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1. Novel viruses of the family Partitiviridae discovered in Saccharomyces cerevisiae

   https://doi.org/10.1371/journal.ppat.1011418  

   Taggart, Nathan T.; Crabtree, Angela M.; Creagh, Jack W.; Bizarria, Rodolfo; Li, Shunji; de la Higuera, Ignacio; Barnes, Jonathan E.; Shipley, Mason A.; Boyer, Josephine M.; Stedman, Kenneth M.; et al (June 2023, PLOS Pathogens)

   Wang, Aiming (Ed.)

   It has been 49 years since the last discovery of a new virus family in the model yeastSaccharomyces cerevisiae. A large-scale screen to determine the diversity of double-stranded RNA (dsRNA) viruses inS.cerevisiaehas identified multiple novel viruses from the familyPartitiviridaethat have been previously shown to infect plants, fungi, protozoans, and insects. MostS.cerevisiaepartitiviruses (ScPVs) are associated with strains of yeasts isolated from coffee and cacao beans. The presence of partitiviruses was confirmed by sequencing the viral dsRNAs and purifying and visualizing isometric, non-enveloped viral particles. ScPVs have a typical bipartite genome encoding an RNA-dependent RNA polymerase (RdRP) and a coat protein (CP). Phylogenetic analysis of ScPVs identified three species of ScPV, which are most closely related to viruses of the genusCryspovirusfrom the mammalian pathogenic protozoanCryptosporidium parvum. Molecular modeling of the ScPV RdRP revealed a conserved tertiary structure and catalytic site organization when compared to the RdRPs of thePicornaviridae. The ScPV CP is the smallest so far identified in thePartitiviridaeand has structural homology with the CP of other partitiviruses but likely lacks a protrusion domain that is a conspicuous feature of other partitivirus particles. ScPVs were stably maintained during laboratory growth and were successfully transferred to haploid progeny after sporulation, which provides future opportunities to study partitivirus-host interactions using the powerful genetic tools available for the model organismS.cerevisiae. 

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2. The Species-Specific Acquisition and Diversification of a K1-like Family of Killer Toxins in Budding Yeasts of the Saccharomycotina

   https://doi.org/10.1371/journal.pgen.1009341  

   Fredericks, Lance R.; Lee, Mark D.; Crabtree, Angela M.; Boyer, Josephine M.; Kizer, Emily A.; Taggart, Nathan T.; Roslund, Cooper R.; Hunter, Samuel S.; Kennedy, Courtney B.; Willmore, Cody G.; et al (February 2021, PLOS Genetics)

   Fay, Justin C. (Ed.)

   Killer toxins are extracellular antifungal proteins that are produced by a wide variety of fungi, including Saccharomyces yeasts. Although many Saccharomyces killer toxins have been previously identified, their evolutionary origins remain uncertain given that many of these genes have been mobilized by double-stranded RNA (dsRNA) viruses. A survey of yeasts from the Saccharomyces genus has identified a novel killer toxin with a unique spectrum of activity produced by Saccharomyces paradoxus . The expression of this killer toxin is associated with the presence of a dsRNA totivirus and a satellite dsRNA. Genetic sequencing of the satellite dsRNA confirmed that it encodes a killer toxin with homology to the canonical ionophoric K1 toxin from Saccharomyces cerevisiae and has been named K1-like (K1L). Genomic homologs of K1L were identified in six non- Saccharomyces yeast species of the Saccharomycotina subphylum, predominantly in subtelomeric regions of the genome. When ectopically expressed in S . cerevisiae from cloned cDNAs, both K1L and its homologs can inhibit the growth of competing yeast species, confirming the discovery of a family of biologically active K1-like killer toxins. The sporadic distribution of these genes supports their acquisition by horizontal gene transfer followed by diversification. The phylogenetic relationship between K1L and its genomic homologs suggests a common ancestry and gene flow via dsRNAs and DNAs across taxonomic divisions. This appears to enable the acquisition of a diverse arsenal of killer toxins by different yeast species for potential use in niche competition. 

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3. 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 transmission 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. 

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4. Visualization of RNA virus infection in a marine protist with a universal biomarker

   https://doi.org/10.1038/s41598-023-31507-w  

   Coy, Samantha R.; Utama, Budi; Spurlin, James W.; Kim, Julia G.; Deshmukh, Harshavardhan; Lwigale, Peter; Nagasaki, Keizo; Correa, Adrienne M. (December 2023, Scientific Reports)

   Abstract Half of the marine virosphere is hypothesized to be RNA viruses (kingdom Orthornavirae ) that infect abundant micro-eukaryotic hosts (e.g. protists). To test this, quantitative approaches that broadly track infections in situ are needed. Here, we describe a technique—dsRNA-Immunofluorescence (dsRIF)—that uses a double-stranded RNA (dsRNA) targeting monoclonal antibody to assess host infection status based on the presence of dsRNA, a replicative intermediate of all Orthornavirae infections. We show that the dinoflagellate Heterocapsa circularisquama produces dsRIF signal ~ 1000 times above background autofluorescence when infected by the + ssRNA virus HcRNAV. dsRNA-positive virocells were detected across > 50% of the 48-h infection cycle and accumulated to represent at least 63% of the population. Photosynthetic and chromosomal integrity remained intact during peak replication, indicating HcRNAV infection does not interrupt these processes. This work validates the use of dsRIF on marine RNA viruses and their hosts, setting the stage for quantitative environmental applications that will accelerate understanding of virus-driven ecosystem impacts. 

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5. Visualizing the translation and packaging of HIV-1 full-length RNA

   https://doi.org/10.1073/pnas.1917590117  

   Chen, Jianbo; Liu, Yang; Wu, Bin; Nikolaitchik, Olga A.; Mohan, Preeti R.; Chen, Jiji; Pathak, Vinay K.; Hu, Wei-Shau (March 2020, Proceedings of the National Academy of Sciences)

   HIV-1 full-length RNA (HIV-1 RNA) plays a central role in viral replication, serving as a template for Gag/Gag-Pol translation and as a genome for the progeny virion. To gain a better understanding of the regulatory mechanisms of HIV-1 replication, we adapted a recently described system to visualize and track translation from individual HIV-1 RNA molecules in living cells. We found that, on average, half of the cytoplasmic HIV-1 RNAs are being actively translated at a given time. Furthermore, translating and nontranslating RNAs are well mixed in the cytoplasm; thus, Gag biogenesis occurs throughout the cytoplasm without being constrained to particular subcellular locations. Gag is an RNA binding protein that selects and packages HIV-1 RNA during virus assembly. A long-standing question in HIV-1 gene expression is whether Gag modulates HIV-1 RNA translation. We observed that despite its RNA-binding ability, Gag expression does not alter the proportion of translating HIV-1 RNA. Using single-molecule tracking, we found that both translating and nontranslating RNAs exhibit dynamic cytoplasmic movement and can reach the plasma membrane, the major HIV-1 assembly site. However, Gag selectively packages nontranslating RNA into the assembly complex. These studies illustrate that although HIV-1 RNA serves two functions, as a translation template and as a viral genome, individual RNA molecules carry out only one function at a time. These studies shed light on previously unknown aspects of HIV-1 gene expression and regulation. 

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