Injury-elicited stressors alter endogenous retrovirus expression in lymphocytes depending on cell type and source lymphoid organ.

0.66 MB·English

by  Lee, Kang-Hoon

Leeetal.BMCImmunology2013,14:2
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RESEARCH ARTICLE Open Access
Injury-elicited stressors alter endogenous
retrovirus expression in lymphocytes depending
on cell type and source lymphoid organ
Kang-Hoon Lee, Debora Lim, Tajia Green, David Greenhalgh and Kiho Cho*
Abstract
Background: Murine leukemia virus-type endogenous retroviruses (MuLV-ERVs) constitute ~10% ofthemouse
genome and are associated with various pathophysiologic processes. Inthis study, we examined whether
MuLV-ERVs’ response to burn-elicitedstressors is specific for certain lymphocyte populations and/or locations of
lymphoid organ.
Results: B-and T-cells, which were sorted from nine lymphoid organs of C57BL/6J mice after burn, were subjected
to MuLV-ERV expression analyses. Overall, the post-burn MuLV-ERV expression pattern was dependent on
lymphocyte type, time after injury, location of lymphoid organ, and MuLV-ERV type. For instance,theMuLV-ERV
expression in T-cells from thethymus and three cervical lymph nodes decreased at3 hours post-burn whilethe
expression of some MuLV-ERVswas augmentedin B-cells derived from themesenteric lymph node. The MuLV-ERV
U3 sequences population of theburn-24 hours group was less diverse in comparisonto the noburnand
burn-3 hours groups. In addition, it was apparent that atthe24 hours time point, the U3 populations ofB-cells
from both noburn and burngroups were less heterogeneous than theT-cells’U3 populations. Using the U3
sequences, some of which were isolated only from specific experimentalgroups (B- vs. T-cells; no burnvs. burn), as
probes, 51 putative MuLV-ERVs,including 16 full-length proviruses, were mappedfollowed by characterization of
their biologic properties.
Conclusion: MuLV-ERVs’ response to burn-elicitedstressorsmay be differentially controlled dependingon
lymphocyte type, location of lymphoidorgan, MuLV-ERV type, and stress duration.
Keywords: B-cell, T-cell, Burn, Stress, Murine leukemia virus-type endogenous retrovirus
Background essentialfor virionassembly[2-4].ExpressionofMuLV-
Proviral sequences of the murine leukemia virus-type ERVsismostlycontrolledbytheirU3promoterslocated
0
endogenous retroviruses (MuLV-ERVs) are genomic onthe5 LTR,inconjunctionwiththetranscriptionma-
footprints reflecting the ancient infection of germ-line chinery that is dynamically and uniquely formed within
cellsandarepresumedtoconstitute~10%ofthemouse individualcells[5].
genome [1]. Although most MuLV-ERV genomes are Recentstudiesprovideevidencethathumanendogen-
reported to be defective primarily due to mutation and ous retroviruses (HERVs) are involved in normal physi-
recombination events, some retain intact proviral gen- ology as well as various disease processes [6]. The
ome features, such as identical long terminal repeats HERV-W envelope protein, called syncytin, is highly
(LTRs) at both ends, which are formed by retroviruses’ expressed in the syncytiotrophoblast layer and plays a
unique replication process, and coding potential for the critical role in placental differentiation [7]. In addition,
major structural genes (gag, pol and env) which are the inflammatory properties of syncytin contribute to
the degeneration of oligodendrocytes responsible for
*Correspondence:[email protected] the development of multiple sclerosis, an autoimmune
DepartmentofSurgery,BurnResearch,ShrinersHospitalsforChildren
disease[8].
NorthernCalifornia,UniversityofCalifornia,Davis,Sacramento,CA95817,
USA
©2013Leeetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Patients with severe burn injury often succumb to sys- without any pooling. The burn experiment was repeated
temic immune disorder in conjunction with internal fourtimes.
organ damage [9]. However, the mechanisms underlying
the post-burn pathologic changes have not yetbeen fully Cellsorting
characterized. The results from recent studies suggest The collected tissues were minced, suspended in a tryp-
that post-burn systemic inflammatory response is asso- sin/EDTA solution, and then passed through a cell
ciated with the activation and/or depletion of specific strainer (BD Biosciences, CA). The cells were washed
lymphocyte subpopulations, such as CD4+CD25+ T regu- with PBS containing 0.1% BSA followed by centrifuga-
latory cells, lymphocytes expressing CD25, CD69 and tion at 1,000 ×g for 5 min at 4°C. Following the removal
CD71, NK-T-cells, and CD8+ T-cells [10-12]. Sepsis- of red blood cells, cells were sorted into B- and T-cells
elicited stressors induce apoptosis in the spleen, resulting using pan-B and pan-TDynabeads (Invitrogen, Carlsbad,
in the depletion of B-cells and accompanying immune CA), respectively, according to the protocols provided
malfunction [13]. However, the central signaling mole- bythemanufacturer.
cules responsible for the post-burn activation and/or de-
pletionoflymphocytesremaintobeelucidated. TotalRNAisolationandRT-PCR
Our recent studies demonstrated that burn-elicited Total RNAswereisolatedfrom thesortedcellsusingan
stressors differentially alter the expression of MuLV-ERVs RNeasy Mini Kit (Qiagen, Valencia, CA) following
in a range of internal organs, including various lymphoid homogenization using a QIAshredder (Qiagen). cDNAs
tissues, in a ERV type- and organ-specific manner [14]. were synthesized using Sensiscript reverse transcriptase
Moreover, some MuLV-ERV envelope proteins were able (Qiagen), RNase inhibitor (Promega, Madison, WI) and
0
to modulate the mRNA expression of inflammatory cyto- an oligo-dT primer (5-GGC CAC GCG TCG ACT
kines such as IL-6 and IL-1β in macrophage cells [15]. In AGT ACT TTT TTT TTT TTT TTT T- 30). A set of
0
this study, we hypothesize that the differential post-burn primers, ERV-U1 (5-CGG GCG ACT CAG TCT ATC
0 0
alterations in MuLV-ERV expression, depending on GG-3) and ERV-U2 (5-CAG TAT CAC CAA CTC
0
lymphocyte types and/or lymphoid organs, may be asso- AAA TC-3), was used to amplify the MuLV-ERV U3
ciatedwithsystemicpathogeneses,includingimmunedis- regions[17].β-actin,servingasanormalizationcontrol,
order. We examined whether MuLV-ERVs’ response to was amplified with the primer set: 50-CCA ACT GGG
0 0
burn-elicited stressors is specific depending on lympho- ACG ACA TGG AA-3 and 5-GTA GAT GGG CAC
0
cyte type and the location of lymphoid organ followed by AGTGTGGG-3.
