scholarly journals A cytorhabdovirus phosphoprotein forms mobile inclusions trafficked on the actin/ER network for viral RNA synthesis

2019 ◽  
Vol 70 (15) ◽  
pp. 4049-4062 ◽  
Author(s):  
Xiao-Dong Fang ◽  
Teng Yan ◽  
Qiang Gao ◽  
Qing Cao ◽  
Dong-Min Gao ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Rna Synthesis ◽  
Plant Viruses ◽  
Viral Rna ◽  
P Bodies ◽  
P Protein ◽  
Intracellular Movement ◽  
L Proteins ◽  
Myosin Xi ◽  
Plant Rhabdovirus

AbstractAs obligate parasites, plant viruses usually hijack host cytoskeletons for replication and movement. Rhabdoviruses are enveloped, negative-stranded RNA viruses that infect vertebrates, invertebrates, and plants, but the mechanisms of intracellular trafficking of plant rhabdovirus proteins are largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, as a model to investigate the effects of the actin cytoskeleton on viral intracellular movement and viral RNA synthesis in a mini-replicon (MR) system. The BYSMV P protein forms mobile inclusion bodies that are trafficked along the actin/endoplasmic reticulum network, and recruit the N and L proteins into viroplasm-like structures. Deletion analysis showed that the N terminal region (aa 43–55) and the remaining region (aa 56–295) of BYSMV P are essential for the mobility and formation of inclusions, respectively. Overexpression of myosin XI-K tails completely abolishes the trafficking activity of P bodies, and is accompanied by a significant reduction of viral MR RNA synthesis. These results suggest that BYSMV P contributes to the formation and trafficking of viroplasm-like structures along the ER/actin network driven by myosin XI-K. Thus, rhabdovirus P appears to be a dynamic hub protein for efficient recruitment of viral proteins, thereby promoting viral RNA synthesis.

EV71 2A Protease inhibits P-body formation to promote viral RNA synthesis

2021 ◽  
Author(s):  
Shanshan Fan ◽  
Zihang Xu ◽  
Pengfei Liu ◽  
Yali Qin ◽  
Mingzhou Chen
Keyword(s):  
Viral Replication ◽  
Rna Synthesis ◽  
Enterovirus 71 ◽  
Viral Rna ◽  
Processing Bodies ◽  
Body Formation ◽  
P Bodies ◽  
Body Components ◽  
2A Protease ◽  
Enterovirus 71 Infection

Several viruses were proved to inhibit the formation of RNA processing bodies (P-bodies); however, knowledge regarding whether enterovirus blocks P-body formation remains unclear, and the detailed molecular mechanisms and functions of picornavirus regulation of P-bodies are limited. Here we show the crucial role of 2A protease in inhibiting P-bodies to promote viral replication during enterovirus 71 infection. Moreover, we found that the activity of 2A protease is essential to inhibit P-body formation, which was proved by the result that infection of EV71-2A C110S , the 2A protease activity-inactivated recombinant virus, failed to block the formation of P-bodies. Furthermore, we showed DDX6, a scaffolding protein of P-bodies, interacted with viral RNA to facilitate viral replication rather than viral translation, by using a Renilla luciferase mRNA reporter system and capturing the nascent RNA assay. Altogether, our data firstly demonstrate that the 2A protease of enterovirus inhibits P-body formation to facilitate viral RNA synthesis by recruiting the P-body components to viral RNA. IMPORTANCE Processing bodies (P-bodies) are constitutively present in eukaryotic cells and play an important role in the mRNA cycle, including regulating gene expression and mRNA degradation. P-bodies are the structure that viruses to manipulate to facilitate their survival. Here, we show that the 2A protease alone was efficient to block P-body formation during enterovirus 71 infection and its activity was essential. When the assembly of P-bodies was blocked by 2A, DDX6 and 4E-T which were required for P-body formation bound to viral RNA to facilitate viral RNA synthesis. We propose a model revealing that EV71 manipulates P-body formation to generate an environment that is conducive to viral replication by facilitating viral RNA synthesis: 2A protease blocked P-body assembly to make it possible for virus to take advantage of P-body components.

scholarly journals Oligomerization of Mumps Virus Phosphoprotein

2015 ◽  
Vol 89 (21) ◽  
pp. 11002-11010 ◽  
Author(s):  
Adrian Pickar ◽  
Andrew Elson ◽  
Yang Yang ◽  
Pei Xu ◽  
Ming Luo ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Mutational Analysis ◽  
Mumps Virus ◽  
Viral Rna ◽  
Large Protein ◽  
Terminal Domain ◽  
P Protein ◽  
Minigenome System ◽  
Oligomerization Domain

