Direct neuronal conversion of microglia/macrophages reinstates neurological function after stroke.

Although generating new neurons in the ischemic injured brain would be an ideal approach to replenish the lost neurons for repairing the damage, the adult mammalian brain retains only limited neurogenic capability. Here, we show that direct conversion of microglia/macrophages into neurons in the brain has great potential as a therapeutic strategy for ischemic brain injury. After transient middle cerebral artery occlusion in adult mice, microglia/macrophages converge at the lesion core of the striatum, where neuronal loss is prominent. Targeted expression of a neurogenic transcription factor, NeuroD1, in microglia/macrophages in the injured striatum enables their conversion into induced neuronal cells that functionally integrate into the existing neuronal circuits. Furthermore, NeuroD1-mediated induced neuronal cell generation significantly improves neurological function in the mouse stroke model, and ablation of these cells abolishes the gained functional recovery. Our findings thus demonstrate that neuronal conversion contributes directly to functional recovery after stroke.

Virological characteristics of the SARS-CoV-2 Omicron BA.2 spike.

Soon after the emergence and global spread of the SARS-CoV-2 Omicron lineage BA.1, another Omicron lineage, BA.2, began outcompeting BA.1. The results of statistical analysis showed that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralization experiments revealed that immunity induced by COVID vaccines widely administered to human populations is not effective against BA.2, similar to BA.1, and that the antigenicity of BA.2 is notably different from that of BA.1. Cell culture experiments showed that the BA.2 spike confers higher replication efficacy in human nasal epithelial cells and is more efficient in mediating syncytia formation than the BA.1 spike. Furthermore, infection experiments using hamsters indicated that the BA.2 spike-bearing virus is more pathogenic than the BA.1 spike-bearing virus. Altogether, the results of our multiscale investigations suggest that the risk of BA.2 to global health is potentially higher than that of BA.1.

Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses.

The NF-κB and interferon antiviral signaling pathways play pivotal roles in inflammatory and innate immune responses. The LUBAC ubiquitin ligase complex, composed of the HOIP, HOIL-1L, and SHARPIN subunits, activates the canonical NF-κB pathway through Met1-linked linear ubiquitination. We identified small-molecule chemical inhibitors of LUBAC, HOIPIN-1 and HOIPIN-8. Here we show that HOIPINs down-regulate not only the proinflammatory cytokine-induced canonical NF-κB pathway, but also various pathogen-associated molecular pattern-induced antiviral pathways. Structural analyses indicated that HOIPINs inhibit the RING-HECT-hybrid reaction in HOIP by modifying the active Cys885, and residues in the C-terminal LDD domain, such as Arg935 and Asp936, facilitate the binding of HOIPINs to LUBAC. HOIPINs effectively induce cell death in activated B cell-like diffuse large B cell lymphoma cells, and alleviate imiquimod-induced psoriasis in model mice. These results reveal the molecular and cellular bases of LUBAC inhibition by HOIPINs, and demonstrate their potential therapeutic uses.

Eligibility of Feline Calicivirus for a Surrogate of Human Norovirus in Comparison with Murine Norovirus, Poliovirus and Coxsackievirus.

Feline calicivirus (FCV) is frequently used as a surrogate of human norovirus. We investigated eligibility of FCV for anti-viral assay by investigating the stability of infectivity and pH sensitivity in comparison with other viruses. We found that infectivities of FCV and murine norovirus (MNV) are relatively unstable in infected cells compared with those of coxsackievirus (CoV) and poliovirus (PoV) , suggesting that FCV and MNV have vulnerability. Western blotting indicated that inactivation of FCV was not due to viral protein degradation. We also demonstrated sensitivity of FCV to low pH, the 50% inhibitory pH value being ca. 3.9. Since human norovirus is thought to persist longer, in infectivity and to be a resistant virus, CoV, which is robust and not restrained in use as PoV, may be more appropriate as a test virus for disinfectants, rather than FCV and MNV.

A Single Amino Acid Substitution within the Paramyxovirus Sendai Virus Nucleoprotein Is a Critical Determinant for Production of Interferon-Beta-Inducing Copyback-Type Defective Interfering Genomes.

