Download Vertebrate Virus-Encoded MicroRNAs and Their Sequence

Jpn. J. Infect. Dis., 64, 357-366, 2011
Invited Review
Vertebrate Virus-Encoded MicroRNAs and
Their Sequence Conservation
Kahori Takane1,2 and Akio Kanai1,2*
1Institute
for Advanced Biosciences, Keio University, Tsuruoka 997-0017; and
Biology Program, Graduate School of Media and Governance,
Keio University, Fujisawa 252-8520, Japan
2Systems
(Received June 27, 2011)
CONTENTS:
1. Introduction
2. Vertebrate virus-encoded miRNAs
3. Nucleotide sequence conservation among viral
miRNAs
4. Viral RNAs targeted by viral miRNAs
5. Host mRNAs targeted by viral miRNAs
6. Conclusion
SUMMARY: An increasing number of studies have reported that approximately 400 microRNAs
(miRNAs), encoded by vertebrate viruses, regulate the expression of both host and viral genes. Many
studies have used computational and/or experimental analyses to identify the target genes of miRNAs,
thereby enabling us to understand miRNA functions. Here, we suggest that important aspects become
apparent when we focus on conserved viral miRNAs, although these miRNA sequences generally show
little similarity among viral species. Reliable viral miRNA–target gene pairs can be efficiently identified
using evolutionary information. In this review, we summarize information on (i) the nucleotide sequence conservation among viral miRNAs and (ii) the RNAs targeted by viral miRNAs. Recent advances in these topics are discussed.
1. Introduction
The discovery of huge amounts of noncoding RNAs
(ncRNAs) has indicated the importance of RNA molecules in many steps of gene regulation (1). Among the
ncRNAs, microRNAs (miRNAs) are very small molecules of approximately 20 ribonucleotides, which function posttranscriptionally by hybridizing to their target
mRNAs. Typically, miRNAs repress the translation or
trigger the degradation of their target mRNAs (2). An
increasing number of studies have shown that not only
animals and plants but also viruses encode miRNAs,
which target both viral and host mRNAs to control their
expression (3–6). Because an understanding of viral
miRNAs is necessary for both basic science and the development of therapeutic agents, we focus on viral
miRNAs in this review. In Fig. 1, we summarize existing
knowledge regarding the regulatory relationships between viruses and host cells mediated by miRNAs. Basically, this figure shows the flow of genetic information
from DNA to protein, which is the ``central dogma'' of
molecular biology. It is noteworthy that both viral and
host miRNAs can control the flow of genetic informa-
Fig. 1. Schematic representation of the regulatory relationships
mediated by miRNA in the genetic information flow between
virus and host cell. The flow of genetic information from DNA
to protein, which is the central dogma of molecular biology, is
shown for both the virus and host cell. A recent study demonstrated that miRNAs are not only encoded in the genomes of
host cells, but also by certain viral genomes, and play important roles in their gene regulation. The arrows indicate the
regulatory interactions that affect the genetic information
flow. Four examples of miRNA-mediated regulatory interactions, represented by bold arrow (A–D), are described in the
text.
*Corresponding author: Mailing address: Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata
997-0017, Japan. Tel: ＋81-235-29-0524, Fax: ＋81-23529-0525, E-mail: akio＠sfc.keio.ac.jp
tion according to the central dogma. As examples,
virus-encoded miRNAs target viral RNAs and host cell
RNAs (regulatory relationships A and B, respectively,
in Fig. 1) (4–6), whereas host cell-encoded miRNAs target viral RNAs (regulatory relationship C in Fig. 1) (4).
It is also noteworthy that a viral protein can interact
with a host protein (regulatory relationship D in Fig. 1).
This article is an Invited Review based on a lecture
presented at the 20th Symposium of the National Institute
of Infectious Diseases, Tokyo, May 21, 2010.
357
A viral protein has been reported to bind the Argonaute
(Ago) protein, one of the most important factors
involved in the functioning of the RNA-induced silencing complex (RISC) (7). Based on these findings, the
miRNA regulatory systems of viruses and their host
cells seem to mediate the conflicts between them.
Understanding this complex regulatory relationship is
important for the development of antiviral measures.
In this paper, we review the recent progress in virusencoded miRNAs, focusing particularly on vertebrate
viruses, and list the known virus-encoded miRNAs. The
nucleotide sequence features of these miRNAs are then
discussed based on sequence conservation analysis. To
understand the functions of these miRNAs, it is necessary to identify their target mRNAs. Therefore, we provide information on the cellular and viral targets of
these viral miRNAs.
2. Vertebrate virus-encoded miRNAs
Five miRNAs encoded by a viral genome were first
reported in the Epstein-Barr virus (EBV) in 2004 (6),
and 400 viral miRNAs encoded by 22 viruses are currently registered in miRBase (release 17.0, April 2011).
