Characterization of Hantavirus N Protein Intracellular Dynamics and Localization

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation and several other crucial processes during hantavirus infection. In this study we have generated fluorescently tagged N protein constructs derived from Puumalavirus, the dominant hantavirus species in Central, Northern and Eastern Europe. We have comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We found a significant spatial correlation of N with vimentin, actin and P-bodies, but not with microtubules. N constructs also co-localized with Gn and Gc, albeit not as strong as the glycoproteins associated with each other. Moreover, we as-sessed oligomerization of N constructs, observing efficient and concentration-dependent multi-merization, with complexes comprising more than 10 individual proteins.

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Protein complex formation in methionine chain-elongation and leucine biosynthesis

Scientific Reports ◽

10.1038/s41598-021-82790-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Li-Qun Chen ◽

Shweta Chhajed ◽

Tong Zhang ◽

Joseph M. Collins ◽

Qiuying Pang ◽

...

Keyword(s):

Protein Complexes ◽

Complete Characterization ◽

Bimolecular Fluorescence Complementation ◽

Chain Elongation ◽

Substrate Channeling ◽

Leucine Biosynthesis ◽

Protein Complex Formation ◽

Genes Encoding ◽

Isopropylmalate Dehydrogenase

AbstractDuring the past two decades, glucosinolate (GLS) metabolic pathways have been under extensive studies because of the importance of the specialized metabolites in plant defense against herbivores and pathogens. The studies have led to a nearly complete characterization of biosynthetic genes in the reference plant Arabidopsis thaliana. Before methionine incorporation into the core structure of aliphatic GLS, it undergoes chain-elongation through an iterative three-step process recruited from leucine biosynthesis. Although enzymes catalyzing each step of the reaction have been characterized, the regulatory mode is largely unknown. In this study, using three independent approaches, yeast two-hybrid (Y2H), coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC), we uncovered the presence of protein complexes consisting of isopropylmalate isomerase (IPMI) and isopropylmalate dehydrogenase (IPMDH). In addition, simultaneous decreases in both IPMI and IPMDH activities in a leuc:ipmdh1 double mutants resulted in aggregated changes of GLS profiles compared to either leuc or ipmdh1 single mutants. Although the biological importance of the formation of IPMI and IPMDH protein complexes has not been documented in any organisms, these complexes may represent a new regulatory mechanism of substrate channeling in GLS and/or leucine biosynthesis. Since genes encoding the two enzymes are widely distributed in eukaryotic and prokaryotic genomes, such complexes may have universal significance in the regulation of leucine biosynthesis.

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Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain

iScience ◽

10.1016/j.isci.2021.102681 ◽

2021 ◽

pp. 102681

Author(s):

Chao Wu ◽

Abraham J. Qavi ◽

Asmaa Hachim ◽

Niloufar Kavian ◽

Aidan R. Cole ◽

...

Keyword(s):

N Protein ◽

Terminal Domain ◽

Functional Consequences

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Characterization of the Physiological State of SecA and SecYEG: Key Components of Bacterial Protein Transport Across the Plasma Membrane

10.14418/wes01.3.103 ◽

2019 ◽

Author(s):

Tithi Banerjee

Keyword(s):

Plasma Membrane ◽

Protein Transport ◽

Physiological State ◽

Bacterial Protein

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Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures

10.1101/2021.06.14.448452 ◽

2021 ◽

Author(s):

Christine Roden ◽

Yifan Dai ◽

Ian Seim ◽

Myungwoon Lee ◽

Rachel Sealfon ◽

...

