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1. Structural Analysis and Whole Genome Mapping of a New Type of Plant Virus Subviral RNA: Umbravirus-Like Associated RNAs

   https://doi.org/10.3390/v13040646  

   Liu, Jingyuan ; Carino, Elizabeth ; Bera, Sayanta ; Gao, Feng ; May, Jared P. ; Simon, Anne E. ( April 2021 , Viruses)

   null (Ed.)

   We report the biological and structural characterization of umbravirus-like associated RNAs (ulaRNAs), a new category of coat-protein dependent subviral RNA replicons that infect plants. These RNAs encode an RNA-dependent RNA polymerase (RdRp) following a −1 ribosomal frameshift event, are 2.7–4.6 kb in length, and are related to umbraviruses, unlike similar RNA replicons that are related to tombusviruses. Three classes of ulaRNAs are proposed, with citrus yellow vein associated virus (CYVaV) placed in Class 2. With the exception of CYVaV, Class 2 and Class 3 ulaRNAs encode an additional open reading frame (ORF) with movement protein-like motifs made possible by additional sequences just past the RdRp termination codon. The full-length secondary structure of CYVaV was determined using Selective 2’ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) structure probing and phylogenic comparisons, which was used as a template for determining the putative structures of the other Class 2 ulaRNAs, revealing a number of distinctive structural features. The ribosome recoding sites of nearly all ulaRNAs, which differ significantly from those of umbraviruses, may exist in two conformations and are highly efficient. The 3′ regions of Class 2 and Class 3 ulaRNAs have structural elements similar to those of nearly all umbraviruses, and allmore »Class 2 ulaRNAs have a unique, conserved 3′ cap-independent translation enhancer. CYVaV replicates independently in protoplasts, demonstrating that the reported sequence is full-length. Additionally, CYVaV contains a sequence in its 3′ UTR that confers protection to nonsense mediated decay (NMD), thus likely obviating the need for umbravirus ORF3, a known suppressor of NMD. This initial characterization lays down a road map for future investigations into these novel virus-like RNAs.« less
2. Genome characterization of fig umbra-like virus

   https://doi.org/10.1007/s11262-021-01867-4  

   Wang, Xupeng ; Olmedo-Velarde, Alejandro ; Larrea-Sarmiento, Adriana ; Simon, Anne E. ; Kong, Alexandra ; Borth, Wayne ; Suzuki, Jon Y. ; Wall, Marisa M. ; Hu, John ; Melzer, Michael ( September 2021 , Virus Genes)

   The complete genome of a new umbra-like virus from edible fig (Ficus carica) was identified by high-throughput sequencing. Based on its similarity to umbra-like virus genome sequences available in GenBank, the proposed name of this new virus is "fig umbra-like virus" (FULV). The genome of full-length FULV-1 consists of 3049 nucleotides organized into three open reading frames (ORFs). Pairwise comparisons showed that the complete nucleotide sequence of the virus had the highest identity (71.3%) to citrus yellow vein-associated virus (CYVaV). In addition, phylogenetic trees based on whole-genome nucleotide sequences and amino acid sequences of the RNA-dependent RNA polymerase showed that FULV forms a monophyletic lineage with CYVaV and other umbra-like viruses. Based on the demarcation criteria of the genus Umbravirus, and lack of two umbravirus ORFs, we propose that FULV is a putative new member of the umbra-like virus clade within the family Tombusviridae.
3. Opium Poppy Mosaic Virus Has an Xrn-Resistant, Translated Subgenomic RNA and a BTE 3′ CITE

   https://doi.org/10.1128/JVI.02109-20  

   Ilyas, Muhammad ; Du, Zhiyou ; Simon, Anne E. ( April 2021 , Journal of Virology)

   Dutch, Rebecca Ellis. (Ed.)

   ABSTRACT Opium poppy mosaic virus (OPMV) is a recently discovered umbravirus in the family Tombusviridae . OPMV has a plus-sense genomic RNA (gRNA) of 4,241 nucleotides (nt) from which replication protein p35 and p35 extension product p98, the RNA-dependent RNA polymerase (RdRp), are expressed. Movement proteins p27 (long distance) and p28 (cell to cell) are expressed from a 1,440-nt subgenomic RNA (sgRNA2). A highly conserved structure was identified just upstream from the sgRNA2 transcription start site in all umbraviruses, which includes a carmovirus consensus sequence, denoting generation by an RdRp-mediated mechanism. OPMV also has a second sgRNA of 1,554 nt (sgRNA1) that starts just downstream of a canonical exoribonuclease-resistant sequence (xrRNA D ). sgRNA1 codes for a 30-kDa protein in vitro that is in frame with p28 and cannot be synthesized in other umbraviruses. Eliminating sgRNA1 or truncating the p30 open reading frame (ORF) without affecting p28 substantially reduced accumulation of OPMV gRNA, suggesting a functional role for the protein. The 652-nt 3′ untranslated region of OPMV contains two 3′ cap-independent translation enhancers (3′ CITEs), a T-shaped structure (TSS) near its 3′ end, and a Barley yellow dwarf virus -like translation element (BTE) in the central region. Only the BTE is functionalmore »in luciferase reporter constructs containing gRNA or sgRNA2 5′ sequences in vivo , which differs from how umbravirus 3′ CITEs were used in a previous study. Similarly to most 3′ CITEs, the OPMV BTE links to the 5′ end via a long-distance RNA-RNA interaction. Analysis of 14 BTEs revealed additional conserved sequences and structural features beyond the previously identified 17-nt conserved sequence. IMPORTANCE Opium poppy mosaic virus (OPMV) is an umbravirus in the family Tombusviridae . We determined that OPMV accumulates two similarly sized subgenomic RNAs (sgRNAs), with the smaller known to code for proteins expressed from overlapping open reading frames. The slightly larger sgRNA1 has a 5′ end just upstream from a previously predicted xrRNA D site, identifying this sgRNA as an unusually long product produced by exoribonuclease trimming. Although four umbraviruses have similar predicted xrRNA D sites, only sgRNA1 of OPMV can code for a protein that is an extension product of umbravirus ORF4. Inability to generate the sgRNA or translate this protein was associated with reduced gRNA accumulation in vivo . We also characterized the OPMV BTE structure, a 3′ cap-independent translation enhancer (3′ CITE). Comparisons of 13 BTEs with the OPMV BTE revealed additional stretches of sequence similarity beyond the 17-nt signature sequence, as well as conserved structural features not previously recognized in these 3′ CITEs.« less
4. Characterization of Two Novel Toti-Like Viruses Co-infecting the Atlantic Blue Crab, Callinectes sapidus, in Its Northern Range of the United States