The miRBase is one of the most useful databases,
providing an integrated interface for comprehensive
miRNA sequence data, annotations, and predicted gene
targets (8). In most cases, just as viral miRNAs are synthesized in host cells, they are thought to be processed
by the host miRNA-processing machinery (9,10). Before
discussing recently reported virus-encoded miRNAs in
detail, we will briefly describe the general miRNA biogenesis pathway in host cells (Fig. 2). Generally, several
RNA-processing steps are required to produce mature
miRNAs. Initially, the primary miRNAs (pri-miRNAs)
are transcribed from their genes in the nucleus (11). PrimiRNAs are processed into approximately 70-nucleotide hairpin RNAs by Drosha, a nuclease of the RNase
III family, and DGCR8 (12,13). The processed RNAs
are called ``precursor miRNAs'' (pre-miRNAs), and are
exported to the cytoplasm by Exportin-5, where the
miRNA duplexes produced are cleaved by another
RNase III nuclease, Dicer (14,15). The mature miRNAs
(``guide strands'') are then incorporated into the
miRNA-induced silencing complex (miRISC) to play a
role in regulating gene expression, whereas the other
strands (``passenger strands'') are immediately degraded, although it has also been reported that both the
guide and passenger strands are potentially functional in
some cases (16).
Two approaches are generally used to identify viral
miRNAs. The first involves experimental validation,
such as molecular cloning and nucleotide sequencing of
the small RNA fraction (6), whereas in the second, a
bioinformatics methodology is used. This approach is
based on computational prediction, such as predicting
the secondary structures of pre-miRNAs from viral genomes, or distinguishing positive from false miRNAs to
identify their nucleotide sequences and structural characteristics using a machine learning technique (17,18). It
has been suggested that high-throughput analyses combined with computational analyses constitute the most
effective approach (19,20). In Table 1, we summarize
the currently known miRNAs encoded by vertebrate
Fig. 2. General miRNA processing pathway. Initially, miRNA
genes are transcribed to produce primary miRNAs (primiRNAs) in nucleus. RNase III enzyme Drosha and its binding
partner DiGeorge-syndrome critical-region protein 8
(DGCR8), also known as Microprocessor, processes primiRNAs into precursor miRNAs (pre-miRNAs). After being
exported to cytoplasm by a complex of Exportin-5 and RanGTP, pre-miRNAs are cleaved into miRNA duplexes by
another RNase III enzyme Dicer with its cofactor transactivating region RNA-binding protein (TRBP). One strand of
miRNA duplexes is incorporated into miRNA-induced silencing complex (miRISC) containing Argonaute 2 (AGO2) for
mRNA degradation or translational repression. Triangles in
both pri-miRNA and pre-miRNA indicate the cutting sites of
RNase III enzymes.
viruses. The number of viral miRNAs ranges from 2 to
68 (approximately 16 per viral genome). Among these
viruses, the Herpesviridae encode large numbers of
miRNAs, and rhesus lymphocryptovirus (RLCV) encodes 68 miRNAs. In contrast, only 1–4 miRNAs are
found in the families Polyomaviridae, Papillomaviridae, Adenoviridae, and Retroviridae. We note that
most viruses encoding miRNAs belong to doublestranded DNA virus families, such as the Herpesviridae,
Polyomaviridae, Papillomaviridae, and Adenoviridae.
