scholarly journals Viral cross-linking and solid-phase purification enables discovery of ribonucleoprotein complexes on incoming RNA virus genomes

2020 ◽  
Author(s):  
Byungil Kim ◽  
Sarah Arcos ◽  
Katherine Rothamel ◽  
Manuel Ascano
Keyword(s):  
Protein Interactions ◽  
Rna Viruses ◽  
Solid Phase ◽  
Rna Virus ◽  
Host Protein ◽  
Host Cells ◽  
Cross Linking ◽  
Viral Genomes ◽  
Ribonucleoprotein Complexes ◽  
Virus Genomes

AbstractThe initial interactions between incoming, pre-replicated RNA virus genomes and host protein factors are important in infection and immunity. Yet there are no current methods to study these crucial events. We established VIR-CLASP (VIRal Cross-Linking And Solid-phase Purification) to identify the primary viral RNA-host protein interactions. First, host cells are infected with 4SU-labeled RNA viruses and irradiated with 365 nm light to crosslink 4SU-labeled viral genomes and interacting proteins from host or virus. The cross-linked RBPs are purified by solid-phase reversible immobilization (SPRI) beads with protein denaturing buffers, and then identified by proteomics. With VIR-CLASP, only the incoming viral genomes are labeled with 4SU, so cross-linking events specifically occur between proteins and pre-replicated viral genomic RNA. Since solid-phase purification under protein-denaturing conditions is used to pull-down total RNA and cross-linked RBPs, this facilitates investigation of potentially all RNA viruses, regardless of RNA sequence. Preparation of 4SU-labeled virus takes ∼7 days and VIR-CLASP takes 1 day.

scholarly journals Native Structure-Based Peptides as Potential Protein–Protein Interaction Inhibitors of SARS-CoV-2 Spike Protein and Human ACE2 Receptor

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2157
Author(s):  
Norbert Odolczyk ◽  
Ewa Marzec ◽  
Maria Winiewska-Szajewska ◽  
Jarosław Poznański ◽  
Piotr Zielenkiewicz
Keyword(s):  
Drug Development ◽  
Protein Interactions ◽  
Rna Virus ◽  
Antiviral Drug ◽  
Host Protein ◽  
Host Cells ◽  
Cell Surface Receptor ◽  
Short Peptides ◽  
Virus Protein ◽  
Positive Strand Rna

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a positive-strand RNA virus that causes severe respiratory syndrome in humans, which is now referred to as coronavirus disease 2019 (COVID-19). Since December 2019, the new pathogen has rapidly spread globally, with over 65 million cases reported to the beginning of December 2020, including over 1.5 million deaths. Unfortunately, currently, there is no specific and effective treatment for COVID-19. As SARS-CoV-2 relies on its spike proteins (S) to bind to a host cell-surface receptor angiotensin-converting enzyme-2(ACE2), and this interaction is proved to be responsible for entering a virus into host cells, it makes an ideal target for antiviral drug development. In this work, we design three very short peptides based on the ACE2 sequence/structure fragments, which may effectively bind to the receptor-binding domain (RBD) of S protein and may, in turn, disrupt the important virus-host protein–protein interactions, blocking early steps of SARS-CoV-2 infection. Two of our peptides bind to virus protein with affinity in nanomolar range, and as very short peptides have great potential for drug development.

scholarly journals Viral crosslinking and solid-phase purification enables discovery of ribonucleoprotein complexes on incoming RNA virus genomes

2020 ◽  
Vol 16 (1) ◽  
pp. 516-531
Author(s):  
Byungil Kim ◽  
Sarah Arcos ◽  
Katherine Rothamel ◽  
Manuel Ascano
Keyword(s):  
Solid Phase ◽  
Rna Virus ◽  
Ribonucleoprotein Complexes ◽  
Virus Genomes

scholarly journals Specificity of Plant Rhabdovirus Cell-to-Cell Movement

2019 ◽  
Vol 93 (15) ◽  
Author(s):  
Xin Zhou ◽  
Wenye Lin ◽  
Kai Sun ◽  
Shuo Wang ◽  
Xueping Zhou ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Core Protein ◽  
Rna Viruses ◽  
Rna Virus ◽  
Cell Movement ◽  
Tomato Mosaic Virus ◽  
Cell Periphery ◽  
Nucleic Acid Binding ◽  
Core Proteins

