normal tissue (N) from
M
64
Two other samples, 118 and l t9, did not show major
differences in RNase L expression between tumor cells and nomal
patient
KA(CroupA)
Control(CDu1 3)
samPl€
Wc werc not able to took for SNP R462qln the germline cells of
our cohort, as such mat€dal was not availablc, Ho$!wr,
Fccnt
i
study comparing gErmlinc with somatic mutrtions of RNxe i
obsewed a slmilar distributlon of homozygous, heterozygous or
wlld type allele frequency in borh tissue types.4
ln conclusion, our results suggest that XMniV k not associated
with sporadic prostate cancer itr Northern Eurcpe, The availability
of an XMRV antibody-screening test should gllatly enhance epidemiological studles of the prevalencc ofXMny' ln larger cohorts
of prcstate cancer patients as well asln the general population,
rYPe conflict oflnterest
lissu! D}lA
Tlss. DNA
DM from pro56@ tls3ua frcm pad.nti with prcrt.G oncq (pCA) was a8lyr.d
foa th! preslncagfa shgb nucl.otidr pollmorphism (SNp).t amino.aid posidon
462 withln thc RN.3! [ grn€. R& Rq Qq wild typ., heErcz!€os .nd homo.ygous
The auth。 ,s dectare they have no cOnlp● ung intcrest
l
Acknowledgement
geno0?e. Especflvely.
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了
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AM.Laas W,Pr“
2 Sttnos KS,SauvageOt].Fedor HL Ditk,D DeMarzo AM,、
ular analys“
N Ett J ttd
t once■
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3acs WB A molec
of prokaryotic and viral DNA sequences in prostate●
ss●
e「 om
mig。 。
rganlsms Pr● ltate 200816813):306-20
3D口 lnerJ.К nckt R BOmanJ.leh“ nen M.CelersStan vA,Sapp M.et al Sero.
bm“ WS hfecい on and nsk
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●亀cetcd study or human papl"。 mavlrus and prostate● rdnoma canc『
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嗣2008:82(6)2705-14
lo unsman A,Mo"n3m Rl.口 SCher、 PlummersI.Casey C.Oein EA.eい l lden● 6‐
pa.e.s hOmo2ygO“
器 嶋な 亮 l網 出 層器 電 霞 盤 明 寵 :11,あ ギ
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2′ ,S′
in■ bonuclease L gene do not occur at a greater Frequen″
in P31ent,with
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urO10」 scheて inlk′ Sttumannsu`e20/21.10117黎 nin,c_an,3chaht`.untveFSitaCmedlzln Berlin,Hindenbu"danln 30,12203 Benin
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Rmrirctogtzoo9,5t92 dot:to.t
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2002:3214):581‐ 3
12 Rokrnan A.lkonen■
の 文献3
Retrovirology
283
`3211187277‐
rus type 2‐ mediated disease“
27識f曽 腑ξ
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28 NuppOnen NN,Waleh NI」 .POndano p.Robbins CM.Ta"mela TL Ves‐
sab RL et J M● ta“ onJ a“ ly● s oF suscep● bnty genls tNAsELIHPCl.
EIAC2′ HPC2.and MSRl in spOradic pro,●
tecancen OЛ 6 07● mosom● Can“ r
2● ●
4`“ 2111年25
Thi5 rrticlo ir
Rqc.ird:
A'c6Ptrd:
t86/t742.4690-6-92
.l Jufl 2009
16 oc6b'r2009
.Eil.bb frcm: htrp:/AMrw.nuovirclogy.com/conllni/6/ l/92
cr:l; licensoe BioM.d Ccnhl Ltd,
This k .n Op€fi Acesr sni<le dliribued undcr d. t.mi of tir. CE lta Comhoro Acrlbuglon Uclnie
which p.mlG uh.stlricbd ure, dirglbudon, rnd [prcducdon In.ny mcdlum, providad tha orlgln.l work
@ 2009 Hohn
(it6rlccrdvf,dfr|m^r 6f'rlc.rr3/teD
0),
l! pDplrlt cllad,
PrOC Na“
Abstract
Brckgroundr A novel gammaretrovlrus named xenotopk mwine leukmio virus*lotcd vlrus
CXMRV) has been recently identifled and found to have a prevalence of 40% in prosato tumor
samples
from American pati€nts carrling
a
homozygous R462Q mutatlonin the RNascL gene. Thls
muation impaiF th€ function of the Innate antivinl t),pe I intcrferon pathway and is a known
susceptibility factor for pfostate ancer. Here, we attempt to measure thc prwalcnce ofXMRV In
prostate cancer cases in Germany and detemine whether an amlogous a$ocladon with the
R462Q polymorphism etsa..
