This presentation is about the role of Human endogenous retroviruses in normal function and disease of man

1. Human endogenous retroviruses and
human genome:Innocent bystanders or
disease determinants?
Naeim Ehtesham
Supervisor: Dr. Khan ahmad

2. 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
2

3. Repetitive mobile sequences
Ref: Molecular cell biology(lodish et all. 2008)
3

4. 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)
4

5. 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
5

6. 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
6

7. Ref: Molecular cell biology(lodish et all. 2008)
7

8. 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
8

9. 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
9

10. 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
10

11. 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.
11

12. 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)
12

13. 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
13

14. 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.
14

15. Generation of retroviral genomic RNA
from integrated retroviral DNA
15
Ref: Molecular cell biology(lodish et all. 2008)

16. Taxonomy of HERVs
 A source of confusion
 Two ways to classify HERVs:
 tRNA specificity
 homologies in the pol region to animal retroviruses
16

17. 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
17

18. 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
18

19. 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
19

20. 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
20

21. 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
21

22. Homologies in the pol region to animal
retroviruses(cont…)
 Class III
 related to Spumaviruses
 includes few HERVs
22

23. 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
23

24. 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
24

25. 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
25

26. 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
26

27. 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
27

28. 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
28

29. Beneficial Functions of HERVs(cont…)
 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
29

30. Beneficial Functions of HERVs(cont…)
 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
30

31. Beneficial Functions of HERVs(cont…)
 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
31

32. Beneficial Functions of HERVs(cont…)
 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
32

33. Beneficial Functions of HERVs(cont…)
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
33

34. Molecular Mechanisms Used by HERVs
to Induce Autoimmune Diseases
 Capacity for moving and insertion
 Expression of HERV-encoded proteins
34

35. 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
35

36. Capacity for moving and insertion (cont..)
 Cis regulatory sequences
 New splice sites
 Alternative transcription initiation sites
 Premature polyadenylations signals
 Other isoforms by alternative splicing
36

37. 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
37

38. 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
38

39. Ref: Oldstone, M.B., Molecular mimicry and immune-
mediated diseases. Faseb j, 1998. 12(13): p. 1255-
65.
39

40. 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
40

41. Expression of HERV-encoded
proteins(cont…)
 may act as superantigens
 cause the expansion of autoreactive T lymphocytes
 some HERV peptides may have immunosuppressive properties
41

42. 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.
42

43. 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
43

44. 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.
44

45. 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
45

46. 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
46

47. 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
47

48. 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.
48

49. 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
49

50. oncogenic mechanisms of HERVs
 Failures in somatic cells’ efficiency to control HERV activity
 Potentially results in genome damage
 Contribute to formation of cancer
50

51. 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
51

52. oncogenic mechanisms of HERVs(cont…)
 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
52

53. oncogenic mechanisms of HERVs(cont…)
 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
53

54. 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.
54

55. Ref:Ruprecht, K., et al., Endogenous retroviruses.
Cellular and Molecular Life Sciences, 2008. 65(21):
p. 3366-3382
55

56. (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
56

57. (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
57

58. 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
58

59. 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
59

60. 60
Ref:Stoye, J.P., Endogenous retroviruses: Still active
after all these years? Current Biology, 2001. 11(22):
p. R914-R916.

61. 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.
61

62. 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
62

63. 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
63

64. 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
64

65. 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
65

66. 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
66

67. 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
67

68. 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
68

69. Active immune therapy(cont…)
 Because of the multitude of HERV-encoded sequences
 Development of a polyvalent (containing many epitopes) vaccine
 Basing only on HERV epitopes may be possible
69

70. Active immune therapy(cont…)
 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
70

71. 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.
71

72. 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
72

73. THANKS FOR YOUR ATTENTION