scholarly journals Visualizing the ribonucleoprotein content of single bunyavirus virions reveals more efficient genome packaging in the arthropod host

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Erick Bermúdez-Méndez ◽  
Eugene A. Katrukha ◽  
Cindy M. Spruit ◽  
Jeroen Kortekaas ◽  
Paul J. Wichgers Schreur
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Rift Valley Fever Virus ◽  
Progeny Virus ◽  
Genome Packaging ◽  
Valley Fever ◽  
A Genome ◽  
A Cell ◽  
Infected Cells

AbstractBunyaviruses have a genome that is divided over multiple segments. Genome segmentation complicates the generation of progeny virus, since each newly formed virus particle should preferably contain a full set of genome segments in order to disseminate efficiently within and between hosts. Here, we combine immunofluorescence and fluorescence in situ hybridization techniques to simultaneously visualize bunyavirus progeny virions and their genomic content at single-molecule resolution in the context of singly infected cells. Using Rift Valley fever virus and Schmallenberg virus as prototype tri-segmented bunyaviruses, we show that bunyavirus genome packaging is influenced by the intracellular viral genome content of individual cells, which results in greatly variable packaging efficiencies within a cell population. We further show that bunyavirus genome packaging is more efficient in insect cells compared to mammalian cells and provide new insights on the possibility that incomplete particles may contribute to bunyavirus spread as well.

scholarly journals Visualizing the RNP content of single bunyavirus virions reveals more efficient genome packaging in the arthropod host

2020 ◽  
Author(s):  
Erick Bermúdez-Méndez ◽  
Eugene Katrukha ◽  
Cindy Spruit ◽  
Jeroen Kortekaas ◽  
Paul Wichgers Schreur
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Rift Valley Fever Virus ◽  
Progeny Virus ◽  
Genome Packaging ◽  
Valley Fever ◽  
A Genome ◽  
A Cell ◽  
Infected Cells

Abstract Bunyaviruses have a genome that is divided over multiple segments. Genome segmentation complicates the generation of progeny virus, since each newly formed virus particle should preferably contain a full set of genome segments in order to disseminate efficiently within and between hosts. Here, we combine immunofluorescence and fluorescence in situ hybridization techniques to simultaneously visualize bunyavirus progeny virions and their genomic content at single-molecule resolution in the context of singly infected cells. Using Rift Valley fever virus and Schmallenberg virus as prototype tri-segmented bunyaviruses, we show that bunyavirus genome packaging is influenced by the intracellular viral genome content of individual cells, which results in greatly variable packaging efficiencies within a cell population. We further show that bunyavirus genome packaging is more efficient in insect cells compared to mammalian cells and provide new insights on the possibility that incomplete particles may contribute to bunyavirus spread as well.

scholarly journals Investigating the Central Dogma of Molecular Biology in the Context of Nuclear Architecture and Cell Cycle

2019 ◽  
Author(s):  
Shivnarayan Dhuppar ◽  
Aprotim Mazumder
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Nuclear Architecture ◽  
Nuclear Lamina ◽  
Gene Copy ◽  
A Cell ◽  
Cycle Dependent

AbstractNuclear architecture is the organization of the genome within a cell nucleus with respect to different nuclear landmarks such as nuclear lamina, matrix or nucleoli. Lately it has emerged as a major regulator of gene expression in mammalian cells. The studies connecting nuclear architecture with gene expression are largely population-averaged and do not report on the heterogeneity in genome organization or in gene expression within a population. In this report we present a method for combining 3D DNA Fluorescence in situ Hybridization (FISH) with single molecule RNA FISH (smFISH) and immunofluorescence to study nuclear architecture-dependent gene regulation on a cell-by-cell basis. We further combine it with an imaging-based cell cycle staging to correlate nuclear architecture with gene expression across the cell cycle. We present this in the context of Cyclin A2 (CCNA2) gene for its known cell cycle-dependent expression. We show that, across the cell cycle, the expression of a CCNA2 gene copy is stochastic and depends neither on its sub-nuclear position—which usually lies close to nuclear lamina—nor on the expression from the other copies.

