Nonstructural Nipah Virus C Protein Regulates both the Early Host Proinflammatory Response and Viral Virulence

ABSTRACTNipah virus (NiV) is a highly lethal paramyxovirus that recently emerged as a causative agent of febrile encephalitis and severe respiratory disease in humans. The ferret model has emerged as the preferred small-animal model with which to study NiV disease, but much is still unknown about the viral determinants of NiV pathogenesis, including the contribution of the C protein in ferrets. Additionally, studies have yet to examine the synergistic effects of the various P gene products on pathogenesis in animal models. Using recombinant NiVs (rNiVs), we examine the sole contribution of the NiV C protein and the combined contributions of the C and W proteins in the ferret model of NiV pathogenesis. We show that an rNiV void of C expression resulted in 100% mortality, though with limited respiratory disease, like our previously reported rNiV void of W expression; this finding is in stark contrast to the attenuated phenotype observed in previous hamster studies utilizing rNiVs void of C expression. We also observed that an rNiV void of both C and W expression resulted in limited respiratory disease; however, there was severe neurological disease leading to 60% mortality, and the surviving ferrets demonstrated sequelae similar to those for human survivors of NiV encephalitis.IMPORTANCENipah virus (NiV) is a human pathogen capable of causing lethal respiratory and neurological disease. Many human survivors have long-lasting neurological impairment. Using a ferret model, this study demonstrated the roles of the NiV C and W proteins in pathogenesis, where lack of either the C or the W protein independently decreased the severity of clinical respiratory disease but did not decrease lethality. Abolishing both C and W expression, however, dramatically decreased the severity of respiratory disease and the level of destruction of splenic germinal centers. These ferrets still suffered severe neurological disease: 60% succumbed to disease, and the survivors experienced long-term neurological impairment, such as that seen in human survivors. This new ferret NiV C and W knockout model may allow, for the first time, the examination of interventions to prevent or mitigate the neurological damage and sequelae experienced by human survivors.

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Nipah Virus C Protein Recruits Tsg101 to Promote the Efficient Release of Virus in an ESCRT-Dependent Pathway

PLoS Pathogens ◽

10.1371/journal.ppat.1005659 ◽

2016 ◽

Vol 12 (5) ◽

pp. e1005659 ◽

Cited By ~ 19

Author(s):

Arnold Park ◽

Tatyana Yun ◽

Frederic Vigant ◽

Olivier Pernet ◽

Sohui T. Won ◽

...

Keyword(s):

Nipah Virus ◽

C Protein ◽

Virus C ◽

Dependent Pathway

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Sendai virus C protein limits NO production in infected RAW264.7 macrophages

Innate Immunity ◽

10.1177/1753425918796619 ◽

2018 ◽

Vol 24 (7) ◽

pp. 430-438 ◽

Cited By ~ 2

Author(s):

Erdenezaya Odkhuu ◽

Takayuki Komatsu ◽

Naoki Koide ◽

Yoshikazu Naiki ◽

Kenji Takeuchi ◽

...

Keyword(s):

Sendai Virus ◽

Virus Multiplication ◽

Inos Expression ◽

No Production ◽

Ns1 Protein ◽

Wild Type ◽

C Protein ◽

Raw264.7 Macrophages ◽

Virus C ◽

Stat Pathway

To suppress virus multiplication, infected macrophages produce NO. However, it remains unclear how infecting viruses then overcome NO challenge. In the present study, we report the effects of accessory protein C from Sendai virus (SeV), a prototypical paramyxovirus, on NO output. We found that in RAW264.7 murine macrophages, a mutant SeV without C protein (4C(–)) significantly enhanced inducible NO synthase (iNOS) expression and subsequent NO production compared to wild type SeV (wtSeV). SeV 4C(-) infection caused marked production of IFN-β, which is involved in induction of iNOS expression via the JAK-STAT pathway. Addition of anti-IFN-β Ab, however, resulted in only marginal suppression of NO production. In contrast, NF-κB, a primarily important factor for transcription of the iNOS gene, was also activated by 4C(–) infection but not wtSeV infection. Induction of NO production and iNOS expression by 4C(–) was significantly suppressed in cells constitutively expressing influenza virus NS1 protein that can sequester double-stranded (ds)RNA, which triggers activation of signaling pathways leading to activation of NF-κB and IRF3. Therefore, C protein appears to suppress NF-κB activation to inhibit iNOS expression and subsequent NO production, possibly by limiting dsRNA generation in the context of viral infection.

