*Corresponding author: Mailing address: Institute for
Ad-vanced Biosciences, Keio University, Tsuruoka, Yamagata
997-0017, Japan. Tel:
＋
81-235-29-0524, Fax:
＋
81-235-29-0525, E-mail: akio
＠
sfc.keio.ac.jp
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.
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 demon-strated that miRNAs are not only encoded in the genomes of host cells, but also by certain viral genomes, and play im-portant roles in their gene regulation. The arrows indicate the regulatory interactions that affect the genetic information flow. Four examples of miRNA-mediated regulatory interac-tions, represented by bold arrow (A–D), are described in the text.
Jpn. J. Infect. Dis., 64, 357-366, 2011
Invited Review
Vertebrate Virus-Encoded MicroRNAs and
Their Sequence Conservation
Kahori Takane
1,2and Akio Kanai
1,2*
1
Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017; and
2Systems Biology Program, Graduate School of Media and Governance,
Keio University, Fujisawa 252-8520, Japan
(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
se-quence conservation among viral miRNAs and (ii) the RNAs targeted by viral miRNAs. Recent
ad-vances in these topics are discussed.
1. Introduction
The discovery of huge amounts of noncoding RNAs
(ncRNAs) has indicated the importance of RNA
mole-cules in many steps of gene regulation (1). Among the
ncRNAs, microRNAs (miRNAs) are very small
mole-cules of approximately 20 ribonucleotides, which
func-tion posttranscripfunc-tionally 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
de-velopment of therapeutic agents, we focus on viral
miRNAs in this review. In Fig. 1, we summarize existing
knowledge regarding the regulatory relationships
be-tween viruses and host cells mediated by miRNAs.
Basi-cally, 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-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
tar-get 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).
Fig. 2. General miRNA processing pathway. Initially, miRNA genes are transcribed to produce primary miRNAs (pri-miRNAs) in nucleus. RNase III enzyme Drosha and its binding partner DiGeorge-syndrome critical-region protein 8 (DGCR8), also known as Microprocessor, processes pri-miRNAs into precursor pri-miRNAs (pre-pri-miRNAs). After being exported to cytoplasm by a complex of Exportin-5 and Ran-GTP, pre-miRNAs are cleaved into miRNA duplexes by another RNase III enzyme Dicer with its cofactor transactivat-ing region RNA-bindtransactivat-ing protein (TRBP). One strand of miRNA duplexes is incorporated into miRNA-induced silenc-ing complex (miRISC) containsilenc-ing 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.
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
silenc-ing complex (RISC) (7). Based on these findsilenc-ings, 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
virus-encoded 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
necessa-ry to identify their target mRNAs. Therefore, we
pro-vide 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
cur-rently 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
syn-thesized 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
bio-genesis 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).
Pri-miRNAs are processed into approximately
70-nucleo-tide 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
degrad-ed, 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
ge-nomes, or distinguishing positive from false miRNAs to
identify their nucleotide sequences and structural
char-acteristics using a machine learning technique (17,18). It
has been suggested that high-throughput analyses
com-bined with computational analyses constitute the most
effective approach (19,20). In Table 1, we summarize
the currently known miRNAs encoded by vertebrate
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)
en-codes 68 miRNAs. In contrast, only 1–4 miRNAs are
found in the families
Polyomaviridae
,
Papillomaviri-dae
,
Adenoviridae
, and
Retroviridae
. We note that
most viruses encoding miRNAs belong to
double-stranded 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
im-munodeficiency virus 1 (HIV-1). This is probably
be-cause 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
ge-nomes 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
Table 1. List of vertebrate virus-encoded miRNAs
Family Subfamily Name No. miRNAs1) Host Reference