ABSTRACTThe mumps virus (MuV) genome encodes a phosphoprotein (P) that is important for viral RNA synthesis. P forms the viral RNA-dependent RNA polymerase with the large protein (L). P also interacts with the viral nucleoprotein (NP) and self-associates to form a homotetramer. The P protein consists of three domains, the N-terminal domain (PN), the oligomerization domain (PO), and the C-terminal domain (PC). While PNis known to relax the NP-bound RNA genome, the roles of POand PCare not clear. In this study, we investigated the roles of POand PCin viral RNA synthesis using mutational analysis and a minigenome system. We found that PNand PCfunctions can betrans-complemented. However, this complementation requires PO, indicating that POis essential for P function. Using thistrans-complementation system, we found that P forms parallel dimers (PNto PNand PCto PC). Furthermore, we found that residues R231, K238, K253, and K260 in POare critical for P's functions. We identified PCto be the domain that interacts with L. These results provide structure-function insights into the role of MuV P.IMPORTANCEMuV, a paramyxovirus, is an important human pathogen. The P protein of MuV is critical for viral RNA synthesis. In this work, we established a novel minigenome system that allows the domains of P to be complemented intrans. Using this system, we confirmed that MuV P forms parallel dimers. An understanding of viral RNA synthesis will allow the design of better vaccines and the development of antivirals.

scholarly journals Ebola virus inclusion body formation and RNA synthesis are controlled by a novel domain of NP interacting with VP35

2020 ◽  
Author(s):  
Tsuyoshi Miyake ◽  
Charlotte M. Farley ◽  
Benjamin E. Neubauer ◽  
Thomas P. Beddow ◽  
Thomas Hoenen ◽  
...  
Keyword(s):  
Life Cycle ◽  
Inclusion Body ◽  
Inclusion Bodies ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Central Domain ◽  
Body Formation ◽  
Inclusion Body Formation

AbstractEbola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection IBs display dynamic properties regarding their size and location. Also, the contents of IBs must transition prior to further viral maturation, assembly and release, implying additional steps in IB function. Interestingly, expression of the viral nucleoprotein (NP) alone is sufficient for generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30 and the RNA polymerase L. Previously we defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here we show that NP-Ct is absolutely required for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for production of infectious virus-like particles and retention of viral RNA within these particles. Furthermore, co-expression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins only VP35 is able to overcome the defect in IB formation caused by deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself, and which is mediated by a newly identified domain of NP, the “central domain” (CD).ImportanceInclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative sense RNA viruses including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.

scholarly journals Newly Identified Phosphorylation Site in the Vesicular Stomatitis Virus P Protein Is Required for Viral RNA Synthesis

2013 ◽  
Vol 88 (3) ◽  
pp. 1461-1472 ◽  
Author(s):  
A. Mondal ◽  
K. G. Victor ◽  
R. S. Pudupakam ◽  
C. E. Lyons ◽  
G. W. Wertz
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rna Synthesis ◽  
Phosphorylation Site ◽  
Viral Rna ◽  
P Protein ◽  
Vesicular Stomatitis

Monomer and Dimer of Chandipura Virus Unphosphorylated P-protein Binds Leader RNA Differently: Implications for Viral RNA Synthesis

2004 ◽  
Vol 339 (5) ◽  
pp. 1089-1101 ◽  
Author(s):  
Soumen Basak ◽  
Smarajit Polley ◽  
Mausumi Basu ◽  
Dhrubajyoti Chattopadhyay ◽  
Siddhartha Roy
Keyword(s):  
Rna Synthesis ◽  
Viral Rna ◽  
P Protein ◽  
Chandipura Virus

scholarly journals Role of the Hypervariable Hinge Region of Phosphoprotein P of Vesicular Stomatitis Virus in Viral RNA Synthesis and Assembly of Infectious Virus Particles

2005 ◽  
Vol 79 (13) ◽  
pp. 8101-8112 ◽  
Author(s):  
Subash C. Das ◽  
Asit K. Pattnaik
Keyword(s):  
Amino Acid ◽  
Vesicular Stomatitis Virus ◽  
Rna Synthesis ◽  
Hinge Region ◽  
Viral Rna ◽  
Insertion Mutagenesis ◽  
Insertion Mutant ◽  
P Protein ◽  
Vesicular Stomatitis

