RESUMO
The tripartite motif (TRIM) protein family is the largest subfamily of E3 ubiquitin ligases, playing a crucial role in the antiviral process. In this study, we found that TRIM72, a member of the TRIM protein family, was increased in neuronal cells and mouse brains following rabies lyssavirus (RABV) infection. Over-expression of TRIM72 significantly reduced the viral titer of RABV in neuronal cells and mitigated the pathogenicity of RABV in mice. Furthermore, we found that TRIM72 over-expression effectively prevents the assembly and/or release of RABV. In terms of the mechanism, TRIM72 promotes the K48-linked ubiquitination of RABV Matrix protein (M), leading to the degradation of M through the proteasome pathway. TRIM72 directly interacts with M and the interaction sites were identified and confirmed through TRIM72-M interaction model construction and mutation analysis. Further investigation revealed that the degradation of M induced by TRIM72 was attributed to TRIM72's promotion of ubiquitination at site K195 in M. Importantly, the K195 site was found to be partially conserved among lyssavirus's M proteins, and TRIM72 over-expression induced the degradation of these lyssavirus M proteins. In summary, our study has uncovered a TRIM family protein, TRIM72, that can restrict lyssavirus replication by degrading M, and we have identified a novel ubiquitination site (K195) in lyssavirus M.
Assuntos
Raiva , Proteínas com Motivo Tripartido , Animais , Camundongos , Lyssavirus/metabolismo , Lyssavirus/patogenicidade , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Vírus da Raiva/metabolismo , Vírus da Raiva/patogenicidade , Raiva/genéticaRESUMO
The nucleolus is a common target of viruses and viral proteins, but for many viruses the functional outcomes and significance of this targeting remains unresolved. Recently, the first intranucleolar function of a protein of a cytoplasmically-replicating negative-sense RNA virus (NSV) was identified, with the finding that the matrix (M) protein of Hendra virus (HeV) (genus Henipavirus, family Paramyxoviridae) interacts with Treacle protein within nucleolar subcompartments and mimics a cellular mechanism of the nucleolar DNA-damage response (DDR) to suppress ribosomal RNA (rRNA) synthesis. Whether other viruses utilise this mechanism has not been examined. We report that sub-nucleolar Treacle targeting and modulation is conserved between M proteins of multiple Henipaviruses, including Nipah virus and other potentially zoonotic viruses. Furthermore, this function is also evident for P3 protein of rabies virus, the prototype virus of a different RNA virus family (Rhabdoviridae), with Treacle depletion in cells also found to impact virus production. These data indicate that unrelated proteins of viruses from different families have independently developed nucleolar/Treacle targeting function, but that modulation of Treacle has distinct effects on infection. Thus, subversion of Treacle may be an important process in infection by diverse NSVs, and so could provide novel targets for antiviral approaches with broad specificity.
Assuntos
Vírus Hendra , Lyssavirus , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Ribossômico , Lyssavirus/genética , Lyssavirus/metabolismo , Ribossomos/metabolismo , Vírus Hendra/genética , Vírus Hendra/metabolismo , Fatores de TranscriçãoRESUMO
Lyssavirus is a kind of neurotropic pathogen that needs to evade peripheral host immunity to enter the central nervous system to accomplish infection. NLRP3 inflammasome activation is essential for the host to defend against pathogen invasion. This study demonstrates that the matrix protein (M) of lyssavirus can inhibit both the priming step and the activation step of NLRP3 inflammasome activation. Specifically, M of lyssavirus can compete with NEK7 for binding to NLRP3, which restricts downstream apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization. The serine amino acid at the 158th site of M among lyssavirus is critical for restricting ASC oligomerization. Moreover, recombinant lab-attenuated lyssavirus rabies (rabies lyssavirus [RABV]) with G158S mutation at M decreases interleukin-1ß (IL-1ß) production in bone-marrow-derived dendritic cells (BMDCs) to facilitate lyssavirus invasion into the brain thereby elevating pathogenicity in mice. Taken together, this study reveals a common mechanism by which lyssavirus inhibits NLRP3 inflammasome activation to evade host defenses.