ABSTRACTPositive-stranded RNA virus movement proteins (MPs) generally lack sequence-specific nucleic acid-binding activities and display cross-family movement complementarity with related and unrelated viruses. Negative-stranded RNA plant rhabdoviruses encode MPs with limited structural and functional relatedness with other plant virus counterparts, but the precise mechanisms of intercellular transport are obscure. In this study, we first analyzed the abilities of MPs encoded by five distinct rhabdoviruses to support cell-to-cell movement of two positive-stranded RNA viruses by usingtrans-complementation assays. Each of the five rhabdovirus MPs complemented the movement of MP-defective mutants of tomato mosaic virus and potato X virus. In contrast, movement of recombinant MP deletion mutants of sonchus yellow net nucleorhabdovirus (SYNV) and tomato yellow mottle-associated cytorhabdovirus (TYMaV) was rescued only by their corresponding MPs, i.e., SYNV sc4 and TYMaV P3. Subcellular fractionation analyses revealed that SYNV sc4 and TYMaV P3 were peripherally associated with cell membranes. A split-ubiquitin membrane yeast two-hybrid assay demonstrated specific interactions of the membrane-associated rhabdovirus MPs only with their cognate nucleoproteins (N) and phosphoproteins (P). More importantly, SYNV sc4-N and sc4-P interactions directed a proportion of the N-P complexes from nuclear sites of replication to punctate loci at the cell periphery that partially colocalized with the plasmodesmata. Our data show that cell-to-cell movement of plant rhabdoviruses is highly specific and suggest that cognate MP-nucleocapsid core protein interactions are required for intra- and intercellular trafficking.IMPORTANCELocal transport of plant rhabdoviruses likely involves the passage of viral nucleocapsids through MP-gated plasmodesmata, but the molecular mechanisms are not fully understood. We have conducted complementation assays with MPs encoded by five distinct rhabdoviruses to assess their movement specificity. Each of the rhabdovirus MPs complemented the movement of MP-defective mutants of two positive-stranded RNA viruses that have different movement strategies. In marked contrast, cell-to-cell movement of two recombinant plant rhabdoviruses was highly specific in requiring their cognate MPs. We have shown that these rhabdovirus MPs are localized to the cell periphery and associate with cellular membranes, and that they interact only with their cognate nucleocapsid core proteins. These interactions are able to redirect viral nucleocapsid core proteins from their sites of replication to the cell periphery. Our study provides a model for the specific inter- and intracellular trafficking of plant rhabdoviruses that may be applicable to other negative-stranded RNA viruses.

scholarly journals An overview of RNA virus-encoded microRNAs

ExRNA ◽  
2019 ◽  
Vol 1 (1) ◽  
Author(s):  
Xihan Li ◽  
Xiaoping Zou
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Viral Replication ◽  
Potential Role ◽  
Rna Viruses ◽  
Rna Virus ◽  
Host Cells ◽  
Basic Pattern ◽  
Double Stranded Dna ◽  
Non Coding Rnas

Abstract MicroRNAs (miRNAs) are a number of small non-coding RNAs playing a regulatory part in gene expression. Many virus-encoded miRNAs have been found, which manifests that viruses as well apply the basic pattern of gene regulation, however, mostly in viruses transcribed from double-stranded DNA genomes. It is still in dispute if RNA viruses could encode miRNAs because the excision of miRNA might result in the cleavage of viral RNA genome. We will focus on the miRNAs encoded by RNA virus and discuss their potential role in viral replication cycle and host cells.

scholarly journals Marine RNA Virus Quasispecies Are Distributed throughout the Oceans

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Marli Vlok ◽  
Andrew S. Lang ◽  
Curtis A. Suttle
Keyword(s):  
Amino Acid ◽  
Rna Viruses ◽  
Rna Virus ◽  
Purifying Selection ◽  
Amino Acid Sequences ◽  
Metagenomic Data ◽  
Spatial And Temporal Patterns ◽  
Data Set ◽  
Virus Diversity ◽  
Virus Genomes

ABSTRACTRNA viruses, particularly genetically diverse members of thePicornavirales, are widespread and abundant in the ocean. Gene surveys suggest that there are spatial and temporal patterns in the composition of RNA virus assemblages, but data on their diversity and genetic variability in different oceanographic settings are limited. Here, we show that specific RNA virus genomes have widespread geographic distributions and that the dominant genotypes are under purifying selection. Genomes from three previously unknown picorna-like viruses (BC-1, -2, and -3) assembled from a coastal site in British Columbia, Canada, as well as marine RNA viruses JP-A, JP-B, andHeterosigma akashiwoRNA virus exhibited different biogeographical patterns. Thus, biotic factors such as host specificity and viral life cycle, and not just abiotic processes such as dispersal, affect marine RNA virus distribution. Sequence differences relative to reference genomes imply that virus quasispecies are under purifying selection, with synonymous single-nucleotide variations dominating in genomes from geographically distinct regions resulting in conservation of amino acid sequences. Conversely, sequences from coastal South Africa that mapped to marine RNA virus JP-A exhibited more nonsynonymous mutations, probably representing amino acid changes that accumulated over a longer separation. This biogeographical analysis of marine RNA viruses demonstrates that purifying selection is occurring across oceanographic provinces. These data add to the spectrum of known marine RNA virus genomes, show the importance of dispersal and purifying selection for these viruses, and indicate that closely related RNA viruses are pathogens of eukaryotic microbes across oceans.IMPORTANCEVery little is known about aquatic RNA virus populations and genome evolution. This is the first study that analyzes marine environmental RNA viral assemblages in an evolutionary and broad geographical context. This study contributes the largest marine RNA virus metagenomic data set to date, substantially increasing the sequencing space for RNA viruses and also providing a baseline for comparisons of marine RNA virus diversity. The new viruses discovered in this study are representative of the most abundant family of marine RNA viruses, theMarnaviridae, and expand our view of the diversity of this important group. Overall, our data and analyses provide a foundation for interpreting marine RNA virus diversity and evolution.

