Early Transcriptional Changes in Rabies Virus-Infected Neurons and Their Impact on Neuronal Functions

Rabies is a zoonotic disease caused by rabies virus (RABV). As rabies advances, patients develop a variety of severe neurological symptoms that inevitably lead to coma and death. Unlike other neurotropic viruses that can induce symptoms of a similar range, RABV-infected post-mortem brains do not show significant signs of inflammation nor the structural damages on neurons. This suggests that the observed neurological symptoms possibly originate from dysfunctions of neurons. However, many aspects of neuronal dysfunctions in the context of RABV infection are only partially understood, and therefore require further investigation. In this study, we used differentiated neurons to characterize the RABV-induced transcriptomic changes at the early time-points of infection. We found that the genes modulated in response to the infection are particularly involved in cell cycle, gene expression, immune response, and neuronal function-associated processes. Comparing a wild-type RABV to a mutant virus harboring altered matrix proteins, we found that the RABV matrix protein plays an important role in the early down-regulation of host genes, of which a significant number is involved in neuronal functions. The kinetics of differentially expressed genes (DEGs) are also different between the wild type and mutant virus datasets. The number of modulated genes remained constant upon wild-type RABV infection up to 24 h post-infection, but dramatically increased in the mutant condition. This result suggests that the intact viral matrix protein is important to control the size of host gene modulation. We then examined the signaling pathways previously studied in relation to the innate immune responses against RABV, and found that these pathways contribute to the changes in neuronal function-associated processes. We further examined a set of regulated genes that could impact neuronal functions collectively, and demonstrated in calcium imaging that indeed the spontaneous activity of neurons is influenced by RABV infection. Overall, our findings suggest that neuronal function-associated genes are modulated by RABV early on, potentially through the viral matrix protein-interacting signaling molecules and their downstream pathways.

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PPEY Motif within the Rabies Virus (RV) Matrix Protein Is Essential for Efficient Virion Release and RV Pathogenicity

Journal of Virology ◽

10.1128/jvi.00889-08 ◽

2008 ◽

Vol 82 (19) ◽

pp. 9730-9738 ◽

Cited By ~ 56

Author(s):

Christoph Wirblich ◽

Gene S. Tan ◽

Amy Papaneri ◽

Peter J. Godlewski ◽

Jan Marc Orenstein ◽

...

Keyword(s):

Viral Replication ◽

Rabies Virus ◽

Rna Synthesis ◽

Matrix Protein ◽

Virus Production ◽

Wild Type Virus ◽

Wild Type ◽

Conserved Sequence ◽

Virion Release ◽

The Matrix

ABSTRACT Late (L) domains containing the highly conserved sequence PPXY were first described for retroviruses, and later research confirmed their conservation and importance for efficient budding of several negative-stranded RNA viruses. Rabies virus (RV), a member of the Rhabdoviridae family, contains the sequence PPEY (amino acids 35 to 38) within the N terminus of the matrix (M) protein, but the functions of this potential L-domain in the viral life cycle, viral pathogenicity, and immunogenicity have not been established. Here we constructed a series of recombinant RVs containing mutations within the PPEY motif and analyzed their effects on viral replication and RV pathogenicity. Our results indicate that the first proline at position 35 is the most important for viral replication, whereas P36 and Y38 have a lesser but still noticeable impact. The reduction in viral replication was most likely due to inhibition of virion release, because initially no major impact on RV RNA synthesis was observed. In addition, results from electron microscopy demonstrated that the M4A mutant virus (PPEY→SAEA) displayed a more cell-associated phenotype than that of wild-type RV. Furthermore, all mutations within the PPEY motif resulted in reduced spread of the recombinant RVs as indicated by a reduction in focus size. Importantly, recombinant PPEY L-domain mutants were highly attenuated in mice yet still elicited potent antibody responses against RV G protein that were as high as those observed after infection with wild-type virus. Our data indicate that the RV PPEY motif has L-domain activity essential for efficient virus production and pathogenicity but is not essential for immunogenicity and thus can be targeted to increase the safety of rabies vaccine vectors.

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Genetic, Behavioral and Environmental Determinants of Male Longevity in Caenorhabditis elegans

Genetics ◽

10.1093/genetics/154.4.1597 ◽

2000 ◽

Vol 154 (4) ◽

pp. 1597-1610 ◽

Cited By ~ 1

Author(s):

David Gems ◽

Donald L Riddle

Keyword(s):

