How can plant DNA viruses evade siRNA-directed DNA methylation and silencing?

Abstract

Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes.

Figure 1

Models for maintenance methylation and RdDM at the plant genome loci. (Based mostly on the findings using the model plant Arabidopsis). The plant dsDNA associated with nucleosomes is depicted as solid lines and the methylated cytosines at one or both strands indicated with black lollypops. Following DNA replication, cytosine methylation at CG, CHG and CHH sites of the newly-synthesized strand (blue) is catalyzed by the maintenance methyltransferates MET1, CMT3, and CMT2, respectively, with the help of co-factors VIM and KYP that recognize hemimethylated dsDNA; CMT3 and CMT2 also bind the repressive histone methylation mark H3K9me2 indicated as grey lollypops. The RNA-directed DNA methylation (RdDM) pathway establishing methylation of dsDNA de novo is catalyzed by DRM2 that interacts with the DRD1-Pol V complex generating a scaffold transcript. The nascent scaffold transcript is targeted by the 24-nt siRNA-AGO4 complex. Following DRM2-catalyzed de novo methylation of both DNA strands, Pol IV with the help of SHH1 (binding H3K9me2) and CLSY1 initiates siRNA biogenesis. The Pol IV transcript is converted by RDR2 to dsRNA. The resulting dsRNA is processed by DCL3 into 24-nt siRNA duplexes. The duplexes are handed over to AGO4 to form the silencing complexes with a single-stranded siRNA guide. This completes an siRNA amplification loop that reinforces RdDM-mediated transcriptional silencing. The chromatin remodeler DDM1 facilitates the access of all the methyltransferases to dsDNA.

Figure 2

Models for RCR and RDR modes of geminivirus DNA replication. (a) RCR. The viral circular ssDNA is released from the virion (yellow) into the nucleus. The host DNA polymerase synthesizes the complementary strand, yielding circular covalently-closed dsDNA. This dsDNA serves as a template for bidirectional transcription of the early leftward (Rep) and the late rightward (coat protein) genes. Viral mRNAs are transported to the cytoplasm. Following translation, Rep moves to the nucleus to initiate replication of the viral dsDNA by a rolling circle replication (RCR) mechanism. Rep (in yellow) nicks the virion strand in the origin of replication and recruits the host DNA polymerase to extend 3′-end of the cleaved virion strand on the complementary strand template. As the extension progresses, the polymerase complex, associated with Rep covalently linked to the 5′-end of the virion strand, displaces the virion strand. After one or more rounds of replication on the circular complementary strand template, Rep nicks and religates the displaced virion strand extended by one or more copies of the newly-synthesized virion strand and thereby releases one or more copies of circular ssDNA. The resulting circles can re-enter the replication cycle or get packaged into virions; (b) The circular covalently-closed dsDNA is invaded by a short viral DNA primer. The primer is extended by the host DNA polymerase on the circular viral template strand. After (or during) one or more rounds of replication, the newly-synthesized linear ssDNA gets fully or partially converted to linear dsDNA by the same (or another) DNA polymerase complex. Thus, RDR generates a heterogeneous population of linear dsDNAs. The long linear dsDNAs that harbor two or more origins of replication are transcribed by Pol II in both orientations to generate viral mRNAs. Following translation, Rep initiates replication of the long linear dsDNA with two or more origins of replication. The replicational release of ssDNA from the multimeric linear dsDNA generates circular ssDNA that can re-enter the replication cycle or get packaged.

Figure 2

Models for RCR and RDR modes of geminivirus DNA replication. (a) RCR. The viral circular ssDNA is released from the virion (yellow) into the nucleus. The host DNA polymerase synthesizes the complementary strand, yielding circular covalently-closed dsDNA. This dsDNA serves as a template for bidirectional transcription of the early leftward (Rep) and the late rightward (coat protein) genes. Viral mRNAs are transported to the cytoplasm. Following translation, Rep moves to the nucleus to initiate replication of the viral dsDNA by a rolling circle replication (RCR) mechanism. Rep (in yellow) nicks the virion strand in the origin of replication and recruits the host DNA polymerase to extend 3′-end of the cleaved virion strand on the complementary strand template. As the extension progresses, the polymerase complex, associated with Rep covalently linked to the 5′-end of the virion strand, displaces the virion strand. After one or more rounds of replication on the circular complementary strand template, Rep nicks and religates the displaced virion strand extended by one or more copies of the newly-synthesized virion strand and thereby releases one or more copies of circular ssDNA. The resulting circles can re-enter the replication cycle or get packaged into virions; (b) The circular covalently-closed dsDNA is invaded by a short viral DNA primer. The primer is extended by the host DNA polymerase on the circular viral template strand. After (or during) one or more rounds of replication, the newly-synthesized linear ssDNA gets fully or partially converted to linear dsDNA by the same (or another) DNA polymerase complex. Thus, RDR generates a heterogeneous population of linear dsDNAs. The long linear dsDNAs that harbor two or more origins of replication are transcribed by Pol II in both orientations to generate viral mRNAs. Following translation, Rep initiates replication of the long linear dsDNA with two or more origins of replication. The replicational release of ssDNA from the multimeric linear dsDNA generates circular ssDNA that can re-enter the replication cycle or get packaged.

Figure 3

Models for the biogenesis of geminiviral and pararetorviral siRNAs. (a) The biogenesis of geminiviral siRNAs is initiated by bi-directional readthrough transcription beyond the poly(A) signals that normally terminate transcription of the viral leftward genes (in begomoviruses, AC1/Rep, AC4, AC2/TrAP and AC3) and the rightward genes (in begomoviruses, AV2 and AV1/CP). The resulting sense and antisense readthrough transcripts (dotted lines) anneal to the complementary viral mRNAs (solid lines with arrowheads) and to each other (in the intergenic region between the transcription start sites). This creates dsRNAs spanning the entire circular viral genome. Every DCL digests these dsRNAs into siRNAs of different sizes, with DCL3 (24-nt), DCL4 (21-nt) and DCL2 (22-nt) being favored (in that order); (b) Pol II transcribes both the discontinuous and the covalently-closed dsDNA forms of pararetrovial dsDNA. Abrupt termination of Pol II transcription at the unrepaired minus-strand DNA gap (Met-tRNA gap), results in production of aberrant 8S RNA lacking poly(A) tail (Leader RNA). This RNA forms a viroid-like secondary structure which can be converted by Pol II to dsRNA. The resulting dsRNA serves as a decoy to engage all the four DCLs in massive production of 21-, 22-, and 24-nt vsRNAs. Pol II-mediated transcription of the covalently-closed circular dsDNA generates pgRNA covering the entire genome as well as antisense transcript(s) (red dotted line). The 35S pgRNA promoter was reported to drive transcription not only in the forward but also in the reverse orientation [79] (indicated with bent lines with arrowheads). The pgRNA and antisense Pol II transcripts form low-abundance dsRNA spanning the entire virus genome. This dsRNA is diced by the four DCLs to generate viral 21, 22 and 24-nt siRNAs.

Figure 4

Model for pararetrovirus replication. Viral circular dsDNA from the virion (in yellow) is released into the nucleus. The gaps at both DNA strands are sealed by the host DNA repair machinery. The resulting covalently-closed dsDNA serves as a template for Pol II transcription generating viral pregenomic RNA (pgRNA). The capped and polyadenylated pgRNA is transported to the cytoplasm for translation of viral proteins including the reverse transcriptase (RT), and for subsequent reverse transcription catalyzed by RT. The resulting dsDNA with discontinuities at both strands can get packaged into a new virion or targeted to the nucleus for the next round of replication.