Human endogenous retroviruses

Human endogenous retroviruses and human genome:Innocent bystanders or disease determinants?

Naeim Ehtesham

Supervisor: Dr. Khan ahmad

Repetitive mobile sequences

Known as transposable elements(TEs) Almost half of the mammalian genome DNA transposons (2.8%) Retroelements (42.2%) DNA transposons amplify without RNA intermediates Retroelements require a reverse transcriptase

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Repetitive mobile sequences

Ref: Molecular cell biology(lodish et all. 2008)

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Retroelements

Two major categories non-viral retroelements (SINE&LINE) Viral retroelements Differ on the absence or presence of 250- to 600 bp direct terminal

repeats (long terminal repeat)

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Retroviruses

Three defined sets of regions of genes: gag, pol, and env flanked by LTRs All exogenous and preserved endogenous retrovirus

strains have the basic genetic order 5-gag-pro-pol-env-3

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Retroviruses(cont…)

The long terminal repeats (LTRs) contain: TATA box promoters enhancers polyadenylation signals cDNA synthesis starts at the primer binding site (PBS) near U5 (5

unique sequence) complementary to a sequence of one particular tRNA

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Retroviruses(cont…)

Found in two forms: exogenous or endogenous or both Endogenous retroviruses: entered the germ line present in the genome of all host cells are inherited through successive generations in the classical

Mendelian fashion

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History of Retrovirus in human

Integration into the human germ line 2 to about 70 million years ago The numerous endogenous retroviruses indicate that large numbers

of exogenous retroviruses must have been circulating earlier in evolution

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History of Retrovirus in human (cont…)

An endogenous retrovirus will establish Fixation of the virus’ sequences in the host genome Endogenization or molecular domestication HERVs amplified during evolution by repeated reintegration of

reverse-transcribed mRNA into DNA

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Ref:Magiorkinis, G., R. Belshaw, and A. Katzourakis, 'There and back again': revisiting the pathophysiological roles of human endogenous retroviruses in the post-genomic era. Philos Trans R Soc Lond B Biol Sci, 2013. 368(1626): p. 20120504.

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Viral retroelements

According to Repbase more than 200 families of LTR-containing are defined

Make up 8% of human chromosomes retrotransposons human endogenous retroviruses (HERVs)

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Viral retroelements (cont…)

HERVs differ from retrotransposons by the presence of env gene HERVs are therefore the most complete retroelements

Ref: Balada, E., J. Ordi-Ros, and M. Vilardell-Tarres, Molecular mechanisms mediated by human endogenous retroviruses (HERVs) in autoimmunity. Rev Med Virol, 2009. 19(5): p. 273-86

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Genomic structure of HERV in detail

Ref:Kim, H.S., Genomic impact, chromosomal distribution and transcriptional regulation of HERV elements. Mol Cells, 2012. 33(6): p. 539-44.

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Generation of retroviral genomic RNAfrom integrated retroviral DNA

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Taxonomy of HERVs

A source of confusion Two ways to classify HERVs: tRNA specificity homologies in the pol region to animal retroviruses

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tRNA specificity

A systematic nomenclature Based on the tRNA specificity one-letter code Specific amino acid (linked to the tRNA) Suffix to the acronym “HERV’’ HERV-K:lysine-specific tRNA as primer

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Homologies in the pol region to animal retroviruses

Belonging to the virus family Retroviridae Contain seven genera Are classified by the International Committee for Taxonomy of

Viruses By morphological and biological features This morphological classification is not used anymore

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Homologies in the pol region to animal retroviruses(cont…)

Animal retroviruses Classification based largely on sequence similarity within pol gene:

Alpharetrovirus Betaretrovirus Deltaretrovirus Gammaretrovirus Epsilonretrovirus Lentivirus Spumavirus

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Homologies in the pol region to animal retroviruses(cont…)HERVs are classified into three classes based on this approach Class I related to Gammaretroviruses are subdivided into six groups

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Homologies in the pol region to animal retroviruses(cont…) Class II related to Betaretroviruses subdivided into 10 groups all class II HERVs use a lysine tRNA to initiate the RT

reaction(HERV-K) Youngest and most active HERV family

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Homologies in the pol region to animal retroviruses(cont…) Class III related to Spumaviruses includes few HERVs

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HERV-K

Exogenous viruses Notably HIV Possess additional non-structural accessory genes Facilitate their replication or impair host defenses Rare in endogenous virus strains, with the exception of HERV-K Rec is functionally related to the HIV protein Rev and the HTLV

protein Rex It shuttles RNA transcripts out of the nucleus

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Ref: Young, G.R., J.P. Stoye, and G. Kassiotis, Are human endogenous retroviruses pathogenic? An approach to testing the hypothesis. Bioessays, 2013. 35(9): p. 794-803

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Are they important ?