In contrast, there have been no reports of RNA viruses
encoding miRNAs, except for Retroviridae human immunodeficiency virus 1 (HIV-1). This is probably because the replication systems of the double-stranded
DNA viruses and RNA viruses differ. In general,
double-stranded DNA viruses replicate their genomes in
the host nuclei, whereas RNA viruses replicate their genomes in the host cytoplasm. As described above, host
nuclear factors such as Drosha and DGCR8 are required
for the pre-miRNA processing step. Therefore, many
358
Table 1. List of vertebrate virus-encoded miRNAs
Family
Herpesviridae
Subfamily
Name
a-Herpesvirinae
Herpes simplex virus-1 (HSV-1)
Herpes simplex virus-2 (HSV-2)
Marek's disease virus-1 (MDV-1)
Marek's disease virus-2 (MDV-2)
Turkey herpesvirus (HVT)
Infectious laryngotracheitis virus (ILTV)
Bovine herpesvirus (BHV1)
Herpes B virus (HBV)
Human cytomegalovirus (HCMV)
Mouse cytomegalovirus (MCMV)
Epstein-Barr virus (EBV)
Rhesus lymphocryptovirus (RLCV)
Kaposi's sarcoma-associated herpesvirus (KSHV)
Rhesus monkey rhadinovirus (RRV)
Mouse gamma herpesvirus-68 (MHV68)
b-Herpesvirinae
g-Herpesvirinae
Polyomaviridae
—
Retroviridae
1):
—
Lentivirinae
Host
Reference
25
24
26
36
28
10
12
3
17
29
44
68
25
11
28
(16)
(18)
(14)
(18)
(17)
(7)
(10)
(3)
(11)
(18)
(25)
(36)
(12)
(7)
(15)
Human
Human
Avian
Avian
Avian
Avian
Cattle
Simian
Human
Murine
Human
Simian
Human
Simian
Murine
50–52
50, 53, 54
55, 56
23, 24
24, 57
24, 58
59
60
17, 61
62, 63
6, 64–66
64, 67, 68
17, 65, 69, 70
71
17
2
2
2
2
2
2
(1)
(1)
(1)
(1)
(1)
(1)
Simian
Simian
Human
Human
Human
Murine
18
72
73
74
74
75
1 (1)
Bandicoot
76
1 (1)
Bandicoot
76
Human adenovirus (AV)
3 (2)
Human
77
Human immunodeficiency virus 1 (HIV-1)
4 (3)
Human
21, 22
Simian virus 40 (SV40)
Simian agent 12 (SA12)
Merkel cell polyomavirus (MCV)
BK polyomavirus (BKV)
JC polyomavirus (JCV)
Murine polyomavirus (PYV)
Bandicoot papillomatosis carcinomatosis virus
type 1 (BPCV1)
Bandicoot papillomatosis carcinomatosis virus
type 2 (BPCV2)
Papillomaviridae
Adenoviridae
No. miRNAs1)
Number of precursor miRNAs is shown in parentheses.
Table 2. Nucleotide sequence conservation of viral miRNAs in
miRBase
viral miRNAs may be found in double-stranded DNA
viruses. However, miRNAs have been reported in
HIV-1 of the family Retroviridae (21,22). This retrovirus can integrate into the host nuclear genome as
double-stranded DNA using the virally encoded enzymes RNA reverse transcriptase and integrase. Therefore, miRNAs are produced from the genome of this
retrovirus as they are from double-stranded DNA
viruses.
3. Nucleotide sequence conservation among
viral miRNAs
As discussed above, the number of studies on viral
miRNAs is increasing. Recent studies have also reported
that viral miRNA sequences are less similar to one
another than are nonviral miRNAs, although the
genomic locations of the viral miRNAs are often conserved (23,24). However, some questions remain regarding how many viral miRNA sequences are conserved in
the same viruses or individual viruses, and whether any
sequence characteristics exist in these miRNAs. To address these questions, we comprehensively analyzed the
nucleotide sequence conservation among viral miRNAs.
For this purpose, we first aligned the 400 viral
miRNA sequences registered in miRBase in all combinations (79,800 pairs in total). The levels of conservation
among these pairs are listed in Table 2. The sequence
similarities of the 79,800 miRNA pairs were calculated.
No. of combinations
(without considering
seed sequence match)
No. of combinations
(with complete match
of seed sequence)
Total
79,800
79,800
Æ30z
Æ40z
Æ50z
Æ60z
Æ70z
Æ80z
Æ90z
100z
56,083
19,078
3,390
277
67
34
17
4
90
89
72
50
34
22
14
4
Numbers of combinations of conserved viral miRNAs in various
viruses are shown with their similarities. In the left column, only
the sequence similarity is considered, whereas in the right
column, both the sequence identity and the seed sequence matches are considered (nucleotides 1–7, 2–8, or 3–9 from the 5? end of
the miRNA).
The ``seed'' sequences include nucleotides 2–8 from the
5? end of the miRNAs, and are important for target
recognition (25). Therefore, we considered two sets of
data: the degree of similarity between sequence pairs
when the seed sequence matches were not considered
and the degree of similarity in sequence pairs when their
seed sequences showed complete matches. As is evident
359
spectively (Table 2). Notably, among the same or related viruses, the viral miRNA sequences are 100z identical for four pairs (bkv-miR-B1-3p and jcv-miR-J1-3p,
ebv-miR-BART1-3p and rlcv-miR-rL1-6, hvt-miR-H93p and hvt-miR-H12-3p, and bpcv1-miR-B1 and bpcv2miR-B1), although the pre-miRNA sequences are not
completely conserved (Fig. 3). This strongly suggests
that both these miRNAs and their possible targets have
been highly conserved, and have influenced viral function throughout viral evolution. For instance, the identical miRNAs of the polyomaviruses (bkv-miR-B1-3p and
jcv-miR-J1-3p) have functions that allow the viruses to
evade immune cell attack (26). Specifically, these two
miRNAs target the 3? untranslated region (3?-UTR) of
ULBP3 mRNA, which encodes a stress-induced ligand
recognized by the killer receptor NKG2D of natural
killer (NK) cells. Focusing on the conserved miRNA–
target gene pairs is also very important for an understanding of the evolution of miRNA-mediated regulation. Recently, we reported that miRNA–target gene
pairs can be effectively identified based on the evolutionary analysis of bilaterian animals (27).
