Engineered RNA viral synthesis of microRNAs

Abstract

MicroRNAs (miRNAs) are short noncoding RNAs that exert posttranscriptional gene silencing and regulate gene expression. In addition to the hundreds of conserved cellular miRNAs that have been identified, miRNAs of viral origin have been isolated and found to modulate both the viral life cycle and the cellular transcriptome. Thus far, detection of virus-derived miRNAs has been largely limited to DNA viruses, suggesting that RNA viruses may be unable to exploit this aspect of transcriptional regulation. Lack of RNA virus-produced miRNAs has been attributed to the replicative constraints that would incur following RNase III processing of a genomic hairpin. To ascertain whether the generation of viral miRNAs is limited to DNA viruses, we investigated whether influenza virus could be designed to deliver functional miRNAs without affecting replication. Here, we describe a modified influenza A virus that expresses cellular microRNA-124 (miR-124). Insertion of the miR-124 hairpin into an intron of the nuclear export protein transcript resulted in endogenous processing and functional miR-124. We demonstrate that a viral RNA genome incorporating a hairpin does not result in segment instability or miRNA-mediated genomic targeting, thereby permitting the virus to produce a miRNA without having a negative impact on viral replication. This work demonstrates that RNA viruses can produce functional miRNAs and suggests that this level of transcriptional regulation may extend beyond DNA viruses.

SUMOylation Affects the Interferon Blocking Activity of the Influenza A Nonstructural Protein NS1 without Affecting Its Stability or Cellular Localization

“…As a similar inability to generate K219R mutants was previously reported by others (30), we considered that this issue likely indicated that the N62H mutation in NS2 was lethal. Therefore, to allow us to test the relevance of NS1 SUMOylation in the absence of changes in the primary sequence of NS2, we followed a previously reported methodology to separate the ORF for NS1 from that for NS2 while still allowing NS2 to be produced as a splicing product of the primary NS mRNA transcript (57). Briefly, starting with PolI-driven constructs for either wt NS1 or NS1K70AK219A, the splicing acceptor site located in NS1 was inactivated by site-directed mutagenesis.…”

Evidence for a cytoplasmic microprocessor of pri-miRNAs

“…Generation of rSINV expressing miR-124 has been described elsewhere (Shapiro et al 2010;Varble et al 2010). Similarly, rSINV122 was generated by cloning the mmu-miR-122 locus (chr18:65, 408,015-65,409,080) into rSINV as previously described (Shapiro et al 2010).…”

Noncanonical cytoplasmic processing of viral microRNAs

“…The primiR-124-2 and the TE12Q constructs have all been described elsewhere (Cheng et al 1996;Perez et al 2009;Varble et al 2010). miR-124 inserts were amplified using previously described primers flanked with BstEII linkers and were cloned directly into the SP6-based DNA plasmid.…”