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>>>> 17 March 2012 <<<<
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Below you will find the Abstract, Introduction-,
Discussion- sections of:
*Identification of XMRV Infection-Associated microRNAs in
Four Cell Types in Culture*
The full text can be found at the address below
~jan van roijen
````
PLOSone
Research Article
Identification of XMRV Infection-Associated
microRNAs in Four Cell Types in Culture
Ketha V. K. Mohan1#, Krishnakumar Devadas2#,
Shilpakala Sainath Rao1, Indira Hewlett2,
Chintamani Atreya1*
1 Section of Cell Biology, Laboratory of Cellular Hematology,
Center for Biologics Evaluation and Research, Food and
Drug Administration, Bethesda, Maryland, United States of
America,
2 Laboratory of Molecular Virology, Center for Biologics
Evaluation and Research, Food and Drug Administration,
Bethesda, Maryland, United States of America
Abstract
Introduction
XMRV is a gammaretrovirus that was thought to be
associated with prostate cancer (PC) and chronic fatigue
syndrome (CFS) in humans until recently. The virus is
culturable in various cells of human origin like the
lymphocytes, NK cells, neuronal cells, and prostate cell
lines. MicroRNAs (miRNA), which regulate gene
expression, were so far not identified in cells infected with
XMRV in culture.
Methods
Two prostate cell lines (LNCaP and DU145) and two primary
cells, Peripheral Blood Lymphocytes [PBL] and
Monocyte-derived Macrophages [MDM] were infected with
XMRV. Total mRNA was extracted from mock- and
virus-infected cells at 6, 24 and 48 hours post infection and
evaluated for microRNA profile in a microarray.
Results
MicroRNA expression profiles of XMRV-infected continuous
prostate cancer cell lines differ from that of virus-infected
primary cells (PBL and MDMs). miR-193a-3p and
miRPlus-E1245 observed to be specific to XMRV infection
in all 4 cell types. While miR-193a-3p levels were down
regulated miRPlus-E1245 on the other hand exhibited varied
expression profile between the 4 cell types.
Discussion
The present study clearly demonstrates that cellular
microRNAs are expressed during XMRV infection of human
cells and this is the first report demonstrating the regulation
of miR193a-3p and miRPlus-E1245 during XMRV infection
in four different human cell types.
Introduction
XMRV is a recently identified gammaretrovirus, closely
related to xenotropic murine leukemia viruses (MLVs), that
was initially detected in familial cases of prostate cancer
tissue using a virus gene array [1].
XMRV was also detected in blood cells of patients with
Chronic Fatigue Syndrome (CFS) and normal healthy
controls [2], [3].
Subsequently, a number of additional studies have failed to
confirm any association of XMRV with CFS or prostate
cancer [4]=96[11]. Indeed, recent reports suggest that XMRV
likely originated as a laboratory contaminant in prostate
xenografts serially passaged through nude mice by the
recombination of endogenous MLVs.
Though the XMRV is of murine origin, it is known to infect
different human cell types like T and B lymphocytes, NK
cells, prostate cancer cell lines, and neuronal cells
[12]=96[15].
Various detection methods like serology, cell culture, and
nucleic-acid based assays have already been used for
detecting XMRV infection [4], [12], [16]=96[19].
However, use of microRNAs (miRNAs) as biomarkers of
XMRV infection has not been reported so far.
MicroRNAs have known to play a critical role in the life cycle
of retroviruses and a few oncogenic viruses such as
reticuloendotheliosis virus strain T (REV-T), Epstein-Barr
virus and Hepatitis C virus (HCV) wherein the viruses
regulate host cells and viral replication through specific
microRNAs [20]=96[23].
MicroRNAs are a class of evolutionarily conserved,
endogenous, small non-coding RNAs that regulate gene
expression and play a role in diverse cellular processes,
including proliferation, differentiation and cell death [24].
As an abundant class of regulatory molecules, there are
hundreds of distinct miRNAs identified in the human
genome to date and hundreds more predicted.
A single miRNA can regulate expression of multiple genes,
and expression of a single gene may be regulated by
several distinct miRNAs, creating complicated regulatory
networks.
It is estimated that roughly 60% of human protein-coding
genes are regulated by miRNAs [25]=96[28].
In this study, we evaluated whether miRNAs are modulated
by XMRV in cultured cells and if so, can they be identified
to see whether a single or a set of miRNAs specific to the
infection can be detected early that could serve as
biomarker(s) of XMRV infection.
Our results demonstrate that
a) two miRNAs, miR-193a-3p and miRPlus-E1245 (a
proprietary sequence of Exiqon Inc, Denmark and named as
such to differentiate from miR-1245) were commonly
regulated among all 4 cell types infected with XMRV used in
the study, and
b) while miR-193a-3p is down regulated, miRPlus-E1245
exhibited varied expression profile in the four cell types
infected with XMRV.
Discussion
The discovery of XMRV and its potential association with PC
and CFS aroused considerable excitement and promise
within the research and clinical community regarding a
possible infectious etiology for at least some cases of these
disease or conditions. [2], [3], [33].
However, recent research findings have not supported any
association between the virus and CFS or prostate cancer
[7], [9], [10], [34]=96[37]. In fact, the virus itself may have
originated as a result of recombination in a laboratory
setting [38], [39].