Let us examine the characteristics of miRNA pairs
with more than 80z sequence similarity. Twenty-two
pairs with this degree of this similarity have completely
matched seed sequences, whereas 34 pairs have this
degree of this similarity if we do not consider the seed
matches (Table 2). Therefore, 12 pairs have Æ80z sequence similarity but incompletely matched seed sequences. Three representative examples of these 12 pairs
are illustrated in Fig. 4. We have identified a rule for the
pattern of nucleotide sequences in the seed region. As
shown in Fig. 4, these miRNA pairs contain nucleotide
mismatches in the seed region. When G–U wobble pairs
are permitted, it is conceivable that these pairs recognize
identical or orthologous target mRNAs. Indeed, it has
been reported that miRNA sequences that form G–U
pairs recognize target mRNAs and reduce their expres-
from this table, the numbers of conserved miRNA pairs
(80z–100z sequence similarity) are relatively small.
We also note that the numbers of conserved miRNA
pairs differ depending on whether we ignore the seed
sequence matches or consider the completely matched
seed sequences. The number of conserved miRNA pairs
(Æ70z sequence similarity) is considerably larger when
we do not consider the seed sequence matches than
when we consider the completely matched seed sequences. Assuming that the seed sequence is important
for the recognition of target mRNAs, the large numbers
of pairs with only 30z–60z sequence similarity are
considered to be false-positive pairs.
Therefore, we focus on the evolutionarily conserved
miRNAs in this review and describe the importance of
analyzing pairs with high sequence similarity. For example, when considering the seed sequence matches, the
number of miRNA pairs with more than 70z, 80z,
and 90z sequence similarities are 34, 22, and 14, re-
Fig. 3. Secondary structures of virus-encoded pre-miRNAs sharing the same mature miRNA sequences. (A–D) All pairs of premiRNAs with 100z identical mature miRNA sequences are
shown. The mature miRNA sequences are shown in bold uppercase letters. Notably, both the secondary structure and
nucleotide sequence of the precursor miRNA differ slightly,
despite the complete match of the mature miRNA sequences in
these four pairs. bkv, polyomavirus BK; jcv, polyomavirus JC;
ebv, Epstein-Barr virus; rlcv, rhesus lymphocryptovirus; hvt,
turkey herpesvirus; bpcv1, bandicoot papillomatosis carcinomatosis virus type 1; bpcv2, bandicoot papillomatosis carcinomatosis virus type 2.
Fig. 4. Three examples of highly conserved miRNAs encoded by
viruses. (A–C) Nucleotide sequences of mature viral miRNAs
with Æ80z conservation are aligned. Conserved nucleotide
residues are indicated with an asterisk. Gaps are inserted for
maximum homology. The miRNA seed region is bracketed.
Note that the miRNA sequences differ in their seed regions but
possibly bind the same target mRNAs when G–U wobble pairs
are permitted. See the text for details. ebv, Epstein-Barr virus;
mdv1, Marek's disease virus 1; hsv1, herpes simplex virus 1.
360
miRNAs play important roles in the maintenance of
viral latency.
miRNAs involved in the avoidance of the host immune system are classified in group II. An example of
this is polyomavirus simian virus 40 (SV40) miRNA
(sv40-miR-S1) downregulation of the expression of the
viral T-antigen, which is a target of the cytotoxic T-lymphocyte response. sv40-miR-S1 accumulates during the
late stages of infection and targets early viral mRNAs
for cleavage, resulting in the reduced expression of the
viral T antigen (18). One possible function of sv40-miRS1 is to allow the virus to escape from the host immune
system, increasing the probability of successful infection.
The group III miRNAs are related to the regulation of
cellular apoptosis, limiting host cell death during viral
proliferation. It has been reported that overexpression
of the EBV LMP1 gene promotes host cell apoptosis
(35). These authors showed that three EBV miRNAs
(miR-BART1-5p, miR-BART16, and miR-BART17-5p)
target the 3?-UTR of LMP1 mRNA and downregulate
the expression of LMP1 protein, reducing the proapoptotic effect. Therefore, EBV miRNAs have an impact
on the host cell-death pathway, enhancing viral survival.
Viral miRNAs associated with the control of viral
replication are classified to group IV. EBV expresses
different replication systems during latent and lytic infection (36). The authors reported that cellular replication factors are recruited during latent infection,
whereas viral replication factors, including viral polymerase BALF5, are required during lytic infection. It
has been shown that miR-BART2 regulates the transition from latent to lytic viral replication (6,81). During
latent infection, EBV miR-BART2 cleaves the 3?-UTR
of the BALF5 mRNA, whereas induction of the lytic
replication cycle causes a reduction in miR-BART2 levels. Another example is the HIV-1 protein Nef. The
viral miRNA miR-N367 suppresses Nef expression
through the regulatory U3 region in the 5? long terminal
repeat (5?-LTR) (22). Because Nef is considered a
regulatory factor for HIV-1 replication, miR-N367 may
control viral replication by limiting Nef expression.