Herpesviridae a-Herpesvirinae Herpes simplex virus-1 (HSV-1) 25 (16) Human 50–52
Herpes simplex virus-2 (HSV-2) 24 (18) Human 50, 53, 54
Marek's disease virus-1 (MDV-1) 26 (14) Avian 55, 56
Marek's disease virus-2 (MDV-2) 36 (18) Avian 23, 24
Turkey herpesvirus (HVT) 28 (17) Avian 24, 57
Infectious laryngotracheitis virus (ILTV) 10 (7) Avian 24, 58
Bovine herpesvirus (BHV1) 12 (10) Cattle 59
Herpes B virus (HBV) 3 (3) Simian 60
b-Herpesvirinae Human cytomegalovirus (HCMV) 17 (11) Human 17, 61
Mouse cytomegalovirus (MCMV) 29 (18) Murine 62, 63
g-Herpesvirinae Epstein-Barr virus (EBV) 44 (25) Human 6, 64–66
Rhesus lymphocryptovirus (RLCV) 68 (36) Simian 64, 67, 68
Kaposi's sarcoma-associated herpesvirus (KSHV) 25 (12) Human 17, 65, 69, 70
Rhesus monkey rhadinovirus (RRV) 11 (7) Simian 71
Mouse gamma herpesvirus-68 (MHV68) 28 (15) Murine 17
Polyomaviridae — Simian virus 40 (SV40) 2 (1) Simian 18
Simian agent 12 (SA12) 2 (1) Simian 72
Merkel cell polyomavirus (MCV) 2 (1) Human 73
BK polyomavirus (BKV) 2 (1) Human 74
JC polyomavirus (JCV) 2 (1) Human 74
Murine polyomavirus (PYV) 2 (1) Murine 75
Papillomaviridae Bandicoot papillomatosis carcinomatosis virus
type 1 (BPCV1) 1 (1) Bandicoot 76
Bandicoot papillomatosis carcinomatosis virus
type 2 (BPCV2) 1 (1) Bandicoot 76
Adenoviridae — Human adenovirus (AV) 3 (2) Human 77
Retroviridae Lentivirinae Human immunodeficiency virus 1 (HIV-1) 4 (3) Human 21, 22
1): Number of precursor miRNAs is shown in parentheses.
Table 2. Nucleotide sequence conservation of viral miRNAs in miRBase
No. of combinations (without considering seed sequence match)
No. of combinations (with complete match of seed sequence) Total 79,800 79,800 Æ30z 56,083 90 Æ40z 19,078 89 Æ50z 3,390 72 Æ60z 277 50 Æ70z 67 34 Æ80z 34 22 Æ90z 17 14 100z 4 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 match-es are considered (nucleotidmatch-es 1–7, 2–8, or 3–9 from the 5?end of the miRNA).
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
retro-virus can integrate into the host nuclear genome as
double-stranded DNA using the virally encoded
en-zymes RNA reverse transcriptase and integrase.
There-fore, 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
con-served (23,24). However, some questions remain
regard-ing how many viral miRNA sequences are conserved in
the same viruses or individual viruses, and whether any
sequence characteristics exist in these miRNAs. To
ad-dress 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
combina-tions (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.
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
Fig. 3. Secondary structures of virus-encoded pre-miRNAs shar-ing the same mature miRNA sequences. (A–D) All pairs of pre-miRNAs with 100zidentical mature miRNA sequences are shown. The mature miRNA sequences are shown in bold up-percase 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 cinomatosis virus type 1; bpcv2, bandicoot papillomatosis car-cinomatosis virus type 2.
Fig. 4. Three examples of highly conserved miRNAs encoded by viruses. (A–C) Nucleotide sequences of mature viral miRNAs with Æ80zconservation 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.
from this table, the numbers of conserved miRNA pairs
(80
z
–100
z
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
(
Æ
70
z
sequence similarity) is considerably larger when
we do not consider the seed sequence matches than
when we consider the completely matched seed
se-quences. Assuming that the seed sequence is important
for the recognition of target mRNAs, the large numbers
of pairs with only 30
z
–60
z
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
exam-ple, when considering the seed sequence matches, the
number of miRNA pairs with more than 70
z
, 80
z
,
and 90
z
sequence similarities are 34, 22, and 14,
re-spectively (Table 2). Notably, among the same or
relat-ed viruses, the viral miRNA sequences are 100
z
identi-cal for four pairs (bkv-miR-B1-3p and jcv-miR-J1-3p,
ebv-miR-BART1-3p and rlcv-miR-rL1-6,
hvt-miR-H9-3p and hvt-miR-H12-hvt-miR-H9-3p, and bpcv1-miR-B1 and
bpcv2-miR-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
func-tion throughout viral evolufunc-tion. For instance, the
identi-cal 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
under-standing of the evolution of miRNA-mediated
regula-tion. Recently, we reported that miRNA–target gene
pairs can be effectively identified based on the
evolu-tionary analysis of bilaterian animals (27).