Caenorhabditis Elegans ◽

Life Span ◽

Wild Type ◽

Neuronal Function ◽

Nematode Caenorhabditis Elegans ◽

Wild Isolate ◽

Young Males ◽

C Elegans ◽

Single Sex ◽

Solitary Males

Abstract Males of the nematode Caenorhabditis elegans are shorter lived than hermaphrodites when maintained in single-sex groups. We observed that groups of young males form clumps and that solitary males live longer, indicating that male-male interactions reduce life span. By contrast, grouped or isolated hermaphrodites exhibited the same longevity. In one wild isolate of C. elegans, AB2, there was evidence of copulation between males. Nine uncoordinated (unc) mutations were used to block clumping behavior. These mutations had little effect on hermaphrodite life span in most cases, yet many increased male longevity even beyond that of solitary wild-type males. In one case, the neuronal function mutant unc-64(e246), hermaphrodite life span was also increased by up to 60%. The longevity of unc-4(e120), unc-13(e51), and unc-32(e189) males exceeded that of hermaphrodites by 70–120%. This difference appears to reflect a difference in sex-specific life span potential revealed in the absence of male behavior that is detrimental to survival. The greater longevity of males appears not to be affected by daf-2, but is influenced by daf-16. In the absence of male-male interactions, median (but not maximum) male life span was variable. This variability was reduced when dead bacteria were used as food. Maintenance on dead bacteria extended both male and hermaphrodite longevity.

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A Replication-Defective Gammaherpesvirus Efficiently Establishes Long-Term Latency in Macrophages but Not in B Cells In Vivo

Journal of Virology ◽

10.1128/jvi.00186-08 ◽

2008 ◽

Vol 82 (17) ◽

pp. 8500-8508 ◽

Cited By ~ 17

Author(s):

Haiyan Li ◽

Kazufumi Ikuta ◽

John W. Sixbey ◽

Scott A. Tibbetts

Keyword(s):

B Cells ◽

Viral Genome ◽

Lytic Replication ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Virus Latency

ABSTRACT Murine gammaherpesvirus 68 (γHV68 or MHV68) is genetically related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), providing a useful system for in vivo studies of the virus-host relationship. To begin to address fundamental questions about the mechanisms of the establishment of gammaherpesvirus latency, we previously generated a replication-defective γHV68 lacking the expression of the single-stranded DNA binding protein encoded by orf6. In work presented here, we demonstrate that this mutant virus established a long-term infection in vivo that was molecularly identical to wild-type virus latency. Thus, despite the absence of an acute phase of lytic replication, the mutant virus established a chronic infection in which the viral genome (i) was maintained as an episome and (ii) expressed latency-associated, but not lytic replication-associated, genes. Macrophages purified from mice infected with the replication-defective virus harbored viral genome at a frequency that was nearly identical to that of wild-type γHV68; however, the frequency of B cells harboring viral genome was greatly reduced in the absence of lytic replication. Thus, this replication-defective gammaherpesvirus efficiently established in vivo infection in macrophages that was molecularly indistinguishable from wild-type virus latency. These data point to a critical role for lytic replication or reactivation in the establishment or maintenance of latent infection in B cells.

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Thrombospondin-4 knockout in hypertension protects small-artery endothelial function but induces aortic aneurysms

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00046.2016 ◽

2016 ◽

Vol 310 (11) ◽

pp. H1486-H1493 ◽

Cited By ~ 11

Author(s):

Teresa Palao ◽

Catarina Rippe ◽

Henk van Veen ◽

Ed VanBavel ◽

Karl Swärd ◽

...

Keyword(s):

Calcium Binding ◽

Matrix Protein ◽

Pressure Overload ◽

Aortic Aneurysms ◽

Endoplasmic Reticulum Stress Response ◽

Extracellular Matrix Protein ◽

Ang Ii ◽

Wild Type ◽

Resistance Arteries ◽

Thrombospondin 4

Thrombospondin-4 (TSP-4) is a multidomain calcium-binding protein that has both intracellular and extracellular functions. As an extracellular matrix protein, it is involved in remodeling processes. Previous work showed that, in the cardiovascular system, TSP-4 expression is induced in the heart in response to experimental pressure overload and infarction injury. Intracellularly, it mediates the endoplasmic reticulum stress response in the heart. In this study, we explored the role of TSP-4 in hypertension. For this purpose, wild-type and TSP-4 knockout ( Thbs4 −/−) mice were treated with angiotensin II (ANG II). Hearts from ANG II-treated Thbs4 −/− mice showed an exaggerated hypertrophic response. Interestingly, aortas from Thbs4 −/− mice treated with ANG II showed a high incidence of aneurysms. In resistance arteries, ANG II-treated wild-type mice showed impaired endothelial-dependent relaxation. This was not observed in ANG II-treated Thbs4 −/− mice or in untreated controls. No differences were found in the passive pressure-diameter curves or stress-strain relationships, although ANG II-treated Thbs4 −/− mice showed a tendency to be less stiff, associated with thicker diameters of the collagen fibers as revealed by electron microscopy. We conclude that TSP-4 plays a role in hypertension, affecting cardiac hypertrophy, aortic aneurysm formation, as well as endothelial-dependent relaxation in resistance arteries.