Among other HIV-1 miRNAs, TAR miRNA is thought
to be processed from the HIV-1 TAR element by the
Dicer enzyme in host cells (21,37). Although the function of TAR miRNA is unclear, it is believed to repress
viral gene expression through the viral LTR (group V).
sion (28). It is noteworthy that this pattern of nucleotide
sequences associated with G–U pairs was observed in 11
of the 12 pairs. Among these 11 pairs, four pairs are encoded by the genomes of related viruses (two by herpes
simplex virus 1 [HSV-1] and herpes simplex virus 2
[HSV-2], and two by EBV and RLCV). Moreover,
seven pairs are encoded by the genomes of identical
viruses (a pair each in EBV, HSV-1, HSV-2, and
Marek's disease virus 1 [MDV-1], and three pairs in turkey herpesvirus [HVT]). When these conserved miRNA
pairs are encoded in the genomes of the same viruses,
they may play a role in a backup system, or may be useful in avoiding host cell attack. If these pairs recognize
the same target sites, the part of nucleotide sequences on
the target sites are restricted to ``G'' or ``U'' (Fig. 4).
Therefore, it is presumed that reliable target sites can be
extracted using this conserved miRNA sequence information.
It has also been reported that one mRNA can have
several different miRNA-binding sites. For instance,
C/EBPb p20 (LIP) mRNA, which encodes a negative
transcriptional regulator of specific cytokines, including
interleukin 6 (IL6) and IL10, has target sites for two
Kaposi's sarcoma-associated herpesvirus (KSHV)
miRNAs (miR-k12-3 and miR-k12-7) (29). These
miRNAs control the expression of LIP mRNA, resulting in the induction of cytokine (IL6 and IL10) secretion
by macrophages. In this situation, IL6 and IL10 play
important roles in KSHV-associated cancer (30,31).
4. Viral RNAs targeted by viral miRNAs
Recent reports have suggested that viral miRNA targets can be categorized into two classes: viral RNA targets (regulatory relationship A in Fig. 1) and cellular
mRNA targets (regulatory relationship B in Fig. 1),
which are described in this section and in Section 5 of
this review, respectively. Viral RNAs regulated by viral
miRNAs are listed in Table 3. Currently, 21 viral
mRNA targets have been experimentally identified, and
this number is increasing. Viral miRNA functions are
categorized into five groups: (I) latent and lytic viral infection, (II) immune evasion, (III) prevention of apoptosis, (IV) viral replication, and (V) others.
More than half of these viral miRNAs are associated
with latent and lytic viral infections (group I). For instance, infectious laryngotracheitis virus (ILTV) miR-I5
targets the transcriptional activator ICP4 mRNA, which
is essential for viral growth and is repressed during latent infection. Therefore, miR-I5 is involved in
modulating the balance between the lytic and latent status of the virus (32). miR-I5 is located antisense to the
ICP4 mRNA in the ILTV genome. With the complete
hybridization of miRNA–mRNA pairs, miR-I5 regulates ICP4 mRNA in an siRNA-like manner, cleaving
ICP4 mRNA rather than inhibiting its translation. In
another example, KSHV-encoded miR-k12-9* suppresses the expression of the viral replication and transcription activator (RTA), which is the major lytic switch
protein, by hybridizing directly with the 3?-UTR of its
mRNA to regulate lytic reactivation (33). A recent
report mentioned that miR-k12-7-5p also targets the
3?-UTR of RTA mRNA to prevent the production of
progeny virus (34). These findings suggest that KSHV
5. Host mRNAs targeted by viral miRNAs
In this section, we focus on recent research that has
described the regulation of cellular mRNAs by viral
miRNAs. Thirty-two cellular mRNA targets of viral
miRNAs are listed in Table 4. The cellular mRNAs
regulated by viral miRNAs can be categorized into six
groups: (I) latent and lytic viral infection, (II) immune
evasion, (III) prevention of apoptosis, (IV) viral replication, (V) cell cycle, and (VI) others. This classification is
essentially the same as that in Section 4, except for
group V.
