Mapping of epitopes and structural analysis of antigenic sites in the nucleoprotein of rabies virus

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 119-127 ◽  
Author(s):  
Hideo Goto ◽  
Nobuyuki Minamoto ◽  
Hiroshi Ito ◽  
Naoto Ito ◽  
Makoto Sugiyama ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Antigenic Site ◽  
Cytoplasmic Inclusion ◽  
The Other ◽  
N Protein ◽  
Antigenic Sites ◽  
Immature Form ◽  
Infected Cells ◽  
Cytoplasmic Inclusion Bodies ◽  
Nucleoprotein N

Linear epitopes on the rabies virus nucleoprotein (N) recognized by six MAbs raised against antigenic sites I (MAbs 6-4, 12-2 and 13-27) and IV (MAbs 6-9, 7-12 and 8-1) were investigated. Based on our previous studies on sites I and IV, 24 consecutively overlapping octapeptides and N- and C-terminal-deleted mutant N proteins were prepared. Results showed that all three site I epitopes studied and two site IV epitopes (for MAbs 8-1 and 6-9) mapped to aa 358–367, and that the other site IV epitope of MAb 7-12 mapped to aa 375–383. Tests using chimeric and truncated proteins showed that MAb 8-1 also requires the N-terminal sequence of the N protein to recognize its binding region more efficiently. Immunofluorescence studies demonstrated that all three site I-specific MAbs and one site IV-specific MAb (7-12) stained the N antigen that was diffusely distributed in the whole cytoplasm; the other two site IV-specific MAbs (6-9 and 8-1) detected only the N antigen in the cytoplasmic inclusion bodies (CIB). An antigenic site II-specific MAb (6-17) also detected CIB-associated N antigen alone. Furthermore, the level of diffuse N antigens decreased after treatment of infected cells with cycloheximide. These results suggest that epitopes at site I are expressed on the immature form of the N protein, but epitope structures of site IV MAbs 6-9 and 8-1 are created and/or exposed only after maturation of the N protein.

scholarly journals Pathogenicity of Different Rabies Virus Variants Inversely Correlates with Apoptosis and Rabies Virus Glycoprotein Expression in Infected Primary Neuron Cultures

1999 ◽  
Vol 73 (1) ◽  
pp. 510-518 ◽  
Author(s):  
Kinjiro Morimoto ◽  
D. Craig Hooper ◽  
Sergei Spitsin ◽  
Hilary Koprowski ◽  
Bernhard Dietzschold
Keyword(s):  
Protein Expression ◽  
G Protein ◽  
Rabies Virus ◽  
Antigenic Site ◽  
Expression Levels ◽  
N Protein ◽  
Adult Mice ◽  
Glycoprotein G ◽  
Neuronal Processes ◽  
Nucleoprotein N

ABSTRACT The mouse-adapted rabies virus strain CVS-24 has stable variants, CVS-B2c and CVS-N2c, which differ greatly in their pathogenicity for normal adult mice and in their ability to infect nonneuronal cells. The glycoprotein (G protein), which has previously been implicated in rabies virus pathogenicity, shows substantial structural differences between these variants. Although prior studies have identified antigenic site III of the G protein as the major pathogenicity determinant, CVS-B2c and CVS-N2c do not vary at this site. The possibility that pathogenicity is inversely related to G protein expression levels is suggested by the finding that CVS-B2c, the less pathogenic variant, expresses at least fourfold-higher levels of G protein than CVS-N2c in infected neurons. Although there is some difference between CVS-B2c- and CVS-N2c-infected neurons in G protein mRNA expression levels, the differential expression of G protein appears to be largely determined by posttranslational mechanisms that affect G protein stability. Pulse-chase experiments indicated that the G protein of CVS-B2c is degraded more slowly than that of CVS-N2c. The accumulation of G protein correlated with the induction of programmed cell death in CVS-B2c-infected neurons. The extent of apoptosis was considerably lower in CVS-N2c-infected neurons, where G protein expression was minimal. While nucleoprotein (N protein) expression levels were similar in neurons infected with either variant, the transport of N protein into neuronal processes was strongly inhibited in CVS-B2c-infected cells. Thus, downregulation of G protein expression in neuronal cells evidently contributes to rabies virus pathogenesis by preventing apoptosis and the apparently associated failure of the axonal transport of N protein.

