Identification of XMRV Infection-Associated microRNAs in Four Cell Types in Culture

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*Identification of XMRV Infection-Associated microRNAs in

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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, land, United States of

America,

2 Laboratory of Molecular Virology, Center for Biologics

Evaluation and Research, Food and Drug Administration,

Bethesda, land, 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]–[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]–[15].

Various detection methods like serology, cell culture, and

nucleic-acid based assays have already been used for

detecting XMRV infection [4], [12], [16]–[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]–[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]–[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

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]–[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]–[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]–[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]–[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]–[23].

In human retroviruses such as HIV-1 and HTLV-1, the role of

microRNAs has already been demonstrated [50]–[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]–[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.