Specifically, it has been postulated that XMRV originated as
a result of recombination between two MLV proviruses in
laboratory mice [40].
These findings appear to raise doubts about the significance
and involvement of XMRV in any human disease or
condition [38]=96[41].
Nonetheless, because at least some studies have
demonstrated that XMRV is a culturable virus and that it
can readily infect cells of human origin [12]=96[15], additional
research efforts will help to further our understanding of
XMRV pathogenesis and provide insights into the modes of
transmission involved in XMRV infection.
It also remains to be seen whether XMRV demonstrates
potential to be transmitted across species [12], [37], [41].
The present study further emphasizes that XMRV can infect
human prostate and hematopoietic cells and the study
clearly demonstrates that microRNAs are regulated during
XMRV infection of these culturable human cells. In fact, the
qPCR results indicate that while all the 4 cell types were
susceptible to XMRV infection with significant increase in
viral titers by 48 h time point it was evident that there was a
distinct difference in infection levels between the 4 cell
types (Fig. 1).
The prostate cell lines (LNCaP and DU145) supported robust
XMRV infection, while the PBLs and MDMs were
moderately infected.
It is interesting to note that the variability in infection status
of the 4 cell types may potentially be dependent on
individual APOBEC levels in each cell type [42]. It has been
shown earlier that XMRV is resistant to human APOBEC
3G (hA3G) and that the levels of hA3G are down-regulated
by XMRV in LNCaP and DU145 cells thereby supporting
efficient viral infection in these cell types [42], [43].
The hA3G is down regulated by the human
immunodeficiency virus-1 (HIV-1) vif protein during infection.
However, since XMRV lacks vif, an alternate mechanism of
hA3G down regulation has been suggested [43]. PBMCs on
the other hand, seemingly possess significantly higher
levels of h3AG and hence are relatively resistant to XMRV
infection [42].
The two microRNAs (miR-193a-3p and miRPlus-E1245) are
moderately regulated in the four cell types. However, it is
interesting to note that within the four cell types,
miR-193a-3p is down regulated over time, while
miRPlus-E1245 however exhibited varied levels of
expression profile between the 4 cell types: up regulation in
MDMs and PBL cell types and down regulation in LNCaP
and DU156 cell types.
Since the miRPlus-E1245 has not been annotated and not
submitted in the miRNA database yet by its discoverer, the
Exiqon Inc., Denmark, it is not feasible at this time to
identify its potential targets. Therefore, we only analyzed
the miR-193a-3p for its tentative mRNA targets by 3
different online programs as indicated in Table 1.
Target Prediction by miRDB, TargetScan and microRNA.org
programs revealed that out of the top 10 mRNA targets that
were identified individually by these 3 different softwares, 1
target mRNA was picked by all three programs and 5
mRNA targets were commonly flagged at least by two
different programs. Of the six predicted mRNA targets for
miR-193a-3p, five mRNA targets were related to
tumorogenesis or suppression.
Interestingly 3 mRNA targets, namely SON DNA binding
domain (SON), Friend Leukemia Virus Integration 1 (FLI1)
and v-erb-erythroblastic leukemia viral oncogene homolog 4
(ERBB4) have been implicated with virus/virus infections. Of
the 3, the FLI1 protein (or its homolog) may have a potential
role in XMRV infection as this protein has already been
implicated in Friend Leukemia Virus which also is a
retrovirus causing tumorigenesis [44], [45].
The human genome was recently analyzed for potential
XMRV genome integration sites and results revealed that
the virus had integration sites in at least 11 of the 23
chromosomes [46]. Hence it is to be seen whether this
particular host mRNA target is being modulated by
miR-193a-3p during XMRV infection. Of the other two, while
the SON protein binds to hepatitis B virus (HBV) DNA and
exhibits sequence similarity to other oncoproteins, the
ERBB4 protein affects mitogenesis and cell differentiation
and furthermore it is known that mutations within this gene
are associated with cancer [47]=96[49].
More pertinently, while the qPCR results revealed robust
infection in two cell types (LNCaP and DU145 cells) and
moderate infection in the other two tested cell types (PBLs
and MDMs), what is common to all 4 cell types is the
regulation of the two miRNAs (miR-193a-3p and
miRPlus-E1245) during XMRV infection regardless of the
level of infectivity, virus titer or dose of the infection. This is
the first report indicating the expression and regulation of
miRs during XMRV infection of human cells. It remains to
be seen whether the same set of miRNAs are up regulated
during infection of murine cells or cell lines.
The current findings reported here certainly demonstrate that
XMRV infection modulates miRNAs in the host cells as is
the case with many other viruses that are pathogenic to
humans [20]=96[23].
In human retroviruses such as HIV-1 and HTLV-1, the role of
microRNAs has already been demonstrated [50]=96[54].
Many of these exquisite studies have clearly shown how
certain miRs up regulate or down regulate certain host
genes/proteins to promote viral infection or disease
pathogenesis [50]=96[52], [54], [55].
In fact, it is now known that HIV-1 and other viruses
themselves code for microRNAs, which play a critical
regulatory role during virus infection [50], [56].
Our studies also demonstrate that miRNA profiles are
different in XMRV-infected prostate cancer cell lines
compared to primary hematopoietic cells, suggesting that
miRNAs could play a role in XMRV infection, and serve as
markers of XMRV infection in cultured cells.
~~~~
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