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Item Adenosine modifications impede SARS-CoV-2 RNA-dependent RNA transcription(Cold Spring Harbor Laboratory, 2024-06) Snyder, Laura R.; Kilde, Ingrid; Nemudryi, Artem; Wiedenheft, Blake; Koutmos, Markos; Koutmou, Kristin S.SARS-CoV-2, the causative virus of the COVID-19 pandemic, follows SARS and MERS as recent zoonotic coronaviruses causing severe respiratory illness and death in humans. The recurrent impact of zoonotic coronaviruses demands a better understanding of their fundamental molecular biochemistry. Nucleoside modifications, which modulate many steps of the RNA life cycle, have been found in SARS-CoV-2 RNA, although whether they confer a pro- or antiviral effect is unknown. Regardless, the viral RNA-dependent RNA polymerase will encounter these modifications as it transcribes through the viral genomic RNA. We investigated the functional consequences of nucleoside modification on the pre-steady state kinetics of SARS-CoV-2 RNA-dependent RNA transcription using an in vitro reconstituted transcription system with modified RNA templates. Our findings show that N6-methyladenosine and 2′-O-methyladenosine modifications slow the rate of viral transcription at magnitudes specific to each modification, which has the potential to impact SARS-CoV-2 genome maintenance.Item Honey bee host-virus interactions at the individual and cellular level(Montana State University - Bozeman, College of Agriculture, 2021) McMenamin, Alexander James; Chairperson, Graduate Committee: Michelle Flenniken; This is a manuscript style paper that includes co-authored chapters.Honey bees are important pollinators of the fruits, nuts and vegetable crops that feed our growing population. Unfortunately, honey bee colony losses have averaged 38% from 2008-2018. These losses are due to a variety of factors, including reduced quality forage, pesticide exposure in agricultural fields, parasites like the Varroa destructor mite, and pathogens. The most diverse group of pathogens effecting honey bees are small RNA viruses. Honey bees have evolved numerous strategies to restrict virus infection, including the RNA interference (RNAi) pathway. Bees infected with a model virus, Sindbis-GFP (SINV) have differential expression of hundreds of genes, including RNAi genes and several heat shock protein (HSP) encoding genes. Therefore, we hypothesized that heat shock proteins are antiviral in honey bees. To induce the heat shock response (HSR), SINV-infected bees were heat shocked at 42°C for 4 hours. Heat shock resulted in a 74-90% reduction in SINV RNA copies as compared to bees maintained at 32°C. Heat shocked and/or virus-infected bees had increased expression of several core HSR protein-encoding genes, but heat shock did not consistently result in the increased expression of RNAi genes (argonaute-2 and dcr-like). This indicates that heat shock proteins are contributing to an antiviral response. SINV-infected bees also had higher expression of a recently identified antiviral gene - bee antiviral protein-1 (bap1). Therefore, we further characterized bap1 using computation approaches including phylogenetic analysis, which determined that this gene is taxonomically restricted to Hymenoptera and Blatella germanica (the German cockroach). Structural predication programs indicated that bap1 is a highly disordered protein. Intriguingly, transcriptome and correlation analyses determined that bap1 was coexpressed with several genes implicated in antiviral immunity (i.e., ago2, tudor-sn and TEP7). Although the precise antiviral function of bap1 remains to be elucidated, we further developed experimental tools that will enable more incisive investigation of bap1 and other antiviral genes. Primary cultures of larval hemocytes (immune cells) and mixed-cell pupal tissue cultures supported productive replication of sacbrood virus, deformed wing virus, and Flock House virus. Infected pupal cell cultures exhibited virus-specific transcriptional responses in bap1, ago2, and dcr-like expression. Together, these data further elucidate honey bee antiviral immunity and provide new tools for studying honey bee host-virus interactions.Item Investigating Virus–Host Interactions in Cultured Primary Honey Bee Cells(MDPI AG, 2021-07) McMenamin, Alexander J.; Parekh, Fenali; Lawrence, Verena; Flenniken, Michelle L.Honey bee (Apis mellifera) health is impacted by viral infections at the colony, individual bee, and cellular levels. To investigate honey bee antiviral defense mechanisms at the cellular level we further developed the use of cultured primary cells, derived from either larvae or pupae, and demonstrated that these cells could be infected with a panel of viruses, including common honey bee infecting viruses (i.e., sacbrood virus (SBV) and deformed wing virus (DWV)) and an insect model virus, Flock House virus (FHV). Virus abundances were quantified over the course of infection. The production of infectious virions in cultured honey bee pupal cells was demonstrated by determining that naïve cells became infected after the transfer of deformed wing virus or Flock House virus from infected cell cultures. Initial characterization of the honey bee antiviral immune responses at the cellular level indicated that there were virus-specific responses, which included increased expression of bee antiviral protein-1 (GenBank: MF116383) in SBV-infected pupal cells and increased expression of argonaute-2 and dicer-like in FHV-infected hemocytes and pupal cells. Additional studies are required to further elucidate virus-specific honey bee antiviral defense mechanisms. The continued use of cultured primary honey bee cells for studies that involve multiple viruses will address this knowledge gap.Item Honey bee antiviral defense mechanisms at the individual and cellular level(Montana State University - Bozeman, College of Agriculture, 2021) Parekh, Fenali Mukesh; Chairperson, Graduate Committee: Michelle Flenniken; Katie F. Daughenbaugh and Michelle L. Flenniken were co-authors of the article, 'Chemical stimulants and stressors impact the outcome of virus infection and immune gene expression in honey bees (Apis mellifera)' in the journal 'Frontiers in immunology' which is contained within this dissertation.; Alexander J. McMenamin was an author