One of the first defenses against infecting pathogens is the innate immune system activated by cellular recognition of pathogen-associated molecular patterns (PAMPs). Although virus-derived RNA species, especially copyback (cb)-type defective interfering (DI) genomes, have been shown to serve as real PAMPs, which strongly induce interferon-beta (IFN-ß) during mononegavirus infection, the mechanisms underlying DI generation remain unclear. Here, for the first time, we identified a single amino acid substitution causing production of cbDI genomes by successful isolation of two distinct types of viral clones with cbDI-producing and cbDI-nonproducing phenotypes from the stock Sendai virus (SeV) strain Cantell, which has been widely used in a number of studies on antiviral innate immunity as a representative IFN-ß-inducing virus. IFN-ß induction was totally dependent on the presence of a significant amount of cbDI genome-containing viral particles (DI particles) in the viral stock, but not on deficiency of the IFN-antagonistic viral accessory proteins C and V. Comparison of the isolates indicated that a single amino acid substitution found within the N protein of the cbDI-producing clone was enough to cause the emergence of DI genomes. The mutated N protein of the cbDI-producing clone resulted in a lower density of nucleocapsids than that of the DI-nonproducing clone, probably causing both production of the DI genomes and their formation of a stem-loop structure, which serves as an ideal ligand for RIG-I. These results suggested that the integrity of mononegaviral nucleocapsids might be a critical factor in avoiding the undesirable recognition of infection by host cells.IMPORTANCE The type I interferon (IFN) system is a pivotal defense against infecting RNA viruses that is activated by sensing viral RNA species. RIG-I is a major sensor for infection with most mononegaviruses, and copyback (cb)-type defective interfering (DI) genomes have been shown to serve as strong RIG-I ligands in real infections. However, the mechanism underlying production of cbDI genomes remains unclear, although DI genomes emerge as the result of an error during viral replication with high doses of viruses. Sendai virus has been extensively studied and is unique in that its interaction with innate immunity reveals opposing characteristics, such as high-level IFN-ß induction and strong inhibition of type I IFN pathways. Our findings provide novel insights into the mechanism of production of mononegaviral cbDI genomes, as well as virus-host interactions during innate immunity.

Induction of necroptotic cell death by viral activation of the RIG-I or STING pathway.

Necroptosis is a form of necrotic cell death that requires the activity of the death domain-containing kinase RIP1 and its family member RIP3. Necroptosis occurs when RIP1 is deubiquitinated to form a complex with RIP3 in cells deficient in the death receptor adapter molecule FADD or caspase-8. Necroptosis may play a role in host defense during viral infection as viruses like vaccinia can induce necroptosis while murine cytomegalovirus encodes a viral inhibitor of necroptosis. To see how general the interplay between viruses and necroptosis is, we surveyed seven different viruses. We found that two of the viruses tested, Sendai virus (SeV) and murine gammaherpesvirus-68 (MHV68), are capable of inducing dramatic necroptosis in the fibrosarcoma L929 cell line. We show that MHV68-induced cell death occurs through the cytosolic STING sensor pathway in a TNF-dependent manner. In contrast, SeV-induced death is mostly independent of TNF. Knockdown of the RNA sensing molecule RIG-I or the RIP1 deubiquitin protein, CYLD, but not STING, rescued cells from SeV-induced necroptosis. Accompanying necroptosis, we also find that wild type but not mutant SeV lacking the viral proteins Y1 and Y2 result in the non-ubiquitinated form of RIP1. Expression of Y1 or Y2 alone can suppress RIP1 ubiquitination but CYLD is dispensable for this process. Instead, we found that Y1 and Y2 can inhibit cIAP1-mediated RIP1 ubiquitination. Interestingly, we also found that SeV infection of B6 RIP3-/- mice results in increased inflammation in the lung and elevated SeV-specific T cells. Collectively, these data identify viruses and pathways that can trigger necroptosis and highlight the dynamic interplay between pathogen-recognition receptors and cell death induction.

Inactivation of pathogenic viruses by plant-derived tannins: strong effects of extracts from persimmon (Diospyros kaki) on a broad range of viruses.

