Provirus Silencing in Stem Cells: The Forbidden
Regulators of Cell Fate
Sandeep Satapathy
Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India.
ABSTR ACT: The cryptic presence of a wide range of retroviruses with varying copy number holds biological significance for host reproduction and
development. Most of the endogenous retroviruses with lost pathogenicity and replication ability still serve as transcriptional regulators of host cellular
genes. These structural and functional features of proviruses present them as alternate promoters and enhancers for several host cellular genes involved
in development and other biological processes. In addition, embryonic stem (ES) cells and induced pluripotent cells are known to effectively silence the
expression of most of these proviruses through repressive epigenetic marks and proviral sequence heterochromatization, which is not a case for those of
differentiated cells. In this review, we aim to dissect the underlying salient features of proviral silencing in embryonic stem cells and analyze the potential
of these proviruses in cell fate determination.
KEY WORDS: endogenous retroviruses, Trim-28, YY1, cell fate, stem cells, histone methylation, histone chaperons, Chaf1a/Chaf1b
CITATION: Satapathy. Provirus Silencing in Stem Cells: The Forbidden Regulators
of Cell Fate. Signal Transduction Insights 2016:5 15–23 doi:10.4137/STI.S12311.
TYPE: Review
RECEIVED: May 31, 2016. RESUBMITTED: July 26, 2016. ACCEPTED FOR
PUBLICATION: July 29, 2016.
COPYRIGHT: © the authors, publisher and licensee Libertas Academica Limited.
This is an open-access article distributed under the terms of the Creative Commons
CC-BY-NC 3.0 License.
CORRESPONDENCE: [email protected]
PEER REVIEW: Six peer reviewers contributed to the peer review report. Reviewers’
reports totaled 1400 words, excluding any confidential comments to the academic
editor.
Paper subject to independent expert single-blind peer review. All editorial decisions
made by independent academic editor. Upon submission manuscript was subject to
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of agreement to article publication and compliance with all applicable ethical and legal
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to human and animal study participants, and compliance with any copyright requirements
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FUNDING: Author discloses no external funding sources.
Provenance: the author was invited to submit this paper.
COMPETING INTERESTS: Author discloses no potential conflicts of interest.
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ACADEMIC EDITOR: Edgar Grinstein, Editor in Chief
Introduction
The biology of retroviruses has been intensely studied and
understood over the past decades. Most of the studies on
retroviruses focus on their associated clinical complications
and pathological manifestations. Distinctively, these viruses
can pass their genetic information (RNA to DNA) in reverse
to the usual direction for flow of biological information, ie,
from DNA to RNA.1 Most of the retroviruses have an RNA
genome, which gets transcribed into a double-stranded DNA
sequence within the host cellular machinery using reverse
transcriptase (RT) and other polymerases (Fig. 1).1 Such a
critical role of RT in infection and virus transmission makes
these genomic entities crucial for grouping of viruses according to the similarity in the transcriptional binding and the
mode of RT functioning.2 Furthermore, these viral DNA
sequences are integrated into the host genome using integrase
(IN1) (Fig. 2).3 These integrated viral genomic entities are
referred to as proviruses4 for their ability to be harbored within
the host genome and passed onto subsequent generations in
a vertical transmission pathway (Fig. 3). Proviruses can be
endogenous retroviruses (ERVs), which have been integrated
into the host genome in the past and passed through germline transmission, or can be exogenous retroviruses, which
are recently exposed to the host and thus initiate the cascade
of viral entry and integration with the host genome (Fig. 3).5
Exogenous retroviruses are mostly infecting or pathogenic
retroviruses that get processed and integrated into the host
genome and ultimately silenced over generations to avoid any
further pathogenic manifestations.6 The flow and selection of
these retroviruses within the host genome has been discussed
in detail elsewhere.7–9
Thus, certain ERVs can replicate or act in trans to several
genes in the chromosome (retrotransposon)10 and insert germline mutations via such events of retrotransposition.11 There
are few other ERVs that are silenced and cannot replicate but
add on to the host transcriptomic diversity by acting as alternate promoters and enhancers.12 Thus, the ability to recombine, alter, and yield the downstream functional products such
as proteins and also act as additional transcriptional regulators make these proviruses interesting.13 With a wide range of
functional interference and regulation exerted by these viral
sequences, it is of relevance to understand the mechanism of
how these viruses have evolved in the host genome for over a
hundred millions of years. Mostly, retroviruses are known
to infect the somatic cells and are thereby negatively selected
and eliminated from the host.14 The proviruses under study
are those that have been vertically transmitted and selected
by natural forces of selection to reside and coevolve with the
host. However, these proviruses are silenced due to accumulating mutations leading to several premature stop codons,
frame-shift mutations, absence of partial functional coding
sequences like env or pol (Fig. 2), and sometimes absence of
the whole functional coding sequence.15 Even for the ERVs
that are gradually silenced over several generations instead of
Signal Transduction Insights 2016:5
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Satapathy
Figure 1. Viral RNA processing and integration into host genome. Retroviruses upon infection convert their single-stranded RNA genome into doublestranded DNA using DNA-dependent DNA polymerases via copying of its own RNA strand using RNA-dependent DNA polymerase. In addition, the viral
dsDNA along with the preintegration complex is integrated into host genome using integrase (IN1).