Let us examine the characteristics of miRNA pairs
with more than 80
z
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
Æ
80
z
quence similarity but incompletely matched seed
se-quences. 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-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
en-coded 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
tur-key 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
use-ful 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
infor-mation.
It has also been reported that one mRNA can have
several different miRNA-binding sites. For instance,
C/EBP
b
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,
result-ing 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
gets can be categorized into two classes: viral RNA
tar-gets (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
in-fection, (II) immune evasion, (III) prevention of
apo-ptosis, (IV) viral replication, and (V) others.
More than half of these viral miRNAs are associated
with latent and lytic viral infections (group I). For
in-stance, infectious laryngotracheitis virus (ILTV) miR-I5
targets the transcriptional activator ICP4 mRNA, which
is essential for viral growth and is repressed during
la-tent infection. Therefore, miR-I5 is involved in
modulating the balance between the lytic and latent
sta-tus 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
regu-lates ICP4 mRNA in an siRNA-like manner, cleaving
ICP4 mRNA rather than inhibiting its translation. In
another example, KSHV-encoded miR-k12-9*
suppress-es the exprsuppress-ession of the viral replication and
transcrip-tion 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
miRNAs play important roles in the maintenance of
viral latency.
miRNAs involved in the avoidance of the host
im-mune 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-lym-phocyte 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-miR-S1 is to allow the virus to escape from the host immune
system, increasing the probability of successful
infec-tion.
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
proapo-ptotic effect. Therefore, EBV miRNAs have an impact
on the host cell-death pathway, enhancing viral
sur-vival.
Viral miRNAs associated with the control of viral
replication are classified to group IV. EBV expresses
different replication systems during latent and lytic
in-fection (36). The authors reported that cellular
replica-tion factors are recruited during latent infecreplica-tion,
whereas viral replication factors, including viral
poly-merase BALF5, are required during lytic infection. It
has been shown that miR-BART2 regulates the
transi-tion from latent to lytic viral replicatransi-tion (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
lev-els. 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
func-tion of TAR miRNA is unclear, it is believed to repress
viral gene expression through the viral LTR (group V).
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
replica-tion, (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
in-volved in viral lytic reactivation (group I). It has been
Table 3. Summary of viral mRNA targets of viral miRNAs Virus
Viral miRNA Viral mRNA target Group1) Host Reference
Family Subfamily Name Name Possible function
Herpesviridae a-Herpesvirinae HSV-1 miR-H2-3p ICP0 Transcriptional activator: thought to have a role in reactivation
I Human 51
HSV-1 miR-H6 ICP4 Transcriptional activator: required for expression of most HSV1 genes during productive infection
I Human 51
HSV-2 miR-I, II ICP34.5 Neurovirulence factor: required to control viral replication in neuronal cells
I, IV Human 53, 78
HSV-2 miR-III ICP0 Transcriptional activator:
important for HSV reactivation I Human 53 MDV1 miR-M4 UL28 DNA packing protein: involved
in cleavage/packing of herpesvirus DNA
I Avian 49
MDV1 miR-M4 UL32 DNA packing protein: involved in cleavage/packing of herpesvirus DNA
I Avian 49