One of the roles of viral miRNAs is the maintainance
viral latency via repression of the cellular factors involved in viral lytic reactivation (group I). It has been
361
Table 3. Summary of viral mRNA targets of viral miRNAs
Virus
Family
Herpesviridae
Viral mRNA target
Subfamily
Name
a-Herpesvirinae
HSV-1
miR-H2-3p
ICP0
HSV-1
miR-H6
ICP4
HSV-2
miR-I, II
ICP34.5
HSV-2
miR-III
ICP0
MDV1
miR-M4
UL28
MDV1
miR-M4
UL32
ILTV
miR-I5
ICP4
b-Herpesvirinae
g-Herpesvirinae
Papillomaviridae
Retroviridae
—
—
Lentivirinae
Name
Possible function
Group1)
Host
Reference
Transcriptional activator:
thought to have a role in
reactivation
Transcriptional activator:
required for expression of most
HSV1 genes during productive
infection
Neurovirulence factor: required
to control viral replication in
neuronal cells
Transcriptional activator:
important for HSV reactivation
DNA packing protein: involved
in cleavage/packing of
herpesvirus DNA
DNA packing protein: involved
in cleavage/packing of
herpesvirus DNA
Transcriptional activator:
essential for viral growth and
repressed during latency
Transcriptional activator:
critical for gene expression and
required for viral replication
Uracil DNA glycosylase:
important for viral replication
DNA polymerase: required for
lytic viral replication
Latent membrane protein:
potent immunogenic viral
antigen recognized by cytotoxic
T cells
Latent membrane protein:
induces cell growth
I
Human
51
I
Human
51
I, IV
Human
53, 78
I
Human
53
I
Avian
49
I
Avian
49
I
Avian
32
I, IV
Human
79
I, IV
Human
80
I, IV
Human
6, 81
II
Human
82
III
Human
35
HCMV miR-UL112-1
IE1/IE72
HCMV miR-UL112-1
UL114
EBV
miR-BART2
BALF5
EBV
miR-BART22
LMP2a
EBV
miR-BART1-5p,
miR-BART16,
miR-BART17-5p
miR-k12-9*,
miR-k12-7-5p
LMP1
RTA
Lytic switch protein: controls
viral reactivation from latency
I
Human
33, 34
SV40
miR-S1
LT-Ag
II
Simian
18
JCV
miR-J1
LT-Ag
II
Human
74
BKV
miR-B1
LT-Ag
II
Human
74
PYV
miR-P1
LT-Ag
Antigen protein: involved in
signaling, cell cycle, and viral
replication. Target of cytotoxic
T lymphocyte response
Antigen protein: involved in
signaling, cell cycle, and viral
replication. Target of cytotoxic
T lymphocyte response
Antigen protein: involved in
signaling, cell cycle, and viral
replication. Target of cytotoxic
T lymphocyte response
Antigen protein: involved in
signaling, cell cycle, and viral
replication. Target of cytotoxic
T lymphocyte response
II
Murine
75
BPCV1 miR-B1
LT-Ag
II
Bandicoot
76
BPCV2 miR-B1
LT-Ag
Antigen protein: involved in
signaling, cell cycle, and viral
replication. Target of cytotoxic
T lymphocyte response
Antigen protein: involved in
signaling, cell cycle, and viral
replication. Target of cytotoxic
T lymphocyte response
II
Bandicoot
76
HIV-1
miR-N367
NEF
IV
Human
22
HIV-1
miR-TAR
LTR
Accessory protein: important,
but not essential for viral
replication
Important for viral replication
V
Human
37
KSHV
Polyomaviridae
Viral miRNA
For abbreviations, see Table 1.
Roman numerals indicate the following possible functions: (I) latent and lytic viral infection, (II) immune evasion, (III) prevention of
apoptosis, (IV) viral replication, and (V) others.