those being silenced spontaneously, the number of replicative
cycle is limited, and thus the chances of retaining pathogenicity get limited. These silenced proviruses have often been
referred to as domesticated,16 tamed,17 or co-opted viruses.18
ERVs are classified as class I, class II, and class III ERVs
based on the relative geological time scale of integration of these
ERVs into the host genome.19 This makes class III ERVs the
most recent ERVs and the ones with relatively minimal mutations or modifications.7 In contrast, class I and class II ERVs
were integrated much earlier than class III ERVs and harbor
several silencing mutations.20 A bias for specific host has been
noted among these proviral elements. Most of these proviruses
are known to be present in basal vertebrates, although few of
them have also been reported in invertebrates, insects, and other
lower animals.21 Approximately 400 different families of proviruses are known to be harboring within the human genome.
These proviruses constitute about 10% of mouse genome and
58% of human genome.22 Such a high prevalence of viral
genomic fragments substantiates for the fact that these proviruses hold certain beneficial contribution to host fitness and
survivability.23
In embryonic cells and induced pluripotent cells (iPSCs),
the ERVs are known to be under transcriptional and posttranscriptional silencing modifications such as regulation of
transcription factor binding and epigenetic repressive marks
such as methylation, acetylation, and sumoylation of histones
as well as the proviral DNA sequences (Fig. 4). 24 These
mechanisms of proviral silencing are known to be unique
to stem cells. 25 Thus, proviral silencing in embryonic cells
and embryonic carcinoma cells has been an important regulation of ERVs and most often studied using mouse model
system. 26 The advantage of proviral silencing in stem cells has
been used for retroviral and lentiviral vector-based transgene
delivery. Such an inherent silencing mechanism of proviruses
may facilitate cellular reprogramming, of which very little is
known till date. 27 In addition, silenced proviruses have been
found to be effective in transgene therapy by overcoming the
interference of the viral transgenes. 27 However, owing to a
dynamic and stochastic pattern of provirus silencing, there
are reports wherein transgenes limit the efficiency of cellular reprogramming and inhibit the differentiation of reprogrammed cells to specific lineages. 28 Furthermore, viral
transgenes can interfere with the cellular transcriptome
and proteome by modulating the expression of noncoding
RNAs, which are known to influence several protein-coding
mRNAs. As a result, these wide-scale interferences of viral
transgenes with host genetic machinery lead to pathogenic or
oncogenic transformations of host cells. 28 Therefore, understanding proviral silencing in stem cells is essential to identify
the provirus silencing machinery and shed light on how the
absence of this process regulates lineage-specific development and
cell fate determination.
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Figure 2. The genomic map of endogenous retroviruses. The map highlights the regulatory regions like 5′-LTR and 3′-LTR along with function gene
products like envelope proteins, ie, env (gp-120/41), pol (reverse transcriptase), gag (group antigens), and rev (codes for Rev protein that exports most of
the unspliced and incompletely spliced mRNAs).