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Determination of the henipavirus phosphoprotein gene mRNA editing frequencies and detection of the C, V and W proteins of Nipah virus in virus-infected cells

Journal of General Virology ◽

10.1099/vir.0.007294-0 ◽

2009 ◽

Vol 90 (2) ◽

pp. 398-404 ◽

Cited By ~ 39

Author(s):

Michael K. Lo ◽

Brian H. Harcourt ◽

Bruce A. Mungall ◽

Azaibi Tamin ◽

Mark E. Peeples ◽

...

Keyword(s):

Nipah Virus ◽

Hendra Virus ◽

Mrna Editing ◽

C Protein ◽

Reading Frame ◽

Frame Sequence ◽

P Gene ◽

Infected Cells ◽

Phosphoprotein Gene

The henipaviruses, Nipah virus (NiV) and Hendra virus (HeV), are highly pathogenic zoonotic paramyxoviruses. Like many other paramyxoviruses, henipaviruses employ a process of co-transcriptional mRNA editing during transcription of the phosphoprotein (P) gene to generate additional mRNAs encoding the V and W proteins. The C protein is translated from the P mRNA, but in an alternate reading frame. Sequence analysis of multiple, cloned mRNAs showed that the mRNA editing frequencies of the P genes of the henipaviruses are higher than those reported for other paramyxoviruses. Antisera to synthetic peptides from the P, V, W and C proteins of NiV were generated to study their expression in infected cells. All proteins were detected in both infected cells and purified virions. In infected cells, the W protein was detected in the nucleus while P, V and C were found in the cytoplasm.

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A Single Deletion in the Membrane-Proximal Region of the Sindbis Virus Glycoprotein E2 Endodomain Blocks Virus Assembly

Journal of Virology ◽

10.1128/jvi.74.9.4220-4228.2000 ◽

2000 ◽

Vol 74 (9) ◽

pp. 4220-4228 ◽

Cited By ~ 31

Author(s):

Raquel Hernandez ◽

Heuiran Lee ◽

Christine Nelson ◽

Dennis T. Brown

Keyword(s):

Sindbis Virus ◽

Virus Assembly ◽

Specific Interaction ◽

Proximal Region ◽

Host Cells ◽

C Protein ◽

Mosquito Cells ◽

Single Deletion ◽

Binding Cleft ◽

Virus C

ABSTRACT The envelopment of the Sindbis virus nucleocapsid in the modified cell plasma membrane involves a highly specific interaction between the capsid (C) protein and the endodomain of the E2 glycoprotein. We have previously identified a domain of the Sindbis virus C protein involved in binding to the E2 endodomain (H. Lee and D. T. Brown, Virology 202:390–400, 1994). The C-E2 binding domain resides in a hydrophobic cleft with C Y180 and W247 on opposing sides of the cleft. Structural modeling studies indicate that the E2 domain, which is proposed to bind the C protein (E2 398T, 399P, and 400Y), is located at a sufficient distance from the membrane to occupy the C protein binding cleft (S. Lee, K. E. Owen, H. K. Choi, H. Lee, G. Lu, G. Wengler, D. T. Brown, M. G. Rossmann, and R. J. Kuhn, Structure 4:531–541, 1996). To measure the critical spanning length of the E2 endodomain which positions the TPY domain into the putative C binding cleft, we have constructed a deletion mutant, ΔK391, in which a nonconserved lysine (E2 K391) at the membrane-cytoplasm junction of the E2 tail has been deleted. This mutant was found to produce very low levels of virus from BHK-21 cells due to a defect in an unidentified step in nucleocapsid binding to the E2 endodomain. In contrast, ΔK391 produced wild-type levels of virus from tissue-cultured mosquito cells. We propose that the phenotypic differences displayed by this mutant in the two diverse host cells arise from fundamental differences in the lipid composition of the insect cell membranes which affect the physical and structural properties of membranes and thereby virus assembly. The data suggest that these viruses have evolved properties adapted specifically for assembly in the diverse hosts in which they grow.

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The C, V and W proteins of Nipah virus inhibit minigenome replication

Journal of General Virology ◽

10.1099/vir.0.83582-0 ◽

2008 ◽

Vol 89 (5) ◽

pp. 1300-1308 ◽

Cited By ~ 34

Author(s):

Katrina Sleeman ◽

Bettina Bankamp ◽

Kimberly B. Hummel ◽

Michael K. Lo ◽

William J. Bellini ◽

...