scholarly journals Potentially translated sequences determine protein-coding potential of RNAs in cellular organisms

2021 ◽  
Author(s):  
Yusuke Suenaga ◽  
Mamoru Kato ◽  
Momoko Nagai ◽  
Kazuma Nakatani ◽  
Hiroyuki Kogashi ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Rna Virus ◽  
Host Cells ◽  
Rna Sequences ◽  
Protein Coding ◽  
Functional Peptides ◽  
Definition Of ◽  
Virus Genomes ◽  
Sequence Characteristics ◽  
Coding Potential

AbstractRNA sequence characteristics determine whether their transcripts are coding or noncoding. Recent studies have shown that, paradoxical to the definition of noncoding RNA, several long noncoding RNAs (lncRNAs) translate functional peptides/proteins. However, the characteristics of RNA sequences that distinguish such newly identified coding transcripts from lncRNAs remain largely unknown. In this study, we found that potentially translated sequences in RNAs determine the protein-coding potential of RNAs in cellular organisms. We defined the potentially translated island (PTI) score as the fraction of the length of the longest potentially translated region among all regions. To analyze its relationship with protein-coding potential, we calculated the PTI scores in 3.4 million RNA transcripts from 100 cellular organisms, including 5 bacteria, 10 archaea, and 85 eukaryotes, as well as 105 positive-sense single-strand RNA virus genomes. In bacteria and archaea, coding and noncoding transcripts exclusively presented high and low PTI scores, respectively, whereas those of eukaryotic coding and noncoding transcripts showed relatively broader distributions. The relationship between the PTI score and protein-coding potential was sigmoidal in most eukaryotes; however, it was linear passing through the origin in three distinct eutherian lineages, including humans. The RNA sequences of virus genomes appeared to adapt to translation systems of host organisms by maximizing protein-coding potential in host cells. Hence, the PTIs determined the protein-coding potential of RNAs in cellular organisms. Additionally, coding and noncoding RNA do not exhibit dichotomous sequence characteristics in eukaryotes, instead they exhibit a gradient of protein-coding potential.

scholarly journals Proteome-wide cross-linking mass spectrometry to identify specific virus capsid-host interactions between tick-borne encephalitis virus and neuroblastoma cells

2021 ◽  
Author(s):  
Sarah V Barrass ◽  
Lauri I A Pulkkinen ◽  
Olli Vapalahti ◽  
Suvi Kuivanen ◽  
Maria Anastasina ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Neuroblastoma Cells ◽  
Encephalitis Virus ◽  
Host Protein ◽  
Cross Linking ◽  
Host Proteins ◽  
Host Interactions ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

Virus-host protein-protein interactions are central to viral infection, but are challenging to identify and characterise, especially in complex systems involving intact viruses and cells. In this work, we demonstrate a proteome-wide approach to identify virus-host interactions using chemical cross-linking coupled with mass spectrometry. We adsorbed tick-borne encephalitis virus onto metabolically-stalled neuroblastoma cells, covalently cross-linked interacting virus-host proteins, and performed limited proteolysis to release primarily the surface-exposed proteins for identification by mass spectrometry. Using the intraviral protein cross-links as an internal control to assess cross-link confidence levels, we identified 22 high confidence unique intraviral cross-links and 59 high confidence unique virus-host protein-protein interactions. The identified host proteins were shown to interact with eight distinct sites on the outer surface of the virus. Notably, we identified an interaction between the substrate-binding domain of heat shock protein family A member 5, an entry receptor for four related flaviviruses, and the hinge region of the viral envelope protein. We also identified host proteins involved in endocytosis, cytoskeletal rearrangement, or located in the cytoskeleton, suggesting that entry mechanisms for tick-borne encephalitis virus could include both clathrin-mediated endocytosis and macropinocytosis. Additionally, cross-linking of the viral proteins showed that the capsid protein forms dimers within tick-borne encephalitis virus, as previously observed with purified C proteins for other flaviviruses. This method enables the identification and mapping of transient virus-host interactions, under near-physiological conditions, without the need for genetic manipulation.

