TRANSLATION, REPLICATION AND TRANSCRIPTOMICS OF THE SIMPLEST PLUS-STRAND RNA PLANT VIRUSES

Abstract

Plus (+)-strand RNA viruses are among the most common pathogens of plants and animals. Furthermore, they present model systems for the study of basic biological processes, including protein translation and RNA replication, and shed light on the versatile roles that RNA structures play in these processes. After cell entry, the next step in the (+)-strand RNA viral life cycle is translation of the viral genome to produce the viral RNA-dependent RNA polymerase (RdRp) and associated replication proteins necessary for viral replication to occur. For many (+)-strand RNA viruses lacking a 5´cap and 3´ poly(A) tail, translation depends upon RNA structural elements within their genomes capable of hijacking the host translation machinery, which for plant viruses are commonly located in their 3´ proximal regions and are termed 3´ cap-independent translation enhancer (CITE) elements. In Chapter 2, I report upon my work characterizing a new subclass of panicum mosaic virus-like translation enhancer (PTE) elements, which bind and co-opt for viral use the host translation initiation factor 4E (eIF4E) – the translation initiation factor normally responsible for binding and recognition of mRNA 5´caps during canonical eukaryotic translation initiation. Thus, PTE 3´CITEs present a novel mechanism for co-opting the critical host factor eIF4E. My work characterizing a new subclass of PTE 3´CITEs further revealed characteristics common among all PTE 3´CITEs pertaining to their mechanism of binding eIF4E.After translation of the necessary viral replication proteins, replication of the viral RNA occurs, which again is in large part mediated by RNA structural elements within the viral genome that can bind to the viral RdRp and/or host factors involved in viral replication. Indeed, RNA structural elements often serve dual roles in viral translation and replication and/or are located proximal to RNA structural elements involved in the alternate function. In Chapter 3, I discuss my work characterizing novel replication elements in the 3´ terminal regions of umbraviruses (family Tombusviridae). The uncovered replication elements appear to be specific to umbraviruses and are located immediately upstream of replication/translation elements that are common throughout the Tombusviridae, lending greater complexity to the already complex 3´ proximal structures of umbraviruses. While the study of (+)-strand RNA viruses has historically focused on their protein-coding transcripts, (+)-strand RNA viruses also commonly produce additional non-coding transcripts, including recombinant defective RNAs, typically containing 5´ and 3´ co-terminal viral genome segments, and (+/-)-foldback RNAs, composed of complementary (+)- and (-)-strand viral sequences joined together. Long non-coding RNAs that accumulate to high levels have also been reported for plant and animal (+)-strand RNA viruses in recent years, and truncations of viral transcripts also commonly arise due to host nuclease activity and/or premature termination of replication elongation by the viral RdRp. The rise of long-read high-throughput sequencing technologies such as nanopore sequencing presents an opportunity to fully map the complexity of (+)-strand RNA viral transcriptomes. In Chapter 4, I present my work performing this analysis, employing direct RNA nanopore sequencing, in which the transcripts present in an RNA sample of interest are directly sequenced. This analysis revealed for the umbra-like virus citrus yellow vein-associated virus (CY1): (i) three novel 5´ co-terminal long non-coding RNAs; (ii) D-RNA population dynamics; (iii) a common 3´ terminal truncation of 61 nt among (+)-strand viral transcripts; (iv) missing 3´ terminal CCC-OH motif in virtually all (-)-strand reads; (v) major timepoint- and tissue-specific differences; and (vi) an abundance of (+/-)-foldback RNAs at later infection timepoints in leaf tissues. This work also sheds light on the current shortcomings of direct RNA nanopore sequencing as a technique. Finally, the importance of RNA structural biology in the study of (+)-strand RNA viruses presents the need for specialized RNA structure drawing software with functionality to easily control the layout of nucleobases, edit base-pairs, and annotate/color the nucleobases and bonds in a drawing. It is through the visual exploration of RNA structures that RNA biologists routinely improve upon the outputs of RNA structure prediction programs and perform crucial phylogenetic analyses among related RNA structures. Large RNA structures, such as whole viral genomes thousands of nucleotides long, can only be studied in their entirety with the aid of RNA structure visualization tools. To this end, I have developed over the course of my doctoral education the 2D RNA structure drawing application RNAcanvas, which is available as a web app and has grown popular among the RNA biology community. RNAcanvas emphasizes graphical mouse-based interaction with RNA structure drawings and has special functionality well suited for the drawing and exploration of large RNA structures, such as automatic layout adjustment and maintenance, complementary sequence highlighting, motif finding, and performance optimizations. Large viral structures such as that of the 2.7 kb CY1 genomic RNA could not have been characterized without the aid of RNAcanvas. In Chapter 5, I present my work developing RNAcanvas.