Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription

ABSTRACT Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and viral genomic RNA (vRNA). Time course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times postinfection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with the translation of viral proteins being closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time course quantitative reverse transcription-PCR analysis suggested that the coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications. IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatiotemporal analysis of HMPV replication and transcription in bronchial epithelial cell-derived immortal cells was performed. HMPV was shown to induce the formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures, such as stress granules and processing bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though inclusion body formation is nuanced, it corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, an understanding of inclusion body formation is crucial for the discovery of innovative therapeutic targets.

Download Full-text

Ubiquitin versus misfolding: The minimal requirements for inclusion body formation

The Journal of Cell Biology ◽

10.1083/jcb.201603095 ◽

2016 ◽

Vol 213 (2) ◽

pp. 147-149 ◽

Cited By ~ 1

Author(s):

Nico P. Dantuma ◽

Florian A. Salomons

Keyword(s):

Neurodegenerative Diseases ◽

Inclusion Body ◽

Protein Misfolding ◽

Inclusion Bodies ◽

Functional Role ◽

Body Formation ◽

Protein Deposits ◽

Cell Biol ◽

Inclusion Body Formation ◽

Characteristic Features

Ubiquitin-containing inclusion bodies are characteristic features of numerous neurodegenerative diseases, but whether ubiquitin plays a functional role in the formation of these protein deposits is unclear. In this issue, Bersuker et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201511024) report that protein misfolding without ubiquitylation is sufficient for translocation into inclusion bodies.

Download Full-text

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

10.1101/2020.04.06.028423 ◽

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.

Download Full-text

Interaction between coxsackievirus B3 infection and α-synuclein in models of Parkinson’s disease

PLoS Pathogens ◽

10.1371/journal.ppat.1010018 ◽

2021 ◽

Vol 17 (10) ◽

pp. e1010018

Author(s):

Soo Jin Park ◽

Uram Jin ◽

Sang Myun Park

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Inclusion Body ◽

Dopaminergic Neurons ◽

Inclusion Bodies ◽

Lewy Bodies ◽

Coxsackievirus B3 ◽

Body Formation ◽

Inclusion Body Formation ◽

Cvb3 Infection

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. PD is pathologically characterized by the death of midbrain dopaminergic neurons and the accumulation of intracellular protein inclusions called Lewy bodies or Lewy neurites. The major component of Lewy bodies is α-synuclein (α-syn). Prion-like propagation of α-syn has emerged as a novel mechanism in the progression of PD. This mechanism has been investigated to reveal factors that initiate Lewy pathology with the aim of preventing further progression of PD. Here, we demonstrate that coxsackievirus B3 (CVB3) infection can induce α-syn-associated inclusion body formation in neurons which might act as a trigger for PD. The inclusion bodies contained clustered organelles, including damaged mitochondria with α-syn fibrils. α-Syn overexpression accelerated inclusion body formation and induced more concentric inclusion bodies. In CVB3-infected mice brains, α-syn aggregates were observed in the cell body of midbrain neurons. Additionally, α-syn overexpression favored CVB3 replication and related cytotoxicity. α-Syn transgenic mice had a low survival rate, enhanced CVB3 replication, and exhibited neuronal cell death, including that of dopaminergic neurons in the substantia nigra. These results may be attributed to distinct autophagy-related pathways engaged by CVB3 and α-syn. This study elucidated the mechanism of Lewy body formation and the pathogenesis of PD associated with CVB3 infection.

Download Full-text

Human metapneumovirus nucleoprotein and phosphoprotein interact and provide the minimal requirements for inclusion body formation

Journal of General Virology ◽

10.1099/vir.0.2008/004051-0 ◽

2008 ◽

Vol 89 (11) ◽

pp. 2698-2708 ◽

Cited By ~ 31

Author(s):

Aaron Derdowski ◽

Timothy R. Peters ◽

Nancy Glover ◽

Ray Qian ◽

Thomas J. Utley ◽

...

Keyword(s):

Inclusion Body ◽

Matrix Protein ◽

Cytoplasmic Inclusion ◽

Human Metapneumovirus ◽

Yeast Two Hybrid ◽

Cytoplasmic Inclusions ◽

Body Formation ◽

Avian Pneumovirus ◽

Inclusion Body Formation ◽

Two Hybrid

Human metapneumovirus (HMPV) is a recently discovered paramyxovirus of the subfamily Pneumovirinae, which also includes avian pneumovirus and human respiratory syncytial virus (HRSV). HMPV is an important cause of respiratory disease worldwide. To understand early events in HMPV replication, cDNAs encoding the HMPV nucleoprotein (N), phosphoprotein (P), matrix protein (M), M2-1 protein and M2-2 protein were cloned from cells infected with the genotype A1 HMPV wild-type strain TN/96-12. HMPV N and P were shown to interact using a variety of techniques: yeast two-hybrid assays, co-immunoprecipitation and fluorescence resonance energy transfer (FRET). Confocal microscopy studies showed that, when expressed individually, fluorescently tagged HMPV N and P exhibited a diffuse expression pattern in the host-cell cytoplasm of uninfected cells but were recruited to cytoplasmic viral inclusion bodies in HMPV-infected cells. Furthermore, when HMPV N and P were expressed together, they also formed cytoplasmic inclusion-like complexes, even in the absence of viral infection. FRET microscopy revealed that HMPV N and P interacted directly within cytoplasmic inclusion-like complexes. Moreover, it was shown by yeast two-hybrid analysis that the N-terminal 28 aa are required for the recruitment to and formation of cytoplasmic inclusions, but are dispensable for binding to HMPV P. This work showed that HMPV N and P proteins provide the minimal viral requirements for HMPV inclusion body formation, which may be a distinguishing characteristic of members of the subfamily Pneumovirinae.