Re3ults: 58t prosutt€ tumor smples were genotyped by real-time PCR with regard to the
RNaseL mutation. DNA and RNA samples from these patlents werc screened for tlu prcsencc bf
XMRVspectfic gog seguencs uslng a hlghly sensitjve nesed PCR and RT-PCR approach,
Furthermore, 146 sen sample from prosate tumor patlents werc tested for XMRV Gag and Env
antibodi$ using a newly dcveloped ELISA usay. In agreementwith erlier data 12,9% f/6 samplc)
were shown to be of tha QQ genotype. However, XMRV specillc seluences were detected at
nelther the DNA nor thq RNA level. Consistent with thls resulq none of thc sen anrly:ed from
prostat€ cancer paticnts conalned XMRV-specific antibodles.
Conclusion: Our r$ults indicat! a much lowcr prevalcnce (or evgn complete absencc) ofXMRV
in prostate tumor paticntr in Germany. Onc posslble reason for this could be a geognphically
restricted incidence of XMRV Infections.
Background
Prostate cancer (PCa) is cunendy the most commonly
diagnosed canca in European males and causes approxi-
mately 80,000 deaths per year [f]. Modern merhods of
diagnosis and ortensive prognms for early detecdoi have
increased lhe drmces for succesfirl tr€agneni in recent
Pego 1 of 11
(pago
nmbq td tv
cna0on
rlr!D$)
│
Ratrcvirolory 2009,6:92
years, but there is still only limited knowledge concerning
susceptibility and putative risk factors for PCa. In addition
to age, the risk factors for developing PCa are thought to
be diet, alcohol consumption,
exposure to ultraviolet
radiation [2], and genetic faaors [3]. One ofthe first stud.
ies to investigal€ the hereditary factos associated with a
predisposition for developing prostate cancer identified
the HPC1 locus (hereditary prostate cancer lorust ) [ ],
which is now known to harbor the RNaseL gene. RNaseL
codes for a endoribonuclease involved in the IFN-regu.
lated antiviral defense pathway (reviewed by l5l). The sig,
of RNroeL gene polymorphisms for rhe
development ofprostate cancer is still under scrutiny, The
R462Q (rs486907) polymorphism for example is implicated in up to I 3% of all US prostate cancer cases [6] and
*rree other variants conuibute to familial prostate cancer
risk in the fapanese population [7], whereas no significanr
association with disease risk could be found in rhe German population [8].
nificance
Recendy, an analysis forviral sequences in prostate cancer
stroma tissues using custom-made microarrays resulted in
the discovery of a new gammaretrovirus named, motropk
murine buhcmiavins-relatcd nru (XMRV), [9,10]. XMRV
was present in eight of twenty (40olo) cases in patients
with familial prostate cancer thatwere homozygous at the
R462Q locus for the QQ allel. On the other hand, the
virus could be deected in only 1.50lo of carriers of the RQ
or RR allels. In subsequent studies involving smaller
cohons of European prostate cancer padents, the prevalence and correlation ofthe QQ-phenotype with the pres- .
ence of XMRV were either far less significant | 1 1 | or the
virus could not be detected ar all [121. Very recently XMRV
was recogqized by immunohistochemistry in 23% of
prostate cancers from US American donors, independent
ofthe R462Q polyniorphism [13].
This present study describes the development and use of
sensitive PCR and KI-PCR assays to tes! DNA and RNA
from 589 PCa tumor sampls obrained from the Charit€
hospital in Berlin (Gemany) for the presence of proviral
XMRV DNA and coresponding viral transcripts. ln addition, we used an ELISA based on recombinant XMRV proteins to screen 146 PCa patient sera forviral Env- and Gag,
spaific antibodies. Neither in the 76
specimens
homozygous for the QQ allele, nor in any of the other
smples could XMRV or a related gammareuovirus be
detected. Furthemore, none of the sera contained antibodies specific for the XMRV Env or Gag proteins.
Methods
Pat|€ntt
Tissue samples were collected from 589 patients undergo-
ing radical prostatectomy for histologically proven primary prostate cancer at the Depanment of Urology,
http:/ rww.Etrovirology.com/contenv6/,1
/92
RelrcviroloW
Charit€ - Univercitdtsmedizin Eerlin, between 2OOO and
2006. lnstitudonal rwiew board approval for this study
like sequences, e.g. MLV DG-75: ln addition, using a
primer spanning the XMRV-specilic deletion 5'-
was obtained and all patients gave their informed consent
prior to surgery. Tissue samplc were obtained immedi.