scholarly journals The S Segment of Rift Valley Fever Phlebovirus (Bunyaviridae) Carries Determinants for Attenuation and Virulence in Mice

2000 ◽  
Vol 74 (3) ◽  
pp. 1538-1543 ◽  
Author(s):  
P. Vialat ◽  
A. Billecocq ◽  
A. Kohl ◽  
M. Bouloy
Keyword(s):  
Rift Valley ◽  
Rift Valley Fever ◽  
Nonstructural Protein ◽  
Rift Valley Fever Virus ◽  
Virulent Strain ◽  
Coding Region ◽  
Valley Fever ◽  
Attenuated Virus ◽  
Infected Cells

ABSTRACT Unlike all the other Rift Valley fever virus strains (Bunyaviridae, Phlebovirus) studied so far, clone 13, a naturally attenuated virus, does not form the filaments composed of the NSs nonstructural protein in the nuclei of infected cells (R. Muller, J. F. Saluzzo, N. Lopez, T. Drier, M. Turell, J. Smith, and M. Bouloy, Am. J. Trop. Med. Hyg. 53:405–411, 1995). This defect is correlated with a large in-frame deletion in the NSs coding region of the S segment of the tripartite genome. Here, we show that the truncated NSs protein of clone 13 is expressed and remains in the cytoplasm, where it is degraded rapidly by the proteasome. Through the analysis of reassortants between clone 13 and a virulent strain, we localized the marker(s) of attenuation in the S segment of this attenuated virus. This result raises questions regarding the role of NSs in pathogenesis and highlights, for the first time in theBunyaviridae family, a major role of the S segment in virulence and attenuation, possibly associated with a defect in the nonstructural protein.

scholarly journals NSs Filament Formation Is Important but Not Sufficient for RVFV Virulence In Vivo

Viruses ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 834 ◽  
Author(s):  
Shufen Li ◽  
Xiangtao Zhu ◽  
Zhenqiong Guan ◽  
Wenfeng Huang ◽  
Yulan Zhang ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Rift Valley Fever ◽  
Reverse Genetics ◽  
Rift Valley Fever Virus ◽  
Domestic Animals ◽  
T7 Rna Polymerase ◽  
Filament Formation ◽  
Valley Fever ◽  
Infected Cells

Rift Valley fever virus (RVFV) is a mosquito-borne phlebovirus that represents as a serious health threat to both domestic animals and humans. The viral protein NSs is the key virulence factor of RVFV, and has been proposed that NSs nuclear filament formation is critical for its virulence. However, the detailed mechanisms are currently unclear. Here, we generated a T7 RNA polymerase-driven RVFV reverse genetics system based on a strain imported into China (BJ01). Several NSs mutations (T1, T3 and T4) were introduced into the system for investigating the correlation between NSs filament formation and virulence in vivo. The NSs T1 mutant showed distinct NSs filament in the nuclei of infected cells, the T3 mutant diffusively localized in the cytoplasm and the T4 mutant showed fragmented nuclear filament formation. Infection of BALB/c mice with these NSs mutant viruses revealed that the in vivo virulence was severely compromised for all three NSs mutants, including the T1 mutant. This suggests that NSs filament formation is not directly correlated with RVFV virulence in vivo. Results from this study not only shed new light on the virulence mechanism of RVFV NSs but also provided tools for future in-depth investigations of RVFV pathogenesis and anti-RVFV drug screening.

scholarly journals A ΩXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence

2015 ◽  
Vol 112 (19) ◽  
pp. 6021-6026 ◽  
Author(s):  
Normand Cyr ◽  
Cynthia de la Fuente ◽  
Lauriane Lecoq ◽  
Irene Guendel ◽  
Philippe R. Chabot ◽  
...  
Keyword(s):  
Rift Valley ◽  
Rift Valley Fever ◽  
Excision Repair ◽  
Rna Virus ◽  
Nonstructural Protein ◽  
Rift Valley Fever Virus ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Valley Fever ◽  
Infected Cells

Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ΩXaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ΩXaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ΩXaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from otherBunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of otherBunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ΩXaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH.