Assuntos
Células Dendríticas , Inflamassomos , Interleucina-1beta , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas da Matriz Viral , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Inflamassomos/metabolismo , Inflamassomos/imunologia , Camundongos , Proteínas da Matriz Viral/metabolismo , Humanos , Interleucina-1beta/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/virologia , Lyssavirus/metabolismo , Lyssavirus/imunologia , Quinases Relacionadas a NIMA/metabolismo , Ligação Proteica , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Células HEK293RESUMO
Mokola virus (MOKV) is a nonsegmented, negative-sense RNA virus that belongs to the Lyssavirus genus and Rhabdoviridae family. MOKV phosphoprotein P is an essential component of the replication and transcription complex and acts as a cofactor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. Here we present a structure for this domain of MOKV P, obtained by expression of full-length P in Escherichia coli, which was subsequently truncated during crystallization. The structure has a high degree of homology with P of rabies virus, another member of Lyssavirus genus, and to a lesser degree with P of vesicular stomatitis virus (VSV), a member of the related Vesiculovirus genus. In addition, analysis of the crystal packing of this domain reveals a potential binding site for the nucleoprotein N. Using both site-directed mutagenesis and yeast two-hybrid experiments to measure P-N interaction, we have determined the relative roles of key amino acids involved in this interaction to map the region of P that binds N. This analysis also reveals a structural relationship between the N-RNA binding domain of the P proteins of the Rhabdoviridae and the Paramyxoviridae.
Assuntos
Lyssavirus/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Fosfoproteínas/química , Proteínas Virais/química , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Lyssavirus/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Relação Estrutura-Atividade , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Viruses are obligatory intracellular parasites that use cell proteins to take the control of the cell functions in order to accomplish their life cycle. Studying the viral-host interactions would increase our knowledge of the viral biology and mechanisms of pathogenesis. Studies on pathogenesis mechanisms of lyssaviruses, which are the causative agents of rabies, have revealed some important host protein partners for viral proteins, especially for most studied species, i.e. Rabies virus. In this review article, the key physical lyssavirus-host protein interactions, their contributions to rabies infection, and their exploitation are discussed to improve the knowledge about rabies pathogenesis.
Assuntos
Interações entre Hospedeiro e Microrganismos/fisiologia , Lyssavirus/metabolismo , Vírus da Raiva/metabolismo , Raiva/metabolismo , Animais , Humanos , Fagocitose/fisiologia , Ligação Proteica/fisiologia , Raiva/transmissãoRESUMO
The phosphoprotein P of non-segmented negative-sense RNA viruses is an essential component of the replication and transcription complex and acts as a co-factor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. We have obtained the structure of the C-terminal domain of P of Mokola virus (MOKV), a lyssavirus that belongs to the Rhabdoviridae family and mapped at the amino acid level the crucial positions involved in interaction with N and in the formation of the viral replication complex. Comparison of the N-RNA binding domains of P solved to date suggests that the N-RNA binding domains are structurally conserved among paramyxoviruses and rhabdoviruses in spite of low sequence conservation. We also review the numerous other functions of this domain and more generally of the phosphoprotein.
Assuntos
Lyssavirus/genética , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Paramyxoviridae/genética , RNA/metabolismo , Rhabdoviridae/genética , Humanos , Lyssavirus/metabolismo , Modelos Biológicos , Modelos Moleculares , Nucleoproteínas/genética , Nucleoproteínas/fisiologia , Paramyxoviridae/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Rhabdoviridae/metabolismo , Relação Estrutura-AtividadeRESUMO
Notwithstanding the availability of effective vaccines, 40 - 60 thousand rabies cases in humans are reported every year. Almost always the disease is fatal because therapeutic treatment of lyssavirus encephalitis has not been developed. Since 1970 the number of reports on rare cases of convalescence including those using experimental treatment protocols has been gradually increasing 20 cases of convalescence, "partial" convalescence or long-term survival of humans (1970-2015) were selected as they were complaint with laboratory criteria of active lyssavirus infection. Children and teenagers were predominant in the analyzed group (85%). The cases were irregularly spread between the continents: Asia - 6 cases, North America - 6 cases, Africa - 2 cases and Europe - 1 case. India and the USA were on the top of the list of countries by the number of described cases. More than 60% humans were infected from dogs, three cases got infection from bats and 2 cases were allegedly associated with an unknown lyssavirus and an unidentified infection source. 70% cases were vaccinated and 10% cases were treated with gamma globulin before the disease onset. Serological tests for detection of antibodies to lyssaviruses in cerebrospinal fluid of infected humans were typically used for diagnostic laboratory verification. Less than 30% IFA and PCR positives were obtained. Lyssaviruses were never detected. Only 4 convalescent patients were treated using experimental protocols. 80% cases demonstrated severe neurological consequences, four (may be more) patients died afterwards within the period from two months to four years. Different perspectives on prospects of Milwaukee protocol use and other therapeutic techniques are given.