scholarly journals A subpopulation of arenavirus nucleoprotein localizes to mitochondria

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Francesca Baggio ◽  
Udo Hetzel ◽  
Lisbeth Nufer ◽  
Anja Kipar ◽  
Jussi Hepojoki
Keyword(s):  
Mammalian Cells ◽  
Cytoplasmic Inclusion ◽  
Mitochondrial Morphology ◽  
Cellular Functions ◽  
Cellular Defense ◽  
Mitochondrial Functions ◽  
Infected Cells ◽  
Cytoplasmic Inclusion Bodies ◽  
Targeting Signal

AbstractViruses need cells for their replication and, therefore, ways to hijack cellular functions. Mitochondria play fundamental roles within the cell in metabolism, immunity and regulation of homeostasis due to which some viruses aim to alter mitochondrial functions. Herein we show that the nucleoprotein (NP) of arenaviruses enters the mitochondria of infected cells, affecting the mitochondrial morphology. Reptarenaviruses cause boid inclusion body disease (BIBD) that is characterized, especially in boas, by the formation of cytoplasmic inclusion bodies (IBs) comprising reptarenavirus NP within the infected cells. We initiated this study after observing electron-dense material reminiscent of IBs within the mitochondria of reptarenavirus infected boid cell cultures in an ultrastructural study. We employed immuno-electron microscopy to confirm that the mitochondrial inclusions indeed contain reptarenavirus NP. Mutations to a putative N-terminal mitochondrial targeting signal (MTS), identified via software predictions in both mamm- and reptarenavirus NPs, did not affect the mitochondrial localization of NP, suggesting that it occurs independently of MTS. In support of MTS-independent translocation, we did not detect cleavage of the putative MTSs of arenavirus NPs in reptilian or mammalian cells. Furthermore, in vitro translated NPs could not enter isolated mitochondria, suggesting that the translocation requires cellular factors or conditions. Our findings suggest that MTS-independent mitochondrial translocation of NP is a shared feature among arenaviruses. We speculate that by targeting the mitochondria arenaviruses aim to alter mitochondrial metabolism and homeostasis or affect the cellular defense.

scholarly journals Interferon-Induced Alterations in the Pattern of Parainfluenza Virus 5 Transcription and Protein Synthesis and the Induction of Virus Inclusion Bodies

2005 ◽  
Vol 79 (22) ◽  
pp. 14112-14121 ◽  
Author(s):  
T. S. Carlos ◽  
R. Fearns ◽  
R. E. Randall
Keyword(s):  
Protein Synthesis ◽  
Inclusion Bodies ◽  
Cytoplasmic Inclusion ◽  
Vero Cells ◽  
Simian Virus ◽  
Parainfluenza Virus ◽  
Antiviral State ◽  
Infected Cells ◽  
Cytoplasmic Inclusion Bodies ◽  
Parainfluenza Virus 5

ABSTRACT Although parainfluenza virus 5 (simian virus 5 [SV5]) circumvents the interferon (IFN) response by blocking IFN signaling and by reducing the amount of IFN released by infected cells, its ability to circumvent the IFN response is not absolute. The effects of IFN on SV5 infection were examined in Vero cells, which do not produce but can respond to IFN, using a strain of SV5 (CPI−) which does not block IFN signaling. Thus, by infecting Vero cells with CPI− and subsequently treating the cells with exogenous IFN, it was possible to observe the effects that IFN had on SV5 infection in the absence of virus countermeasures. IFN rapidly (within 6 h) induced alterations in the relative levels of virus mRNA and protein synthesis and caused a redistribution of virus proteins within infected cells that led to the enhanced formation of virus cytoplasmic inclusion bodies. IFN induced a steeper gradient of mRNA transcription from the 3′ to the 5′ end of the genome and the production of virus mRNAs with longer poly(A) tails, suggesting that the processivity of the virus polymerase was altered in cells in an IFN-induced antiviral state. Additional evidence is presented which suggests that these findings also apply to the replication of strains of SV5, parainfluenza virus type 2, and mumps virus that block IFN signaling when they infect cells that are already in an IFN-induced antiviral state.