scholarly journals A Genome-Wide RNA Interference Screen Identifies a Role for Wnt/β-Catenin Signaling during Rift Valley Fever Virus Infection

2016 ◽  
Vol 90 (16) ◽  
pp. 7084-7097 ◽  
Author(s):  
Brooke Harmon ◽  
Sara W. Bird ◽  
Benjamin R. Schudel ◽  
Anson V. Hatch ◽  
Amy Rasley ◽  
...  
Keyword(s):  
Rna Interference ◽  
Wnt Signaling ◽  
Rift Valley ◽  
Rift Valley Fever ◽  
Rift Valley Fever Virus ◽  
Fever Virus ◽  
Valley Fever ◽  
Rnai Screening ◽  
Genome Wide ◽  
A Genome

ABSTRACTRift Valley fever virus (RVFV) is an arbovirus within theBunyaviridaefamily capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, we found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was preactivated, was reduced with knockdown of β-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses La Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. We propose a model where bunyaviruses activate Wnt-responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication. The findings in this study should aid in the design of efficacious host-directed antiviral therapeutics.IMPORTANCERVFV is a mosquito-borne bunyavirus that is endemic to Africa but has demonstrated a capacity for emergence in new territories (e.g., the Arabian Peninsula). As a zoonotic pathogen that primarily affects livestock, RVFV can also cause lethal hemorrhagic fever and encephalitis in humans. Currently, there are no treatments or fully licensed vaccines for this virus. Using high-throughput RNAi screening, we identified canonical Wnt signaling as an important host pathway regulating RVFV infection. The beneficial role of Wnt signaling was observed for RVFV, along with other disparate bunyaviruses, indicating a conserved bunyaviral replication mechanism involving Wnt signaling. These studies supplement our knowledge of the fundamental mechanisms of bunyavirus infection and provide new avenues for countermeasure development against pathogenic bunyaviruses.

scholarly journals In Situ Hybridization for Detection of Rift Valley Fever Virus

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Kaitlynn Schuck ◽  
Lieza Odendaal ◽  
Deepa Upreti ◽  
William C. Wilson ◽  
A. Sally Davis
Keyword(s):  
In Situ Hybridization ◽  
Rift Valley ◽  
Rift Valley Fever ◽  
Rift Valley Fever Virus ◽  
Fever Virus ◽  
Valley Fever

scholarly journals The Phagocytic Code Regulating Phagocytosis of Mammalian Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Tom O. J. Cockram ◽  
Jacob M. Dundee ◽  
Alma S. Popescu ◽  
Guy C. Brown
Keyword(s):  
Mammalian Cells ◽  
The Body ◽  
Psychiatric Disease ◽  
Target Cells ◽  
Immune Disease ◽  
Neuronal Synapses ◽  
Galectin 3 ◽  
A Cell ◽  
Infected Cells ◽  
Dying Cells

Mammalian phagocytes can phagocytose (i.e. eat) other mammalian cells in the body if they display certain signals, and this phagocytosis plays fundamental roles in development, cell turnover, tissue homeostasis and disease prevention. To phagocytose the correct cells, phagocytes must discriminate which cells to eat using a ‘phagocytic code’ - a set of over 50 known phagocytic signals determining whether a cell is eaten or not - comprising find-me signals, eat-me signals, don’t-eat-me signals and opsonins. Most opsonins require binding to eat-me signals – for example, the opsonins galectin-3, calreticulin and C1q bind asialoglycan eat-me signals on target cells - to induce phagocytosis. Some proteins act as ‘self-opsonins’, while others are ‘negative opsonins’ or ‘phagocyte suppressants’, inhibiting phagocytosis. We review known phagocytic signals here, both established and novel, and how they integrate to regulate phagocytosis of several mammalian targets - including excess cells in development, senescent and aged cells, infected cells, cancer cells, dead or dying cells, cell debris and neuronal synapses. Understanding the phagocytic code, and how it goes wrong, may enable novel therapies for multiple pathologies with too much or too little phagocytosis, such as: infectious disease, cancer, neurodegeneration, psychiatric disease, cardiovascular disease, ageing and auto-immune disease.

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