an investigation of the biologic characteristics of the rele-
vantMuLV-ERVs. Measurementofrelativepurityofsortedcellpopulations
The relative purity of the sorted cell populations was
Methods measured by real-time RT-PCR using CD20 (B-cell anti-
0
Animalexperiments gen) primers (5-AAA ACC TCC AGG AAG AGT TTG
0 0
Theexperimentalprotocolsinvolvingmicewereapproved GTC-3 and 5-CGA TCT CAT TTT CCA CTG GCA
0
by the Animal Use and Care Administrative Advisory AG-3)for B-cells andThy1(Thymuscell antigen-1)pri-
0 0 0
Committee of the University of California, Davis. Female mers (5-TCC TGC TCT CAG TCT TGC-3 and 5-
0
C57BL/6J mice (12 weeks old) were purchased from AGT AGT CGC CCT CAT CCT TG-3) for T-cells.
Jackson Laboratory-West(Sacramento, CA). The baseline Real-time RT-PCR was performed using the Brilliant-III
MuLV-ERV expression patterns were examined in nine SYBR Green QPCR kit from Agilent Technologies (La
lymphoidorgans(spleen,thymus, andsevenlymphnodes Jolla, CA) and the results were analyzed using the
[axillary, inguinal, mesenteric and pooled cervicals {man- MxPro (v.4.10) program (Agilent Technologies). Cell
dibular, accessory mandibular, superficial parotid, and type specific mRNA levels (CD20 and Thy1) were calcu-
deepcervical}]).Threemiceweresacrificedfortissue col- lated from each sorted cell population with the level of
lectionbycervical dislocationwithout anytreatment.The β-actin as a normalization control, using the same pri-
burn protocol has been described previously [16]. Briefly, mer set as above. The relative purity of each cell popula-
undergeneralanesthesia,an~18%totalbodysurfacearea tion was calculated with the assumption that there are
flameburnwasgeneratedontheshavedbackofmicefol- only B- and T-cells in each sorted cell population and
lowed by resuscitation with saline and buprenorphine. CD20 and Thy1 are exclusively expressed in B-cells and
Control (no burn) mice were treated the same as burn T-cells,respectively.
mice but without the flame burn. The three mice from
each experimental group were sacrificed by CO inhal- Densitometryandstatisticalanalysis
2
ation at 3 hours and 24 hours after burn followed by the The density of the PCR products was measured using
collection of the same nine lymphoid organs as above KODAK Molecular Imaging Software Version 4.5.1
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(Eastman Kodak Company, Rochester, NY). All optical b) no burn-B-cells, burn-24 hours-B-cells, no burn-T-
densities were measured based on mass values with the cells,andburn-24hours-T-cells.
™
Hi-Lo DNA marker (a gift from Minnesota Molecular
Inc., Minneapolis, MN). The density of each MuLV-ERV InsilicomappingofputativeMuLV-ERVsand
U3 band was normalized to its corresponding β-actin characterizationoftheirbiologicalproperties
value to obtain the relative intensity of each U3 band. The C57BL/6J mouse genome database from the Na-
Differences in the relative intensity of the matching tional Center for Biotechnology Information (NCBI) was
MuLV-ERV U3 bands between no burn and burn were surveyed for putative MuLV-ERVs with a greater than
calculated as fold changes. Statistical significance (p< 98% identity using the 100 unique U3 sequences as min-
0.05) of fold changes between the matching pairs (no ing probes and the Advanced BLAST program (NCBI).
burn and burn) of the individual U3 bands was deter- The coding potential of each MuLV-ERV was deter-
mined using the data from four burn experiments and mined using the Vector NTI program (Invitrogen).
Student’st-test. Three polypeptide genes (gag, pol, and env) were noted
as intact (+), partial (P), or defective (−) relative to the
Cloningandsequencing references (AY219567.2 and AF033811). Each MuLV-
Eighty-eight MuLV-ERV U3 PCR amplicons/bands, ERV was mapped with the chromosomal location and
which had contrasting densities between individual no strandorientation.Theprimerbindingsite(PBS)ofeach
burn and burn pairs, were selected for sequence ana- MuLV-ERV was identified by examination of a stretch of
0
lyses. The PCR amplicons were purified using the QIA- 18 nucleotides immediately downstream of the 5 LTR
quick Gel Extraction kit (Qiagen) and then cloned into region [22]. In addition, the annotated gene(s) within
W
the pGEM -T Easy vector (Promega). Three clones 100 Kb upstream and downstream of each MuLV-ERV
were prepared from each U3 PCR amplicon/band. The locus,ifany, weremapped.
plasmid DNAs were prepared using a QIAprep Spin
Miniprep kit (Qiagen) for sequencing analysis. Sequen- Results and discussion
cing was performed at a core sequencing facility at the Lymphocytetype-specificMuLV-ERVexpressionpatterns
University of California, Davis. inlymphoidorgansofnormalmice
Prior to the investigation of post-burn alterations in
Alignmentandphylogeneticanalyses MuLV-ERV expression in B- and T-cells of different
From a total of 260 U3 sequences, 100 unique U3 lymphoid organs (spleen, thymus, axillary lymph node
sequenceswereidentifiedusingSeqmanProVersion8.1.2 [LN], inguinal LN, mesenteric LN, and cervical LNs),
(DNASTAR, Madison, WI). Multiple sequence alignment baselineMuLV-ERV expression levels were examined in
ofthe100uniqueU3swasperformedusingtheClustalW B- and T-cells isolated from ~12 week old female
method in MegAlign (DNASTAR), and a phylogenetic C57BL/6J mice. The relative purity of the sorted cell
tree was generated by the HYPERTREE program [18]. populations was determined by measuring CD20(for B-
Seven U3 sequences, representing the seven phylogenetic cells)andThy1(forT-cells)levelsbyreal-timeRT-PCR.
