scholarly journals Subcellular localization of nucleocapsid protein of SFTSV and its assembly into the ribonucleoprotein complex with L protein and viral RNA

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sithumini M. W. Lokupathirage ◽  
Yoshimi Tsuda ◽  
Kodai Ikegame ◽  
Kisho Noda ◽  
Devinda S. Muthusinghe ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Subcellular Localization ◽  
Critical Role ◽  
Viral Proteins ◽  
Viral Rna ◽  
Host Cells ◽  
N Protein ◽  
L Protein ◽  
Translational Activity ◽  
Antiviral Targets

AbstractSevere fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus that causes novel zoonotic diseases in Asian countries including China, Japan, South Korea, and Vietnam. In phleboviruses, viral proteins play a critical role in viral particle formation inside the host cells. Viral glycoproteins (GPs) and RNA-dependent RNA polymerase (RdRp) are colocalized in the Golgi apparatus and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The nucleocapsid (N) protein was widely expressed in the cytoplasm, even in cells coexpressing GP. However, the role of SFTSV N protein remains unclear. The subcellular localization of SFTSV structural proteins was investigated using a confocal microscope. Subsequently, minigenome and immunoprecipitation assays were carried out. The N protein interacts with viral RNA (vRNA) and further shows translational activity with RdRp which is L protein and localized in the ERGIC and Golgi apparatus when co-expressed with GP. On the other hand, mutant N protein did not interact with vRNA either localized in the ERGIC or Golgi apparatus. The interaction between the N protein of SFTSV and vRNA is important for the localization of viral proteins and viral assembly. This study provides useful insights into the life cycle of SFTSV, which will lead to the detection of antiviral targets.

scholarly journals Characterization of a Replicating Mammalian Orthoreovirus with Tetracysteine-Tagged μNS for Live-Cell Visualization of Viral Factories

2017 ◽  
Vol 91 (22) ◽  
Author(s):  
Luke D. Bussiere ◽  
Promisree Choudhury ◽  
Bryan Bellaire ◽  
Cathy L. Miller
Keyword(s):  
Nonstructural Protein ◽  
Critical Role ◽  
Viral Proteins ◽  
Live Cell ◽  
Host Cells ◽  
Live Cells ◽  
Virus Protein ◽  
Mammalian Orthoreovirus ◽  
Virus Proteins

ABSTRACT Within infected host cells, mammalian orthoreovirus (MRV) forms viral factories (VFs), which are sites of viral transcription, translation, assembly, and replication. The MRV nonstructural protein μNS comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells, but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced μNS. We set out to develop a method to overcome this obstacle by utilizing the 6-amino-acid (CCPGCC) tetracysteine (TC) tag and FlAsH-EDT2 reagent. The TC tag was introduced into eight sites throughout μNS, and the capacity of the TC-μNS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-μNS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore, the rTC-μNS viruses were utilized to infect cells and examine VF dynamics using live-cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in-depth studies of VF dynamics and host cell interactions. IMPORTANCE MRV has historically been used as a model to study the double-stranded RNA (dsRNA) Reoviridae family, the members of which infect and cause disease in humans, animals, and plants. During infection, MRV forms VFs that play a critical role in virus infection but remain to be fully characterized. To study VFs, researchers have focused on visualizing the nonstructural protein μNS, which forms the VF matrix. This work provides the first evidence of recovery of replicating reoviruses in which VFs can be labeled in live cells via introduction of a TC tag into the μNS open reading frame. Characterization of each recombinant reovirus sheds light on μNS interactions with viral proteins. Moreover, utilizing the TC-labeling FlAsH-EDT2 biarsenical reagent to visualize VFs, evidence is provided of dynamic VF movement and interactions at least partially dependent on intact microtubules.

scholarly journals Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Interacts with Nsp9 and Cellular DHX9 To Regulate Viral RNA Synthesis

2016 ◽  
Vol 90 (11) ◽  
pp. 5384-5398 ◽  
Author(s):  
Long Liu ◽  
Jiao Tian ◽  
Hao Nan ◽  
Mengmeng Tian ◽  
Yuan Li ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Critical Role ◽  
Continuous Extension ◽  
Rna Helicase ◽  
Viral Rna ◽  
Respiratory Syndrome Virus ◽  
Nascent Transcript ◽  
Multifunctional Protein ◽  
N Protein