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Vaccinia Virus Envelope H3L Protein Binds to Cell Surface Heparan Sulfate and Is Important for Intracellular Mature Virion Morphogenesis and Virus Infection In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.74.7.3353-3365.2000 ◽

2000 ◽

Vol 74 (7) ◽

pp. 3353-3365 ◽

Cited By ~ 183

Author(s):

Chi-Long Lin ◽

Che-Sheng Chung ◽

Hans G. Heine ◽

Wen Chang

Keyword(s):

Cell Surface ◽

Vaccinia Virus ◽

Heparan Sulfate ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Virion Morphogenesis

ABSTRACT An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L−) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L− mutant virus. IMV from the H3L− mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L− mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.

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TLR4 regulates rabies virus-induced humoral immunity through recruitment of cDC2 to lymph organs

Journal of Virology ◽

10.1128/jvi.00829-21 ◽

2021 ◽

Author(s):

Chen Chen ◽

Chengguang Zhang ◽

Haoqi Li ◽

Zongmei Wang ◽

Yueming Yuan ◽

...

Keyword(s):

Immune Response ◽

Immune Responses ◽

Rabies Virus ◽

Humoral Immunity ◽

Humoral Immune Response ◽

Critical Role ◽

Wild Type ◽

Humoral Immune ◽

Specific Igg ◽

Molecular Patterns

Rabies, caused by rabies virus (RABV), is fatal to both humans and animals around the world. Effective clinical therapy for rabies has not been achieved, and vaccination is the most effective means of preventing and controlling rabies. Although different vaccines, such as live attenuated and inactivated vaccines, can induce different immune responses, different expression of pattern recognition receptors (PRRs) also causes diverse immune responses. Toll-like receptor 4 (TLR4) is a pivotal PRR that induces cytokine production and bridges innate and adaptive immunity. Importantly, TLR4 recognizes various virus-derived pathogen-associated molecular patterns (PAMPs) and virus-induced damage-associated molecular patterns (DAMPs), usually leading to the activation of immune cells. However, the role of TLR4 in the humoral immune response induced by RABV has not been revealed yet. Based on TLR4-deficient ( TLR4 -/- ) and wild-type (WT) mouse models, we report that TLR4-dependent recruitment of the conventional type-2 dendritic cells (CD8α - CD11b + cDC2) into secondary lymph organs (SLOs) is critical for antigen presentation. cDC2-initiated differentiation of Tfh cells promotes the proliferation of germinal centre (GC) B cells, the formation of GCs, and the production of plasma cells (PCs), all of which contribute to the production of RABV-specific IgG and virus-neutralizing antibodies (VNAs). Collectively, our work demonstrates that TLR4 is necessary for the recruitment of cDC2 and for the induction of RABV-induced humoral immunity, which is regulated by the cDC2-Tfh-GC B axis. IMPORTANCE Vaccination is the most efficient method to prevent rabies. TLR4, a well-known immune sensor, plays a critical role in initiating innate immune response. Here, we found that TLR4 deficiency ( TLR4 -/- ) mice suppressed the induction of humoral immune response after immunization with rabies virus (RABV), including reduced production of VNAs and RABV-specific IgG, compared with that occurred in wild-type (WT) mice. As a consequence, TLR4 -/- mice exhibited higher mortality than WT mice after challenge with virulent RABV. Importantly, further investigation found that TLR4 signaling promoted the recruitment of cDC2 (CD8α + CD11b - ), a subset of cDCs known to induce CD4 + T cell immunity through their MHC-II presentation machinery. Our results imply that TLR4 is indispensable for an efficient humoral response to rabies vaccine, which provides new insight into the development of novel rabies vaccines.

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Involvement of the N-Terminal Deubiquitinating Protease Domain of Human Cytomegalovirus UL48 Tegument Protein in Autoubiquitination, Virion Stability, and Virus Entry

Journal of Virology ◽

10.1128/jvi.02766-15 ◽

2016 ◽

Vol 90 (6) ◽

pp. 3229-3242 ◽

Cited By ~ 11

Author(s):

Young-Eui Kim ◽

Se Eun Oh ◽

Ki Mun Kwon ◽

Chan Hee Lee ◽

Jin-Hyun Ahn

Keyword(s):

Human Cytomegalovirus ◽

Virus Entry ◽

Terminal Region ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Viral Growth ◽