Assuntos
Convalescença , Lyssavirus , Raiva , Animais , Cães , Encefalite Viral/diagnóstico , Encefalite Viral/epidemiologia , Encefalite Viral/genética , Encefalite Viral/terapia , Humanos , Lyssavirus/genética , Lyssavirus/metabolismo , Raiva/diagnóstico , Raiva/epidemiologia , Raiva/genética , Raiva/terapiaRESUMO
Phosphoprotein (P) and matrix protein (M) cooperate to undermine the immune response to rabies virus (RABV) infections. While P is involved in the modulation of the Jak-Stat pathway through the cytoplasmic retention of interferon (IFN)-activated STAT1 (pSTAT1), M interacts with the RelAp43-p105-ABIN2-TPL2 complex, to efficiently inhibit the nuclear factor-κB (NF-κB) pathway. Using transfections, protein-complementation assays, reverse genetics and DNA ChIP, we identified a role of M protein in the control of Jak-Stat signaling pathway, in synergy with the P protein. In unstimulated cells, both M and P proteins were found to interact with JAK1. Upon type-I IFN stimulation, the M switches toward pSTAT1 interaction, which results in an enhanced capacity of P protein to interact with pSTAT1 and restrain it in the cytoplasm. Furthermore, the role for M-protein positions 77, 100, 104 and 110 was also demonstrated in interaction with both JAK1 and pY-STAT1, and confirmed in vivo. Together, these data indicate that M protein cooperates with P protein to restrain in parallel, and sequentially, NF-κB and Jak-Stat pathways.
Assuntos
Lyssavirus/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/metabolismo , Animais , Citoplasma/metabolismo , Células HeLa , Humanos , Imunidade Inata , Interferon Tipo I/metabolismo , Janus Quinase 1/metabolismo , Lyssavirus/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , NF-kappa B/metabolismo , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Proteínas da Matriz Viral/genética , Proteínas Virais/genética , VirulênciaRESUMO
Rabies is an acute viral encephalomyelitis in warm-blooded vertebrates, caused by viruses belonging to Rhabdovirus family and genus Lyssavirus. Although rabies is categorised as a neglected disease, the rabies virus (RABV) is the most studied amongst Lyssaviruses which show nearly identical infection patterns. In efforts to improving post-exposure prophylaxis, several anti-rabies monoclonal antibodies (mAbs) targeting the glycoprotein (G protein) sites I, II, III and G5 have been characterized. To explore cross-neutralization capacity of available mAbs and discover new possible B-cell epitopes, we have analyzed all available glycoprotein sequences from Lyssaviruses with a focus on sequence variation and conservation. This information was mapped on the structure of a representative G protein. We proposed several possible cross-neutralizing B-cell epitopes (GUVTTTF, WLRTV, REECLD and EHLVVEEL) in complement to the already well-characterized antigenic sites. The research could facilitate development of novel cross-reactive mAbs against RABV and even more broad, against possibly all Lyssavirus members.
Assuntos
Reações Cruzadas/imunologia , Glicoproteínas/imunologia , Lyssavirus/imunologia , Lyssavirus/metabolismo , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/virologia , Proteínas Virais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Sequência Conservada , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Glicoproteínas/química , Glicoproteínas/genética , Lyssavirus/classificação , Lyssavirus/genética , Modelos Moleculares , Testes de Neutralização , Filogenia , Conformação Proteica , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
The oligomeric structure and the fusion activity of lyssavirus glycoprotein (G) was studied by comparing G from Mokola virus (GMok) and rabies virus (PV strain) (GPV), which are highly divergent lyssaviruses. G expressed at the surface of BSR cells upon either plasmid transfection or virus infection are shown to be mainly trimeric after cross-linking experiments. However, solubilization by a detergent (CHAPS) and analysis in sucrose sedimentation gradient evidenced that GMok trimer is less stable than GPV trimer. A chimeric glycoprotein (G Mok-PV) associating the N-terminal half of GMok to the C-terminal half part of GPV formed trimers with an intermediate stability, indicating that the G C-terminal domain is essential in trimer stability. A cell to cell fusion assay revealed that GMok (and not G Mok-PV) was able to induce fusion at a higher pH (0.5 pH unit) than GPV. Such differences in the oligomeric structure stability and in the fusion activity of lyssavirus glycoproteins may partly account for the previously reported differences of their immunogenic and pathogenic properties.