CCCCAACMAGCCACTCCAAAA.3'we were able ro dis
tinguish between XMRV and DG75 sequences. Second
round PCR reaction was performed at an elevated annealing temperature of 64'C for 3 5 cydes.
ately after surgery snap.frozen in liquid niuogen and
stored at .80'C. Histopatlologic classification ofthe sam.
ples was based on the World Health Organization and
1997 TNM classification guidelines (lntemational Union
Against Cancer, 1997). The patient's median age was 63
years (range 43 - 80). The serum PSA levels were measured
prior to surgery and ranged from 0.1 to 100 n6/ml
(median 7.5 ng/nl). 405 of 589 patients (690lo) had
organ.confined disease (pT2) while the remaining 3lolo
had non organ-confined disease (pT3 and pT4). Using the
Gleason-score (GS) system, the sample population was
divided into low.gnde tumon (GS 2"6, n - 282), inrermediate case (GS 7, n = tZ5), and high.grade prostate carcinomas (CS 8-10, n - 68).
A
nested-PCR stategy was used to detea XMRV-specific
viral RNA8 in the total RNA of prostate tissue sample in
which rhe first round RT.PCR was performed as described
above sing h-For and In-Rey followed by a quantitative
real.time PCR published by Donger al., 2007 [i, l4]. As an
intemal control, a human GAPDH spicific primer and
probe set were included in which the primers for the outer
RT-PCR were tie same as for the inner PCR: foruard 5 -
CGCGATGCTGCCGCTGAGTAG3' serse S'-TCGTG
CACACCCATGACCA-3' and the probe 5 -YAK-TICAC.
.CACCATGGAGAAGGCTCGG.Edipse Dark quenc}er-3'
[1s ].
Nuclclc ocld lsolotlon
Frozen tissues were mechanically sliced and imrnediately
lysed in DNA- or RNAJysis buffer, column-purified, and
pl)
according to rhe manufacturss
instructions (QIAamp DNA Mini Kit, RNeasy Mini Kir"
QIAGEN GmbH, Hilden, Germany). The ODruo7rro ratio
and nucleic acid concentrations were determined usine
the Nanodrop-1000 instrumenr (Peqlab Biotechnologii
GmbH, Erlangen, Gemany). In addition, RNA samples
were checked for integrity using a Bioanalzyer-2100 (Agilent Technologies, Inc., Santa Clara CA, United States).
No additional maco-/mico-dissections were performed
on the prostate dssues because viral nucleic acids were
eluted (50-200
expected to be present preferentially
in the stromal com-
panmen$.
Dlagnoaic PCR
A nested PCR was dweloped for the detection of XMRV
sequences thal amplifies regions upstream ofthe gag stan
codon, harboring the unique 24 nt delerion [101. First, we
constructed by fusion-PcR a syntletlc gene reprsenting
the region from nudeotide I ro 8OO of the MLV DG-75
(Genbank acc. number 4F2.t065). This fragment was
cloned into the pCR4-TOPO vector (lnviuogen, Karlsruhe, Cemany), and the identity of the fragrnent was
confimed by sequencing. The same procedure was used
to done a conesponding 800 nt fragment for use as a pos.
itiv€ control of rhe XMRV genome (Genbank acc. num.
8F18s282). Conditions of first round PCR for the detec.
tion of pro)'iral sequences were: 100 ng patient DNA,
primer Out-For 5'-CCGTGTTCCCAATAMGCCT-3', OutRw 5 -TGAC,ATCC{CAGACTCGTTG-3', (30 sec @ 94"C,
30 sec @ 60'C,30 sec @ 72'C) 20 cycl6. Using 1/106
'
of the fint reacdon and primer In-For S'-GCAGCCCTCCGAGACCTG3' and In-Rev 5'-CGGCGCCGTITCGGCG-3'the second round PCR is able to detect mvXMRV-
Page 2 ofll
ωage nmoernd 7orcrallon ρ
"刺
'
2OOg , A:92
RNoscL g€notyPrng
A real-drie PCR s€tup designed by Olfen LandqrIIB MOL
BIOL, Berlin was used for RNaseL genotyping of tumor
samples which detecls rhe single nucleotide polymorphism G1385A (rs486907) responsible for the R462Q
mutation. PCR was caried out with R462Q_F CCTATTMGATCTITTCTGGTIGCAG, R462Q-A GGAACATGT.