Have been regarded as selfish or junk DNA It remains unclear whether they are really all ‘‘junk’’ Like all genes they are subject to natural selection Must not be considered as parasites True symbionts Majority of retrotransposition events are either neutral or

deleterious The latter will be eliminated by negative selection

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Expression of HERVs

Although many HERVs show mutations and deletions, some are transcriptionally active and produce functional proteins

Some HERV mRNAs and a few HERV-encoded proteins are expressed in placental or embryonic tissues

Principally regulated by their individual LTRs Cytokines such as TNF-α Regulate HERV expression In a temporal and tissue-specific fashion

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Beneficial Functions of HERVs

Enhancement and promotion of gene expression HERV-E LTR enhancer for endothelin B receptor and apolipoprotein C-Ⅰ HERV-H LTR enhancer activities in embryonic and hematopoietic cells

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Beneficial Functions of HERVs(cont…)

In embryonic stage: Trophoblast specific human growth factor pleiotrophin

(PTN) is under the control of a HERV LTR envelope glycoproteins are anchored in the

membrane initiate the fusion of viral and cellular membranes

during the infection In syncytiotrophoblasts but not in cytotrophoblasts high levels of HERV-W envelope proteins named syncytin- 1 and syncytin-2

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In embryonic stage (cont…): cell–cell fusogenic activity mediated by HERV Env

proteins contributes to the physiological placenta

morphogenesis mediating fusion of cytotrophoblasts to

syncytiotrophoblasts ERVs have been central in the radiation of placental

mammals

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In embryonic stage (cont…): Placentas from the viewpoint of mother allogeneic organs reasons for maternal tolerance: poorly understood immunological tolerance has to be effective to prevent allogeneic

rejection of the fetus

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In embryonic stage (cont…): immunosuppressive property of retroviral Env proteins syncytin-2 not for syncytin-1

Downregulation of placental syncytin expression Abnormal intracellular localization of placental contribute to the etiology of pre-eclampsia

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In embryonic stage (cont…): Conclusion: HERVs be instrumental in: safe-guarding placenta morphogenesis physiology fetal–maternal tolerance Similar fusogenic and immunosuppressive

endogenous retrovirus proteins were recently detected in all rodents as well as in sheep

suggests positive selection over millions of years

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Other beneficial advantageous: The telomerase is derived from RT HERV LTRs often contain binding sites for the p53 HERV LTRs account for over 30% of all p53 binding sites May have been co-opte as regulatory sequences to expand the p53

transcriptional network Contribute to the anti-oncogenic function of the stress-responsive

p53

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Molecular Mechanisms Used by HERVs to Induce Autoimmune Diseases

Capacity for moving and insertion

Expression of HERV-encoded proteins

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Capacity for moving and insertion

Espicially in MHC class II region and complement cascade Alter the structure and function Disrupt genes Leading to knock-outs Truncated proteins

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Capacity for moving and insertion (cont..)

Cis regulatory sequences New splice sites Alternative transcription initiation sites Premature polyadenylations signals Other isoforms by alternative splicing

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Capacity for moving and insertion (cont..)

LTR events fall into three main classes Specifically augment transcription in a particular tissue (often

occurs in the placenta) Confer widespread non-specific transcription New inserted LTRs may harbor promoter sequences Have become converted as the main gene promoter

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Expression of HERV-encoded proteins

Would be considered as “foreign” Could trigger B-cells to produce antibodies against them Cross-react with other proteins of our bodies Molecular mimicry mechanism

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Ref: Oldstone, M.B., Molecular mimicry and immune-mediated diseases. Faseb j, 1998. 12(13): p. 1255-65.