1):
362
Table 4. Summary of cellular mRNA targets of viral miRNAs
Virus
Family
Herpesviridae
Cellular mRNA target
Subfamily
Name
a-Herpesvirinae MDV1
MDV1
MDV1
MDV1
MDV1
MDV1
MDV1
b-Herpesvirinae MCMV
HCMV
HCMV
HCMV
HCMV
g-Herpesvirinae
EBV
EBV
EBV
EBV
KSHV
KSHV
KSHV
KSHV
KSHV
KSHV
KSHV
KSHV
KSHV
KSHV
KSHV
KSHV
Polyomaviridae
—
BKV
JCV
Retroviridae
Lentivirinae
HIV-1
HIV-1
Viral miRNA
mdv1-miR-M3
Name
SMAD2
Possible function
A critical factor in the
transforming growth factor b
signal pathway
mdv1-miR-M4
PU.1
—
mdv1-miR-M4
GPM6B
—
mdv1-miR-M4
RREB1
—
mdv1-miR-M4
c-Myb
—
mdv1-miR-M4
MAP3K7IP2
—
mdv1-miR-M4
C/EBP
—
miR-M23-2
CXCL16
Chemokine expressed in both
soluble and transmembrane
forms
miR-US25-1
CCNE2
G1/S cyclin E2
miR-US25-1
H3F3B
H3 histone, family 3B
miR-US25-1
TRIM28
Transcriptional silencer
miR-UL112-1
MICB
Stress-induced ligand of the
natural killer cell activating
receptor NKG2D
miR-BHRF1-3
CXCL11
CXC chemokine ligand for
CXCR3
miR-BART2-5p MICB
Stress-induced ligand of the
natural killer cell activating
receptor NKG2D
miR-BART5
PUMA
Induces apoptosis in response to
a wide variety of stimuli
miR-BART6
Dicer
RNase III enzyme, which is a
component of the miRNA
processing pathway
miR-k12-1
P21
A key inducer of cell-cycle arrest
An inhibitor of the NF-kB
miR-k12-1
IkBa
complex
miR-k12-3
NFIB
Activates the promoter of the
viral RTA gene
miR-k12-7
MICB
Stress-induced ligand of the
natural killer cell activating
receptor NKG2D
miR-k12-4-5p
Rbl2
A known repressor of DNA
methyltransferase 3a and 3b
mRNA
miR-k12-10a
TWEAKR
Tumor-necrosis-factor-like
weak inducer of apoptosis
receptor
miR-k12-11
Fos
—
miR-k12-11
BACH1
Transcriptional repressor of
heme oxygenase 1, which
promotes cell survival
miR-k12-3,
C/EBPb p20 An isoform of C/EBPb known
miR-k12-7
(LIP)
to function as a negative
transcriptional regulator
miR-k12-1,
MAF
Cellular transcription factor,
miR-k12-6-5p,
which has a role in tissue
miR-k12-11
specification and the terminal
differentiation of a wide variety
of cell types
miR-k12-5,
BCLAF-1
Bcl2-associated transcription
miR-k12-9,
factor
miR-k12-10
miR-k12-1,
THBS1
Potent inhibitor of blood vessel
miR-k12-3-5p,
growth
miR-k12-6-3p,
miR-k12-11
miR-B1-3p
ULBP3
Stress-induced ligand recognized
by the killer receptor NKG2D
miR-J1-3p
ULBP3
Stress-induced ligand recognized
by the killer receptor NKG2D
TAR miRNA
ERCCI
Involved in serum-starvationinduced apoptosis
TAR miRNA
IER3
Involved in serum-starvationinduced apoptosis
Group1)
Host
Reference
III
Avian
83
VI
VI
VI
VI
VI
VI
II
Avian
Avian
Avian
Avian
Avian
Avian
Murine
49
49
49
49
49
49
42
V
VI
VI
II
Human
Human
Human
Human
47
47
47
39
II
II
Human
Human
41
40
III
Human
43
I
Human
84
V
IV
Human
Human
46
45
I
Human
38
II
Human
40
VI
Human
48
III
Human
85
VI
VI
Human
Human
86
87
VI
Human
29
VI
Human
88
VI
Human
89
VI
Human
90
II
Human
26
II
Human
26
III
Human
44
III
Human
44
For abbreviations, see Table 1.
Roman numerals indicate the following functions: (I) latent and lytic viral infection, (II) immune evasion, (III) prevention of apoptosis,
(IV) viral replication, (V) cell cycle, and (VI) others.
1):
363
Finally, the ``others'' of group VI include an interesting example. Viral miRNA regulates DNA methylation
at several sites in both the viral and host genomes. Viral
genomic methylation by viral miRNA has been described. The KSHV miRNA miR-k12-4-5p reduces the
expression of retinoblastoma (Rb)-like protein 2 (Rbl2)
mRNA. Rbl2 is a repressor of DNA methyltransferases
3a and 3b (DNMT); consequently, this miRNA increases the number of methylated DNA sites in the viral
genome (48). The promoter region of the viral RTA
gene is methylated, which helps to maintain the latent
status of the virus.
In Sections 4 and 5, we discussed viral and cellular
mRNA regulation by individual viral miRNAs.
However, it should be noted that the same viral miRNA
sometimes targets both viral and cellular mRNAs. For
instances, the MDV1 miRNA miR-M4, which is known
to be an orthologue of the host miR-155, targets cellular
mRNAs (of PU.1, GPM6B, RREB1, c-Myb,
MAP3K7IP2, and C/EBP) and viral RNAs (of UL28
and UL32) (49). Furthermore, HCMV miR-UL112-1
regulates both viral RNAs (IE1/IE72 and UL114) and a
cellular mRNA (MICB), as described above (39,40). Because the number of virus-encoded miRNAs is generally
small, it is conceivable that the same viral miRNAs have
evolved to regulate both host and viral genes for efficient viral infection. However, this is observed in a
minority of cases, with only a dozen miRNA target
genes having been identified to date. With advances in
molecular virology, it is probable that more examples of
miRNAs that target both viral and cellular mRNAs will
be identified.
reported that the KSHV miRNA miR-k12-3 targets the
3?-UTR of nuclear factor I/B (NFIB) mRNA and downregulates its expression (38). NFIB enhances the
promoter activity of the viral RTA gene, which is involved in KSHV reactivation. Therefore, miR-k12-3
stabilizes viral latency through the regulation of NFIB.