   https://doi.org/10.3389/fmicb.2022.855750  

   Zhao, Mingli ; Xu, Lan ; Bowers, Holly ; Schott, Eric J. ( March 2022 , Frontiers in Microbiology)

   The advancement of high throughput sequencing has greatly facilitated the exploration of viruses that infect marine hosts. For example, a number of putative virus genomes belonging to the Totiviridae family have been described in crustacean hosts. However, there has been no characterization of the most newly discovered putative viruses beyond description of their genomes. In this study, two novel double-stranded RNA (dsRNA) virus genomes were discovered in the Atlantic blue crab ( Callinectes sapidus ) and further investigated. Sequencing of both virus genomes revealed that they each encode RNA dependent RNA polymerase proteins (RdRps) with similarities to toti-like viruses. The viruses were tentatively named Callinectes sapidus toti-like virus 1 (CsTLV1) and Callinectes sapidus toti-like virus 2 (CsTLV2). Both genomes have typical elements required for −1 ribosomal frameshifting, which may induce the expression of an encoded ORF1–ORF2 (gag-pol) fusion protein. Phylogenetic analyses of CsTLV1 and CsTLV2 RdRp amino acid sequences suggested that they are members of two new genera in the family Totiviridae . The CsTLV1 and CsTLV2 genomes were detected in muscle, gill, and hepatopancreas of blue crabs by real-time reverse transcription quantitative PCR (RT-qPCR). The presence of ~40 nm totivirus-like viral particles in all three tissues was verified by transmissionmore »electron microscopy, and pathology associated with CsTLV1 and CsTLV2 infections were observed by histology. PCR assays showed the prevalence and geographic range of these viruses, to be restricted to the northeast United States sites sampled. The two virus genomes co-occurred in almost all cases, with the CsTLV2 genome being found on its own in 8.5% cases, and the CsTLV1 genome not yet found on its own. To our knowledge, this is the first report of toti-like viruses in C. sapidus . The information reported here provides the knowledge and tools to investigate transmission and potential pathogenicity of these viruses.« less
5. The shape of aroma: Measuring and modeling citrus oil gland distribution

   https://doi.org/10.1002/ppp3.10333  

   Amézquita, Erik J. ; Quigley, Michelle Y. ; Ophelders, Tim ; Seymour, Danelle ; Munch, Elizabeth ; Chitwood, Daniel H. ( September 2022 , PLANTS, PEOPLE, PLANET)

   Societal Impact Statement Citrus are intrinsically connected to human health and culture, preventing human diseases like scurvy and inspiring sacred rituals. Citrus fruits come in a stunning number of different sizes and shapes, ranging from small clementines to oversized pummelos, and fruits display a vast diversity of flavors and aromas. These qualities are key in both traditional and modern medicine and in the production of cleaning and perfume products. By quantifying and modeling overall fruit shape and oil gland distribution, we can gain further insight into citrus development and the impacts of domestication and improvement on multiple characteristics of the fruit. Summary - Citrus come in diverse sizes and shapes, and play a key role in world culture and economy. Citrus oil glands in particular contain essential oils which include plant secondary metabolites associated with flavor and aroma. Capturing and analyzing nuanced information behind the citrus fruit shape and its oil gland distribution provide a morphology-driven path to further our insight into phenotype–genotype interactions. We investigated the shape of citrus fruit of 51 accessions based on 3D X-ray computed tomography (CT) scan reconstructions. Accessions include members of the three ancestral citrus species as well as related genera, and several interspecificmore »hybrids. We digitally separate and compare the size of fruit endocarp, mesocarp, exocarp, and oil gland tissue. Based on the centers of the oil glands, overall fruit shape is approximated with an ellipsoid. Possible oil gland distributions on this ellipsoid surface are explored using directional statistics. - There is a strong allometry along fruit tissues; that is, we observe a strong linear relationship between the logarithmic volume of any pair of major tissues. This suggests that the relative growth of fruit tissues with respect to each other follows a power law. We also observe that on average, glands distance themselves from their nearest neighbor following a square root relationship, which suggests normal diffusion dynamics at play. - The observed allometry and square root models point to the existence of biophysical developmental constraints that govern novel relationships between fruit dimensions from both evolutionary and breeding perspectives. Understanding these biophysical interactions prompts an exciting research path on fruit development and breeding.« less