1220Biochemical Society Transactions (2008) Volume 36, part 6
Regulation of hepatitis C virus by microRNA-122
Catherine L. Jopling1
Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.
Most metazoan miRNAs (microRNAs) bind to sites in the 3?-UTRs (untranslated regions) of mRNA targets
and negatively regulate protein synthesis. The liver-specific miR-122, however, exerts a positive effect on
HCV (hepatitis C virus) RNA levels by binding directly to a site in the 5?-UTR of the viral RNA. HCV translation
and RNA stability are unaffected, and therefore miR-122 is likely to act at the level of viral replication. The
miR-122-binding site in HCV RNA was examined to determine whether the nature of the site is responsible
for the unusual mode of action for a miRNA. When the site was placed in the 3?-UTR of a reporter mRNA,
miR-122 repressed translation, and therefore the location of the miR-122-binding site dictates its effect
on gene expression. Additionally, a second binding site for miR-122 was identified in the HCV 5?-UTR, and
miR-122 binding to both sites in the same viral RNA was found to be necessary for viral replication. The two
sites are adjacent and are separated by a short spacer, which is largely conserved between HCV genotypes.
The binding site requirements for miR-122 to positively regulate HCV replication provide an insight into this
unusual mode of miRNA action.
miRNAs (microRNAs) are non-coding RNAs expressed by
a wide range of eukaryotic organisms that are important in
the control of gene expression . Several hundred miRNAs
are expressed in mammals, initially as part of large precursor
transcripts, which subsequently undergo nuclear and cyto-
plasmic processing to yield 21–23 nt single-stranded mature
miRNA molecules. miRNAs can mediate cleavage of exactly
complementary RNA targets, but in metazoan systems most
miRNAs have been shown to function by binding with
imperfect complementarity to 3?-UTR (untranslated region)
sites as part of a complex of proteins known as the miRNP.
This binding resulted in inhibition of gene expression, which
occurred at the level of translation initiation or by a post-
initiation block in different studies. Localization of mRNA
to processing (P) bodies and subsequent degradation has also
been observed, and it is likely that all three mechanisms of
repression may function in different circumstances .
In higher organisms miRNAs show a high degree of
specificity of expression, both according to tissue type and
developmental stage. miR-122 was identified as a highly
liver-specific miRNA and was found to account for approx.
70% of total liver miRNA content, with approx. 66000
copies per cell .
HCV (hepatitis C virus) is a hepatotropic positive-sense
RNA virus that establishes persistent infections in liver tissue
. Infection can eventually lead to cirrhosis of the liver
and hepatocellular carcinoma. Current antiviral therapies are
frequently ineffective, and a greater understanding of the
Key words: hepatitis C virus (HCV), internal ribosome entry site (IRES), microRNA, miR-122, viral
Abbreviations used: HCV, hepatitis C virus; IFN, interferon; IRES, internal ribosome entry site;
miRNA, microRNA; TOP, terminal oligopyrimidine tract; UTR, untranslated region.
examined liver cell lines and found that miR-122 is expressed
in Huh7 cells, which are supportive of HCV replication, but
not in a different human liver cell line, HepG2 . Huh7
cells stably expressing a dicistronic HCV replicon, in which
the EMCV (encephalomyocarditis virus) IRES (internal
ribosome entry site) directs translation of the complete HCV
to occur , were also examined, and miR-122 was expressed
in these cells. We were interested to see what impact the
presence of high levels of miR-122 had on HCV replication,
and used a 2?-O-methylated antisense oligonucleotide to
sequester miR-122 in Huh7 cells. Both in Huh7 cells stably
containing the dicistronic genotype 1b HCV replicon de-
scribed above, and in cells transiently expressing a full-length
type 1a HCV replicating RNA , sequestration of miR-122
resulted in a dramatic reduction in HCV RNA levels .
Mutagenesis indicated that miR-122 binds to a site in the
5?-UTR of HCV RNA and that its effect on HCV is due
to this binding. The miR-122–binding site we identified is
in an unstructured region of the 5?-UTR, upstream of the
HCV IRES, and is conserved across all six genotypes of
the virus (Figure 1A, seed match 1). We observed no effect
of miR-122 binding on HCV translation or RNA stability,
and concluded that miR-122 positively regulates HCV at the
level of viral replication .
The miR-122-binding site from HCV
mediates repression of gene expression
when placed in a heterologous 3?-UTR
The discovery of this novel mode of action for a miRNA
suggested that particular features of miR-122 or its binding
site in HCV might cause this unusual mode of regulation.
A major determinant of productive miRNA binding has
been found to be exact Watson–Crick complementarity to
nt 2–7 of the miRNA, known as the ‘seed’. This seed match
C ?The Authors Journal compilation
C ?2008 Biochemical SocietyBiochem. Soc. Trans. (2008) 36, 1220–1223; doi:10.1042/BST0361220
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