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Expression of HERV-encoded proteins(cont…) Env proteins from members of the class II HERVs modulating the expression of several cytokines can act on T lymphocytes skew the immunological network towards a Th1 or Th2 response

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Expression of HERV-encoded proteins(cont…) may act as superantigens cause the expansion of autoreactive T lymphocytes some HERV peptides may have immunosuppressive properties

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REF:Brodziak, A., et al., The role of human endogenous retroviruses in the pathogenesis

of autoimmune diseases. Med Sci Monit, 2012. 18(6): p. Ra80-8.

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Expression of HERV-encoded proteins(cont…) Surface unit (ENVSU) of the HERV-W Env: specifically activate cells of the innate immune system production of major proinflammatory cytokines such as IL-1β, IL-

6, or TNF-α

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Ref:Balada, E., J. Ordi-Ros, and M. Vilardell-Tarres, Molecular mechanisms mediated by human endogenous retroviruses (HERVs) in autoimmunity. Rev Med Virol, 2009. 19(5): p. 273-86.

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Disregulation of HERVs in autoimmune diseases Systemic Lupus Erythematosus Insulin-Dependent Diabetes Mellitus Systemic sclerosis Graves’ disease Autoimmune Addison’s disease (AAD) Rheumatoid Arthritis Multiple Sclerosis

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HERVs and Rheumatoid Arthritis

In a study 50% of synovial samples were shown to have proviral HERV-L

DNA A significantly higher level of HERV-K gag mRNA in both

peripheral cells and synovial fluid cells An association between the HERV-K viral load in plasma and the

disease severity

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HERVs and Multiple Sclerosis

A different env superantigen is associated with MS Encoded by the MS-associated HERV-W (MSRV) As part of the multicopy HERV-W group MSRV RNA in the CSF at the onset of MS seems to be indicative

of a poor prognosis Transcripts levels of the HERV-W env protein , syncytin-1,

increased in brain and glial cells

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Syncytin-1-mediated neuropathogenesis in multiple sclerosis

Antony, J.M., et al., Human endogenous retroviruses and multiple sclerosis: innocent bystanders or disease determinants? Biochim Biophys Acta, 2011. 1812(2): p. 162-76.

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HERVs and Multiple Sclerosis(cont…)

At a genetic level: HERV-K env genotype variation seems to influence genetic

susceptibility to MS HSV-1, VZV and EBV are associated with MS HERVs and herpes viruses seem to have complex interactions They seem to directly transactivate particular sequences of HERVs

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oncogenic mechanisms of HERVs

Failures in somatic cells’ efficiency to control HERV activity Potentially results in genome damage Contribute to formation of cancer

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oncogenic mechanisms of HERVs(cont…)

General or more specific(re)activation of HERV sequences by hypomethylation

Expression of HERV encoded oncogenes such as Rec and NP9 Inactivation of tumor suppressor genes by de novo insertion or

translocation Mediating ectopic recombination

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Regulation of nearby (proto-) oncogenes or growth factors by LTRs Potential of Env proteins to induce cell fusions (Syncytin-1) Contribute to tumor progression or even aid in metastasizing

processes

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An additional aspect: Env proteins of the mouse leukemia virus, the Mason-Pfizer

monkey virus and HERV-W have strong immunosuppressive properties

immunosuppressive domain is localized in the transmembrane (TM) portion

advantageous in syncytiotrophoblasts at the fetal–maternal interface help tumor cells evade an antitumoral immune response Possibly preventing activity of the innate immune system in

clearing tumors

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Ref:Mullins, C.S. and M. Linnebacher, Human endogenous retroviruses and cancer: causality and therapeutic possibilities. World J Gastroenterol, 2012. 18(42): p. 6027-35.