16
Signal Transduction Insights 2016:5
Provirus silencing in stem cells
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Figure 3. A schematic model of retroviral integration into host genome.
The process of integration depends on the selection of specific host
target site on chromosome and further downstream integration of viral
genome. The functional product expressed from such a recombinant
genomic element leads to modified protein product or gives rise to
regulatory features like premature stop codons, mutation in coding
sequences, or cryptic splice sites.
Salient Features of Provirus Expression
The proviral genetic elements within the host genome and
their expression pattern are summarized as follows:
•
•
•
•
Viral genetic entities are integrated within the host
genome at gene fragile sites or cell-specific sites of integration. These integrated genetic elements influence
the expression of their neighboring genes on the same
chromosome.29
Although these elementary regulatory sequences are
retained in proviruses, yet in most cases, there is a
loss of functional nucleocaspids or packaging signal,
which further ceases viral replicability and horizontal
transmission.30
Domesticated proviruses with lost replicative ability can
be reactivated by functional protein product of spontaneous exogenous retrovirus infection and, thus, may exhibit
transient replication ability.31
The viral sequences regulate the expression of several
genes through their flanking regulatory regions, ie,
long-terminal repeat (LTR) region containing insulators,
enhancers, and promoters along with other regions such
as primer binding sites (PBS), packaging signals (ψ), and
the polyurine tract signals.32
Overall, these viral transgenes have served as transposable elements and also as fixed transgenes in the host
genetic network. These genetic elements add more diversity and alter the host genetic map leading to an active
genetic rewiring of host genome, 32 often termed as “Restless
genome.” These transgenes are known to be highly sensitive to external stimulus and thus form an important part
in understanding the systemic biology. 32 For example, the
activation of ERVs by spontaneous infection or the activation of exogenous viruses possesses another dimension of
viral transgene-mediated regulation of host genes and systemic responses. 31 Even viruses with defective genetic entities or those that are silenced can be reactivated by using
the polymerases of active virus infection. 32 In addition, viral
genetic fragments devoid of replicative ability or replicationdeficient ERVs are known to be duplicated by mere errors
during host genomic replication, 32 especially human ERVs K
(HERVK), also known as the most recently acquired retroviruses with multiple copies of open reading frames, resulting
in HERVK-encoded viral protein. However, recent work by
Grow et al 33 has identified that the presence of DNA hypomethylation at LTR regions followed by transactivation by
Oct-4 binding in the HERVK genetic inserts leads to reactivation of these transgenes in human embryos and germline
cells. These widespread mechanisms of viral integration, replication, or duplication of retroviruses have presented these
“proviruses of interest” for their role in evolution, fitness,
and adaptability. However, much interest has been focused
recently on understanding the unique expression pattern of
these proviruses in stem cells in comparison to that of the
differentiated cells.
Endogenous Retrovirus Family and Diversity
The conventional classification of the viruses using the International Community of Taxonomy of Virus (ICTV) relies
on factors such as the presence of oncogenes, viral core,
and accessory gene elements and the range of their host.19,33
Unfortunately, most of the ERVs (largely present as fragments
within the host genome) cannot be mapped to their infectious exogenous virus counterparts and, thus, do not find a
place in the conventional ICTV classification. These ERVs
also vary among themselves depending on their primer binding sites as these are important sites for the start of reverse
transcription.19,33 Different viruses use different tRNA primers (tRNA Pro/tRNAGlu, etc.) specific to their PBS sequences.33
Copy number of ERVs also plays a crucial role in the silencing
of these ERVs and their expression in differentiated cells.34
ERVs with high copy number are known to be gradually
silenced than those of ERVs with low copy numbers, which
are silenced spontaneously.34 Thus, the expression of different families of ERVs not only varies according to their PBS
or other regulatory sequences but also varies according to
their classes (ie, class I, class II, or class III) and their relative copy number in the host genome. Interestingly, it is also
known that the proviral integration within the host genome
is skewed for host preference, and thus, the classification of
Signal Transduction Insights 2016:5
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Figure 4. Proviral silencing mechanism in ES cells. The silencing machinery of provirus in embryonic cells involves DNA/histone methylation-,
deacetylation-, and sumoylation-based epigenetic modifications in a Trim-28-mediated pathway for Pro-specific PBS and in a YY1-dependent pathway
for alternate PBS.