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis.IMPORTANCEIt is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of longer viral sgmRNAs and gRNA. Our data here provide some new insights into the discontinuous to continuous extension of PRRSV RNA synthesis and also offer a new potential anti-PRRSV strategy targeting the N-Nsp9 and/or N-DHX9 interaction.

scholarly journals Characterization of a replicating mammalian orthoreovirus with tetracysteine tagged μNS for live cell visualization of viral factories

2017 ◽  
Author(s):  
Luke D. Bussiere ◽  
Promisree Choudhury ◽  
Bryan Bellaire ◽  
Cathy L. Miller
Keyword(s):  
Critical Role ◽  
Viral Proteins ◽  
Live Cell ◽  
Host Cells ◽  
Live Cells ◽  
Virus Protein ◽  
Structural Protein ◽  
Mammalian Orthoreovirus ◽  
Virus Proteins

AbstractWithin infected host cells, mammalian orthoreovirus (MRV) forms viral factories (VFs) which are sites of viral transcription, translation, assembly, and replication. MRV non-structural protein, μNS, comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced μNS. We set out to develop a method to overcome this obstacle by utilizing the 6 amino-acid (CCPGCC) tetracysteine (TC)-tag and FlAsH-EDT2 reagent. The TC-tag was introduced into eight sites throughout μNS, and the capacity of the TC-μNS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC-tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-μNS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore the rTC-μNS viruses were utilized to infect cells and examine VF dynamics using live cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs, and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in depth studies of VF dynamics and host cell interactions.ImportanceMRV has historically been used as a model to study the double-stranded RNA (dsRNA)Reoviridaefamily, which infect and cause disease in humans, animals, and plants. During infection, MRV forms VFs that play a critical role in virus infection, but remain to be fully characterized. To study VFs, researchers have focused on visualizing the non-structural protein μNS which forms the VF matrix. This work provides the first evidence of recovery of replicating reoviruses in which VFs can be labeled in live cells via introduction of a TC-tag into the μNS open reading frame. Characterization of each recombinant reovirus sheds light on μNS interactions with viral proteins. Moreover, utilizing the TC labeling FlAsH-EDT2 biarsenical reagent to visualize VFs, evidence is provided of dynamic VF movement and interactions at least partially dependent on intact microtubules.

scholarly journals Comprehensive analysis of the host-virus interactome of SARS-CoV-2

2021 ◽  
Author(s):  
Zhen Chen ◽  
Chao Wang ◽  
Xu Feng ◽  
Litong Nie ◽  
Mengfan Tang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Drug Targets ◽  
Affinity Purification ◽  
Functional Characterization ◽  
Viral Proteins ◽  
Host Cells ◽  
Virus Protein ◽  
N Protein ◽  
Protein Protein Interaction ◽  
Human Coronaviruses

Host-virus protein-protein interaction is the key component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lifecycle. We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity labeling strategies and identified 437 human proteins as the high-confidence interacting proteins. Functional characterization and further validation of these interactions elucidated how distinct SARS-CoV-2 viral proteins participate in its lifecycle, and discovered potential drug targets to the treatment of COVID-19. The interactomes of two key SARS-CoV-2 encoded viral proteins, NSP1 and N protein, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein-protein interactions that may explain differences in disease pathology. This comprehensive interactome of coronavirus disease-2019 provides valuable resources for understanding and treating this disease.

scholarly journals Roles of the Non-Structural Proteins of Influenza A Virus

Pathogens ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 812
Author(s):  
Wenzhuo Hao ◽  
Lingyan Wang ◽  
Shitao Li
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Rna Virus ◽  
Critical Role ◽  
Viral Proteins ◽  
Host Cells ◽  
Structural Proteins ◽  
Host Interactions ◽  
New Findings ◽  
Seasonal Epidemics

Influenza A virus (IAV) is a segmented, negative single-stranded RNA virus that causes seasonal epidemics and has a potential for pandemics. Several viral proteins are not packed in the IAV viral particle and only expressed in the infected host cells. These proteins are named non-structural proteins (NSPs), including NS1, PB1-F2 and PA-X. They play a versatile role in the viral life cycle by modulating viral replication and transcription. More importantly, they also play a critical role in the evasion of the surveillance of host defense and viral pathogenicity by inducing apoptosis, perturbing innate immunity, and exacerbating inflammation. Here, we review the recent advances of these NSPs and how the new findings deepen our understanding of IAV–host interactions and viral pathogenesis.