scholarly journals All Roads Lead to Cytosol: Trypanosoma cruzi Multi-Strategic Approach to Invasion

2021 ◽  
Vol 11 ◽  
Author(s):  
Gabriel Ferri ◽  
Martin M. Edreira
Keyword(s):  
Host Cell ◽  
Protein Interactions ◽  
Developmental Stages ◽  
Parasitophorous Vacuole ◽  
Host Protein ◽  
Host Cells ◽  
Signaling Cascades ◽  
Complex Life Cycle ◽  
Strategic Approach ◽  
Gain Access

T. cruzihas a complex life cycle involving four developmental stages namely, epimastigotes, metacyclic trypomastigotes, amastigotes and bloodstream trypomastigotes. Although trypomastigotes are the infective forms, extracellular amastigotes have also shown the ability to invade host cells. Both stages can invade a broad spectrum of host tissues, in fact, almost any nucleated cell can be the target of infection. To add complexity, the parasite presents high genetic variability with differential characteristics such as infectivity. In this review, we address the several strategiesT. cruzihas developed to subvert the host cell signaling machinery in order to gain access to the host cell cytoplasm. Special attention is made to the numerous parasite/host protein interactions and to the set of signaling cascades activated during the formation of a parasite-containing vesicle, the parasitophorous vacuole, from which the parasite escapes to the cytosol, where differentiation and replication take place.

scholarly journals Analysis of viral RNA-host protein interactomes enables rapid antiviral drug discovery

2021 ◽  
Author(s):  
Shaojun Zhang ◽  
Wenze Huang ◽  
Lili Ren ◽  
Xiaohui Ju ◽  
Mingli Gong ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Ebola Virus ◽  
Rna Viruses ◽  
Antiviral Drug ◽  
Antiviral Agent ◽  
Viral Rna ◽  
Host Protein ◽  
Host Responses ◽  
Interaction Patterns ◽  
Global Public Health

RNA viruses including SARS-CoV-2, Ebola virus (EBOV), and Zika virus (ZIKV) constitute a major threat to global public health and society. The interactions between viral genomes and host proteins are essential in the life cycle of RNA viruses and thus provide targets for drug development. However, viral RNA-host protein interactions have remained poorly characterized. Here we applied ChIRP-MS to profile the interactomes of human proteins and the RNA genomes of SARS-CoV-2, EBOV, and ZIKV in infected cells. Integrated interactome analyses revealed interaction patterns that reflect both common and virus-specific host responses, and enabled rapid drug screening to target the vulnerable host factors. We identified Enasidenib as a SARS-CoV-2 specific antiviral agent, and Trifluoperazine and Cepharanthine as broad spectrum antivirals against all three RNA viruses.

scholarly journals Berbamine inhibits the infection of SARS-CoV-2 and flaviviruses by compromising TPRMLs-mediated endolysosomal trafficking of viral receptors

2020 ◽  
Author(s):  
Lihong Huang ◽  
Huanan Li ◽  
Terrence Tsz-Tai Yuen ◽  
Zuodong Ye ◽  
Qiang Fu ◽  
...  
Keyword(s):  
Viral Entry ◽  
Rna Viruses ◽  
Rna Virus ◽  
Antiviral Agents ◽  
Host Cells ◽  
Lethal Challenge ◽  
Benzylisoquinoline Alkaloids ◽  
The World ◽  
Positive Sense ◽  
Viral Receptors

Abstract Positive-sense single-stranded ((+)ss) RNA viruses are among the leading causes of human and animal infectious diseases in the world, but so far, no effective antiviral agents are available to treat these infections. Here we found that several bis- benzylisoquinoline alkaloids (e.g. berbamine), potently inhibited the infection of coronaviruses (e.g. SARS-CoV-2 and MERS-CoV), flaviviruses (e.g. JEV, ZIKV and DENV), and enteroviruses (e.g. EV-A71) in host cells. Moreover, berbamine protected mice from lethal challenge of JEV. We also found that berbamine inhibited TRPMLs (Ca2+ permeable non-selective cation channels in endosomes and lysosomes), which compromised the endolysosomal trafficking of viral receptors, such as ACE2 and DPP4. This led to the increased secretion of these receptors via extracellular vesicles and the concomitant decrease in their levels at the plasma membrane, thereby preventing (+)ss RNA viruses from entering the host cells. In summary, these results indicate that bis- benzylisoquinoline alkaloids such as berbamine, can act as a pan-anti-(+)ss RNA virus drug by inhibiting TPRMLs to prevent viral entry.

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