MicroRNA-Guided Processing Impairs
            <i>Plum Pox Virus</i>
            Replication, but the Virus Readily Evolves To Escape This Silencing Mechanism

Simón‐Mateo, Carmen; Garcı́a, Juan Antonio

doi:10.1128/jvi.80.5.2429-2436.2006

Cited by 137 publications

(93 citation statements)

References 61 publications

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“…S1 in the supplemental material) (22) resulting from miR159-guided cleavage. This might suggest that miR159 more rapidly and efficiently targets its homologous sequence than does miR171, or this could be partly due to much lower endogenous levels of miR171 present in the N. benthamiana leaf tissue compared to miR159 (28).…”

Section: Discussionmentioning

confidence: 99%

Tomato Bushy Stunt Virus Recombination Guided by Introduced MicroRNA Target Sequences

Kumar

Uratsu

Dandekar

et al. 2009

J Virol

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Previously we described Tomato bushy stunt virus (TBSV) vectors, which retained their capsid protein gene and were engineered with magnesium chelatase (ChlH) and phytoene desaturase (PDS) gene sequences from Nicotiana benthamiana. Upon plant infection, these vectors eventually lost the inserted sequences, presumably as a result of recombination. Here, we modified the same vectors to also contain the plant miR171 or miR159 target sequences immediately 3 of the silencing inserts. We inoculated N. benthamiana plants and sequenced recombinant RNAs recovered from noninoculated upper leaves. We found that while some of the recombinant RNAs retained the microRNA (miRNA) target sites, most retained only the 3 10 and 13 nucleotides of the two original plant miRNA target sequences, indicating in planta miRNA-guided RNA-induced silencing complex cleavage of the recombinant TBSV RNAs. In addition, recovered RNAs also contained various fragments of the original sequence (ChlH and PDS) upstream of the miRNA cleavage site, suggesting that the 3 portion of the miRNA-cleaved TBSV RNAs served as a template for negative-strand RNA synthesis by the TBSV RNA-dependent RNA polymerase (RdRp), followed by template switching by the RdRp and continued RNA synthesis resulting in loss of nonessential nucleotides.Several plant viruses have been developed as tools for various biotechnology applications, including expression platforms for protein production in plants (1, 2, 6) and as gene silencing systems as part of reverse genetics approaches toward understanding host plant gene function (4, 5). For both of these applications, nonviral sequences conferring the desired function are cloned into the virus genome in order to be expressed during replication in plants. One advantage of using viruses engineered with nonviral sequences is flexibility in manipulating these "extra" sequences, which are not essential for viral replication or movement (1). However, recombinant viruses also tend to lose these sequences, causing instability at the insertion site and resulting in loss of function of the recombinant viral vector. The relatively high error rates of viral replicases (7,14,24) and the propensity for recombination events (9) contribute to the instability often seen with some viral vector systems.Recombination events in RNA viruses typically result in joining of two noncontiguous RNA segments (16). These could be sequences from two separate RNA molecules or distant regions of the same molecule. Retention by viruses of favorable sequences is selection driven and eliminates sequences that are unnecessary or negatively affect fitness (11, 31), hence making recombination critical to virus evolution (13, 29). Although phylogenetic analyses predict that recombination events have affected evolution for essentially all groups of RNA viruses (3), some viruses appear to be more prone to recombination than others. For example, plantinfecting supergroup II viruses of the family Tombusviridae appear to undergo frequent recombination, as is supported by the ...

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Section: Discussionmentioning

confidence: 99%

Tomato Bushy Stunt Virus Recombination Guided by Introduced MicroRNA Target Sequences

Kumar

Uratsu

Dandekar

et al. 2009

J Virol

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“…Lecellier et al (2005) found that a cellular miRNA, miR-32, mediated antiviral defense in human cells, and regulated primate foamy virus type I (PFV-1) proliferation. Several mammalian viruses, including the herpesvirus family, such as Epstein-Barr virus (EBV), simian virus 40 (SV40), and Kaposi sarcoma-associated virus, have been found to code miRNAs (Pfeffer et al, , 2005Cai et al, 2005;Grey et al, 2005;Omoto and Fujii, 2005;Sullivan et al, 2005;Sullivan and Ganem, 2005a,b;Cai and Cullen, 2006;Jiang et al, 2006;Schuetz and Sarnow, 2006;Simon-Mateo and Garcia, 2006). Although the function of these coding miRNAs is still unknown, it may play a role in human disease development.…”