branches, were subjected to a multiple alignment analysis The relative purity of the sorted B- and T-cell popula-
to examine the U3 sequence features of direct repeats (1/ tions was greater than 93%, except for the B-cell popu-
1*, 4/4*, 5/5*, and 6/6*), insertion of 190 nucleotides, and lation in the thymus (~65%). Throughout the lymphoid
unique region (2) based on the protocol by Tomonaga organs examined, the MuLV-ERV expression pattern,
0
etal.[17,19]. which was examined by RT-PCR amplification of the 3
U3 regions, was substantially different between B- and
Profilingoftranscriptionregulatoryelements T-cells (Figure 1). The expression pattern of an intense
The 100 unique MuLV-ERV U3 sequences were surveyed ~500 bp and two (~750 bp and ~450 bp) lighter U3
for putative transcription regulatory elements using the bands was shared by B-cells derived from all lymphoid
MatInspector program (Genomatix, Munich, Germany) organs examined. In contrast, four distinct U3 bands,
within the vertebrate matrix group with a core similarity ranging from ~500 bp to ~750 bp, were amplified in T-
higherthan0.95[20]. cells with variable intensity depending on the source
lymphoid organs. It was interesting to note that the
DeterminationofpopulationdiversityofU3sequences ~500 bp U3 band was more intense in B-cells from all
withineachexperimentalgroup four LN samples compared to B-cells from the spleen
Using the MEGA4 program [21], the population diver- and thymus. The findings from this study suggest that
sity of U3 sequences was measured in each experimental the expression of MuLV-ERVs is differentially regulated
groupandcomparedamongdifferentgroupsintwoana- depending on the lymphocyte type, source lymphoid
lyses: a) no burn, burn-3 hours, and burn-24 hours, and organ,andERVtype.
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Figure1MuLV-ERVexpressioninB-andT-cellsderivedfromlymphoidorgansofC57BL/6Jmice.MuLV-ERVexpressionwasexaminedin
B-andT-cellsisolatedfromthespleen,thymus,andsevendifferentlymphnodesoffemaleC57BL/6Jmicewithoutanytreatment.Aschematic
drawingofatypicalMuLV-ERVproviralstructureindicatestherelativelocationsofasetofprimersusedforamplificationoftheU3regions.
DifferentialMuLV-ERVexpressionpatternswereobservedinB-andT-cellsderivedfromvariouslymphoidorgans.LN(lymphnode),SM(sizemarker).
Burn-elicitedalterationsinMuLV-ERVexpressionin the thymus and three cervical LNs (mandibular, accessory
lymphocytesdependingonlymphocytetype,source mandibular, and superficial parotid), but not in theT-cells
lymphoidorgan,andERVtype fromthedeepcervicalLN(Figure2).Incontrast,therewas
The effects of burn-elicited stressors on the expression of an increase in intensity of one to two U3 bands in theT-
MuLV-ERVsintheB-andT-cellsfromthespleen,thymus, cellsfromthethymusandsuperficialparotidcervicalLNat
and seven lymph nodes were examined at 3 hours and 24hourspost-burnwhentherewasadecreaseinU3bands
0
24 hours after injury by amplifying the 3 U3 regions. At intheT-cellsofthespleenandmesentericLN.
3hourspost-burn, theMuLV-ERV expression, represented In B-cells, there was an induction of two (a and b
byfourdistinctU3bands,wasdecreasedintheT-cellsfrom bands) of the three U3 bands in the mesenteric LN at
Figure2Post-burnalterationsintheexpressionofMuLV-ERVsinB-andT-cellsisolatedfromlymphoidorgansofC57BL/6Jmice.Post-
burn(3hoursand24hours)changesintheexpressionofMuLV-ERVsweremeasuredintheB-andT-cellsisolatedfromninedifferentlymphoid
organs.Theresults,whichwerepresentedasafoldchange(burn/noburn)fortheindividualMuLV-ERVU3bands(a,b,andcforB-cells;d,e,f,
andgforT-cells),revealedauniquepatternforeachexperimentalgroup(cell/organ/time).SPL(spleen),THY(thymus),LN(lymphnode),AXI
(axillaryLN),ING(inguinalLN),MES(mesentericLN),MAN(mandibularcervicalLN),ACC(accessorymandibularcervicalLN),SUP(superficial
parotidcervicalLN),DEE(deepcervicalLN).Ablackarrowindicatesunmeasuredsamplesduetothelowexpressionlevel.Errorbarsindicate
standarderrorofthemean.*P<0.05.
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3hours post-burnwhiletwo bands (aand c) weredown
regulated in the B-cells of the mandibular cervical LN.
However, at 24 hours post-burn, the expression levels
of all three U3 bands were increased in the B-cells from
the mandibular cervical LN. Also at the 24 hours time
point, post-burn increases in one or two U3 bands were
also observed in the B-cells of the spleen, thymus, axil-
lary LN, inguinal LN, and/or accessory mandibular
cervicalLN.
Theresultsfromthisstudysuggestthatthelymphocyte
type (B- or T-cell), source lymphoid organ, and duration
afterinjuryplayaroleinpost-burnchanges (inductionor
repression)intheexpressionofMuLV-ERVs.Itispossible
that the post-burn differential expression of MuLV-ERVs
resulted from changes in the composition of the B- and
T-cell sub-populations (e.g. naïve vs. effector cells), which
have different transcription environments, instead of
(and/or in addition to) the affected MuLV-ERVs’ altered
transcriptional potentials. It was interesting to note that
Figure3Phylogeneticrelatednessof100uniqueMuLV-ERVU3
the post-burn increase in the MuLV-ERV expression in
sequences.Phylogeneticrelatednessofthe100uniqueMuLV-ERV
theB-cellsfromthemesentericLNparallelstheprolifera-
U3sequenceswasdeterminedbyamultiplealignmentanalysis.The
tion of specific B-cell populations in the mesenteric LN MuLV-ERVU3sequencesineachbranchwereanalyzedfortheir
andspleenafterburn[23]. presenceinaspecificlymphocytetype(B-orT-cell)and/orstress
condition(noburnorburn).Sevenbrancheswereformed,primarily
basedonthesizeoftheU3sequences.Numberofsequencesis
MuLV-ERVU3sequencesidentifiedinspecificlymphocyte
inparentheses.