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Ref:Ruprecht, K., et al., Endogenous retroviruses. Cellular and Molecular Life Sciences, 2008. 65(21): p. 3366-3382

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(re)activation of HERV sequences by hypomethylation

Maintenance of methylation patterns and status play a central role in HERV transcriptional control

In healthy somatic and mature germ cells HERV sequences are generally (hyper-) methylated

HERV transcriptional activity is mainly restricted to germ cell development

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(re)activation of HERV sequences by hypomethylation(cont…)

Responsible for a high(er) retroelement expression in germ cell tumors

Can be found in testicular germ cell cancer, teratocarcinomas, colorectal, breast and ovarian cancer

Active retrotranspositions may cause DNA strand-breaks Lead to an activation of check-point signaling, e.g., TP53 Occur especially in tumors with defect check-points and TP53

mutations

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Expression of HERV encoded oncogenes such as Rec and NP9

2 accessory viral proteins Exclusively in HER-K Product of alternative splicing of the env gene Rec and Np9 bind to the promyelocytic leukemia zinc finger

protein (PLZF) Both a tumor suppressor and a transcriptional

suppressor of the c-myc proto-oncogene Increased of c-Myc protein and proteins regulated downstream of c-

Myc

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Mediating ectopic recombination

Produce chromosomal anomalies and gene rearrangement A hallmark of most tumors Recombination between the thousands of HERV loci may lead to

gain-of function sequences

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Ref:Stoye, J.P., Endogenous retroviruses: Still active after all these years? Current Biology, 2001. 11(22): p. R914-R916.

Ref:Romanish, M.T., C.J. Cohen, and D.L. Mager, Potential mechanisms of endogenous retroviral-mediated genomic instability in human cancer. Seminars in Cancer Biology, 2010. 20(4): p. 246-253.

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THERAPEUTIC STRATEGIES

Assuming that in normal physiology of adult tissues HERVs do not play a vital role

HERV sequences are of significance in tumor formation, development and metastasis

HERVs recommend themselves as prime targets for tumor therapy

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Inhibition

Tremendous success in HIV control with infected people treated by antiretroviral combination therapies

Reverse expression of HERV sequences in tumor cells Analyzis the effect of a reverse transcription inhibitor (Abcavir) on

prostate cancer cell Lines showed a strong anti-proliferative capacity even triggered senescence in the cancer cells

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Inhibition(cont…)

Direct targeting of HERV by RNA interference Therapeutical use of natural inhibitors of retroviruses such as

APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like) or tripartite motif (TRIM) 5-alpha protein

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Passive immune therapy

HERV-K Env protein expression was substantially higher in breast cancer

A higher rate of lymph node metastasis was associated with HERV-K-positive tumors

Designation monoclonal antibody (mAb) recognizing a HERV-K Env protein

Inhibited tumor growth and induced apoptosis of breast cancer cells in vitro

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Passive immune therapy(cont…)

This treatment resulted in an over-expression of several proteins involved in the apoptotic signaling pathways

Passive immune therapies may well be applied in combination with active immune therapies

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Active immune therapy

The ideal cancer therapeutic agent: discriminate between cancer and normal cells (specificity) potent enough to kill small or large numbers of tumor cells

(sensitivity) ideal cancer immunotherapy should be able to prevent recurrence of

the tumor (durability) this durability in prevention is due to persistent recognition of

tumor antigens by lymphocytes

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Active immune therapy(cont…)

tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs)

TAAs expressed in normal tissue but to a much higher extent in malignant cells

TSA are truly specifically expressed in tumor cells alone Most of the features an ideal TSA have been assigned to HERV

encoded proteins necessity of expression for maintenance of the cancer cells’

transformed state they might indeed be ideal targets for tumor immunotherapy

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Because of the multitude of HERV-encoded sequences Development of a polyvalent (containing many epitopes) vaccine Basing only on HERV epitopes may be possible

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Actual bioinformatics approaches Identification of immunogenic core epitopes shared between

different HERV copy ORFs active in different tumor entities Design a universal HERV-based vaccine

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Ref:Mullins, C.S. and M. Linnebacher, Human endogenous retroviruses and cancer: causality and therapeutic possibilities. World J Gastroenterol, 2012. 18(42): p. 6027-35.

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Conclusions and Outlook

Rapidly increasing knowledge of transposable elements in the creation, modulation, regulation and inactivation of eukaryotic genes

stop describing them as ‘‘junk’’ DNA. They can shape the structure, function and networking of the

human genome with their promoters, enhancers, polyadenylation signals and polymerases

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THANKS FOR YOUR ATTENTION