Functional Mapping of AGO-Associated Zika Virus-Derived Small Interfering RNAs in Neural Stem Cells

Abstract

Viral interfering RNA (viRNA) has been identified from several viral genomes via directly deep RNA sequencing of the virus-infected cells, including zika virus (ZIKV). Once produced by endoribonuclease Dicer, viRNAs are loaded onto the Argonaute (AGO) family proteins of the RNA-induced silencing complexes (RISCs) to pair with their RNA targets and initiate the cleavage of target genes. However, the identities of functional ZIKV viRNAs and their viral RNA targets remain largely unknown. Our recent study has shown that ZIKV capsid protein interacted with Dicer and antagonized its endoribonuclease activity, which requires its histidine residue at the 41st amino acid. Accordingly, the engineered ZIKV-H41R loss-of-function (LOF) mutant virus no longer suppresses Dicer enzymatic activity nor inhibits miRNA biogenesis in NSCs. By combining AGO-associated RNA sequencing, deep sequencing analysis in ZIKV-infected human neural stem cells (NSCs), and miRanda target scanning, we defined 29 ZIKV derived viRNA profiles in NSCs, and established a complex interaction network between the viRNAs and their viral targets. More importantly, we found that viRNA production from the ZIKV mRNA is dependent on Dicer function and is a limiting factor for ZIKV virulence in NSCs. As a result, much higher levels of viRNAs generated from the ZIKV-H41R virus-infected NSCs. Therefore, our mapping of viRNAs to their RNA targets paves a way to further investigate how viRNAs play the role in anti-viral mechanisms, and perhaps other unknown biological functions.

Regulatory Role of Host MicroRNAs in Flaviviruses Infection

“…Only when loaded into Argonaute (AGO), siRNAs and miRNAs perform their functions. Hence, Zeng et al (2021) constructed AGO-associated viral siRNA profiles and completed a network between ZIKV siRNAs and their probable viral RNA (vRNA) targets via AGO-associated RNA sequencing. Twenty-nine siRNAs and 114 potential targets were obtained from the ZIKV genome, and further functional characterization of ZIKV siRNAs need to be verified.…”

“…In addition, the virus itself produces a variety of non-coding RNAs, such as subgenomic flaviviral RNAs (sfRNAs) and viral small RNAs (vsRNAs), the latter also include viral small interfering RNAs (siRNAs) and virus-encoded miRNAs. All of them can interact with cellular and viral RNA to regulate flavivirus replication ( Schuessler et al, 2012 ; Hussain and Asgari, 2014 ; Zeng et al, 2021 ).…”

Neuroimmune Evasion of Zika Virus to Facilitate Viral Pathogenesis

“…Once produced, vsiRNAs are loaded onto AGO family proteins of the RISCs to initiate the cleavage of host target genes and cognate viral RNAs (Guo et al, 2019). ZIKV infection has been demonstrated to induce abundant vsiRNAs in NSCs and hNPCs (Xu et al, 2019;Zeng et al, 2021). Zeng et al reported not only that vsiRNA is a limiting factor for ZIKV infection in NSCS, but also that the total 29 vsiRNAs across the ZIKV genome through AGO-associated RNA-seq.…”

“…Once produced, vsiRNAs are loaded onto AGO family proteins of the RISCs to initiate the cleavage of host target genes and cognate viral RNAs ( Guo et al., 2019 ). ZIKV infection has been demonstrated to induce abundant vsiRNAs in NSCs and hNPCs ( Xu et al., 2019 ; Zeng et al., 2021 ). Zeng et al.…”

“…More importantly, the production of these vsiRNAs is demonstrated to be dependent on Dicer. The vsiRNA-p18, the most abundant one within the identified 29 vsiRNAs, was detectable in ZIKV-infected NSCs while its physiologically antiviral effect remains unknown ( Zeng et al., 2021 ). Similar evidence was also shown by Xu et al.…”