Modulation of human cytomegalovirus replication by micro-RNA 132 (miR132), micro-RNA 145 (mi145) and micro-RNA 212 (miR212)
The present invention related to miR145, miR132, miR212, and the genes or gene products regulated by these miRNAs. miR145 is downregulated in cells infected with HCMV. This downregulation modulates expression of miR145 target genes, including IRS-I. Transfection of cells with a miR145 agent, such as a miR145 mimetic, reduces HCMV replication and protein expression. miR132 and miR212 are upregulated in cells infected with HCMV. This upregulation modulates expression of miR132 and miR212 target genes, including MeCP2 and RICS. Transfection of cells with a miR132 and/or a miR212 antagonist reduces HCMV replication and protein expression. Accordingly, the invention provides methods of attenuating HCMV replication by modulating, for example, miR145, miR132, and/or miR212, and targets thereof. Also provided are methods of detecting an HCMV infection, and compositions and kits useful for attenuating HCMV replication.
1. A method of inhibiting HCMV replication in a cell infected with HCMV, comprising contacting the cell with
a miR145 agent in an amount effective to decrease the level of one or more miR145 targets,
such that HCMV replication is inhibited.
2. The method of claim 1 , wherein the miR145 agent is selected from the group consisting of a miR145 mimic, a synthetic miR145 oligonucleotide, and an expression vector encoding miR145.
3. The method of claim 1 , wherein the miR145 agent comprises a nucleic acid molecule comprising SEQ ID NO:1 or SEQ ID NO:2.
4. The method of claim 1 , wherein the level of one or more targets is determined by measuring the level of expression of a polypeptide encoded by the target, and comparing the level of expression to a suitable control.
5. The method of claim 4 , wherein measuring the level of expression of a polypeptide encoded by the target is performed using a method selected from the group consisting of Western blot, ELISA, or antibody microarray.
6. The method of claim 1 , wherein the level of one or more targets is determined by measuring the level of expression of an RNA corresponding to the target, and comparing the level of expression to a suitable control.
7. The method of claim 6 , wherein the RNA is an mRNA.
8. The method of claim 6 , wherein measuring the level of expression of the target is performed using a method selected from the group consisting of Northern blot, quantitative Real Time PCR (qRT-PCR), or microarray.
9. The method of claim 1 , wherein the miR145 target is IRS-1.
10. The method of claim 1 , wherein the cell is in an organism.
11. The method of claim 1 , wherein the miR145 target is selected based on having sequence complementarity with all or a portion of SEQ ID NO:1 or SEQ ID NO:2.
12. The method of claim 11 , wherein the miR145 target has a region of 6-8 contiguous nucleotides that are complementary to the seed region of miR145.
13. The method of claim 12 , wherein the 6-8 contiguous nucleotides are located within the 3′UTR of the miRNA target, or within an open reading frame of the miRNA target.
14. The method of claim 1 , further comprising contacting the cell with an additional therapeutic agent.
15. The method of claim 14 , wherein the additional therapeutic agent is an antiviral agent.
16. The method of claim 15 , wherein the antiviral agent is selected from the group consisting of Ganciclovir, Valganciclovir, Cidofovir, Foscarnet, Formivirsen, Acyclovir, Valacyclovir, CMX001, Artesunate, BAY-384766, T-611, GW-275175X, and Maribavir.
17. The method of claim 1 , wherein the cell infected with HCMV is a fibroblast cell.