Section: Functions Of Micrornas In Animalsmentioning

confidence: 99%

MicroRNAs and their regulatory roles in animals and plants

Zhang

Wang

Pan

2006

Journal Cellular Physiology

493

307

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microRNAs (miRNAs) are an abundant class of newly identified endogenous non-protein-coding small RNAs. They exist in animals, plants, and viruses, and play an important role in gene silencing. Translational repression, mRNA cleavage, and mRNA decay initiated by miRNA-directed deadenylation of targeted mRNAs are three mechanisms of miRNA-guided gene regulation at the posttranscriptional levels. Many miRNAs are highly conserved in animals and plants, suggesting that they play an essential function in plants and animals. Lots of investigations indicate that miRNAs are involved in multiple biological processes, including stem cell differentiation, organ development, phase change, signaling, disease, cancer, and response to biotic and abiotic environmental stresses. This review provides a general background and current advance on the discovery, history, biogenesis, genomics, mechanisms, and functions of miRNAs.

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“…Although miRNAs have been shown to be involved in developmental regulation and many other cellular processes (3), the possibility that miRNA-guided RNA silencing could play a role in host defense against virus infection has been proposed (25). Indeed, the antiviral function of natural miRNAs has been described in animal systems (23), and more recently the plant miRNA pathway was also shown to be implicated in impairing the replication of an engineered potyvirus bearing an miRNA target (36).…”

mentioning

confidence: 99%

Artificial MicroRNA-Mediated Virus Resistance in Plants

Fang

2007

J Virol

305

164

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RNA silencing in plants is a natural defense system against foreign genetic elements including viruses. This natural antiviral mechanism has been adopted to develop virus-resistant plants through expression of virusderived double-stranded RNAs or hairpin RNAs, which in turn are processed into small interfering RNAs (siRNAs) by the host's RNA silencing machinery. While these virus-specific siRNAs were shown to be a hallmark of the acquired virus resistance, the functionality of another set of the RNA silencing-related small RNAs, microRNAs (miRNAs), in engineering plant virus resistance has not been extensively explored. Here we show that expression of an artificial miRNA, targeting sequences encoding the silencing suppressor 2b of Cucumber mosaic virus (CMV), can efficiently inhibit 2b gene expression and protein suppressor function in transient expression assays and confer on transgenic tobacco plants effective resistance to CMV infection. Moreover, the resistance level conferred by the transgenic miRNA is well correlated to the miRNA expression level. Comparison of the anti-CMV effect of the artificial miRNA to that of a short hairpin RNA-derived small RNA targeting the same site revealed that the miRNA approach is superior to the approach using short hairpin RNA both in transient assays and in transgenic plants. Together, our data demonstrate that expression of virus-specific artificial miRNAs is an effective and predictable new approach to engineering resistance to CMV and, possibly, to other plant viruses as well.RNA silencing is a natural regulatory mechanism of eukaryotes acting against invasive nucleic acids or modulating endogenous gene expression (4, 41). Silencing is initiated when double-stranded RNAs (dsRNAs) or hairpin RNAs (hpRNAs) are processed into 21-to 24-nucleotide (nt) small interfering RNA (siRNA) or microRNA (miRNA) duplexes by dsRNAspecific RNase III-type Dicer enzymes. These small RNAs (sRNAs) are then incorporated into the RNA-induced silencing complex to guide the degradation or translation repression of cRNA targets (4, 39). Unlike animals, higher plants have evolved diversified RNA silencing pathways to generate different sRNA classes with specialized functions (9, 40). For example, Arabidopsis thaliana encodes four Dicer-like (DCL) proteins (DCL1 to DCL4). While DCL1 mainly processes imperfectly base-paired fold-back precursors to produce miRNAs (3), the other three DCL proteins are responsible for generating various endogenous siRNAs from perfect dsRNAs, such as stressrelated 24-nt natural-antisense-transcript siRNAs by DCL2 (6), 24-nt repeat-associated siRNAs that guide heterochromatin formation by DCL3 (46), and 21-nt trans-acting (ta-) siRNAs that control some aspects of plant development by DCL4 (17, 45). However, there is some functional redundancy among the four Arabidopsis DCL proteins. For instance, DCL1 is also capable of producing 21-nt ta-siRNAs in the dcl2 dcl3 dcl4 triple mutant (7), and DCL2 can generate 22-nt ta-siRNAs when the DCL4 activity is compromised (17,45). I...

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MicroRNA-Guided Processing Impairs Plum Pox Virus Replication, but the Virus Readily Evolves To Escape This Silencing Mechanism

Cited by 137 publications

References 61 publications

Tomato Bushy Stunt Virus Recombination Guided by Introduced MicroRNA Target Sequences

Tomato Bushy Stunt Virus Recombination Guided by Introduced MicroRNA Target Sequences

MicroRNAs and their regulatory roles in animals and plants

Artificial MicroRNA-Mediated Virus Resistance in Plants

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