scholarly journals Minimal Elements Required for the Formation of Respiratory Syncytial Virus Cytoplasmic Inclusion Bodies In Vivo and In Vitro

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Marie Galloux ◽  
Jennifer Risso-Ballester ◽  
Charles-Adrien Richard ◽  
Jenna Fix ◽  
Marie-Anne Rameix-Welti ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Liquid Phase ◽  
Inclusion Bodies ◽  
Cytoplasmic Inclusion ◽  
Cytoplasmic Inclusion Bodies ◽  
Syncytial Virus ◽  
P Proteins ◽  
Nucleoprotein N ◽  
Liquid Liquid Phase Separation

ABSTRACT Infection of host cells by the respiratory syncytial virus (RSV) is characterized by the formation of spherical cytoplasmic inclusion bodies (IBs). These structures, which concentrate all the proteins of the polymerase complex as well as some cellular proteins, were initially considered aggresomes formed by viral dead-end products. However, recent studies revealed that IBs are viral factories where viral RNA synthesis, i.e., replication and transcription, occurs. The analysis of IBs by electron microscopy revealed that they are membrane-less structures, and accumulated data on their structure, organization, and kinetics of formation revealed that IBs share the characteristics of cellular organelles, such as P-bodies or stress granules, suggesting that their morphogenesis depends on a liquid-liquid phase separation mechanism. It was previously shown that expression of the RSV nucleoprotein N and phosphoprotein P of the polymerase complex is sufficient to induce the formation of pseudo-IBs. Here, using a series of truncated P proteins, we identified the domains of P required for IB formation and show that the oligomeric state of N, provided it can interact with RNA, is critical for their morphogenesis. We also show that pseudo-IBs can form in vitro when recombinant N and P proteins are mixed. Finally, using fluorescence recovery after photobleaching approaches, we reveal that in cellula and in vitro IBs are liquid organelles. Our results strongly support the liquid-liquid phase separation nature of IBs and pave the way for further characterization of their dynamics. IMPORTANCE Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants, elderly, and immunocompromised people. No vaccine or efficient antiviral treatment is available against this virus. The replication and transcription steps of the viral genome are appealing mechanisms to target for the development of new antiviral strategies. These activities take place within cytoplasmic inclusion bodies (IBs) that assemble during infection. Although expression of both the viral nucleoprotein (N) and phosphoprotein (P) allows induction of the formation of these IBs, the mechanism sustaining their assembly remains poorly characterized. Here, we identified key elements of N and P required for the scaffolding of IBs and managed for the first time to reconstitute RSV pseudo-IBs in vitro by coincubating recombinant N and P proteins. Our results provide strong evidence that the biogenesis of RSV IBs occurs through liquid-liquid phase transition mediated by N-P interactions.

scholarly journals Definition of the immune evasion-replication interface of rabies virus P protein

2021 ◽  
Vol 17 (7) ◽  
pp. e1009729
Author(s):  
Jingyu Zhan ◽  
Angela R. Harrison ◽  
Stephanie Portelli ◽  
Thanh Binh Nguyen ◽  
Isshu Kojima ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Immune Evasion ◽  
Structural Data ◽  
Direct Analysis ◽  
Multifunctional Protein ◽  
N Protein ◽  
Binding Interface ◽  
P Protein ◽  
Definition Of ◽  
Nucleoprotein N

Rabies virus phosphoprotein (P protein) is a multifunctional protein that plays key roles in replication as the polymerase cofactor that binds to the complex of viral genomic RNA and the nucleoprotein (N protein), and in evading the innate immune response by binding to STAT transcription factors. These interactions are mediated by the C-terminal domain of P (PCTD). The colocation of these binding sites in the small globular PCTD raises the question of how these interactions underlying replication and immune evasion, central to viral infection, are coordinated and, potentially, coregulated. While direct data on the binding interface of the PCTD for STAT1 is available, the lack of direct structural data on the sites that bind N protein limits our understanding of this interaction hub. The PCTD was proposed to bind via two sites to a flexible loop of N protein (Npep) that is not visible in crystal structures, but no direct analysis of this interaction has been reported. Here we use Nuclear Magnetic Resonance, and molecular modelling to show N protein residues, Leu381, Asp383, Asp384 and phosphor-Ser389, are likely to bind to a ‘positive patch’ of the PCTD formed by Lys211, Lys214 and Arg260. Furthermore, in contrast to previous predictions we identify a single site of interaction on the PCTD by this Npep. Intriguingly, this site is proximal to the defined STAT1 binding site that includes Ile201 to Phe209. However, cell-based assays indicate that STAT1 and N protein do not compete for P protein. Thus, it appears that interactions critical to replication and immune evasion can occur simultaneously with the same molecules of P protein so that the binding of P protein to activated STAT1 can potentially occur without interrupting interactions involved in replication. These data suggest that replication complexes might be directly involved in STAT1 antagonism.