In Section 4, we described how KSHV miRNAs (miRk12-9* and miR-12-7-5p) directly target and suppress
the expression of viral RTA. These data together suggest that KSHV miRNAs suppress both viral and cellular RTA-mediated factors to maintain viral latency.
As mentioned in Section 4, some viral miRNAs can
potentially allow the virus to evade the host immune system by targeting viral mRNAs. It is also true that cellular mRNAs are targeted by viral miRNAs for the same
purpose (group II). In fact, translation of the mRNA of
MICB, which is the stress-induced ligand recognized by
NKG2D on NK cells, is repressed by viral miRNAs, allowing the virus to evade the host immune response
(39,40). It is noteworthy that the downregulation of
MICB expression mediated by viral miRNAs has been
confirmed in three types of herpesviruses (HCMV miRUL112-1, EBV miR-BART2-5p, and KSHV miR-k127), although these three miRNAs have no nucleotide sequence conservation. In another example, polyomavirus
miRNAs are able to evade the host immune system by
targeting the ULBP3 ligand, as described Section 3 (26).
The chemokines CXCL11 and CXCL16 are also suppressed by EBV miR-BHRF1-3 and mouse cytomegalovirus (MCMV) miR-M23-2 miRNAs, respectively
(41,42). These studies indicate that the targeting of
ligands by viral miRNAs is a major viral strategy for
avoiding the host immune system, at least in the polyomaviruses and herpesviruses.
Viral miRNAs involved in the resistance to cellular
apoptosis are classified in group III. A recent study
demonstrated that miR-BART5 targets and represses
the expression of proapoptotic PUMA (p53 upregulated
modulator of apoptosis) mRNAs, protecting the virus
from cellular apoptosis (43). HIV-1 TAR miRNA is also
reported to attenuate host apoptosis (44). Briefly, TAR
miRNA downregulates the expression of both ERCC1
(excision repair cross complementing group 1) and IER3
(intermediate early response 3) mRNAs, the products of
which are known to induce apoptosis in response to serum starvation.
Viral replication is regulated by viral miRNAs of
group IV via the cellular NF-kB pathway. It has been
shown that the deletion of the KSHV miRNA cluster
reduces NF-kB activity, resulting in increased lytic replication. Detailed research has shown that miR-k1, in
particular, represses the expression of IkBa mRNA,
which encodes an inhibitor of the NF-kB complex (45).
The host cell cycle is regulated by the viral miRNAs of
group V. KSHV miRNA attenuates p21-mediated cellcycle arrest and miR-k1 represses p21 mRNA, a key inducer of cell-cycle arrest (46). Another example of host
cell-cycle regulation is HCMV miR-US25-1 targeting
and downregulation of the expression of cyclin E2
(CCNE2) mRNA (47). Cyclin E protein is expressed in
the G1 phase, and binds to and activates CDK2 protein,
resulting in progression to the S phase. Therefore, the
inhibition of CCNE2 mRNA by miR-US25-1 may block
cell-cycle progression.
6. Conclusion
To determine the functions of viral miRNAs, it is
important to consider the following two characteristics:
(i) the nucleotide sequence conservation among viral
miRNAs, and (ii) the target mRNAs of the viral
miRNAs. By focusing on the miRNA sequences that are
highly conserved among these viruses, reliable miRNA–
target genes can be identified. It is noteworthy that
different miRNAs can bind to the same mRNAs. Therefore, it is necessary to consider not only the highly conserved miRNAs but also other miRNAs that may bind
to the same mRNAs. Viral miRNAs tend to regulate
functions important for viral survival, such as the evasion of the host immune system and the regulation of
viral replication, by targeting both host and viral
mRNAs. Furthermore, one viral miRNA can regulate
both host and viral mRNAs because there are only a
limited number of viral miRNAs. Finally, we proposed
that these two characteristics could contribute to the development of therapeutic applications. Developing
drugs from these conserved miRNAs could allow us to
(a) target several viruses by antagonizing these conserved miRNAs, and (b) effectively repress viral growth
by targeting genes essential for viral survival.
Acknowledgments The authors would like to thank the members
of the RNA Group at the Institute for Advanced Biosciences, Keio
University, Japan, for their helpful discussions.
K. Takane was supported by a Grant-in-Aid from the Japan Society
for the Promotion of Science. This work was also supported by
364
research funds from the Yamagata Government and Tsuruoka City,
Japan.
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Conflict of interest None to declare.
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