- HCV interaction with the host miRNA machinery
1) HCV interaction with the host miRNA machinery
The HCV life cycle is dependent on miR-122
The central focus of my laboratory research is the study of virus-host interactions between HCV and the infected cell. In particular we are interested in the interactions between HCV and the micro RNA pathway of the cell. Micro RNAs (miRNAs) are endogenously expressed small regulatory RNAs that bind to the 3’ untranslated regions of cellular mRNAs and regulates their translation and stability. There are over 1500 identified human cellular miRNAs that directly regulate about one third of all human genes. Recent links between miRNA regulatory changes and cancer attests to their importance in general biology.
Hepatitis C virus is unique among viruses in that it appears to usurp elements of the miRNA pathway to promote its own replication. Evidence suggests that a liver specific miRNA, miR-122, is required for high levels of HCV RNA accumulation, and recent evidence suggests that it stabilises the HCV genome. This is an unusual function for a miRNA, which normally suppresses translation and promotes degradation of RNAs. HCV use miR-122 in a unique way to promote RNA genome stability. MicroRNA-122 binds to two tandem binding sites in the 5’ untranslated region of the HCV genome (Figure 1).
Figure 1: Schematic representation of the binding sites for miR-122 on the HCV 5’ untranslated region and the unusual binding pattern between miR-122 and the HCV genome that is required for efficient HCV RNA accumulation in infected cells. (Adapted from Macklin et al. 2011, PNAS 108, 3193-8)
The influence of miR-122 on the HCV life cycle requires the cellular miRNA machinery
Canonical activity of miR-122 in host cell processes
miR-122 is a liver specific microRNA whose functions include regulation of cholesterol metabolism, however, alterations to miR-122 expression often correlate with liver tumor formation suggesting a role in cancer development. In normal cells, miR-122 and other miRNAs function as part of a protein complex. In the normal celluar miRNA pathway, Dicer and TRBP are involved in processing of almost all cellular miRNAs, and Ago2 is the central player in the miRNA effector complex called the RNA induced Silencing Complex (RISC) (Figure 2).
Figure 2: Model of canonical miRNA processing, RISC complex loading, and RISC binding to the 3’ UTR of mRNA. Pre-miRNAs are processed by Dicer and TRBP into mature miRNA duplexes. Mature duplex miRNAs are transferred to an Ago containing RISC. The miRNA guide strand is unwound and discarded. the passenger strand and Ago2 bind to the 3’UTR of targeted mRNA and mark them for degradation or translation suppression. In Dicer independent non-canonical processing pre-miRNAs are processed by Ago2.
Host proteins Dicer, TRBP and Ago2 are involved in the canonical activity of miRNAs and in the activity of miR-122 in promoting the HCV life cycle
Our work has identified that the miRNA pathway proteins have similar roles in the activity of miR-122 in promoting the HCV life cycle (Figure 3). For the activity of miR-122 to promote HCV replication, we have found that Dicer and TRBP are necessary for the processing of miR-122 hairpin precursor RNAsand that Ago2/RISC is required for the activity of miR-122, including miR-122 duplex inwinding and binding to the HCV 5’UTR. We hypothesize that the primary role of miR-122 is to target RISC, or a similar protein complex to the HCV 5’UTR, and that the proteins, primarily Argonautes, perform the mechanism of stabilization. Alternatively, the RISC complex may be required simply to process miR-122, but that following binding to the 5’UTR of the HCV genome, miR-122 can function to stabilize the HCV genome, through simply masking the end of the genome from host degradation enzymes.
Figure 3: Model of pre-miR-122 processing and transport to the HCV 5’UTR. Processing of miR-122 resembles canonical processing and activity of miRNAs and includes pre-miR-122 processing by Dicer and TRBP, RISC loading of mature miR-122 duplexes, and strand selection and HCV RNA binding by Ago2. (Zhang et al. 2012, Virology. 43:479-488)
a) Determine the role of Ago2 in miR-122 augmentation of the HCV life cycle
Our future goal is to determine the mechanism of action of miR-122 and in defining the role of Ago2 (and other Ago proteins, Ago1, 3 and 4) in the process. We have recently described miR-122 independent HCV replication of some engineered HCV RNAs. Thus, miR-122 is not essential for replication of HCV RNA per se. However, since miR-122 independent RNA accumulation levels are 10 to 100 fold lower than miR-122 dependent replication then miR-122 is required to sustain high levels of viral RNA in the cell. This system also provides a model system in which to compare aspects of the host and of the virus in situations in which replication is dependent or independent of miR-122, and thus identify miR-122 induced changes.
b) Identify other host proteins involved in the activity of miR-122
In addition, the establishment of a system in which RNA accumulation of HCV is independent of miR-122 provides us with a model to identify host cell proteins that play a role in the activity of miR-122 in HCV replication. We will use this system in conjunction with siRNA knockdown to identify cellular and viral genes required for the activity of miR-122, but that are not required for miR-122 independent replication.