typeand/orinjury(noburn/burn)condition
Three clones from each of the 88 MuLV-ERV U3 ampli-
cons/bands, which showed contrasting expression levels saturationcloningprotocolwouldfindthatsomecelltype
between the individual no burn and burn pairs, were and/or burn-specific U3 sequences are not exclusively
sequenced to identify lymphocyte type- and/or injury present in one group over the other. In this case, a statis-
(no burn/burn)-specific U3 sequences. From a total of ticalevaluationmaybeneededtodeterminethespecificity
260MuLV-ERVU3sequences cloned from the entire set of the U3 sequences in regard to the lymphocyte type
of MuLV-ERV U3 bands examined, 100 unique U3 and/orburn-elicitedstressors.
sequences, ranging from 346 nucleotides to 615 nucleo-
tides in size, were identified (Additional file 1: Table S1). CharacteristicsofsevenU3sequencesrepresentingthe
An initial phylogenetic analysis resulted in seven distinct phylogeneticbranches
branches of U3 sequences, primarily segregated by their Seven representative U3 sequences, which were chosen
size (group I [largest U3s]~group VII [smallest U3s]) fromthesevenmainphylogeneticbranches,weresubjected
(Figure 3). Group III (440 bp length) had the most num- to an alignment analysis using reference sequences to
ber of U3 sequences (40) followed by group VII (346 bp characterizetheirsequencefeatures,suchasdirectrepeats,
length) (25 sequences) then group I (greater than 603 bp unique sequence, and an insertion of 190 nucleotides.
length)(19sequences).TherewereonlytwoU3sequences TheseU3sequencefeatureswereutilizedaskeyparameters
in groups IVand VI (433 bp and 361 bp in size, respect- for the in silico determination of the MuLV-ERV tropism
ively). The group II U3 sequences (5) were isolated only traits[19].TheU3sequencesofthegroupsIandIIhadan
from T-cells and the group VI U3 sequences (2) were insertion of 190 nucleotides, which is consistent with their
unique for B-cells. On the other hand, the group IV U3 relatively large size compared to the other five branches
sequences (2) were identified only from no burn control (Table 1). There was a well-conserved TATA box in all
mice. The other four branches (groups I, III, V, and VII) sevenU3sequences;however,eachU3sequenceretaineda
consisted of U3 sequences isolated from both lymphocyte unique profile in regard to the direct repeats (1/1*, 4/4*,
typesderivedfrombothnoburnandburnmice. 5/5*, and 6/6*) and the unique sequence (2). The specific
Theresultsobtainedfrom thisstudysuggestthat some featuresoftheindividualU3sequencesweretranslatedinto
MuLV-ERVs are selectively expressed depending on the tropism traits of the respective putative MuLV-ERVs
lymphocyte type (B- or T-cell) and/or presence of burn- [17,19]. However, the sequences features of the group VI
elicited stressors.However,itispossiblethatanexpanded U3sequencesweretoodiversetopredictitstropismtrait.
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Table1SequencecharacteristicsandputativetropismtraitsofsevenMuLV-ERVU3sequencesrepresentingtheseven
mainphylogeneticbranches
Repeat/uniqueregion Insertion
Group U3Probe Size(bp) TATA Tropism
1/1* 2 4/4* 5/5* 6/6* (190bp)
I B -T -1 603 +/+ + + + +/+ + + P-II
3H TH
III B -T -3 556 +/+ (cid:129) + + +/+ + + P-I
3H IN
III B -T -7 440 +/+ + + +/+ +/+ + (cid:129) X-II
3H TH
IV NB -T -3 433 +/+ (cid:129) +/+ +/+ + + (cid:129) X-II
24H SC
V B -T -6 392 +/+ + + +/+ +/+ + (cid:129) X-I
3H TH
VI B -B -9 361 +/+ (cid:129) (cid:129) +/+ +/+ + (cid:129) (cid:129)
3H ME
VII B -T -11 346 +/+ + +/+ (cid:129) +/+ + (cid:129) X-II
3H TH
Sequencecharacteristics:directrepeats(1/1*,4/4*,5/5*,and6/6*),uniqueregion(2),insertionof190nucleotides,andTATAbox;+/+(directrepeat),+(single
copy),(cid:129)(absence).
Table2DifferentialtranscriptionpotentialsofMuLV-ERV
Evidenceforpost-burnincreaseintheexpressionof
U3-promotersequences
specificMuLV-ERVs
Occurrence
The population diversity of the U3 sequences (a total of TRE Consensusmotif
Burn/Noburn
260) was measured in each experimental group and
compared among the groups in two different analyses DMRT ctttcttGTAActtttgagaa 68 / 1
[21]: i) no burn, burn-3 hours, and burn-24 hours, and BRNF tctggatctatTGATttga 35 / 4
ii) no burn-B-cells, burn-24 hours-B-cells, no burn-T- BCL6 ctctttcTGGAaactga 31 / 3
cells, and burn-24 hours-T-cells. The results from the TF2D gctctggtactttttcatcccttgcaaaatGGCGttact 27 / 1
first diversity analysis revealed that the U3 population of PTF1 gccaTCTGttcttggccctga 28 / 1
the burn-24 hours group was substantially less heteroge-
MYT1 tgagAGCTcagct 27 / 2
neous compared to the other two groups (no burn and
AIRE gtatttctcggtcatttGGGGaaactg 19 / 0
burn-3 hours) (Figure 4). In contrast, it appears that
ZNFP cacaCACCtggtc 22 / 0
there was an increase in diversity among the U3 popula-
NRF1 gggGCCCaggcgcttga 21 / 1
tion intheburn-3hoursgroup.Thefinding ofdecreased
Occurrence
U3 population diversity within the burn-24 hours group TRE Consensusmotif
B-cell/T-cell
PAX2 gttattcggggaacctgAAACtg 32 / 13
HAML ctgaGTGGttagttc 35 / 8
NKXH acttttgaGAACttagctc 33 / 10
HOMF taatctTTAActgttaatc 16 / 37
SORY gttctaaaCAATgtctattttca 14 / 28
HEAT gagagggtctaatGAAGgttcaaca 10 / 30
MAZF atggGAGGggtac/gtacGAGGagagg 8 / 18
MEF2 acaatgtCTATtttcaagaaatg 5 / 19
GATA tttAGATctaaac 6 / 16
CART gggtcTAATgaaggttcaaca 8 / 16
HOXF gggtcTAATgaaggttcaa 7 / 17
LHXF gagagggtcTAATgaaggttcaa 7 / 16
FXRE AGGTgcttgacca 5 / 19
GRHL cacacaGGTTcaa 7 / 16
NKX6 atctTTAActgttaa 6 / 16
CEBP aaggttcaGCAAtgg 6 / 15
Figure4DecreasedpopulationdiversityofMuLV-ERVU3
sequencesat24hoursafterburn.TheU3populationdiversity FKHD tagttcTAAAcaatgtc 5 / 16
withineachexperimentalgroupwasmeasured[21]andtherelevant PDX1 gaaagagtcTAATgaaggt 5 / 15
values,indicatingdifferentdiversitylevels,werepresentedas
RXRF cacagGTTCaaggagtggccagagc 5 / 16
nucleotidediversityandTajimastatisticsscores.Therewasamarked
decreaseintheU3populationdiversityat24hoursafterburn NumbersindicatetheoccurrencefrequencyofspecificTREs(transcription
comparedtonoburn. regulatoryelements)withinthe100uniqueU3-promotersequences.Please
referto(Additionalfile3:TableS3)forthecompletelistofTREabbreviations.