Keyword(s):

Phase Separation ◽

Rna Structure ◽

Nucleocapsid Protein ◽

Rna Binding ◽

Genome Packaging ◽

Rna Motifs ◽

Double Stranded Rna ◽

N Protein ◽

Binding Domains

Betacoronavirus SARS-CoV-2 infections caused the global Covid-19 pandemic. The nucleocapsid protein (N-protein) is required for multiple steps in the betacoronavirus replication cycle. SARS-CoV-2-N-protein is known to undergo liquid-liquid phase separation (LLPS) with specific RNAs at particular temperatures to form condensates. We show that N-protein recognizes at least two separate and distinct RNA motifs, both of which require double-stranded RNA (dsRNA) for LLPS. These motifs are separately recognized by N-protein's two RNA binding domains (RBDs). Addition of dsRNA accelerates and modifies N-protein LLPS in vitro and in cells and controls the temperature condensates form. The abundance of dsRNA tunes N-protein-mediated translational repression and may confer a switch from translation to genome packaging. Thus, N-protein's two RBDs interact with separate dsRNA motifs, and these interactions impart distinct droplet properties that can support multiple viral functions. These experiments demonstrate a paradigm of how RNA structure can control the properties of biomolecular condensates.

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Identification and characterization of host proteins bound to dengue virus 3′ UTR reveal an antiviral role for quaking proteins

RNA ◽

10.1261/rna.064006.117 ◽

2018 ◽

Vol 24 (6) ◽

pp. 803-814 ◽

Cited By ~ 12

Author(s):

Kuo-Chieh Liao ◽

Vanessa Chuo ◽

Wy Ching Ng ◽

Suat Peng Neo ◽

Julien Pompon ◽

...

Keyword(s):

Dengue Virus ◽

Host Proteins ◽

Antiviral Role ◽

Identification And Characterization

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Substrate Proteins Take Shape at an Improved Bacterial Translocon

Journal of Bacteriology ◽

10.1128/jb.00618-18 ◽

2018 ◽

Vol 201 (1) ◽

Cited By ~ 2

Author(s):

Donald Oliver

Keyword(s):

Protein Transport ◽

Protein Translocation ◽

Membrane Vesicles ◽

Transport Models ◽

Content Type ◽

Rate Limiting ◽

Substrate Proteins

ABSTRACTCharacterization of Sec-dependent bacterial protein transport has often relied on anin vitroprotein translocation system comprised in part ofEscherichia coliinverted inner membrane vesicles or, more recently, purified SecYEG translocons reconstituted into liposomes using mostly a single substrate (proOmpA). A paper published in this issue (P. Bariya and L. Randall, J Bacteriol 201:e00493-18, 2019, https://doi.org/10.1128/JB.00493-18) finds that inclusion of SecA protein during SecYEG proteoliposome reconstitution dramatically improves the number of active translocons. This experimentally useful and intriguing result that may arise from SecA membrane integration properties is discussed here. Furthermore, determination of the rate-limiting transport step for nine different substrates implicates the mature region distal to the signal peptide in the observed rate constant differences, indicating that more nuanced transport models that respond to differences in protein sequence and structure are needed.

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Characterization of the interaction between hepatitis C virus NS5B and the human oestrogen receptor alpha

Journal of General Virology ◽

10.1099/vir.0.039396-0 ◽

2012 ◽

Vol 93 (4) ◽

pp. 780-785 ◽

Cited By ~ 4

Author(s):

Julia Hillung ◽

Elena Ruiz-López ◽

Itxaso Bellón-Echeverría ◽

Pilar Clemente-Casares ◽

Antonio Mas

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Oestrogen Receptor ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Host Factors ◽

Host Proteins ◽

Receptor Alpha ◽

Oestrogen Receptor Alpha

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is part of the viral replicative complex and plays a crucial role in HCV replication. It has been described that NS5B interacts with cellular proteins, and that interactions between NS5B and host proteins are crucial for viral replication. Some of the host factors involved in the HCV replication cycle include the oestrogen receptor alpha (ESR1), protein kinases (c-Src) and chaperones (Hsp70). In this report, we determine the requirements for the interplay between NS5B and the domain C of ESR1 (ESR1C) by using Förster Resonance Energy Transfer. NS5B–ESR1C and ESR1C–ESR1C interactions are dependent on ionic strength, indicating that contacts are mainly electrostatic. Additionally, NS5B residues involved in NS5B oligomerization were also essential for NS5B–ESR1C interaction. The study of the interactions among viral and host factors will provide data to establish innovative therapeutic strategies and the development of new antiviral drugs.

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