Internal Region ◽

Deubiquitinating Protease

ABSTRACTHuman cytomegalovirus (HCMV) protein pUL48 is closely associated with the capsid and has a deubiquitinating protease (DUB) activity in its N-terminal region. Although this DUB activity moderately increases virus replication in cultured fibroblast cells, the requirements of the N-terminal region of pUL48 in the viral replication cycle are not fully understood. In this study, we characterized the recombinant viruses encoding UL48(ΔDUB/NLS), which lacks the DUB domain and the adjacent nuclear localization signal (NLS), UL48(ΔDUB), which lacks only the DUB, and UL48(Δ360–1200), which lacks the internal region (amino acids 360 to 1200) downstream of the DUB/NLS. While ΔDUB/NLS and Δ360–1200 mutant viruses did not grow in fibroblasts, the ΔDUB virus replicated to titers 100-fold lower than those for wild-type virus and showed substantially reduced viral gene expression at low multiplicities of infection. The DUB domain contained ubiquitination sites, and DUB activity reduced its own proteasomal degradation intrans. Deletion of the DUB domain did not affect the nuclear and cytoplasmic localization of pUL48, whereas the internal region (360–1200) was necessary for cytoplasmic distribution. In coimmunoprecipitation assays, pUL48 interacted with three tegument proteins (pUL47, pUL45, and pUL88) and two capsid proteins (pUL77 and pUL85) but the DUB domain contributed to only pUL85 binding. Furthermore, we found that the ΔDUB virus showed reduced virion stability and less efficiently delivered its genome into the cell than the wild-type virus. Collectively, our results demonstrate that the N-terminal DUB domain of pUL48 contributes to efficient viral growth by regulating its own stability and promoting virion stabilization and virus entry.IMPORTANCEHCMV pUL48 and its herpesvirus homologs play key roles in virus entry, regulation of immune signaling pathways, and virion assembly. The N terminus of pUL48 contains the DUB domain, which is well conserved among all herpesviruses. Although studies using the active-site mutant viruses revealed that the DUB activity promotes viral growth, the exact role of this region in the viral life cycle is not fully understood. In this study, using the mutant virus lacking the entire DUB domain, we demonstrate that the DUB domain of pUL48 contributes to viral growth by regulating its own stability via autodeubiquitination and promoting virion stability and virus entry. This report is the first to demonstrate the characteristics of the mutant virus with the entire DUB domain deleted, which, along with information on the functions of this region, is useful in dissecting the functions associated with pUL48.

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Amino Acid Substitution at Position 95 in Rabies Virus Matrix Protein Affects Viral Pathogenicity

Journal of Veterinary Medical Science ◽

10.1292/jvms.11-0151 ◽

2011 ◽

Vol 73 (10) ◽

pp. 1363-1366 ◽

Cited By ~ 7

Author(s):

Naoto ITO ◽

Tetsuo MITA ◽

Kenta SHIMIZU ◽

Yuki ITO ◽

Tatsunori MASATANI ◽

...

Keyword(s):

Amino Acid ◽

Rabies Virus ◽

Amino Acid Substitution ◽

Matrix Protein

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Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission

Journal of Virology ◽

10.1128/jvi.02364-15 ◽

2015 ◽

Vol 90 (3) ◽

pp. 1169-1177 ◽

Cited By ~ 14

Author(s):

Jason Porta ◽

Vidya Mangala Prasad ◽

Cheng-I Wang ◽

Wataru Akahata ◽

Lisa F. P. Ng ◽

...

Keyword(s):

Chikungunya Virus ◽

Neutralizing Antibody ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Virus Like Particles ◽

Fab Fragments ◽

Cell Transmission ◽

Viral Surface

ABSTRACTChikungunya virus is a positive-stranded RNA alphavirus. Structures of chikungunya virus-like particles in complex with strongly neutralizing antibody Fab fragments (8B10 and 5F10) were determined using cryo-electron microscopy and X-ray crystallography. By fitting the crystallographically determined structures of these Fab fragments into the cryo-electron density maps, we show that Fab fragments of antibody 8B10 extend radially from the viral surface and block receptor binding on the E2 glycoprotein. In contrast, Fab fragments of antibody 5F10 bind the tip of the E2 B domain and lie tangentially on the viral surface. Fab 5F10 fixes the B domain rigidly to the surface of the virus, blocking exposure of the fusion loop on glycoprotein E1 and therefore preventing the virus from becoming fusogenic. Although Fab 5F10 can neutralize the wild-type virus, it can also bind to a mutant virus without inhibiting fusion or attachment. Although the mutant virus is no longer able to propagate by extracellular budding, it can, however, enter the next cell by traveling through junctional complexes without being intercepted by a neutralizing antibody to the wild-type virus, thus clarifying how cell-to-cell transmission can occur.IMPORTANCEAlphaviral infections are transmitted mainly by mosquitoes. Chikungunya virus (CHIKV), which belongs to theAlphavirusgenus, has a wide distribution in the Old World that has expanded in recent years into the Americas. There are currently no vaccines or drugs against alphaviral infections. Therefore, a better understanding of CHIKV and its associated neutralizing antibodies will aid in the development of effective treatments.

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