ABSTRACT The phosphoprotein (P protein) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNA-dependent RNA polymerase and has multiple functions residing in its different domains. In the present study, we examined the role of the hypervariable hinge region of P protein in viral RNA synthesis and recovery of infectious VSV by using transposon-mediated insertion mutagenesis and deletion mutagenesis. We observed that insertions of 19-amino-acid linker sequences at various positions within this region affected replication and transcription functions of the P protein to various degrees. Interestingly, one insertion mutant was completely defective in both transcription and replication. Using a series of deletion mutants spanning the hinge region of the protein, we observed that amino acid residues 201 through 220 are required for the activity of P protein in both replication and transcription. Neither insertion nor deletion had any effect on the interaction of P protein with N or L proteins. Infectious VSVs with a deletion in the hinge region possessed retarded growth characteristics and exhibited small-plaque morphology. Interestingly, VSV containing one P protein deletion mutant (PΔ7, with amino acids 141 through 200 deleted), which possessed significant levels of replication and transcription activity, could be amplified only by passage in cells expressing the wild-type P protein. We conclude that the hypervariable hinge region of the P protein plays an important role in viral RNA synthesis. Furthermore, our results provide a previously unidentified function for the P protein: it plays a critical role in the assembly of infectious VSV.

scholarly journals Conserved Motifs in a Tombusvirus Polymerase Modulate Genome Replication, Subgenomic Transcription, and Amplification of Defective Interfering RNAs

2015 ◽  
Vol 89 (6) ◽  
pp. 3236-3246 ◽  
Author(s):  
Chaminda D. Gunawardene ◽  
Karolina Jaluba ◽  
K. Andrew White
Keyword(s):  
Rna Synthesis ◽  
Mutational Analysis ◽  
Rna Virus ◽  
Plant Viruses ◽  
Comparative Sequence Analysis ◽  
Terminal Region ◽  
Viral Rna ◽  
Genome Replication ◽  
Conserved Motifs ◽  
Content Type

ABSTRACTThe replication of plus-strand RNA virus genomes is mediated by virally encoded RNA-dependent RNA polymerases (RdRps). We have investigated the role of the C-proximal region in the RdRp of tomato bushy stunt virus (TBSV) in mediating viral RNA synthesis. TBSV is the prototype species in the genusTombusvirus, familyTombusviridae, and its RdRp is responsible for replicating the viral genome, transcribing two subgenomic mRNAs, and supporting replication of defective interfering RNAs. Comparative sequence analysis of the RdRps of tombusvirids identified three highly conserved motifs in their C-proximal regions, and these sequences were subsequently targeted for mutational analysis in TBSV. The results revealed that these motifs are important for (i) synthesizing viral genomic RNA and subgenomic mRNAs, (ii) facilitating plus- and/or minus-strand synthesis, and (iii) modulatingtrans-replication of a defective interfering RNA. These motifs were also found to be conserved in other plant viruses as well as in a fungal and insect virus. The collective findings are discussed in relation to viral RNA synthesis and taxonomy.IMPORTANCELittle is currently known about the structure and function of the viral polymerases that replicate the genomes of RNA plant viruses. Tombusviruses, the prototype of the tombusvirids, have been used as model plus-strand RNA plant viruses for understanding many of the steps in the infectious process; however, their polymerases remain poorly characterized. To help address this issue, the function of the C-terminal region of the polymerase of a tombusvirus was investigated. Three conserved motifs were identified and targeted for mutational analysis. The results revealed that these polymerase motifs are important for determining what type of viral RNA is produced, facilitating different steps in viral RNA production, and amplifying subgenomic RNA replicons. Accordingly, the C-terminal region of the tombusvirus polymerase is needed for a variety of fundamental activities. Furthermore, as these motifs are also present in distantly related viruses, the significance of these results extends beyond tombusvirids.

scholarly journals Ebola Virus Inclusion Body Formation and RNA Synthesis Are Controlled by a Novel Domain of Nucleoprotein Interacting with VP35

2020 ◽  
Vol 94 (16) ◽  
Author(s):  
Tsuyoshi Miyake ◽  
Charlotte M. Farley ◽  
Benjamin E. Neubauer ◽  
Thomas P. Beddow ◽  
Thomas Hoenen ◽  
...  
Keyword(s):  
Life Cycle ◽  
Inclusion Body ◽  
Inclusion Bodies ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Central Domain ◽  
Body Formation ◽  
Inclusion Body Formation

ABSTRACT Ebola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection, IBs display dynamic properties regarding their size and location. The contents of IBs also must transition prior to further viral maturation, assembly, and release, implying additional steps in IB function. Interestingly, the expression of the viral nucleoprotein (NP) alone is sufficient for the generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30, and the RNA polymerase L. We previously defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here, we show that NP-Ct is necessary for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for the production of infectious virus-like particles (VLPs), and that defective VLPs with NP-Ct deletions are significantly reduced in viral RNA content. Furthermore, coexpression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins, only VP35 is able to overcome the defect in IB formation caused by the deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself and that is mediated by a newly identified functional domain of NP, the central domain. IMPORTANCE Inclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative-sense RNA viruses, including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.

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