ILTV miR-I5 ICP4 Transcriptional activator: essential for viral growth and repressed during latency
I Avian 32
b-Herpesvirinae HCMV miR-UL112-1 IE1/IE72 Transcriptional activator: critical for gene expression and required for viral replication
I, IV Human 79
HCMV miR-UL112-1 UL114 Uracil DNA glycosylase:
important for viral replication I, IV Human 80 g-Herpesvirinae EBV miR-BART2 BALF5 DNA polymerase: required for
lytic viral replication I, IV Human 6, 81 EBV miR-BART22 LMP2a Latent membrane protein:
potent immunogenic viral antigen recognized by cytotoxic T cells
II Human 82
EBV miR-BART1-5p, miR-BART16, miR-BART17-5p
LMP1 Latent membrane protein:
induces cell growth III Human 35
KSHV miR-k12-9*,
miR-k12-7-5p RTA Lytic switch protein: controlsviral reactivation from latency I Human 33, 34
Polyomaviridae — SV40 miR-S1 LT-Ag Antigen protein: involved in signaling, cell cycle, and viral replication. Target of cytotoxic T lymphocyte response
II Simian 18
JCV miR-J1 LT-Ag Antigen protein: involved in signaling, cell cycle, and viral replication. Target of cytotoxic T lymphocyte response
II Human 74
BKV miR-B1 LT-Ag Antigen protein: involved in signaling, cell cycle, and viral replication. Target of cytotoxic T lymphocyte response
II Human 74
PYV miR-P1 LT-Ag Antigen protein: involved in signaling, cell cycle, and viral replication. Target of cytotoxic T lymphocyte response
II Murine 75
Papillomaviridae — BPCV1 miR-B1 LT-Ag Antigen protein: involved in signaling, cell cycle, and viral replication. Target of cytotoxic T lymphocyte response
II Bandicoot 76
BPCV2 miR-B1 LT-Ag Antigen protein: involved in signaling, cell cycle, and viral replication. Target of cytotoxic T lymphocyte response
II Bandicoot 76
Retroviridae Lentivirinae HIV-1 miR-N367 NEF Accessory protein: important, but not essential for viral replication
IV Human 22
HIV-1 miR-TAR LTR Important for viral replication V Human 37
For abbreviations, see Table 1.
1): Roman numerals indicate the following possible functions: (I) latent and lytic viral infection, (II) immune evasion, (III) prevention of
Table 4. Summary of cellular mRNA targets of viral miRNAs Virus
Viral miRNA Cellular mRNA target Group1) Host Reference
Family Subfamily Name Name Possible function
Herpesviridae a-Herpesvirinae MDV1 mdv1-miR-M3 SMAD2 A critical factor in the transforming growth factorb signal pathway
III Avian 83
MDV1 mdv1-miR-M4 PU.1 — VI Avian 49
MDV1 mdv1-miR-M4 GPM6B — VI Avian 49
MDV1 mdv1-miR-M4 RREB1 — VI Avian 49
MDV1 mdv1-miR-M4 c-Myb — VI Avian 49
MDV1 mdv1-miR-M4 MAP3K7IP2 — VI Avian 49
MDV1 mdv1-miR-M4 C/EBP — VI Avian 49
b-Herpesvirinae MCMV miR-M23-2 CXCL16 Chemokine expressed in both soluble and transmembrane forms
II Murine 42
HCMV miR-US25-1 CCNE2 G1/S cyclin E2 V Human 47
HCMV miR-US25-1 H3F3B H3 histone, family 3B VI Human 47
HCMV miR-US25-1 TRIM28 Transcriptional silencer VI Human 47
HCMV miR-UL112-1 MICB Stress-induced ligand of the natural killer cell activating receptor NKG2D
II Human 39
g-Herpesvirinae EBV miR-BHRF1-3 CXCL11 CXC chemokine ligand for
CXCR3 II Human 41
EBV miR-BART2-5p MICB Stress-induced ligand of the natural killer cell activating receptor NKG2D
II Human 40
EBV miR-BART5 PUMA Induces apoptosis in response to
a wide variety of stimuli III Human 43 EBV miR-BART6 Dicer RNase III enzyme, which is a
component of the miRNA processing pathway
I Human 84
KSHV miR-k12-1 P21 A key inducer of cell-cycle arrest V Human 46 KSHV miR-k12-1 IkBa An inhibitor of the NF-kB
complex IV Human 45
KSHV miR-k12-3 NFIB Activates the promoter of the
viralRTAgene I Human 38
KSHV miR-k12-7 MICB Stress-induced ligand of the natural killer cell activating receptor NKG2D
II Human 40
KSHV miR-k12-4-5p Rbl2 A known repressor of DNA methyltransferase 3a and 3b mRNA
VI Human 48
KSHV miR-k12-10a TWEAKR Tumor-necrosis-factor-like weak inducer of apoptosis receptor
III Human 85
KSHV miR-k12-11 Fos — VI Human 86
KSHV miR-k12-11 BACH1 Transcriptional repressor of heme oxygenase 1, which promotes cell survival