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Table3GenomicmapandcharacteristicsofputativeMuLV-ERVsharboringtheuniqueU3sequences
Location Virus ORF Occurrence
No Virus Chr Ori PBS Reference
From To size(bp) gag pol env frequency
1 MuERV-49 1 133539144 133548197 + 9054 Q - + + U/U I,II,III,IV
2 MuERV-31 1 166153938 166159683 + 5746 Q + - - U/T I,II,IV
3 MuERV-30 1 184187251 184196231 + 8981 Q + P + U/T I,II,IV
4 MuERV-33 1 193757492 193766472 + 8981 Q + P - U/T I,II,IV
5 MuERV-35 2 57066823 57075803 - 8981 Q + + + U/T I,II,IV
6 MuERV-3 2 156182292 156189341 - 7050 Q + - + U/U III,IV
7 MuERV-48 3 66900000 66907716 + 7717 Q + - - U/U I,II,III,IV
8 MuERV-55 3 151986104 151995146 + 9043 Q + + + U/U I,II,III,IV
9 MuERV-29 4 101535762 101544743 - 8982 Q + P + U/T I,II,IV
10 MuERV-28 4 107818639 107827621 - 8983 Q + + + U/T I,II,IV
11 MuERV-2 4 132651872 132657538 + 5667 Q - - - U/U I,II,III,IV
12 MuERV-6 4 133715306 133720617 + 5312 Q + - - U/U I,II,III,IV
13 MuLV_ERV * 4 145686198 145694956 - 8759 Q + + + U/U thisstudy
4-5
14 XMV9 4 146592937 146601658 + 8722 Q + + + U/U V
15 L-3-9.5 5 23206397 23215063 - 8667 P + + + U/U I,II
16 MuLV_ERV * 5 23721126 23727971 - 6846 Q + - + U/U thisstudy
5-2
17 MuERV-27 5 33864636 33873616 + 8981 Q - + + U/T I,II,IV
18 MuERV-18 5 44525599 44534580 + 8982 Q - + + U/T I,II,IV
19 MuERV-19 5 77359541 77368522 + 8982 Q + + + U/T I,II,IV
20 MuERV-26 7 7022622 7031603 - 8982 Q P - - U/T I,II,IV
21 MuERV-24 7 30400226 30408924 + 8699 Q + + P U/T I,II,IV
22 MuERV-25 7 31473659 31482639 + 8981 Q + P - U/T I,II,IV
23 MuERV-34 7 123575623 123581296 + 5674 Q + - - U/T I,II,IV
24 MuERV-10 8 44397084 44405817 - 8734 Q + + + U/T I,II,IV
25 MuERV-11 8 87649754 87656141 - 6388 Q + P - U/T I,II,IV
26 MuERV-1 8 93410190 93415857 + 5668 Q - - - U/U I,II,III,IV
27 MuERV-12 8 122413465 122420940 - 7476 Q - - + U/T I,II,IV
28 MuERV-8 9 41713568 41720351 - 6784 Q - + + U/U I,II,IV
29 iLN(III)-1a 9 62286981 62295739 + 8759 Q + + + U/U I
30 K-4-11.10a 10 8263933 8271066 - 7134 Q - - - U/T III
31 MuERV-53 10 22454084 22463128 + 9045 Q + + + U/U I,II,III,IV
32 MuERV-17 10 41156454 41165434 + 8981 Q + + + U/T I,II,IV
33 BM-a-2.11e 11 5905655 5911518 + 5864 Q - - - B/B III
34 MuERV-22 11 6649425 6658394 - 8970 Q + - + U/T I,II,IV
35 MuERV-23 11 820301 8829280 + 8980 Q + + + U/T I,II,IV
36 MuERV-50 11 60392151 60399510 - 7360 Q - P - U/U I,II,III,IV
37 MuERV-51 11 76362310 76371354 + 9045 Q + + + U/U I,II,III,IV
38 MuERV-38 11 88751494 88758280 - 6787 Q + - + U/T I,II,IV
39 MuERV-37 12 20704753 20710513 + 5761 Q - - - U/T I,II,IV
40 MuERV-16 12 55875211 55884192 + 8982 Q + + + U/T IV
41 MuERV-5 13 67985825 67994511 - 8687 Q + + + U/U I,II,III,IV
42 MuERV-21 13 99756230 99765210 - 8981 Q + P + U/T I,II,IV
43 MuERV-7 14 54912048 54917591 - 5544 Q + - - U/U IV
44 L-1-2.14 14 55149203 55157025 - 7823 Q P + + U/U I,II
45 MuERV-15 15 76390247 76397432 - 7186 Q - - + U/T I,II,IV
46 U-1-5.16 16 76135288 76144269 - 8982 Q - P - U/T I,II
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Table3GenomicmapandcharacteristicsofputativeMuLV-ERVsharboringtheuniqueU3sequences(Continued)
47 MuERV-14 16 93697702 93706682 - 8981 Q - + + U/T I,IV
48 MuERV-20 18 82862943 82871822 + 8880 Q - + + U/T I,II,IV
49 MuERV-32 19 38449512 38456573 + 7062 Q - P - U/T I,II,IV
50 MuERV-9 19 61001210 61009936 - 8727 Q + + + U/U I,II,IV
51 MuERV-13 X 15051174 15060154 + 8981 Q + P + U/T I,II,IV
Chr(chromosome),Ori(orientation),PBS(primerbindingsite),Q(tRNAglutamine),P(tRNAproline),ORF(openreadingframe),+(intact/full-length),-(defective),U
(bothB-andT-cells;bothnoburnandburn),B(B-cell),T(T-cell),referencesI[14],II[33],III[31],IV[30]andV[32].*MuLV-ERVnewlyidentifiedinthisstudy.The
namesofMuLV-ERVs,whichhavebeenreportedinpreviousstudies,areadaptedfromtherelevantreferenceinbold.