Tannins, plant-derived polyphenols and other related compounds, have been utilized for a long time in many fields such as the food industry and manufacturing. In this study, we investigated the anti-viral effects of tannins on 12 different viruses including both enveloped viruses (influenza virus H3N2, H5N3, herpes simplex virus-1, vesicular stomatitis virus, Sendai virus and Newcastle disease virus) and non-enveloped viruses (poliovirus, coxsachievirus, adenovirus, rotavirus, feline calicivirus and mouse norovirus). We found that extracts from persimmon (Diospyros kaki), which contains ca. 22% of persimmon tannin, reduced viral infectivity in more than 4-log scale against all of the viruses tested, showing strong anti-viral effects against a broad range of viruses. Other tannins derived from green tea, acacia and gallnuts were effective for some of the viruses, while the coffee extracts were not effective for any of the virus. We then investigated the mechanism of the anti-viral effects of persimmon extracts by using mainly influenza virus. Persimmon extracts were effective within 30 seconds at a concentration of 0.25% and inhibited attachment of the virus to cells. Pretreatment of cells with the persimmon extracts before virus infection or post-treatment after virus infection did not inhibit virus replication. Protein aggregation seems to be a fundamental mechanism underlying the anti-viral effect of persimmon tannin, since viral proteins formed aggregates when purified virions were treated with the persimmon extracts and since the anti-viral effect was competitively inhibited by a non-specific protein, bovine serum albumin. Considering that persimmon tannin is a food supplement, it has a potential to be utilized as a safe and highly effective anti-viral reagent against pathogenic viruses.

Passage of a Sendai virus recombinant in embryonated chicken eggs leads to markedly rapid accumulation of U-to-C transitions in a limited region of the viral genome.

The P gene of paramyxoviruses is unique in producing not only P but also "accessory" C and/or V proteins. Successful generation of C- or V-deficient recombinant viruses using a reverse genetics technique has been revealing their importance in viral pathogenesis as well as replication. As for Sendai virus (SeV), the C proteins, a nested set of four polypeptides C', C, Y1, and Y2, have been shown to exert multiple functions in escaping from the host innate immunity, inhibiting virus-induced apoptosis, promoting virus assembly and budding, and regulating viral RNA synthesis. In this study, we subjected the 4C(-) recombinant lacking expression of all four C proteins to serial passages through eggs, and found the rapid emergence of a C-recovered revertant virus. Unlike the SeV strains or the recombinants reported previously or tested in this study, this was caused by an exceptionally quick accumulation of U-to-C transitions in a limited region of the 4C(-) genome causing recovery of the C protein expression. These results suggest that a lack of C proteins could lead unexpectedly to strong selective pressures, and that the C proteins might play more critical roles in SeV replication than ever reported.

Inhibition of interferon regulatory factor 3 activation by paramyxovirus V protein.

The V protein of Sendai virus (SeV) suppresses innate immunity, resulting in enhancement of viral growth in mouse lungs and viral pathogenicity. The innate immunity restricted by the V protein is induced through activation of interferon regulatory factor 3 (IRF3). The V protein has been shown to interact with melanoma differentiation-associated gene 5 (MDA5) and to inhibit beta interferon production. In the present study, we infected MDA5-knockout mice with V-deficient SeV and found that MDA5 was largely unrelated to the innate immunity that the V protein suppresses in vivo. We therefore investigated the target of the SeV V protein. We previously reported interaction of the V protein with IRF3. Here we extended the observation and showed that the V protein appeared to inhibit translocation of IRF3 into the nucleus. We also found that the V protein inhibited IRF3 activation when induced by a constitutive active form of IRF3. The V proteins of measles virus and Newcastle disease virus inhibited IRF3 transcriptional activation, as did the V protein of SeV, while the V proteins of mumps virus and Nipah virus did not, and inhibition by these proteins correlated with interaction of each V protein with IRF3. These results indicate that IRF3 is important as an alternative target of paramyxovirus V proteins.

Optineurin with amyotrophic lateral sclerosis-related mutations abrogates inhibition of interferon regulatory factor-3 activation.

Optineurin has been shown to be involved in primary open-angle glaucoma. We recently found that optineurin is involved in familial amyotrophic lateral sclerosis (ALS). On the other hand, optineurin has been shown to inhibit transcription factors related to innate immunity such as NF-κB and interferon regulatory factor-3 (IRF3). In the present study, the effect of ALS-associated optineurin mutations on IRF3 activation was investigated. Optineurin inhibited IRF3 activation induced by melanoma differentiation-associated gene 5 or Toll-IL-1 receptor domain-containing adaptor-inducing interferon-ß. The inhibition was abrogated by mutations related to ALS but not by a mutation related to glaucoma. Reporter assay indicated that the JAK-STAT signaling pathway was not affected by optineurin. These results show that ALS-related optineurin is involved in the IRF3 activation pathway. Pathogenesis of ALS may be associated with some kind of innate immunity, especially that against virus infection, through IRF3 activation.