scholarly journals Functional Characterization of Negri Bodies (NBs) in Rabies Virus-Infected Cells: Evidence that NBs Are Sites of Viral Transcription and Replication

2009 ◽  
Vol 83 (16) ◽  
pp. 7948-7958 ◽  
Author(s):  
Xavier Lahaye ◽  
Aurore Vidy ◽  
Carole Pomier ◽  
Linda Obiang ◽  
Francis Harper ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Inclusion Bodies ◽  
Functional Characterization ◽  
Cytoplasmic Inclusion ◽  
Viral Transcription ◽  
Microtubule Network ◽  
Spherical Structures ◽  
Cytoplasmic Inclusion Bodies ◽  
P Proteins ◽  
Transcription And Replication

ABSTRACT Rabies virus infection induces the formation of cytoplasmic inclusion bodies that resemble Negri bodies found in the cytoplasm of some infected nerve cells. We have studied the morphogenesis and the role of these Negri body-like structures (NBLs) during viral infection. The results indicate that these spherical structures (one or two per cell in the initial stage of infection), composed of the viral N and P proteins, grow during the virus cycle before appearing as smaller structures at late stages of infection. We have shown that the microtubule network is not necessary for the formation of these inclusion bodies but is involved in their dynamics. In contrast, the actin network does not play any detectable role in these processes. These inclusion bodies contain Hsp70 and ubiquitinylated proteins, but they are not misfolded protein aggregates. NBLs, in fact, appear to be functional structures involved in the viral life cycle. Specifically, using in situ fluorescent hybridization techniques, we show that all viral RNAs (genome, antigenome, and every mRNA) are located inside the inclusion bodies. Significantly, short-term RNA labeling in the presence of BrUTP strongly suggests that the NBLs are the sites where viral transcription and replication take place.

scholarly journals Mapping and structural analysis of B-cell epitopes on the morbillivirus nucleoprotein amino terminus

2007 ◽  
Vol 88 (4) ◽  
pp. 1231-1242 ◽  
Author(s):  
S. C. Bodjo ◽  
O. Kwiatek ◽  
A. Diallo ◽  
E. Albina ◽  
G. Libeau
Keyword(s):  
Antigenic Site ◽  
Variable Region ◽  
Antigenic Structure ◽  
Competitive Binding Assay ◽  
Amino Terminal ◽  
Antigenic Sites ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Nucleoprotein N ◽  
N Proteins

By analysing the antigenic structure of the morbillivirus nucleoprotein (N) using a competitive-binding assay of monoclonal antibodies (mAbs), six different antigenic sites were identified previously. By using Pepscan methodology complemented by analysis of truncated N proteins, a better characterization of five of these antigenic sites was provided: I, II, III, IV and VI. mAbs specific to Rinderpest virus, defining antigenic sites II, III and IV, and those common to four morbilliviruses, delineating sites I and VI, were analysed in the present study. It was found that all but one mapped to the same region, between aa 120 and 149 of N. However, the mAb 3-1 epitope was located in the carboxy-terminal region (aa 421–525). This result may indicate the high immunogenicity of the amino-terminal variable region, at least in the mouse. It was surprising that the epitope of mAb 33-4, antigenic site VI, which recognized all morbilliviruses so far tested, was located in one of the two non-conserved regions between morbillivirus N proteins. It is shown that the conserved amino acid motif 126EAD128----131F-------148EN149 is critical for epitope constitution and recognition.

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