Skip to main content
Premium Trial:

Request an Annual Quote

New Data Refute Role for RNAi in Mammalian Viral Defense

Premium

NEW YORK (GenomeWeb) – As debate continues over the existence of RNAi in mammals, a new report out of Mount Sinai Hospital indicates that the gene-silencing mechanism is not part of the mammalian response to viruses, which instead is mediated exclusively by interferon (IFN) induction.

Although the findings shouldn't be viewed as absolute proof that RNAi does not exist in mammals, they do suggest that if it does, "it has no measurable effect on the physiological response to virus," according to Benjamin tenOever, senior author of the study.

While the use of small RNAs to fend off viruses can be found in plants, arthropods, and nematodes through RNAi and in bacteria via CRISPR-Cas9, it has long been believed such a system is lacking in humans and other mammals.

However, the discovery of the mammalian microRNA pathway, which uses a small RNA to guide a degradative nuclease to an endogenous gene target in a manner similar to RNAi, helped reignite the controversy. Further complicating matters was the publication of two papers appearing last year in Science suggesting that RNAi acts as an antiviral mechanism in all mammalian cells or in stem cells prior to differentiation, respectively.

In an effort to shed more light on the issue, tenOever and his colleagues took advantage of a relatively harmless recombinant RNA virus known as vesicular stomatitis virus (VSV), and introduced modifications that enable it to either disrupt the RNA-induced silencing complex — a key component of the RNAi mechanism — or IFN-mediated responses.

In the first instance, VSV was engineered to express a pox virus protein, dubbed VP55, that has been shown to add nontemplated adenosines specifically to RNAs that are associated with RISC, resulting in their rapid degradation.

In the other, they added to the virus a nonstructural protein called NS1, which is derived from influenza that is used to inhibit host immune responses primarily by limiting IFN production and blocking the effects of IFN-induced proteins.

The Mount Sinai team began by testing VSV-VP55 in culture, according to their study, which appeared in Cell Reports. In these experiments, VSV-NS1 was found to block the production of IFN in IFN-competent fibroblasts, resulting in enhanced viral replication, while VSV-VP55 infection led to the degradation of all cellular miRNAs.

Meanwhile, in fibroblasts where IFN signaling was impaired, VSV-NS1 replicated to levels comparable to VSV-VP55 and control VSV. A similar phenotype was observed in Dicer-deficient cells.

They next examined VSV-VP55 in the context of an artificial cellular RNAi system, infecting cells and then transfecting them with siRNAs against the virus. As predicted, the cells failed to mount a gene-silencing response.

"Given the lack of an in vitro phenotype following disruption of small RNA silencing, we next sought to determine whether RNAi contributes to the physiological response to virus infection in vivo," the scientists wrote in their paper.

In mice, VSV-NS1 was found to replicate significantly better than control virus — a finding that tenOever told Gene Silencing News was expected. Surprisingly, however, VSV-VP55 was found to replicate at a diminished level compared with wild-type VSV.

"Although these results argue against a role for RNAi in mammals, they are in agreement with a recent publication concerning virus-induced polyribosylation and shutdown of RISC, which also would argue against RNAi functioning in mammals during times of stress," the team wrote in Cell Reports.

Noting that some in the field have argued that the failure to identify a mammalian antiviral RNAi response was due to the ancillary role of small RNA silencing in comparison to IFN, tenOever and his team performed additional testing in mice where IFN signaling has been knocked out.

In such animals, VSV-NS1 no longer has an advantage over wild-type VSV as neither has to contend with an IFN response. As was expected, no significant differences in viral titers were observed in animals infected with either virus.

The researchers then compared VSV-VP55 with control VSV in these animals, conjecturing that if there is an RNAi mechanism underlying IFN, it would come into play since the IFN system had been disrupted.

However, VSV-VP55 failed to replicate to titers exceeding wild-type VSV "strongly suggest[ing] that a functional antiviral RNAi system is not a physiological contributor to mammalian antiviral defenses," they wrote.

"It is a very difficult, if not impossible, thing to prove a negative," tenOever noted. "So I don't think [this paper] will squash the controversy once and for all. But I do think it is a unique and very strong piece of evidence that our mammalian [antiviral] system is entirely based on interferon."

In addition to helping answer a question that has persisted in the RNAi space for years, the findings also lend additional support to the value of viruses for the delivery of therapeutic RNA, he added.

"The lack of interplay between mammalian viruses and the small RNA machinery … makes these vectors ideal for the delivery of small RNAs," tenOever and his colleagues concluded in their paper. "The ability to deliver siRNAs is a critical need in the medical field, and the recent discovery that RNA viruses can also be engineered to encode siRNAs and utilize the small RNA host machinery makes this a provocative option for future therapeutics."

The Scan

Interfering With Invasive Mussels

The Chicago Tribune reports that researchers are studying whether RNA interference- or CRISPR-based approaches can combat invasive freshwater mussels.

Participation Analysis

A new study finds that women tend to participate less at scientific meetings but that some changes can lead to increased involvement, the Guardian reports.

Right Whales' Decline

A research study plans to use genetic analysis to gain insight into population decline among North American right whales, according to CBC.

Science Papers Tie Rare Mutations to Short Stature, Immunodeficiency; Present Single-Cell Transcriptomics Map

In Science this week: pair of mutations in one gene uncovered in brothers with short stature and immunodeficiency, and more.