Analysis of interaction of Sendai virus V protein and melanoma differentiation-associated gene 5.

Sendai virus (SeV), a pneumotropic virus of rodents, has an accessory protein, V, and the V protein has been shown to interact with MDA5, inhibiting IRF3 activation and interferon-ß production. In the present study, interaction of the V protein with various IRF3-activating proteins including MDA5 was investigated in a co-immunoprecipitation assay. We also investigated interaction of mutant V proteins from SeVs of low pathogenicity with MDA5. The V protein interacted with at least retinoic acid inducible gene I, inhibitor of κB kinase epsilon and IRF3 other than MDA5. However, only MDA5 interacted with the V protein dependently on the C-terminal V unique (Vu) region, inhibiting IRF3 reporter activation. The Vu region has been shown to be important for viral pathogenicity. We thus focused on interaction of the V protein with MDA5. Point mutations in the Vu region destabilized the V protein or abolished the interaction with MDA5 when the V protein was stable. The V-R320G protein was highly stable and interacted with MDA5, but did not inhibit activation of IRF3 induced by MDA5. Viral pathogenicity of SeV is related to the inhibitory effect of the V protein on MDA5, but is not always related to the binding of V protein with MDA5.

[Envelope virus assembly and budding].

For many enveloped viruses, viral matrix and retroviral Gag proteins are able to bud from the cell surface by themselves in the form of lipid-enveloped, virus-like particles (VLPs), suggesting that these proteins play important roles in viral assembly and budding. The major three-types of L-domain motifs, PPxY, P(T/S)AT, and YP(x)(n)L have been identified within these proteins. Many viruses have been shown to commonly utilize cellular ESCRT pathway via direct interaction between the L-domains and the components of the pathway for efficient viral budding. However, for many enveloped viruses, L-domain motifs have not yet been identified, and the involvement of the ESCRT pathway in virus budding is still unknown. Among such viruses, we have been focusing on Sendai virus (SeV) and shown that (i) SeV M functionally and physically interact with a component of the ESCRT complex, Alix/AIP1, although budding of M-VLPs does not seem to be dependent on the pathway; (ii) one of the accessory proteins of SeV, C, also interact with Alix/AIP1, and recruit it to the plasma membrane for efficient budding of M-VLPs; (iii) the C protein regulate balance of viral genome and antigenome RNA synthesis for optimized production of infectious virus particles. These results demonstrate a unique mechanism for budding of SeV as well as a novel mechanism of regulated synthesis of viral genome RNAs for efficient production of infectious particles.

Modifications of the PSAP region of the matrix protein lead to attenuation of vesicular stomatitis virus in vitro and in vivo.

The matrix (M) protein of vesicular stomatitis virus (VSV) is a multi-functional protein involved in virus assembly, budding and pathogenesis. The (24)PPPY(27) late (L) domain of the M protein plays a key role in virus budding, whereas amino acids downstream of the PPPY motif contribute to host protein shut-off and pathogenesis. Using a panel of (37)PSAP(40) recombinant viruses, it has been demonstrated previously that the PSAP region of M does not possess L-domain activity similar to that of PPPY in BHK-21 cells. This study reports the unanticipated finding that these PSAP recombinants were attenuated in cell culture and in mice compared with control viruses. Indeed, PSAP recombinant viruses exhibited a small-plaque phenotype, reduced CPE, reduced levels of activated caspase-3, enhanced production of IFN-beta and reduced titres in the lungs and brains of infected mice. In particular, recombinant virus M6PY>A4-R34E was the most severely attenuated, exhibiting little or no CPE in cell culture and undetectable titres in the lungs and brains of infected mice. These findings indicate an important role for the PSAP region (aa 33-44) of the M protein in the pathology of VSV infection and may have implications for the development of VSV as a vaccine and/or oncolytic vector.

Two different conformations of rabies virus glycoprotein taken under neutral pH conditions.