viruses is also based on the range of host that they can infect. 35
Different classes of ERVs mostly infect vertebrates, and it has
been known that such a host preference is not a result of competitive exclusion of the hosts. Thus, the selection of host, copy
number, and frequency of proviral expression holds mutualistic benefits in the host–virus relations. Table 1 summarizes
different classes of ERVs as per the ICTV system of virus
classification.
Class I ERVs in humans are known as HERVs and contain the largest families of ERVs in human (around 19 families) with copy number varying from one to several hundreds.12
However, most of the class I ERVs have been widely defective due to different mutations and silencing mechanism and,
thus, are partially or fully intact. Class II HERVs (with minimum 10 independent families) is also referred as HERVK
superfamily.12 In comparison to class I and class III HERVs,
this is the least populated class of ERVs. In contrast, class
II ERVs are the most populated ERVs in mouse justifying
18
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the host-specific bias of integration of proviruses. Class III
HERVs are the second most populated human ERVs consisting of four families of ERVs with a copy number of 200–500.12
Proviral Silencing Machinery in Embryonic
Stem Cells
The silencing of ERVs is heralded by the binding of a zinc
finger DNA-binding protein, ie, Zfp809, to the proviral DNA
sequence at the primer binding site.25 The binding of Zfp809 is
dependent on the recognition of specific PBS sequence, which
is tRNAPro in most cases. Subsequently, Zfp809 recruits tripartite motif-containing 28 (Trim-28), also known as KAP-1
or Tifb-1.25 Trim-28 is the universal regulator of proviral
silencing via recruitment of several chromatin remodeling
factors to the proviral DNA sequence and epigenetic repressive marks on the proviral DNA or histones (Fig. 4). 36 The
most common repressive marks include DNA methylation by
DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b)
Provirus silencing in stem cells
Table 1. Classification of ERVs as per the International Community of Taxonomy of Virus (ICTV).
ENDOGENOUS RETROVIRUS
GENERA
PROTOTYPE-MEMBER
CRITERIA FOR CLASSIFICATION
HOST
Alpharetroviruses
Avian Leukaemia Virus (ALV)
Rous Sarcoma Virus (RSV)
Identified in the Gallus genes of
game birds and classified into
10 subgroups based on envelope
specialities.
Gallus gallus (domestic chicken),
red jungle fowl
Betaretrovirus-related
ERVs
Mammalian Type-B retroviruses
Mammalian Type-D retroviruses/
MPMV (Mason-Pfizer monkey
virus) or SRV-3
Simian retroviruses (SRV-1/2/4/5)
Jaagsiekte sheep retrovirus (JSRV)
Intracisternal A-type particle (IAP)
Transmission vertically from
mother to pup (via milk), sag (an
accessory gene encoding super
antigen) and the ability to induce
tumuors by oncogenes
Captive and Wild primates
including ungulates, yellow
baboons, brush tailed possums,
spectangled langurs, squirrel
monkeys
Domestic sheep
Mice, Chinese and Syrian
hamsters and brown rats
Gammaretrovirus-related
ERVs
Murine Leukaemia Virus (MuLV)
Gibbon ape Leukaemia Virus
(GaLV)
Feline leukaemia virus (FLV)
Porcine endogenous viruses
(PERVs)
Reticuloendothelosis viruses
(REVs)
Spleen Necrosis Virus (SNV)
Simple genomic organisation,
non­defective viruses without
accessory genes, many
replication defective viruses with
coded oncogenes, aetiology of
leukaemia and sarcomas within
mice
More than one vertebrate
class like mice, humans, apes,
gibbons, feline and porcine