scholarly journals Colocalization and Membrane Association of Murine Hepatitis Virus Gene 1 Products and De Novo-Synthesized Viral RNA in Infected Cells

1999 ◽  
Vol 73 (7) ◽  
pp. 5957-5969 ◽  
Author(s):  
Stephanie T. Shi ◽  
Jennifer J. Schiller ◽  
Amornrat Kanjanahaluethai ◽  
Susan C. Baker ◽  
Jong-Won Oh ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Rna Synthesis ◽  
De Novo ◽  
Hepatitis Virus ◽  
Viral Proteins ◽  
Viral Rna ◽  
Membrane Structures ◽  
Murine Hepatitis Virus ◽  
Physical Evidence ◽  
Infected Cells

ABSTRACT Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (pol) gene, is first translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases. Genetic complementation analyses suggest that the majority of the gene 1 products are required for viral RNA synthesis. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofluorescent-staining studies with four polyclonal antisera to localize various MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation experiments showed that these antisera detected proteins representing the two papain-like proteases and the 3C-like protease encoded by open reading frame (ORF) 1a, the putative polymerase (p100) and a p35 encoded by ORF 1b, and their precursors. De novo-synthesized viral RNA was labeled with bromouridine triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antisera colocalized with newly synthesized viral RNA in the cytoplasm, particularly in the perinuclear region of infected cells. Several cysteine and serine protease inhibitors, i.e., E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, suggesting that the processing of the MHV gene 1 polyprotein is tightly associated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA colocalized with the Golgi apparatus in HeLa cells. However, in murine 17CL-1 cells, the viral proteins and viral RNA did not colocalize with the Golgi apparatus but, instead, partially colocalized with the endoplasmic reticulum. Our results provide clear physical evidence that several MHV gene 1 products, including the proteases and the polymerase, are associated with the viral RNA replication-transcription machinery, which may localize to different membrane structures in different cell lines.

scholarly journals A Temperature-Sensitive Lesion in the N-Terminal Domain of the Rotavirus Polymerase Affects Its Intracellular Localization and Enzymatic Activity

2017 ◽  
Vol 91 (7) ◽  
Author(s):  
Allison O. McKell ◽  
Leslie E. W. LaConte ◽  
Sarah M. McDonald
Keyword(s):  
Enzymatic Activity ◽  
Rna Synthesis ◽  
Viral Proteins ◽  
Viral Rna ◽  
Host Cells ◽  
Temperature Sensitive ◽  
Mutant Form ◽  
Interaction Sites ◽  
Terminal Domain ◽  
Infected Cells

ABSTRACT Temperature-sensitive (ts) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11-tsC, has a mutation that maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39°C compared to that at 31°C. In the present study, we sequenced all 11 genes of SA11-tsC, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP4, VP7, NSP1, and NSP2). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside the SA11-tsC genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affects the ability of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant form as a green fluorescent protein (GFP) fusion protein (VP1L138P-GFP) (i) in wild-type SA11-infected cells or (ii) in uninfected cells along with viroplasm-forming proteins NSP2 and NSP5. We found that VP1L138P-GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31°C but not at 39°C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1L138P) in vitro and found that it synthesized less RNA at 39°C than at 31°C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11-tsC and raise important questions about the role of leucine 138 in supporting key protein interactions and the catalytic function of the VP1 polymerase. IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside host cells. Previously, a mutant simian RV (SA11-tsC) that replicates worse at higher temperatures was identified. This virus has an amino acid mutation in VP1, which is the enzyme responsible for copying the viral RNA genome. The mutation is located in a poorly understood region of the polymerase called the N-terminal domain. In this study, we determined that the mutation reduces the ability of VP1 to properly localize within infected cells at high temperatures, as well as reduced the ability of the enzyme to copy viral RNA in a test tube. The results of this study explain the temperature sensitivity of SA11-tsC and shed new light on functional protein-protein interaction sites of VP1.

scholarly journals SARS CoV-2 nucleocapsid protein forms condensates with viral genomic RNA

2020 ◽  
Author(s):  
Amanda Jack ◽  
Luke S. Ferro ◽  
Michael J. Trnka ◽  
Eddie Wehri ◽  
Amrut Nadgir ◽  
...  
Keyword(s):  
Phase Separation ◽  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Viral Rna ◽  
Host Cells ◽  
Drug Candidate ◽  
N Protein ◽  
Intrinsically Disordered

AbstractThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes COVID-19, a pandemic that seriously threatens global health. SARS CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with unstructured RNA, it forms mesh like-structures with viral RNA strands that contain secondary structure elements. Cross-linking mass spectrometry identified an intrinsically-disordered region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. By screening 1,200 FDA approved drugs in vitro, we identified a kinase inhibitor nilotinib, which affects the morphology of N condensates in vitro and disrupts phase separation of the N protein in vivo. These results indicate that the N protein compartmentalizes viral RNA in infected cells through liquid-liquid phase separation, and this process can be disrupted by a possible drug candidate.

scholarly journals Reovirus Low-Density Particles Package Cellular RNA

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1096
Author(s):  
Timothy W. Thoner ◽  
Xiang Ye ◽  
John Karijolich ◽  
Kristen M. Ogden
Keyword(s):  
Rna Polymerase ◽  
Viral Proteins ◽  
Viral Rna ◽  
Low Density ◽  
Rna Packaging ◽  
Genome Packaging ◽  
Double Stranded Rna ◽  
Viral Genomes ◽  
Mammalian Orthoreovirus ◽  
Insight Into

Packaging of segmented, double-stranded RNA viral genomes requires coordination of viral proteins and RNA segments. For mammalian orthoreovirus (reovirus), evidence suggests either all ten or zero viral RNA segments are simultaneously packaged in a highly coordinated process hypothesized to exclude host RNA. Accordingly, reovirus generates genome-containing virions and “genomeless” top component particles. Whether reovirus virions or top component particles package host RNA is unknown. To gain insight into reovirus packaging potential and mechanisms, we employed next-generation RNA-sequencing to define the RNA content of enriched reovirus particles. Reovirus virions exclusively packaged viral double-stranded RNA. In contrast, reovirus top component particles contained similar proportions but reduced amounts of viral double-stranded RNA and were selectively enriched for numerous host RNA species, especially short, non-polyadenylated transcripts. Host RNA selection was not dependent on RNA abundance in the cell, and specifically enriched host RNAs varied for two reovirus strains and were not selected solely by the viral RNA polymerase. Collectively, these findings indicate that genome packaging into reovirus virions is exquisitely selective, while incorporation of host RNAs into top component particles is differentially selective and may contribute to or result from inefficient viral RNA packaging.

scholarly journals Effects of Gag Mutation and Processing on Retroviral Dimeric RNA Maturation

2006 ◽  
Vol 80 (3) ◽  
pp. 1242-1249 ◽  
Author(s):  
William Fu ◽  
Que Dang ◽  
Kunio Nagashima ◽  
Eric O. Freed ◽  
Vinay K. Pathak ◽  
...  
Keyword(s):  
Viral Protein ◽  
Leukemia Virus ◽  
Viral Rna ◽  
Host Cells ◽  
Protein Cleavage ◽  
Rna Packaging ◽  
Virus Rna ◽  
Rna Dimer ◽  
Rna Maturation ◽  
Hiv 1

ABSTRACT After their release from host cells, most retroviral particles undergo a maturation process, which includes viral protein cleavage, core condensation, and increased stability of the viral RNA dimer. Inactivating the viral protease prevents protein cleavage; the resulting virions lack condensed cores and contain fragile RNA dimers. Therefore, protein cleavage is linked to virion morphological change and increased stability of the RNA dimer. However, it is unclear whether protein cleavage is sufficient for mediating virus RNA maturation. We have observed a novel phenotype in a murine leukemia virus capsid mutant, which has normal virion production, viral protein cleavage, and RNA packaging. However, this mutant also has immature virion morphology and contains a fragile RNA dimer, which is reminiscent of protease-deficient mutants. To our knowledge, this mutant provides the first evidence that Gag cleavage alone is not sufficient to promote RNA dimer maturation. To extend our study further, we examined a well-defined human immunodeficiency virus type 1 (HIV-1) Gag mutant that lacks a functional PTAP motif and produces immature virions without major defects in viral protein cleavage. We found that the viral RNA dimer in the PTAP mutant is more fragile and unstable compared with those from wild-type HIV-1. Based on the results of experiments using two different Gag mutants from two distinct retroviruses, we conclude that Gag cleavage is not sufficient for promoting RNA dimer maturation, and we propose that there is a link between the maturation of virion morphology and the viral RNA dimer.

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