We previously reported that the rabies virus glycoprotein (G) takes either of two different conformations (referred to as B and C forms) under neutral pH conditions, that could be differentiated by their reactivity to a monoclonal antibody (mAb), #1-30-44, that recognizes the acid-sensitive conformational epitope, and the formation taken is dependent on two separate regions containing Lys-202 and Asn-336 of the protein (Kankanamge et al., Microbiol. Immunol., 47, 507-519, 2003). Semi-quantitative antibody-binding assays demonstrated that only one-third to one-fourth of mature G proteins on the cell surface were taking the 1-30-44 epitope-positive B form even at pH 7.4. The ratio of B to C varied, depending on the environmental pH, but did not decrease to zero even at pH 5.8-6.2, preserving a certain content (about 15-20%) of B form. Immunoprecipitation studies demonstrated that a portion of G proteins were intimately associated with a dimer form of matrix (M) protein in terms of resistance to treatment with a mixture of 1% deoxycholate and 1% Nonidet P-40, and seemed to preserve the B form even at lower pHs. Similar results were also obtained with the virion-associated G proteins, including the intimate association of a portion of the G proteins with the M protein dimer. From these results, we assume that a certain portion of the rabies virion-associated G proteins are associated with a dimer form of M protein, keeping the 1-30-44 epitope-positive B conformation under various pH conditions, which might possibly assure the virion's recognition of host cell receptor molecules in the body.

Further studies on the mechanism of rabies virus neutralization by a viral glycoprotein-specific monoclonal antibody, #1-46-12.

We previously reported that a conformational epitope-specific monoclonal antibody (mAb; #1-46-12) neutralized the rabies virus by binding only a small number (less than 20) of the antibody molecules per virion, while a linear epitope-specific mAb (#7-1-9) required more than 250 IgG molecules for the neutralization. We also isolated both the epitope-negative (R-31) and-positive (R-61) escape mutants that resisted mAb #1-46-12. Co-infection studies with wild type (wt) and R-61 mutant have shown that although the infectivity of R-61 mutant was not affected by the binding of about 300 IgG molecules per virion, incorporation of a small number of wt G protein into the R-61 virion resulted in dramatic loss of the resistance. In this study, we further investigated properties of the mutant G proteins. The R-61 G protein lost reactivity to the mAb when solubilized, even keeping a trimer form, suggesting that membrane-anchorage is essential for the maintenance of its epitope-positive conformation. On the other hand, incorporation of wt G proteins into the R-31 virions did not affect their resistance to the mAb very much. Although we have not so far found the presumed conformational changes induced by the mAb-binding, we think that these results are not inconsistent with our previously proposed novel model (referred to as a domino effect model) for the virus neutralization by mAb #1-46-12 other than a classical spike-blocking model, which implicates successive spreading of the postulated antibody-induced conformational changes of G protein to the neighboring spikes until abolishing the host cell-binding ability of the virion.

Functional characterization of Ebola virus L-domains using VSV recombinants.

VSV recombinants containing the overlapping L-domain sequences from Ebola virus VP40 (PTAPPEY) were recovered by reverse-genetics. Replication kinetics of M40-WT, M40-P24L, and M40-Y30A were indistinguishable from VSV-WT in BHK-21 cells, whereas the double mutant (M40-P2728A) was defective in budding. Insertion of the Ebola L-domain region into VSV M protein was sufficient to alter the dependence on host proteins for efficient budding. Indeed, M40 recombinants containing a functional PTAP motif specifically incorporated endogenous tsg101 into budding virions and were dependent on tsg101 expression for efficient budding. Thus, VSV represents an excellent negative-sense RNA virus model for elucidating the functional aspects and diverse host interactions associated with the L-domains of Ebola virus.

High resolution for single-strand conformation polymorphism analysis by capillary electrophoresis.

Since the successful completion of the Human Genome Project, increasing concern is being directed toward the polymorphic aspect of the genome and its clinical relevance. A form of single-strand DNA-conformation polymorphism analysis (SSCP) employing nondenaturing slab-gel electrophoresis (SGE) is applicable to the genetic diagnosis of bladder cancer from urine samples. To bring this technique into routine clinical practice, the use of capillary electrophoresis (CE) is naturally favorable in terms of speed and automation. However, the resolving power of SSCP, a prerequisite basis for reliability required in diagnostics, remains as a challenge for CE systems. We thus focused on this topic and conducted studies on CE instruments equipped with a single capillary or an array of multiple capillaries, using the resolution (Rs) as a quantitative scale for the resolving power. Polymer concentration and buffer are shown to be the decisive parameters. High Rs values of >2.5 are achieved for representative SNPs markers under the optimized conditions, without sacrificing such intrinsic advantages of CE over SGE as the 10-fold quicker migration time and operation that is reproducible, continuous, and automatic. The strategies presented broaden the limits of CE in both the current and related applications.

Budding of PPxY-containing rhabdoviruses is not dependent on host proteins TGS101 and VPS4A.