Birds like ducks, chickens,
geese, pheasants and turkeys
Trager Ducks
Epsilonretrovirus-related
ERVs
Walley dermal sarcoma viruses
(WDSV)
Walley epidermal hyperplasia
viruses type I and II (WEHV I
and II)
Snakehead retrovirus (SnRV)
Perch hyperplasia virus (PHV)
Large complex genomes (upto
13 kb in length), contain single
accessory gene product similar to
cellular gene with duplicated copy
of cyclins
Fish, Perch, Snake, African
clawed toad
Spumavirus-related ERVS
Feline Spumavirus (FeSV)
Bovine Spumavirus (BSV)
Chimpanzee foamy virus (CFV)/
Human foamy virus (HFV)
Lack any definitive association
with diseases
Birds (tinamou),reptiles (SpEV
within tuatara), amphibians
(within caecilians) and mammals
like humans
Deltaretrovirus/Lentivirus
related ERVS
Human Immunodeficiency Virus-1
(HIV-1)
Human Immunodeficiency Virus-2
(HIV-2)
Complex genome organisation,
slow, smouldering and chronic
disease pattern and encode
at-least two trans­acting regulatory
Tat and Rev proteins in lentivirus/
Tat and Rex in delta retrovirus)
along with gag and pol proteins
Mammals like primates, domestic
and wild felids and domestic
ungulates like goats, sheep, cattle
and horse (lentivirus)
Mammals including primates,
cattles (delta retroviruses)
Notes: The classification lists seven genera of ERVs, including Alpharetroviruses, Betaretrovirus-related ERVs, Gammaretrovirus-related ERVs, Epsilonretrovirusrelated ERVs, Spumavirus-related ERVS, and Delta retrovirus/Lentivirus-related ERVS.13,19
as well as histone methylation by H3K9 methyltransferase,
ie, ESET/SETDB1, 24 H3K9 methyltransferase G9, 37
H3K4 methyltransferase Suv39h1/Suv39h2, 38 and H3K20
methyltransferase Suv420h1 (Fig. 4).15 It has been well understood that the proviral gene silencing occurs via both a DNA
methylation-dependent and -independent manner (histone
methylation). For most cases of histone methylation, ESETmediated H3K9me3 is known to be crucial and indispensable
for proviral silencing.24 G9-mediated H3K9me2 is known to
be the other arm for H3K9 methylation, which acts in response
to be spontaneous and transient activation of retroviruses upon
exogenous virus infections. However, G9-mediated methylation is dispensable for prolonged proviral silencing. 37 Apart
from methylation, it has been recently shown that events of
deacetylation and sumoylation are crucial in silencing the
ERVs in embryonic stem (ES) cells.39 Yang et al39 through a
genome wide screen of mouse ES cells have shown the role of
histone chaperons Chaf1a and Chaf1b and sumoylation factors like Sumo2, Ubei 2 and few others in Trim-28-mediated
proviral silencing (Fig. 4).
ES cells, which otherwise exhibit a large open chromatin for expression of a wide range of genes required for
development, can still harbor local heterochromatized structure spanning the ERV sequences leading to a silencing of
these ERVs in undifferentiated or stem cell populations. ERV
silencing is also achieved by local heterochromatization of the
proviral genetic fragments.40 This process of heterochromatin formation is guided by HP1 (heterochromatin-associated
protein 1)40 and PcG I and II proteins (polycomb group I
and II proteins).41 These proteins lead to the H3K27me3
Signal Transduction Insights 2016:5
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Satapathy
enrichment of class I and class II ERVs and KDM1A (lysinespecific demethylase-1A) enrichment of class III ERVs.42
Although the Trim-28-dependent silencing machinery
depends on upstream recognition of specific PBS (tRNA Pro).
