NEW YORK (GenomeWeb) – Researchers from the Icahn School of Medicine at Mount Sinai have designed and generated a synthetic vector based on a poxvirus gene that allows them to eliminate cellular microRNA (miRNA) populations, and have used the tool to learn more about the role miRNAs play in viral infections.
MiRNAs have the capacity to fine-tune protein expression which helps regulate cell maintenance and differentiation. The regulatory pathway is known to often malfunction in the development of cancer, but its role in the response to virus infection has remained largely unknown and difficult to study. The Mount Sinai researchers designed and generated a synthetic vector that rapidly eliminates total cellular miRNA populations in any cell or tissue and used this tool to determine the global role of miRNAs in both cell biology and the response to virus infection.
Previous profiling of small RNA in poxvirus-infected cells demonstrated that this virus family rapidly tailed and degraded miRNAs. The vaccinia virus protein, VP55, has been shown to recapitulate this tailing and degradation of miRNAs. The researchers verified the use of VP55 to rapidly degrade specific miRNAs and incorporated the protein with a codon-optimized EGFP-VP55 construct into a nonreplicative adenovirus 5 vector, creating the AdV-VP55 vector. After further testing, their data demonstrated that the AdV-VP55 vector specifically disrupts miRNA stability which provides a unique tool to study miRNA biology.
In the study published this week in Cell Host & Microbe, the researchers then used the synthetic vector that they developed to look at the physiological role of miRNA during viral infections and antiviral response.
The researchers applied their AdV-VP55 tool to primary cell models both ex vivo and in vivo (in mice) to better reflect the physiological response mediated by type I interferon cytokines (IFN-I). Control cells, treated with AdV-GFP and dsRNA, demonstrated a robust response of 1,548 differentially expressed genes as measured by quantitative RT-PCR. This same stimulation in the absence of miRNAs only statistically impacted 12 of the 1,548 genes.
Initially, the researchers found prolonged IFN-I stimulation increased the differentially regulated genes from 12 to 78, however, they noted that the genes impacted by the loss of miRNAs did not include central mediators in the IFN-I response.
To further test this, they looked at the impact of miRNA depletion in the absence of virus-related signaling to define what genes might be impacted if cell infection persisted beyond the acute phase by using mRNA sequencing to compare VP-55-mediated loss of miRNA silencing at either 1 or 9 days post-AdV administration.
The researchers found that despite the lack of major changes in RNA levels of the components of the intrinsic antiviral defense, loss of miRNA function had a significant impact on an expansive list of chemokines, including those responsible for recruitment of antigen-presenting cells and neutrophils (IL8, CXCL1, CXCL2, CXCL6, CCL2, and CCL7), immune regulators of hematopoiesis (IL1B, IL11, IL33, CSF1, and CSF2), and proinflammatory cytokines such as IL6.
Taken together, the findings indicate that while the loss of miRNAs had a negligible impact on the cell's immediate reaction to a virus or the short-term biology of the cell, sustained depletion had dramatic results on gene expression that was coupled to a burst of cytokines. The researchers concluded in the paper that miRNA function is limited to modulating the biology of the cell over long periods of time.
The researchers' miRNA-depletion vector could be used to look at miRNA functions in any tissue or cell, Benjamin tenOever, co-author on the paper and professor of microbiology at the Icahn School of Medicine at Mount Sinai, said in a statement. The researchers stated that having a tool that can manipulate miRNA response provides scientists with an unprecedented platform to reprogram healthy cells to treat diseases such as cancer where these pathways have malfunctioned.