Viral matrix proteins of several enveloped RNA viruses play important roles in virus assembly and budding and are by themselves able to bud from the cell surface in the form of lipid-enveloped, virus-like particles (VLPs). Three motifs (PT/SAP, PPxY, and YxxL) have been identified as late budding domains (L-domains) responsible for efficient budding. L-domains can functionally interact with cellular proteins involved in vacuolar sorting (VPS4A and TSG101) and endocytic pathways (Nedd4), suggesting involvement of these pathways in virus budding. Ebola virus VP40 has overlapping PTAP and PPEY motifs, which can functionally interact with TSG101 and Nedd4, respectively. As for vesicular stomatitis virus (VSV), a PPPY motif within M protein can interact with Nedd4. In addition, M protein has a PSAP sequence downstream of the PPPY motif, but the function of PSAP in budding is not clear. In this study, we compared L-domain functions between Ebola virus and VSV by constructing a chimeric M protein (M40), in which the PPPY motif of VSV M is replaced by the L domains of VP40. The budding efficiency of M40 was 10-fold higher than that of wild-type (wt) M protein. Overexpression of a dominant negative mutant of VPS4A or depletion of cellular TSG101 reduced the budding of only M40-containing VLPs but not that of wt M VLPs or live VSV. These findings suggest that the PSAP motif of M protein is not critical for budding and that there are fundamental differences between PTAP-containing viruses (Ebola virus and human immunodeficiency virus type 1) and PPPY-containing viruses (VSV and rabies virus) regarding their dependence on specific host factors for efficient budding.

Characterization of a slow-migrating component of the rabies virus matrix protein strongly associated with the viral glycoprotein.

We investigated multiple forms of rabies virus matrix (M) protein. Under non-reducing electrophoretic conditions, we detected, in addition to major bands of monomer forms (23- and 24-kDa) of M protein, an M antigen-positive slow-migrating minor band (about 54 kDa) in both the virion and infected cells. Relative contents of the 54-kDa and monomer components in the virion were about 20-30% and 70-80% of the whole M protein, respectively, while the content of the 54-kDa component was smaller (about 10-20% of the total M protein) in the cell than in the virion. The 54-kDa components could be extracted from the infected cells with sodium deoxycholate, but they were quite resistant to extraction with 1% nonionic detergents by which most monomer components were solubilized. The 54-kDa component was precipitated more efficiently than the monomer by a monoclonal antibody (mAb; #3-9-16), which recognized a linear epitope located at the N-terminal of the M protein. The mAb #3-9-16 coprecipitated the viral glycoprotein (G), which was demonstrated to be due to strong association between the G and 54-kDa component of the M protein. Monomers and the 54-kDa polypeptide migrated to the same isoelectric point (pI) in twodimensional (2-D) gel electrophoresis, implicating that the 54-kDa component was composed of component(s) of the same pI as that of the M protein monomers. From these results, we conclude that the M antigen-positive 54-kDa polypeptide is a homodimer of M protein, taking an N-terminal-exposed conformation, and is strongly associated with the viral glycoprotein. Possible association with a membrane microdomain of the cell will be discussed.

Mapping of the low pH-sensitive conformational epitope of rabies virus glycoprotein recognized by a monoclonal antibody #1-30-44.

Monoclonal antibody (mAb) #1-30-44 recognized an acid-sensitive conformational epitope of rabies virus glycoprotein (G). The antigenicity of G protein exposed on the cell surface was lost when the infected cells were exposed to pH 5.8. By comparing the deduced amino acid sequence of G protein between the HEP-Flury strain and the epitope-negative CVS strain as well as the mAb-resistant escape mutants, two distant sites that contained Lys-202 and Asn-336 were shown to be involved in the epitope formation. Lys-202 is located in the so-called neurotoxin-like sequence, while Asn-336 is included in antigenic site III and is very near the amino acid at position 333, which is known to affect greatly the neuropathogenicity of rabies virus when changed. Consistent with this finding, antigenicity of a neurovirulent revertant of the HEP-Flury strain, in which Gln-333 of G protein was replaced by Arg, was also affected as shown by its greatly decreased reactivity with mAb #1-30-44 compared to that of the original avirulent HEP virus. Based on these results, we hypothesize that the neurotoxin-like domain and some amino acids in antigenic site III come into contact with each other to form a conformational epitope for mAb #1-30-44, and such a configuration would be lost when exposed to acidic conditions to perform a certain low pH-dependent function of G protein.