Most of the ERVs are also known to use alternate PBS for the
binding of primers for initiation of reverse transcription. However, all such ERVs are also effectively silenced in embryonic
cells by Trim-28-independent ERV silencing.42 Schlesinger
et al have shown that another zinc finger DNA-binding protein Ying Yang 1 (YY1) can repress proviral silencing for such
ERVs using alternate PBS recognition, and even Rex1 (a YY1
family protein) is known to be important for proviral silencing. YY1 is known to bind to the regulatory region within the
LTR of ERVs (Fig. 4).42 In addition, Schlesinger et al have
shown that in ES cells, YY1 can partially interact with Trim28 and mediate proviral silencing, and such an interaction is
lost in differentiated cells.42
Interestingly, a 3′D4Z4 insulator sequence along with
HS4 insulators (in the LTR region) protects the ERVs from
silencing and repressive epigenetic marks, leading to a variable and persistent expression of ERVs for a limited number
of cell cycles.43 3′D4Z4 does not target integration sites on
specific chromosomal location but confers protection from
proviral silencing.43 Therefore, a critical understanding of the
proviral silencing machinery aims to highlight the role of the
LTR regions flanking the ERV genetic elements in cell fate
determination and regulation of cellular development in comparison to their undifferentiated counterparts.
Pattern of Proviral Silencing
The retroviral silencing machinery in ES cells is composed
of different subunits with several factors regulated in a cellspecific manner. The role of PBS in proviral expression
has been studied by Taichi et al44 and Baghi et al45 using
Moloney murine leukemia virus in mouse.46 It is known
that the retroviral silencing is dependent primarily on the
recognition of primer binding site (PBS) of the ERVs.46
Therefore, viruses using similar primer for recognition of
PBS are classified together as a group of related viruses and,
thus, are expected to be under a similar regulatory mechanism for expression or silencing in different cell types. The
proviral silencing machinery in ES cells inhibits the proviral gene expression by exerting transcriptional repression through insulator sequences of LTR region, inhibition
of transcription factor recruitment and binding to proviral
promoters/enhancers, as well as induction of local heterochromatization of gene harboring the proviral sequence.47
In addition to this transcriptional inhibition, several
posttranscriptional modifications such as methylation,
deacetylation, sumoylation, and characteristic histone marks
regulate the pattern of proviral silencing.
The pattern of proviral silencing involves a mixed population of ES cells, which include cells with spontaneously
silenced ERVs, cells with moderate expression of ERVs, and
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cells with high expression of ERVs (rare population also
known as escapees) (Fig. 5).48 For any population of embryonic cells reactivated by exogenous retrovirus infection, the
silencing of all ERVs may not be all at the same time but may
be gradually silenced over a few generation of cell passages
(in vitro), and thus, in between this process, there is always a
chance to recover all three types of ES cells with a spectrum
of ERV expression, ie, from a silenced or almost negligible
ERV expression with moderate-to-high ERV (rare) expressing cells.48 In addition, the repressive DNA/histone marks
seen in ES cells leading to proviral silencing is seen across
all the subpopulation of cells such as those with completely
silenced ERVs to those with moderate and high ERV expressions.48 Therefore, the recruitment of the methyltransferases,
acetyl transferases, or other sumoylation factors is spontaneous upon exogenous retrovirus infection, although the silencing of ERVs is attained in a more gradual manner. Thus,
when the ERVs were integrated for the first time into the host
genome, the silencing of the ERVs was rather gradual than the
anticipated spontaneous and global silencing of ERVs. This
hints for a possible selection of gradual silencing machinery
in response to the beneficial or detrimental effects of the ERV
expression.
In addition, it has been appreciated that the silencing
pattern of ERVs is greatly influenced by the copy number of
the ERVs.49 Viruses integrated into the host genome with
high copy number may also lead to a gradual silencing effect
on ERVs, although the histone/DNA epigenetic marks are
enriched spontaneously.49
Thus, the mode of retroviral silencing is more local,
stochastic, and asynchronous than that of a global and synchronous
retroviral silencing (Fig. 5).48 The herald of retroviral silencing
machinery may not be understood as block and silence but must
be viewed as block and progressing toward silence. Therefore,
the silencing of ERVs aims at attaining an effective cell type
and virus copy number-dependent silencing pattern, which is
largely follows a local, stochastic, and nonsynchronous model.
Understanding the mechanism of transgene silencing can
facilitate targeted gene therapy or virus-based cellular reprogramming. Interestingly, both the types of provirus silencing
dependent and independent on the cell type are known to use
a broad strategy of provirus silencing machinery. As discussed
earlier, this broad choice of silencing machinery involves using
both PBS-binding tRNA primers and PBS-independent
silencing machinery.