wasconfirmedbyitsnegativescorefortheTajima’sNeu- It is probable that theTREs (e.g., BRNF, AIRE, NRF1),
trality test in comparison to the positive values of the which were mapped on the U3 sequences almost exclu-
other two groups [24]. The results from the second di- sively derived from burn mice, are closely associated
versity analysis demonstrated that there is a decrease in with the modulation of MuLV-ERV expression in con-
U3 population diversity in both burn-24 hours-B-cells junction with a transcription environment dynamically
and burn-24 hours-T-cells groups in comparison to the formulated byburn-elicited stressors.
no burn controls. Furthermore, it was apparent that at
the 24 hours time point, the U3 population in B-cells Insilicocloningandcharacterizationofputative
from both no burn and burn were less diverse in com- MuLV-ERVsharboringtheuniqueU3sequences
parisontoT-cells’U3population. To investigate the biological properties of the putative
Thefindingsfrom thesediversityanalyses indicatethat MuLV-ERVs harboring the U3 sequences identified in
burn-elicited stressors lead to the induction of specific this study, the C57BL/6J mouse genome was surveyed to
MuLV-ERVs in a time after injury-dependent manner, maptheprovirallociusingthe100uniqueU3sequences
and the population of MuLV-ERVs expressed in T-cells as mining probes. From the in silico mining experiment,
ismore heterogeneousthanthe population inB-cells. 49 ERVs were found with two identical LTR sequences
at both ends and two, MuERV-49 and MuERV-26,
DifferentialtranscriptionpotentialsofMuLV-ERV retained slightly different LTR sequences due to a few
U3-promotersequences point mutations. Forty-nine of the 51 putative MuLV-
0
The U3 sequence on the 5 LTR, which is presumed to ERVsidentified from thissurvey,including15full-length
0
be identical to the U3 of the 3 LTR, serves as an ERV proviruses retaining a set of intact coding potentials for
promoter. In this study, the transcription potentials of gag, pol and env polypeptides, have been reported in
the 100 unique U3-promoter sequences were surveyed previous studies [14,30-33]. The two newly identified
for transcription regulatory elements (TREs). NineTREs MuLV-ERVs are: one full-length provirus (8,759 nucleo-
were predominantly present in the U3 sequences isolated tides) with intact coding potentials and one defective
frombothB-andT-cellsofburnmiceatthe3hoursand/ provirus (6,846 nucleotides) (Table 3). An analysis of the
or 24 hours time points compared to the U3 sequences primer binding site revealed that both MuLV-ERVs had
from no burn mice (Table 2 and Additional file 2: Table the tRNAGln priming sequence. All 51 putative MuLV-
S2). Among the nine TREs are BRNF (Brn POU domain ERVs were mapped using the U3 probes derived from
factors), AIRE (Autoimmune regulatory element binding both no burn and burn mice, except for BM-a-2.11e
factor), and NRF1 (Nuclear respiratory factor 1), which [31], which was identified using a U3 probe unique for
havebeenreportedtobelinkedtovariousstressresponses B-cells of burn mice. On the other hand, 30 putative
[25,26]. Furthermore, it appears that threeTREs occurred MuLV-ERV loci were retrieved using U3 probes exclu-
more often in the U3 sequences of B-cells than T-cells sively isolated from T-cells. Future studies focusing on
whereas the U3 sequences from T-cells had a higher the biological properties of these putative MuLV-ERVs
frequency of 16 other TREs in comparison to B-cells would be of interest: 1) effects (e.g., cytotoxicity) of
(Table 2 and Additional file 2: Table S2). Among the 16 their replication on immune cells in conjunction with
TREs,MAZF(Mycassociatedzincfingers),GATA(GATA infection followed by random integration into the
binding factors) and MEF2 (myocyte-specific enhancer genome and 2) functions (e.g., regulation of inflamma-
bindingfactor)havebeenreportedtobeinvolvedinT-cell tory mediators) of their gene products (gag, pol, and
activation [27-29]. A total of 122 TREs, including the 28 env polypeptides).
TREsdescribedabove,wereidentifiedfromthe100unique
sequences, and the comprehensive TRE profiles were IdentificationofgenesneighboringtheMuLV-ERVloci
summarized in Additional file 2: Table S2 and Additional The U3 promoter and the enhancer sequences embedded
file3:TableS3. in MuLV-ERVs can affect the transcriptional activities of
Leeetal.BMCImmunology2013,14:2 Page9of10
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the neighboring genes via the promotion/repression of
identifiedintheU3sequencesderivedfromspecificexperimental
transcription,and alternative splicingand polyadenylation groups:burngroup(green),B-cellgroup(yellow),andT-cellgroup(blue).
[34]. In this study, we identified the genes located within Additionalfile3:TableS3.Abbreviations:transcriptionregulatory
100KbupstreamanddownstreamofeachMuLV-ERVin- elements.
tegrationsite.Ofatotalof131genesidentified,ninegenes Additionalfile4:TableS4.Annotatedgenesneighboring100Kb
upstreamanddownstreamoftheindividualMuLV-ERVintegrationloci.
had MuLV-ERVs integrated into their introns (Additional
Chr(Chromosomenumber),Ori(Orientation).Grayhighlightindicatesthe
file 4: Table S4). It will be interesting to examine how the annotatedgenesharboringanERVintheintron.*MuLV-ERVnewly
expression patterns (depending on lymphocyte type or identifiedinthisstudy.PleaserefertoTable3fortheMuLV-ERVnames.
burn-elicitedstress)oftheseprovirusesarecorrelatedwith
theexpressionoftherelevantgenes,forexample,therela- Abbreviations
HERV:Humanendogenousretroviruses;LN:Lymphnode;LTR:Longterminal
tionship between BM-a-2.11e MuLV-ERV, which was
repeat;MuLV-ERVs:Murineleukemiavirus-typeendogenousretroviruses;
identified using a U3 probe uniquely identified from the PBS:Primerbindingsite;TREs:Transcriptionregulatoryelements.