Download Full-text

Ubiquilin/Dsk2 promotes inclusion body formation and vacuole (lysosome)-mediated disposal of mutated huntingtin

Molecular Biology of the Cell ◽

10.1091/mbc.e16-01-0026 ◽

2016 ◽

Vol 27 (13) ◽

pp. 2025-2036 ◽

Cited By ~ 16

Author(s):

Kun-Han Chuang ◽

Fengshan Liang ◽

Ryan Higgins ◽

Yanchang Wang

Keyword(s):

Neurodegenerative Diseases ◽

Inclusion Body ◽

Inclusion Bodies ◽

Misfolded Proteins ◽

Functional Study ◽

Polyglutamine Expansion ◽

Body Formation ◽

Functional Conservation ◽

Inclusion Body Formation ◽

Short Time

Ubiquilin proteins contain a ubiquitin-like domain (UBL) and ubiquitin-associated domain(s) that interact with the proteasome and ubiquitinated substrates, respectively. Previous work established the link between ubiquilin mutations and neurodegenerative diseases, but the function of ubiquilin proteins remains elusive. Here we used a misfolded huntingtin exon I containing a 103-polyglutamine expansion (Htt103QP) as a model substrate for the functional study of ubiquilin proteins. We found that yeast ubiquilin mutant ( dsk2Δ) is sensitive to Htt103QP overexpression and has a defect in the formation of Htt103QP inclusion bodies. Our evidence further suggests that the UBL domain of Dsk2 is critical for inclusion body formation. Of interest, Dsk2 is dispensable for Htt103QP degradation when Htt103QP is induced for a short time before noticeable inclusion body formation. However, when the inclusion body forms after a long Htt103QP induction, Dsk2 is required for efficient Htt103QP clearance, as well as for autophagy-dependent delivery of Htt103QP into vacuoles (lysosomes). Therefore our data indicate that Dsk2 facilitates vacuole-mediated clearance of misfolded proteins by promoting inclusion body formation. Of importance, the defect of inclusion body formation in dsk2 mutants can be rescued by human ubiquilin 1 or 2, suggesting functional conservation of ubiquilin proteins.

Download Full-text

Interaction Between Coxsackievirus B3 Infection and α-Synuclein in Parkinson’s Disease

10.21203/rs.3.rs-145815/v1 ◽

2021 ◽

Author(s):

Soo Jin Park ◽

Uram Jin ◽

Sang Myun Park

Keyword(s):

Parkinson’S Disease ◽

Inclusion Body ◽

Dopaminergic Neurons ◽

Lewy Body ◽

Inclusion Bodies ◽

Lewy Bodies ◽

Coxsackievirus B3 ◽

Body Formation ◽

Inclusion Body Formation ◽

Cvb3 Infection

Abstract BackgroundParkinson's disease (PD) is one of the most common neurodegenerative disease. PD is pathologically characterized by the death of midbrain dopaminergic neurons and the accumulation of intracellular protein inclusions called Lewy bodies or Lewy neurites. The major component of Lewy bodies is α-synuclein (α-syn). Prion-like propagation of α-syn has emerged as a novel mechanism in the progression of PD. Targeting this mechanism could enable the development of disease-modifying therapies for patients with PD. Nevertheless, the initial triggers of LB formation leading to acceleration of the process remain elusive.MethodsTo evaluate α-syn function in viral replication, we infected coxsackievirus B3 (CVB3) to α-syn overexpressed neurons or α-syn transgenic (TG) mice. We then performed biochemical and histological analyses to evaluate interaction between CVB3 and α-syn in Lewy body formation.ResultsWe demonstrated that CVB3 infection can induce α-syn-associated inclusion body formation in neurons as a trigger. The inclusion bodies contained clustered organelles, including damaged mitochondria with α-syn fibrils. α-Syn overexpression accelerated inclusion body formation and induced more concentric inclusion bodies. In brains from CVB3 infected mice, α-syn aggregates in the cell body of midbrain neurons were observed. Additionally, α-syn overexpression favored CVB3 replication and related cytotoxicity. α-Syn transgenic mice had a low survival rate, enhanced CVB3 replication, and further neuronal cell death, including dopaminergic neurons in the substantia nigra. These results may be due to the different usage of autophagy between CVB3 and α-syn. ConclusionsThis study elucidated the mechanism of Lewy body formation and the pathogenesis of PD associated with CVB3 infection.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.02100-19 ◽

2020 ◽

Vol 94 (16) ◽

Cited By ~ 3

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.

Download Full-text