Provirus in Cell Fate Specification
and Determination
Embryonic cells are known to be under wide range of epigenetic regulatory network facilitating the differentiation
of these cells into different cell types.50 Often such cells
are under bivalent histone modifications of both activating
nature and repressing nature, leading to selective regulation
of transcription. This ensures a stage-specific transcriptional
Provirus silencing in stem cells
Figure 5. Asynchronous provirus silencing in embryonic cells in comparison to that of differentiated cells. The ERVs are known to be silenced in a local,
asynchronous, and gradual process in embryonic cells depending on the site of integration, the class of ERVs, and the copy number of these ERVs. The
population of ES cells includes cells with spontaneously silenced ERVs, cells with moderate expression of ERVs, and cells with high expression of ERVs
(escapees). In comparison, differentiated cells exhibit a variable ERV expression guided by the nearby genes and copy number.
network rewiring and the onset of differentiation from undifferentiated progenitor cells.50
The relevance of proviruses in regulating the nearby host
cellular genes is essential to understand that silenced proviruses in stem cells can downregulate the expression of the
nearby genes and, reciprocally in differentiated cells, these
proviral elements can regulate the host cellular genes sitting
close to them in order to specify and determine the cell fate.51
For example, the use of 5-aza′-2′-deoxycytidine (blocking
DNA methyltransferase) suppressed the methylation of proviruses and blocked the expression of the nearby host cellular genes. In addition, the importance of the chromosomal
site of integration for proviral silencing has been shown in
several other reports.52–54 A change in ERV expression can
modulate the epigenetic drift in stem cells and, thus, play a
major role in cell lineage specification and fate determination.
Thus, it is reasonable to predict the role of proviruses in cell
fate determination via host cellular genes neighboring these
viral elements.
The ability of the ERVs to act as retrotransposons can
regulate the expression of host cellular genes. Retrotransposons have been widely appreciated for their role as controlling elements as they can generate variable expression of host
genes arising from accumulating mutations, alternations
in coding sequences of host genes, and activation of several
proto-oncogenes.55 For example, retrotransposon-based activity of ERVs has been widely studied for targeting salivary
amylases.56
ERVs can also act as alternate enhancers and promoters for
host cellular genes. ERV sequences acting as promoters can
induce tissue-specific gene expression as well as code for specific regulatory elements like (long noncoding RNAs). 37 Such
an ERV-based regulation of host cellular genes is known to be
self-regulatory and acts in trans to other host cellular genes.
For example, the “solo” LTR of mouse ERVL is known to
act as an alternate promoter of nearby genes.57 Mouse-sex
limited protein (Slp) gene is known to harbor ERVs close to
its transcription start site controlling the expression of genes
important for placental development and pregnancy.58 Human
ERV-H can regulate cell fate by interacting with Oct-4 and
acting as promoter enhancers via LTR7 for nearby genes to
maintain pluripotency.58 In mouse and human genomes,
ERV1 is known to carry transcription factor-binding site
for pluripotency-associated genes such as Oct-4 and Nanog,
exerting regulation of host cell pluripotency.58 Statistically,
about 30% of 5′-capped human mRNA transcripts and 25%
of the coding genes (at their 3′-UTR) harbor repetitive ERV
elements.58 Such an ERV-mediated regulation of host cellular genes usually occurs in smaller magnitude, although it
affects a wide range of host genes. Given that several ERVs are
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Satapathy
present in high copy numbers, each ERV can act as alternate
promoter and enhancer for several host cellular genes.
Several ERVs and ERV-associated gene regulators are
known to be crucial for host development and cell fate decision.