B-cells of burn mice, and Camk2b (calcium/calmodulin-
Competinginterests
dependent protein kinase II-beta) locus. The Camk2b ex-
Thereisnoconflictingfinancialinterest.
pression is associated withT-cell activation and cytotoxic
T-cell activity [35,36]. It is possible that the burn-elicited Authors’contributions
ThisstudywasconceivedandmanagedbyKC.DGGparticipatedinscientific
stressors may affect the promoter and/or enhancer activ-
discussions.KHL,DL,andTGperformedtheexperiments.KHLgeneratedthe
ities of certain MuLV-ERVs paralleling modulation of the figuresanddraftedthemanuscript.Allauthorsreadandapprovedthefinal
expressionofadjacentgenesinaspecificlymphocytetype. manuscript.
Acknowledgement
Conclusion ThisstudywassupportedbygrantsfromShrinersofNorthAmerica(No.
86800toKCandNo.84302toKHL[postdoctoralfellowship])andthe
It is expected that burn injury elicits a range of stress
NationalInstitutesofHealth(R01GM071360toKC).
signals that are directly linked to pathologic changes in
the immune system and other organs [37]. The majority Received:19April2012Accepted:11December2012
Published:5January2013
of the underlying mechanisms regarding how these stres-
sors contribute to post-burn pathogenesis still remain to References
becharacterized.Thefindingsfromthisstudysuggestthat 1. WaterstonRH,Lindblad-TohK,BirneyE,RogersJ,AbrilJF,AgarwalP,
AgarwalaR,AinscoughR,AlexanderssonM,AnP,etal:Initialsequencing
certain MuLV-ERVs are differentially modulated depend-
andcomparativeanalysisofthemousegenome.Nature2002,
ing on the lymphocyte type, location of lymphoid organ, 420:520–562.
and duration after injury in conjunction with the burn- 2. MayerJ,SauterM,RaczA,SchererD,Mueller-LantzschN,MeeseE:An
almost-intacthumanendogenousretrovirusKonhumanchromosome
elicited stressors. As a result of activation of certain 7.NatGenet1999,21:257–258.
MuLV-ERVs following burn injury, the gene products 3. Mueller-LantzschN,SauterM,WeiskircherA,KramerK,BestB,BuckM,
and/or newly produced virus particles of the activated GrasserF:HumanendogenousretroviralelementK10(HERV-K10)
encodesafull-lengthgaghomologous73-kDaproteinandafunctional
ERVs may participate in specific burn pathogeneses, such protease.AIDSResHumRetroviruses1993,9:343–350.
asimmunedisorderandcytotoxicity,inacelltype-specific 4. VogtVM:RetroviralVirionsandGenomes.InRetroviruses.EditedbyCoffin
manner. In fact, our laboratory has reported that some JM,HughesSH,VarmusHE.ColdSpringHarbor(NY):ColdSpringHarbor
LaboratoryPress;1997:27–69.
MuLV-ERVs are activated in the liver of mice after burn
5. LavieL,KitovaM,MaldenerE,MeeseE,MayerJ:CpGmethylationdirectly
injurywhichresultedinevidenthepaticdamage[33].Fur- regulatestranscriptionalactivityofthehumanendogenousretrovirus
ther investigations are needed to elucidate the link be- familyHERV-K(HML-2).JVirol2005,79:876–883.
6. MoyesDL,MartinA,SawcerS,TempertonN,WorthingtonJ,GriffithsDJ,
tween lymphocyte type-specific post-burn MuLV-ERV
VenablesPJ:ThedistributionoftheendogenousretrovirusesHERV-K113
activation and systemic inflammatory disorder as well as andHERV-K115inhealthanddisease.Genomics2005,86:337–341.
theotheraspectsofburnpathogeneses. 7. FrendoJL,OlivierD,CheynetV,BlondJL,BoutonO,VidaudM,RabreauM,
Evain-BrionD,MalletF:DirectinvolvementofHERV-WEnvglycoprotein
inhumantrophoblastcellfusionanddifferentiation.MolCellBiol2003,
23:3566–3574.
Additional files
8. MameliG,AstoneV,ArruG,MarconiS,LovatoL,SerraC,SotgiuS,BonettiB,
DoleiA:Brainsandperipheralbloodmononuclearcellsofmultiple
Additionalfile1:TableS1.Sourcesofthe100MuLV-ERVU3 sclerosis(MS)patientshyperexpressMS-associatedretrovirus/HERV-W
sequencesanalyzedinthisstudy.Yellowandgrayhighlightsindicate endogenousretrovirus,butnotHumanherpesvirus6.JGenVirol2007,
uniqueMuLV-ERVU3sequencesandcloneswithoutsequence 88:264.
information,respectively.MAN(mandibularcervicalLN),ACC(accessory 9. PurcellEM,DolanSM,KriynovichS,MannickJA,LedererJA:Burninjury
mandibularcervicalLN),SUP(superficialparotidcervicalLN),DEE(deep inducesanearlyactivationresponsebylymphnodeCD4+T-cells.
cervicalLN)LN(lymphnode),SM(sizemarker). Shock2006,25:135.
10. SchneiderDF,GlennCH,FaunceDE:Innatelymphocytesubsetsandtheir
Additionalfile2:TableS2.Theprofileoftranscriptionregulatory
immunoregulatoryrolesinburninjuryandsepsis.JBurnCareRes2007,
elementsin100uniqueMuLV-ERVU3-promotersequences.Thenumber
28:365–379.
intheboxindicatestheoccurrencefrequencyofeachtranscription
11. MaldonadoMD,VenturoliA,FrancoA,Nunez-RoldanA:Specificchanges
regulatoryelement(TRE).ColoredboxesindicateTREspredominantly
inperipheralbloodlymphocytephenotypefromburnpatients.Probable
Leeetal.BMCImmunology2013,14:2 Page10of10
http://www.biomedcentral.com/1471-2172/14/2
originofthethermalinjury-relatedlymphocytopenia.Burns1991, 35. MackKD,VonGoetzM,LinM,VenegasM,BarnhartJ,LuY,LamarB,StullR,
17:188–192. SilvinC,OwingsP,etal:Functionalidentificationofkinasesessentialfor
12. BarlowY:Tlymphocytesandimmunosuppressionintheburnedpatient: T-cellactivationthroughageneticsuppressionscreen.ImmunolLett
areview.Burns1994,20:487–490. 2005,96:129–145.
