Interpretation of IF 31.745 High Scored Articles ∣ Micro Battlefield: The War Between Viruses and Hosts in Cells

On October 12, 2021, Zhou Xi's team from Wuhan Institute of Virology/State Key Laboratory of Virology, Chinese Academy of Sciences and Lulu Team from the Ministry of Education of Medical Molecular Virology/Key Laboratory of Health and Health Commission of Fudan University jointly published in "Immunity" magazine titled The research paper "Inhibition of viral suppressor of RNAi proteins by designerpeptides protects from enteroviral infection in vivo" reveals for the first time that the rational design of polypeptide inhibitors to specifically target enterovirus RNAi suppressors can effectively break the VSR effect of the virus. The inhibition of RNAi fully releases the antiviral immune function of RNAi, which is a strong proof that the RNAi immune mechanism still exists in mammals. Due to the good antiviral activity and high safety exhibited by this immune mechanism in cells and animals, its potential for clinical development is extremely high.

Immune war: both sides participating in war at any time, a battlefield with frequent means

Since the beginning of life, it is not the kind hello and me, but the cruel battlefield of your life and death. Either you get out of my turf or you stay and be a part of me.

From macro-scale spines, horns, claws, and teeth, to phagocytosis, dissolution, and poisoning at the cellular level, to the cutting, interference, and degradation of molecular structures; from in vitro to in vivo, from extracellular to intracellular: Welcome to Difen A battle of life and death—a battle of immunity that starts at any time.

RNAi - a natural and efficient antiviral immune mechanism

In this unrestricted war, there is a natural (the main business regulates gene expression, and the side business is against virus invasion), efficient, stable and ubiquitous (highly conserved) immune mechanism - RNAi (RNA interference), which is considered to be in the The most important antiviral function in fungi, plants and invertebrates. In short, the double-stranded RNA (dsRNA) produced by virus replication is recognized by the RNAi pathway Dicer protein and cleaved into virus-derived small interfering RNA (viral siRNA, vsiRNA), and then assembled into the RNA-induced silencing complex (RNA-induced silencing complex). complex, RISC), thereby targeting the degradation of viral RNA. Correspondingly, most viruses have also evolved corresponding antagonistic mechanisms, that is, to escape RNAi antiviral immunity by encoding viral RNAi suppressors (Viral Suppressor of RNAi, VSR).

Discovery of RNAi immune mechanisms in mammals

However, since the vsiRNA formed by the cleavage of viral RNA has not been found in mammalian cells, the RNAi immune mechanism is considered to be an "evolutionary relic" that was abandoned after mammals developed other antiviral mechanisms. However, since 2017, a number of related studies have found a variety of VSRs derived from viruses, confirming that the RNAi immune mechanism does not disappear in mammalian cells, but is inhibited by viruses. At the same time, with the discovery of VSR proteins encoded by a variety of important human viruses, the molecular mechanism of its antagonism of RNAi pathway has been revealed.

VSR-targeted peptides: a new direction for drug development

In this study, the research team innovatively designed two VSR-targeting peptides (VSR-targeting peptides, VTP), P1 and P2, based on the A1 and A2 helical sequences of the enterovirus EV-A713A VSR protein. By detecting the accumulation of EV-A71 RNA in the selected cell lines, it was shown that both P1 and P2 effectively inhibited virus replication in a dose-dependent manner.

Then, according to the experimental results, a series of peptides were further synthesized with reference to the P2 sequence with better effect, and it was found that the D-reverse transcription-transformation modified isoform (ER-DRI) of the peptide ER had the best inhibitory effect on viral RNA replication. In order to confirm that this series of VTPs target and inactivate EV-A71 3AVSR and unlock the antiviral activity of RNAi, total RNA from infected cells was extracted and deep-sequenced. The results showed that the detected vsiRNAs were indeed derived from EV-A71. Among them, the purification and extraction of EV-A71 virus RNA in infected cells was performed using Qiagen and Foregene Viral RNA Isolation Kit.(This kit uses the spin column and formula developed by Foregene, which can efficiently extract high-purity and high-quality viral RNA from samples such as plasma, serum, cell-free body fluid, and cell culture

supernatant. The kit specifically adds Linear Acrylamide, which can easily capture small amounts of RNA from the samples. RNA-Only Column can efficiently bind RNA. The kit can process a large number of samples at the same time.

The entire kit does not contain RNase, so the purified RNA will not be degraded. Buffer viRW1 and Buffer viRW2 can ensure that the obtained viral nucleic acid free of protein, nuclease or other impurities, which can be used directly for downstream molecular biology experiments.)

Vrial RNA Isolation Kit

At the same time, VTP could not exert antiviral effect on EV-A71 virus with mutation and deletion of 3A-VSR function, as well as in Dicer1 or AGO2-deficient cells infected with EV-A71 virus. It is proved that its mechanism of action is indeed targeting 3A-VSR and depends on the RNAi pathway.

To investigate the preclinical potential of ER-DRI, the research team evaluated its antiviral effect in EV-A71 virus-infected mice by intraperitoneal injection of fluorescently labeled peptides. The results showed that ER-DRI also stimulated the RNAi antiviral immune response in mice, inhibited the replication of EV-A71, improved the clinical symptoms of infected mice, and reduced the death of mice caused by infection. At the same time, the acute toxicity test showed that ER-DRI had no obvious harm to the health of mice.

The RNAi immune mechanism unlocked by VTPs is powerful enough to mediate efficient antiviral activity, clearly demonstrating the physiological importance and therapeutic feasibility of RNAi as antiviral immunity in mammals. In addition, this study has demonstrated that VSRs are druggable targets, providing a novel strategy to target VSRs for antiviral therapy, and peptides like ER-DRI hold considerable promise as first-in-class VTDs for enterovirus infections.

Article Link: Inhibition of viral suppressor of RNAi proteins by designer peptides protects from enteroviral infection in vivo - PubMed (nih.gov)

Product Link: Viral RNA Isolation Kit