Approximately 25% of mouse ERVL copies are known to be
activated during embryogenesis and is known to affect cellular
genes (307 protein-coding genes), leading to the expression of
around 625 chimeric transcripts.58 Such an activated MERVL
expression during gastrulation is also associated with epigenetic
marks like H3K4 me3 and downregulation of KDM1A, which
otherwise demethylates histones.59 Although most of the ERVs
are silenced in embryonic cells, certain ERVs are selectively
upregulated for their role in host cell development. LINE-1
(long interspersed nuclear elements) forms the group of rare
ERVs, which are upregulated in ES cells in comparison to differentiated cells.60 In addition it has been shown that ERVs are
found in approximately one-third of p53 protein-binding sites
and, thus, pose their relevance for cellular development.61
Gene therapies using retroviral or lentiviral vector system are known to induce host genotoxicity due to insertional
mutagenesis.62 However, the impact and relative scale of genotoxicity is different for undifferentiated and somatic cells.63 In
a study, Zheng et al64 have compared the retrovirus-mediated
insertional mutagenesis in T-cell-committed lymphoid cell
lines and in iPSCs. The results indicate that retroviral silencing in iPSCs results in downregulation of major host cellular genes and, therefore, fail to commit to specific lineage,
whereas T-cell lymphoid cell lines show upregulated expression of host cellular genes neighboring upregulated ERVs.64
In another study, Ramos-Mejía et al have shown that the
escapees or ERVs that escape retroviral silencing can prevent
differentiation of iPSCs into cord blood CD34 + progenitor
cells.65 This indicates that retroviral transgene expression can
potentially limit cell differentiation or lineage specification.
Reactivated ERVs can also determine cell fate. Reactivation of ERVs is dependent upon spontaneous exogenous retrovirus infection, recombination with exogenous
retroviruses during infection and alteration in their genetic
regulatory elements induced by external factors. For example,
infection by avian leukemia virus in provirus-harboring cells
leads to recombination leading to newer pathogenic variants,
modulation of host immune response upon actual infection, as
well as destruction of specific target cells upon viral infection.66
There are reports of neoplastic transformations in certain cells
due to insertional activation of oncogenes mediated by ERVs.67
Interestingly, most of the ERVs implicated for pathogenicity or
deleterious functionality upon reactivation belong to the most
recent class of retroviruses integrated into the host genome.
Conclusion
Retroviral genome poses a diverse aspect of evolutionary biology in case of complex organisms. These retroviruses are well
known for their fast evolving capacity and their coevolution
pattern with respect to their vertebrate hosts mainly humans.
22
Signal Transduction Insights 2016:5
The presence of ERVs pertains to a more complex biological
question than what is being currently investigated through a
binary simplification of the case, ie, whether the proviruses are
active or silenced. The large time scale of integration of these
retroviruses into the host genome and the effective silencing of
these ERVs during vertical transmission suggest for an intertwined host–provirus relationship. The mutual benefit of such
a co-opted relationship gets substantiated by the fact that these
ERVs have a wider role in diversifying the transcriptional
network of the host. The fitness of host, especially humans,
largely depends on the ability to maintain different cellular
populations, including stem cells, undifferentiated progenitor cells, and terminally differentiated cells. This presents a
situation of stage-specific and cell origin-dependent mechanism of cell fate determination that requires an undulating
transcriptional landscape enriched with a wide variety of epigenetic modifications (both activating nature and repressing
nature).The potency of proviruses to rewire the transcriptional
regulatory network through the LTR region to act as alternate promoters and enhancers and regulate the expression of
noncoding RNAs can be viewed as pivotal in pluripotency
maintenance, lineage specification, and cell fate commitment.
However, such a hypothesis does not apply to all the proviruses; there is diversity among different ERVs belonging to
different classes.
This review aims to understand (a) the evolutionary diversity of retroviruses, (b) identification and characterization of
their potential to act as transcriptional regulatory elements,
(c) their skewed host preference for vertebrates, and (d) the
long time scale of coevolution with the complex organisms.
The intricate relationship between the host cellular genes and
residing proviruses has been critically analyzed in this study
with special focus on their role in host cell fate determination and development. An effective proviral regulation can
render improved gene therapy, control random gene expression associated with diseases, and develop a fine tuning of
proviral gene expression for using these as markers of cell fate
(ie, pluripotency and differentiation).
Author Contributions
Conceived and designed the experiments: SS. Analyzed the
data: SS. Wrote the first draft of the manuscript: SS. Made
critical revisions and approved final version: SS. Author
reviewed and approved of the final manuscript.
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