2) GB-Virus-B, a close relative to HCV, is also dependent on miR-122
In a collaboration with the laboratory if Dr. Peter Sarnow, and with a Post-doctoral Researcher Dr. Selena Sagan at Stanford University, we have discovered that another virus, GBV-B, a close relative, and potential surrogate model for HCV, is also dependent on miR-122 for RNA accumulation. The pattern of binding of miR-122 to the 5’UTR of GBV-B is slightly different from that of HCV so provides an alternative model to study the mechanism of action of miR-122. In addition, a mutant GBV-B can replicate efficiently in a miR-122 independent manner. Interestingly the GBV-B mutant (delta 4-29) contained a 25 base pair deletion in the 5’UTR that perfectly removed both miR-122 binding sites.
Figure 4: The GB-Virus-B 5’ untranslated region sequence, miR-122 binding sites and model for miR-122 binding. A) The 5’UTR sequence of the GBV-B genome showing miR-122 binding sites in bold. B) Model for the pattern of miR-122 hybridization with the GBV-B genome C) sequence of GBV-B mutant delta 4-29 which has both miR-122 sites deleted but is capable of efficient RNA accumulation. (Sagan et al. Manuscript in Preparation).
To use GBV-B as an alternative model system in which to study miR-122 dependent and miR-122 independent virus replication, and to study the mechanism of action of miR-122.
Wilson JA* and Sagan SM*, "Hepatitis C virus and human miR-122: insights from the bench to the clinic", 2014, Current Opinion in Virology 7, 11-18: (Invited Review),
Thibault PA , Wilson JA*, "Transient Replication of Hepatitis C Virus Sub- Genomic RNA in Murine Cell Lines Is Enabled by miR-122 and Varies with Cell Passage.",2014, PloS one: 9(2)
Grebely J*, Bilodeau M , Feld JJ , Bruneau J , Fischer B , Raven JF , Roberts E , Choucha N , Myers RP , Sagan SM , Wilson JA , Bialystok F , Tyrrell DL, Houghton M , Krajden M ,, "The Second Canadian Symposium on hepatitis C virus: a call to action.", Canadian journal of gastroenterology = Journal canadien de gastroenterologie, 2013, 27(11)
Wilson JA*, Huys A, "miR-122 promotion of the hepatitis C virus life cycle: sound in the silence.", Wiley interdisciplinary reviews. RNA. WIREs-RNA, 2013, 4(6) (Invited Review).
Thibault PA , Wilson JA*, "Targeting miRNAs to treat Hepatitis C Virus infections and liver pathology: Inhibiting the virus and altering the host.", Pharmacological research: the official journal of the Italian Pharmacological Society, 2013, 75 (Invited Review)
Sagan SM , Sarnow P , Wilson JA*, "Modulation of GB virus B RNA abundance by microRNA-122: dependence on and escape from microRNA-122 restriction.", Journal of virology, 2013, 87(13)
Thibault PA , Huys A , Dhillon P , Wilson JA*, "MicroRNA-122-dependent and - independent replication of Hepatitis C Virus in Hep3B human hepatoma cells.", Virology, 2013, 436(1)
Huys A , Thibault PA , Wilson JA*, "Modulation of hepatitis C virus RNA accumulation and translation by DDX6 and miR-122 are mediated by separate mechanisms.", 2013, PloS one, 8(6)
Zhang C , Huys A , Thibault PA , Wilson JA*, "Requirements for human Dicer and TRBP in microRNA-122 regulation of HCV translation and RNA abundance.", Virology, 2012, 433(2)
Wilson JA*, Zhang C, Huys A, Richardson CD, "Human Ago2 is required for efficient microRNA 122 regulation of hepatitis C virus RNA accumulation and translation.", 2011, Journal of virology, 85(5)