and Verana Lawrence and Michelle L. Flennikenwas were co-authors of the article, 'Investigating virus-host interactions in cultured primary honey bee cells' in the journal 'Insects' which is contained within this dissertation.; Katie F. Daughenbaugh and Michelle L. Flenniken were co-authors of the article, 'Honey bee antiviral response to flock house virus infection' which is contained within this dissertation.; This dissertation contains an article of which Fenali Mukesh Parekh is not the main author.Honey bees are important pollinators of fruit, nut, and vegetable crops that constitute a large proportion of the human diet. Unfortunately, annual honey bee colony losses are high, averaging 38% from 2008-2018 in the United States. Honey bee colony losses are attributed to multiple factors, including pathogens and chemical exposure. Virus incidence and abundance have been associated with colony losses. The majority of honey bee viruses are positive-sense single stranded RNA viruses. Honey bees antiviral defense include RNA interference (RNAi), a double-stranded RNA (dsRNA) triggered sequence-specific post-transcriptional silencing mechanism and a non-sequence specific dsRNA-triggered pathway. In addition, signal transduction cascades include the Toll, Imd, and Jak/STAT pathways that promote the expression of honey bee immune response genes that are also induced in response to virus infections. To investigate the impact of chemical exposure on honey bee immune responses and virus infections, we infected bees with a panel of viruses including two model viruses (i.e., Flock House virus (FHV) and Sindbis-GFP) and a naturally infecting honey bee virus, deformed wing virus (DWV) and fed them sucrose syrup containing either thyme oil, a beekeeper applied fungicide Fumagilin-B ®, or the insecticide clothianidin. We determined that bees fed thyme oil augmented sucrose syrup exhibited greater expression of key immune genes, i.e., ago2, dcr-like, abaecin, hymenoptaecin, and vitellogenin and reduced virus abundance compared to virus-infected bees fed sucrose syrup. Whereas, virus-infected honey bees fed diets containing fumagillin or clothianidin exhibited reduced expression of key immune genes and higher virus abundance suggesting that chemical stressors act as immunosuppressors in honey bees. To understand the interplay of viruses and host cell gene expression more precisely, we cultured primary honey bee cells derived from larvae (i.e., hemocytes, immune cells) or pupae (i.e., mixed cell population including epithelial cells, adipocytes, muscle cells, hemocytes) and demonstrated that these cells supported replication of sacbrood virus, DWV, and FHV. Expression of select immune genes, including bap1, ago2, and dcr-like, in virus-infected honey bee cells was similar to expression in individual bees and varied for each virus. Together, these data further our understanding of the honey bee antiviral defense network and provide new tools for studying honey bee host-virus interactions.Item Understanding resistance and transcriptional responses to potato virus Y infection in potato plants(Montana State University - Bozeman, College of Agriculture, 2021) Ross, Brian Thomas; Chairperson, Graduate Committee: Michelle Flenniken; Nina Zidack and Michelle L. Flenniken were co-authors of the article, 'Extreme resistance to viruses in potato and soybean' in the journal 'Frontiers in plant science' which is contained within this dissertation.; Nina Zidack and Michelle L. Flenniken were co-authors of the article, 'Transcriptional responses to potato virus Y infection in resistant and susceptible potato cultivars' submitted to the journal 'Cultivars' which is contained within this dissertation.The potato is one of the world's most important crops. Cultivation of potatoes occurs on every continent except Antarctica and in a wide variety of climates. Potatoes are susceptible to a multitude of pathogens that can decrease yield and market quality. Viruses are particularly problematic for potato growers, as most potato production involves the replanting of tubers grown the previous year. Because virus-infected potato plants can harbor virus in their tubers, these tubers can in turn be the source of infection in the next generation of plants. Strains of Potato virus Y are the most economically burdensome viruses for potato growers worldwide. In field settings, Potato virus Y is primarily transmitted to plant by aphids feeding on leaves, but PVY can also be transmitted mechanically through infected plant sap. The use of insecticides and the application of mineral oil to leaves can help limit aphid populations and prevent infection to an extent but are generally both less effective and more environmentally impactful than genetic antiviral resistance mechanisms. The incorporation of genes that provide durable resistance to Potato virus Y into commercial potatoes is a major focus of potato breeders. One form of resistance, called extreme resistance, is characterized by a lack of symptoms and little to no virus replication occurring at the site of infection, but the molecular mechanisms of this response are not well understood. A comprehensive analysis of the extreme resistance literature indicates that movement of the resistance protein from the cytoplasm to the nucleus of the cell directly after virus infection may be a key aspect of this immune response. The downstream, transcriptional aspects of the extreme resistance response are also not well understood. We analyzed the gene expression from a Potato virus Y-resistant potato variety, Payette Russet, and a commonly grown susceptible variety, Russet Burbank, at a series of time points after virus infection using RNA sequencing. Results of these analyses indicate that an immune response likely occurs in Payette Russet quickly after virus inoculation. These analyses also indicate that the virus-susceptible variety, Russet Burbank, exhibits changes in gene expression that are similar to other susceptible potato varieties during asymptomatic or tolerant infection. Furthering our understanding of the molecular mechanisms controlling resistance and severity of virus infections will help inform future breeding and genetic engineering efforts, which require detailed knowledge of the mechanisms of virus resistance.