13. HotchkissRS,TinsleyKW,SwansonPE,SchmiegRE,HuiJJ,ChangKC, 36. LiuX,YaoM,LiN,WangC,ZhengY,CaoX:CaMKIIpromotesTLR-
OsborneDF,FreemanBD,CobbJP,BuchmanTG:Sepsis-inducedapoptosis triggeredproinflammatorycytokineandtypeIinterferonproductionby
causesprogressiveprofounddepletionofBandCD4+Tlymphocytesin directlybindingandactivatingTAK1andIRF3inmacrophages.Blood
humans1.JImmunol2001,166:6952–6963. 2008,112:4961–4970.
14. LeeYK,ChewA,PhanH,GreenhalghDG,ChoK:Genome-wideexpression 37. DeitchEA:Roleofthegutlymphaticsysteminmultipleorganfailure.
profilesofendogenousretrovirusesinlymphoidtissuesandtheir CurrOpinCritCare2001,7:92.
biologicalproperties.Virology2008,373:263–273.
15. LeeYK,ChewA,GreenhalghDG,ChoK:Tropism,cytotoxicity,and doi:10.1186/1471-2172-14-2
inflammatorypropertiesoftwoenvelopegenesofmurineleukemia Citethisarticleas:Leeetal.:Injury-elicitedstressorsalterendogenous
virustype-endogenousretrovirusesofC57BL/6Jmice.MediatorsInflamm retrovirusexpressioninlymphocytesdependingoncelltypeand
2011,2011:509604. sourcelymphoidorgan.BMCImmunology201314:2.
16. ChoK,AdamsonLK,GreenhalghDG:InductionofmurineAIDSvirus-
relatedsequencesafterburninjury.JSurgRes2002,104:53–62.
17. TomonagaK,CoffinJM:Structuresofendogenousnonecotropicmurine
leukemiavirus(MLV)longterminalrepeatsinwildmice:implicationfor
evolutionofMLVs.JVirol1999,73:4327–4340.
18. BinghamJ,SudarsanamS:Visualizinglargehierarchicalclustersin
hyperbolicspace.Bioinformatics2000,16:660–661.
19. TomonagaK,CoffinJM:Structureanddistributionofendogenous
nonecotropicmurineleukemiavirusesinwildmice.JVirol1998,
72:8289–8300.
20. QuandtK,FrechK,KarasH,WingenderE,WernerT:MatIndand
MatInspector:newfastandversatiletoolsfordetectionofconsensus
matchesinnucleotidesequencedata.NucleicAcidsRes1995,
23:4878–4884.
21. TamuraK,DudleyJ,NeiM,KumarS:MEGA4:molecularevolutionary
geneticsanalysis(MEGA)softwareversion4.0.MolBiolEvol2007,
24:1596–1599.
22. NikbakhtKN,OuCY,BooneLR,GloverPL,YangWK:Nucleotidesequence
analysisofendogenousmurineleukemiavirus-relatedproviralclones
revealsprimer-bindingsitesforglutaminetRNA.JVirol1985,54:889–893.
23. KotadiaBK,RavindranathTM,ChoudhryMA,HaqueF,Al-GhoulW,Sayeed
MM:EffectsofpentoxyfyllineonmesentericlymphnodeT-cellsinarat
modelofthermalinjury.Shock2003,20:517–520.
24. TajimaF:DNApolymorphisminasubdividedpopulation:theexpected
numberofsegregatingsitesinthetwo-subpopulationmodel.
Genetics1989,123:229–240.
25. KovacsKJ,SawchenkoPE:Sequenceofstress-inducedalterationsin
indicesofsynapticandtranscriptionalactivationinparvocellular
neurosecretoryneurons.JNeurosci1996,16:262–273.
26. SulimanHB,CarrawayMS,Welty-WolfKE,WhortonAR,PiantadosiCA:
Lipopolysaccharidestimulatesmitochondrialbiogenesisviaactivationof
nuclearrespiratoryfactor-1.JBiolChem2003,278:41510–41518.
27. DuncanDD,StupakoffA,HedrickSM,MarcuKB,SiuG:AMyc-associated
zincfingerproteinbindingsiteisoneoffourimportantfunctional
regionsintheCD4promoter.MolCellBiol1995,15:3179–3186.
28. TingCN,OlsonMC,BartonKP,LeidenJM:TranscriptionfactorGATA-3is
requiredfordevelopmentoftheT-celllineage.Nature1996,384:474–478.
29. VolkerJL,RamehLE,ZhuQ,DeCaprioJ,HansenU:Mitogenicstimulation
ofrestingT-cellscausesrapidphosphorylationofthetranscriptionfactor
LSFandincreasedDNA-bindingactivity.GenesDev1997,11:1435–1446.
30. KwonDN,LeeYK,GreenhalghDG,ChoK:Lipopolysaccharidestress
inducescell-typespecificproductionofmurineleukemiavirustype-
endogenousretroviralvirionsinprimarylymphoidcells.JGenVirol2011, Submit your next manuscript to BioMed Central
92:292–300. and take full advantage of:
31. KwonDN,GreenhalghDG,ChoK:Cloningandcharacterizationof
endogenousretrovirusesassociatedwithpostinjurystresssignalsin
• Convenient online submission
lymphoidtissues.Shock2009,32:80.
32. StoyeJP,KaushikN,JeremiahS,BestS:Geneticmapoftheregion • Thorough peer review
surroundingtheretrovirusrestrictionlocus,Fv1,onmousechromosome • No space constraints or color figure charges
4.MammGenome1995,6:31–36.
33. LeeYK,ChewA,FitzsimonL,ThomasR,GreenhalghD,ChoK:Genome- • Immediate publication on acceptance
widechangesinexpressionprofileofmurineendogenousretroviruses • Inclusion in PubMed, CAS, Scopus and Google Scholar
(MuERVs)indistantorgansafterburninjury.BMCGenomics2007,8:440.
• Research which is freely available for redistribution
34. SeltenG,CuypersHT,BernsA:Proviralactivationoftheputative
oncogenePim-1inMuLVinducedT-celllymphomas.EMBOJ1985,
4:1793. Submit your manuscript at
www.biomedcentral.com/submit