Keyword(s):

Measles Virus ◽

Sequence Similarity ◽

Nipah Virus ◽

Amino Acid Sequence Similarity ◽

Dependent Manner ◽

Highly Pathogenic ◽

C Protein ◽

P Gene ◽

Dose Dependent ◽

Cat Expression

Nipah virus (NiV) is a recently emergent, highly pathogenic, zoonotic paramyxovirus of the genus Henipavirus. Like the phosphoprotein (P) gene of other paramyxoviruses, the P gene of NiV is predicted to encode three additional proteins, C, V and W. When the C, V and W proteins of NiV were tested for their ability to inhibit expression of the chloramphenicol acetyltransferase (CAT) reporter gene in plasmid-based, minigenome replication assays, each protein inhibited CAT expression in a dose-dependent manner. The C, V and W proteins of NiV also inhibited expression of CAT from a measles virus (MV) minigenome, but not from a human parainfluenzavirus 3 (hPIV3) minigenome. Interestingly, the C and V proteins of MV, which have previously been shown to inhibit MV minigenome replication, also inhibited NiV minigenome replication; however, they were not able to inhibit hPIV3 minigenome replication. In contrast, the C protein of hPIV3 inhibited minigenome replication of hPIV3, NiV and MV. Although there is very limited amino acid sequence similarity between the C, V and W proteins within the paramyxoviruses, the heterotypic inhibition of replication suggests that these proteins may share functional properties.

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Sendai virus C protein physically associates with Stat1

Genes to Cells ◽

10.1046/j.1365-2443.2001.00442.x ◽

2001 ◽

Vol 6 (6) ◽

pp. 545-557 ◽

Cited By ~ 74

Author(s):

Kenji Takeuchi ◽

Takayuki Komatsu ◽

Junko Yokoo ◽

Atsushi Kato ◽

Tatsuo Shioda ◽

...

Keyword(s):

Sendai Virus ◽

C Protein ◽

Virus C

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The disordered N-terminal region of dengue virus capsid protein contains a lipid-droplet-binding motif

Biochemical Journal ◽

10.1042/bj20112219 ◽

2012 ◽

Vol 444 (3) ◽

pp. 405-415 ◽

Cited By ~ 55

Author(s):

Ivo C. Martins ◽

Francisco Gomes-Neto ◽

André F. Faustino ◽

Filomena A. Carvalho ◽

Fabiana A. Carneiro ◽

...

Keyword(s):

Dengue Virus ◽

Capsid Protein ◽

Specific Treatment ◽

Viral Disease ◽

Structural Features ◽

Terminal Region ◽

Binding Motif ◽

C Protein ◽

Force Microscopy ◽

Virus C

Dengue is the major arthropod-borne human viral disease, for which no vaccine or specific treatment is available. We used NMR, zeta potential measurements and atomic force microscopy to study the structural features of the interaction between dengue virus C (capsid) protein and LDs (lipid droplets), organelles crucial for infectious particle formation. C protein-binding sites to LD were mapped, revealing a new function for a conserved segment in the N-terminal disordered region and indicating that conformational selection is involved in recognition. The results suggest that the positively charged N-terminal region of C protein prompts the interaction with negatively charged LDs, after which a conformational rearrangement enables the access of the central hydrophobic patch to the LD surface. Taken together, the results allowed the design of a peptide with inhibitory activity of C protein–LD binding, paving the way for new drug development approaches against dengue.

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Targeting of the Sendai Virus C Protein to the Plasma Membrane via a Peptide-Only Membrane Anchor

Journal of Virology ◽

10.1128/jvi.02465-06 ◽

2007 ◽

Vol 81 (7) ◽

pp. 3187-3197 ◽

Cited By ~ 16

Author(s):

Jean-Baptiste Marq ◽

Albert Brini ◽

Daniel Kolakofsky ◽

Dominique Garcin

Keyword(s):

Plasma Membrane ◽

Sendai Virus ◽

Intracellular Localization ◽

Membrane Targeting ◽

Membrane Anchor ◽

C Protein ◽

Cellular Antiviral Response ◽

Α Helix ◽

Virus C ◽

Cellular Compartments

ABSTRACT Several cellular proteins are synthesized in the cytosol on free ribosomes and then associate with membranes due to the presence of short peptide sequences. These membrane-targeting sequences contain sites to which lipid chains are attached, which help direct the protein to a particular membrane domain and anchor it firmly in the bilayer. The intracellular concentration of these proteins in particular cellular compartments, where their interacting partners are also concentrated, is essential to their function. This paper reports that the apparently unmodified N-terminal sequence of the Sendai virus C protein (MPSFL KK IL K L R G RR . . .; letters in italics represent hydrophobic residues; underlined letters represent basic residues, which has a strong propensity to form an amphipathic α-helix in a hydrophobic environment) also function as a membrane targeting signal and membrane anchor. Moreover, the intracellular localization of the C protein at the plasma membrane is essential for inducing the interferon-independent phosphorylation of Stat1 as part of the viral program to prevent the cellular antiviral response.

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