GGAAAATGAGGAAG and the probes R462Q_(A) YAKATTTGCCCAAAArcTCCTGTCATGBBQ and Ra62Q_(c)
FAM.ATITGCCCGAMIGTCCTGICATGBBQ following
two-step protocol with 95.C for 20 sec md 60.C for I
min. Positive controls were constructed by fusion PCR"
staning with 40 mer oligonucleotides, ofthe two 292 bp
fragments corresponding to the "R'- and "Q' versions of
$e RNAseL genomic region and doning these into the
pCR4-TOPO vector (lnvitrogen). For each PC& positive
control plasmids containing the R- or Q-sequence were
induded.
a
Rscombladnt Protclns, lmmunlzqtlon
Recombinant protein! of XMRV pr65 (Gag) and gpTO
(Env/SU) were generated for immunization and for the
ELISA assays. For XMRV Env/SU, a fragmenr containing
the amino acids l-245 of the surface unit (gp7o) h?s
amplified, cloned in pETl6b vector (Novagm, Gibbslown, USA) and *pressed in Bl21 E. coli. For XMRV Gag
(pr65), two fragments (amino acids 1.272 and 259-535)
that overlap by 14 amino acids were constructed. The
eeressed proteim were aftinity purified using a Ni-NIA
column and eluted in 8 M urea, and proteins for immunization were subsequently dialyzed against phosphate
buffered saline. BALB/c mice were immunized with tlre
recombinant fragrrents of the Envelope or Gag proteins,
and sera were collected throughout the period of four
htp://ww,retovirology.com/conbnU6/1192
immunizatjons. All animal enperimentr were performed
in accordance with institutional and state guidelines.
EL'SA
TWo weeks after the last immunization the mice were
bled, ud serum antibodies were memred by solid phase
enzymeJinked iminunsorbent assay (ELISA): Briefly,
bacterially epressed md purified (via Hls-tag) protein
fngments wse coated ovemight on Probind-96.well
plates (Be(ton Dickinson Labware Europe, h Pont de
Clai:l France) at room temperature in equimolar
amounts. The plates were blocked with 2% Mawel milk
powds in phosphate buffred saline (PBS) for 2 h at
37"C, washed thr€e times wi& PBS, 0.050/6 Tt{een 20 and
serial diluted mouse seni or patient ien at a 1:2OO dilution in PBS with 2olo milk powder and 0.05% TWeen2O
added into each well. Afterincubation for I hour at 37.C,
each well wro again washed three times and a 1: 1OOO dilution of a goat anti-mouse I8G-HRP con ugate (Sigma
Aldrich, Munich, Gemany) in PBS, 2% milk powder,
0.050/o TWeen' 20 (Serva Heiddberg Germany) was
added. After further inobation for I hour at 37.C each
well wro again washed three times. The chromogen orthophenylendiamin (OPD) in 0.05 M phosphare-citrate
buffa, pH 5.0 containing 4 pl of t 30o/o colution of rhe
hydrogen peroxide substnt€.per l0 ml was then added:
Afts 5-10 minutes the color developmot was stopped by
addltion ofsulphuric acid and the absorbance at 492 nm/
620 nm was measured in a micoplate reader.
Patient sera were tested for )OvlRV-Gag or .Env binding
andbodies in the same way, using a goat anti-human IgCHRP coniugate as secondary antibody (Stgrna Aldridr,
Munlch, Germany). Out of the 146 sera sarnples only
flom 30 patients the concponding nudeic acids were
induded in the 589 DN/y'RNA samples.
lnmunofluorctccaca mlcrct opl
Cells were grown on gelatine (0.370 coldwater flsh gelatine in distilled water) coated glas slidc in l2-well plates
and 24 h after seedingwere transfected using Polyfect Rea-
gent (QiaSen) with the full length moleolar done
pCDNA3.1-VP62 or with the pTH-XMRV-coEnv or pTH-
)(MRV-coGAG plasmids containing codon optimized synthetic full-length genes ofthe XMRV gny orgagunder control of the CMV prorioter. 48 h aft€r tansfection the cells
were fixed with 20lo fomaldehyde (Sigrna) in pBS, Cells
were rimed briefly in PBS, permeabilized rl'irh 0.5% Triton X-100 in PBS for 15 min and washed 3 times wirh
PBS.
After 30 min inobation with blocking buffer
(2026
Maruel nilk powder in PBS) cells were incrbated for 60
min at 37'C witlr the mouse or human antisera diluted
1:200 in blocking buffer. The slides were washed extensively with PB,S. The secondary antibodies conjugated to
fluorophores were added for 30 min. After thorough
@agc
Pag. 3 ot 11
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