Identification of RISC-Associated Adenoviral MicroRNAs, a Subset of Their Direct Targets, and Global Changes in the Targetome upon Lytic Adenovirus 5 Infection

Abstract

Adenoviruses encode a set of highly abundant microRNAs (mivaRNAs), which are generated by Dicer-mediated cleavage of the larger noncoding virus-associated RNAs (VA RNAs) I and II. We performed deep RNA sequencing to thoroughly investigate the relative abundance of individual single strands of mivaRNA isoforms in human A549 cells lytically infected with human adenovirus 5 (Ad5) at physiologically relevant multiplicities of infection (MOIs). In addition, we investigated their relative abundance in the endogenous RNA-induced silencing complexes (RISCs). The occupation of endogenous RISCs by mivaRNAs turned out to be pronounced but not as dominant as previously inferred from experiments with AGO2-overexpressing cells infected at high MOIs. In parallel, levels of RISC-incorporated mRNAs were investigated as well. Analysis of mRNAs enriched in RISCs in Ad5-infected cells revealed that only mRNAs with complementarity to the seed sequences of mivaRNAs derived from VA RNAI but not VA RNAII were overrepresented among them, indicating that only mivaRNAs derived from VA RNAI are likely to contribute substantially to the posttranscriptional downregulation of host gene expression. Furthermore, to generate a comprehensive picture of the entire transcriptome/targetome in lytically infected cells, we determined changes in cellular miRNA levels in both total RNA and RISC RNA as well, and bioinformatical analysis of mRNAs of total RNA/RISC fractions revealed a general, genome-wide trend toward detargeting of cellular mRNAs upon infection. Lastly, we identified the direct targets of both single strands of a VA RNAI-derived mivaRNA that constituted one of the two most abundant isoforms in RISCs of lytically infected A549 cells. IMPORTANCE Viral and cellular miRNAs have been recognized as important players in virus-host interactions.This work provides the currently most comprehensive picture of the entire mRNA/miRNA transcriptome and of the complete RISC targetome during lytic adenovirus infection and thus represents the basis for a deeper understanding of the interplay between the virus and the cellular RNA interference machinery. Our data suggest that, at least in the model system that was employed, lytic infection by Ad5 is accompanied by a measurable global net detargeting effect on cellular mRNAs, and analysis of RISC-associated viral small RNAs revealed that the VA RNAs are the only source of virus-encoded miRNAs. Moreover, this work allows to assess the power of individual viral miRNAs to regulate cellular gene expression and provides a list of proven and putative direct targets of these miRNAs, which is of importance, given the fact that information about validated targets of adenovirus-encoded miRNAs is scarce.

Adenovirus VA RNA: An essential pro-viral non-coding RNA

“…Yet among mRNAs isolated from mivaRNA-containing RISC during Ad5 infection, seed sequences corresponding to mivaRNA II are underrepresented (Bellutti et al, 2015). Furthermore, both 3 -mivaRNA I (A) and (G) more efficiently knock down reporter gene expression than mivaRNAs derived from VA RNA II (Bellutti et al, 2015). Together these findings illustrate a disconnection between mivaRNA abundance in RISC and the likelihood that a given mivaRNA can knock down gene expression.…”

“…While these mivaRNAs are the predominant RISC-associated miRNAs of viral origin during Ad5 infection (Bellutti et al, 2015), a small fraction of adenoviral miRNAs are thought to originate from RNAs other than VA RNA (Zhao et al, 2013). While one group reported that approximately 80% of Ago-2-containing RISC complexes late in AdV infection contain mivaRNAs (Xu et al, 2007), others report a significantly lower proportion, 15% (Bellutti et al, 2015).…”

“…While these mivaRNAs are the predominant RISC-associated miRNAs of viral origin during Ad5 infection (Bellutti et al, 2015), a small fraction of adenoviral miRNAs are thought to originate from RNAs other than VA RNA (Zhao et al, 2013). While one group reported that approximately 80% of Ago-2-containing RISC complexes late in AdV infection contain mivaRNAs (Xu et al, 2007), others report a significantly lower proportion, 15% (Bellutti et al, 2015). Though this disparity may be attributed to differences in experimental approach, for example overexpression of Ago-2 resulting in reduced VA RNA competition with cellular RNAs (Xu et al, 2007), the extraordinary level to which VA RNA accumulates (10 8 copies/cell) (Mathews and Shenk, 1991) makes a substantial effect on the cellular miRNA population highly likely (Fig.…”

“…For example, despite being expressed at 20-fold lower abundance, VA RNA II is the origin of the majority of RISC-associated mivaRNAs (∼69%) (Andersson et al, 2005;Aparicio et al, 2006). Yet among mRNAs isolated from mivaRNA-containing RISC during Ad5 infection, seed sequences corresponding to mivaRNA II are underrepresented (Bellutti et al, 2015). Furthermore, both 3 -mivaRNA I (A) and (G) more efficiently knock down reporter gene expression than mivaRNAs derived from VA RNA II (Bellutti et al, 2015).…”

“…This preference for 5 -VA RNA I (A) strand incorporation, in spite of mivaRNA I (G) representing more than 75% of the total VA RNA pool (Vennstrom et al, 1978a), is determined by 5 -end heterogeneity, as 5 -mivaRNA I (A) is three nucleotides longer than 5 -mivaRNA I (G) due to their different transcriptional start sites (Harris and Roeder, 1978;Xu et al, 2009). Although 3 -mivaRNA I (A) is more likely to be found in RISC, the 3 -strand activity is only about twice that of the 5 strand (Bellutti et al, 2015). Curiously, a conflicting report shows that the 3 -mivaRNA I (A)-associated RISC is only 2% as active as a RISC assembled upon the 5 -mivaRNA I (A) in vitro (Xu et al, 2009).…”