VI Human 87
KSHV miR-k12-3,
miR-k12-7 C/EBPb(LIP) p20 An isoform of C/EBPbto function as a negativeknown transcriptional regulator
VI Human 29
KSHV miR-k12-1, miR-k12-6-5p, miR-k12-11
MAF Cellular transcription factor, which has a role in tissue specification and the terminal differentiation of a wide variety of cell types
VI Human 88
KSHV miR-k12-5, miR-k12-9, miR-k12-10
BCLAF-1 Bcl2-associated transcription
factor VI Human 89
KSHV miR-k12-1, miR-k12-3-5p, miR-k12-6-3p, miR-k12-11
THBS1 Potent inhibitor of blood vessel
growth VI Human 90
Polyomaviridae — BKV miR-B1-3p ULBP3 Stress-induced ligand recognized
by the killer receptor NKG2D II Human 26 JCV miR-J1-3p ULBP3 Stress-induced ligand recognized
by the killer receptor NKG2D II Human 26
Retroviridae Lentivirinae HIV-1 TAR miRNA ERCCI Involved in
serum-starvation-induced apoptosis III Human 44
HIV-1 TAR miRNA IER3 Involved in
serum-starvation-induced apoptosis III Human 44
For abbreviations, see Table 1.
1): Roman numerals indicate the following functions: (I) latent and lytic viral infection, (II) immune evasion, (III) prevention of apoptosis,
reported that the KSHV miRNA miR-k12-3 targets the
3?-UTR of nuclear factor I/B (NFIB) mRNA and
down-regulates its expression (38). NFIB enhances the
promoter activity of the viral
RTA
gene, which is
in-volved in KSHV reactivation. Therefore, miR-k12-3
stabilizes viral latency through the regulation of NFIB.
In Section 4, we described how KSHV miRNAs
(miR-k12-9* and miR-12-7-5p) directly target and suppress
the expression of viral RTA. These data together
sug-gest that KSHV miRNAs suppress both viral and
cellu-lar 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
sys-tem by targeting viral mRNAs. It is also true that
cellu-lar 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,
al-lowing 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
miR-UL112-1, EBV miR-BART2-5p, and KSHV
miR-k12-7), although these three miRNAs have no nucleotide
se-quence 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
sup-pressed by EBV miR-BHRF1-3 and mouse
cytomegalo-virus (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
poly-omaviruses 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
se-rum starvation.
Viral replication is regulated by viral miRNAs of
group IV via the cellular NF-
k
B pathway. It has been
shown that the deletion of the KSHV miRNA cluster
reduces NF-
k
B activity, resulting in increased lytic
repli-cation. Detailed research has shown that miR-k1, in
particular, represses the expression of I
k
B
a
mRNA,
which encodes an inhibitor of the NF-
k
B complex (45).
The host cell cycle is regulated by the viral miRNAs of
group V. KSHV miRNA attenuates p21-mediated
cell-cycle arrest and miR-k1 represses p21 mRNA, a key
in-ducer 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 G
1phase, 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.
Finally, the ``others'' of group VI include an
interest-ing example. Viral miRNA regulates DNA methylation
at several sites in both the viral and host genomes. Viral
genomic methylation by viral miRNA has been
de-scribed. 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
in-creases 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).
Be-cause 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
ef-ficient 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.
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.
There-fore, it is necessary to consider not only the highly
con-served 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
eva-sion 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
de-velopment of therapeutic applications. Developing
drugs from these conserved miRNAs could allow us to
(a) target several viruses by antagonizing these
con-served 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
research funds from the Yamagata Government and Tsuruoka City, Japan.
Conflict of interest
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