Assuntos
Antígenos Virais , Glicoproteínas/química , Lyssavirus/patogenicidade , Fusão de Membrana , Vírus da Raiva/patogenicidade , Proteínas do Envelope Viral/química , Animais , Fusão Celular , Linhagem Celular , Cricetinae , Dimerização , Glicoproteínas/metabolismo , Concentração de Íons de Hidrogênio , Lyssavirus/metabolismo , Vírus da Raiva/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteínas do Envelope Viral/metabolismoRESUMO
The glycoprotein G of lyssaviruses is the major determinant of virus pathogenicity and serves as a target for immunological responses to virus infections. However, assessment of the exact contribution of lyssavirus G proteins to observed differences in the pathogenicity of lyssavirus species is challenging, since the direct comparison of natural lyssaviruses does not allow specific ascription to individual virus proteins or domains. Here we describe the generation and characterization of recombinant rabies viruses (RABV) that express chimeric G proteins comprising of a RABV cytoplasma domain fused to transmembrane and ectodomain G sequences of a virulent RABV (challenge virus standard; CVS-11) or two European bat lyssaviruses (EBLV- and EBLV-2). These "envelope-switched" recombinant viruses were recovered from cDNAs. Similar growth kinetics and protein expression in neuroblastoma cell cultures and successful targeting of primary neurons showed that the chimeric G proteins were able to replace the authentic G protein in a RABV based virus vector. Inoculation of six week old CD-1 mice by the intracranial (i. c.) route of infection further demonstrated that all recombinant viruses were able to spread in the brain and to induce disease. The "envelope-switched" RABV therefore represent an important tool to further investigate the influence of lyssavirus ectodomains on virus tropism, and pathogenicity.
Assuntos
Glicoproteínas/genética , Lyssavirus/genética , Lyssavirus/metabolismo , Vírus da Raiva/genética , Vírus da Raiva/patogenicidade , Proteínas Virais/genética , Replicação Viral/genética , Animais , Antígenos Virais/metabolismo , Linhagem Celular , Células Cultivadas , Córtex Cerebral/virologia , Regulação da Expressão Gênica , Hipocampo/virologia , Imuno-Histoquímica , Camundongos , Neurônios/virologia , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Infecções por Rhabdoviridae/mortalidade , Infecções por Rhabdoviridae/virologiaRESUMO
By comparing three expression vectors for the rabies virus (Rv) minigenome, we show that the characteristic of the Rv RNA is important for efficient rescue despite its not being crucial for replication. Moreover, we show that the coexpression of the viral proteins from helper Rv and Mokola virus could rescue the Rv minigenome while Rv-related European bat lyssavirus 1 could not, suggesting that the signals controlling transcription and replication are conserved in the distantly related Rv and Mokola virus.
Assuntos
Vírus Defeituosos/genética , Vetores Genéticos , Genoma Viral , Lyssavirus/genética , Vírus da Raiva/metabolismo , Animais , DNA Complementar/genética , Vírus Auxiliares/genética , Vírus Auxiliares/metabolismo , Humanos , Lyssavirus/metabolismo , RNA Catalítico/metabolismo , RNA Viral/genética , Vírus da Raiva/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Lyssaviruses, the causative agents of rabies encephalitis, are distributed in seven genotypes. The phylogenetically distant rabies virus (PV strain, genotype 1) and Mokola virus (genotype 3) were used to develop a strategy to identify functional homologous interactive domains from two proteins (P and N) which participate in the viral ribonucleoprotein (RNP) transcription-replication complex. This strategy combined two-hybrid and green fluorescent protein-reverse two-hybrid assays in Saccharomyces cerevisiae to analyze protein-protein interactions and a reverse genetic assay in mammalian cells to study the transcriptional activity of the reconstituted RNP complex. Lyssavirus P proteins contain two N-binding domains (N-BDs), a strong one encompassing amino acid (aa) 176 to the C terminus and a weak one in the 189 N-terminal aa. The N-terminal portion of P (aa 52 to 189) also contains a homomultimerization site. Here we demonstrate that N-P interactions, although weaker, are maintained between proteins of the different genotypes. A minimal transcriptional module of the P protein was obtained by fusing the first 60 N-terminal aa containing the L protein binding site to the C-terminal strong N-BD. Random mutation of the strong N-BD on P protein identified three highly conserved K residues crucial for N-P interaction. Their mutagenesis in full-length P induced a transcriptionally defective RNP. The analysis of homologous interactive domains presented here and previously reported dissections of the P protein allowed us to propose a model of the functional interaction network of the lyssavirus P protein. This model underscores the central role of P at the interface between L protein and N-RNA template.
Assuntos
RNA Polimerases Dirigidas por DNA , Lyssavirus/metabolismo , Fosfoproteínas/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Genótipo , Lyssavirus/química , Lyssavirus/genética , Dados de Sequência Molecular , Mutação , Proteínas do Nucleocapsídeo/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , Ribonucleoproteínas/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Using a yeast two-hybrid human brain cDNA library screen, the cytoplasmic dynein light chain (LC8), a 10-kDa protein, was found to interact strongly with the phosphoprotein (P) of two lyssaviruses: rabies virus (genotype 1) and Mokola virus (genotype 3). The high degree of sequence divergence between these P proteins (only 46% amino acid identity) favors the hypothesis that this interaction is a common property shared by all lyssaviruses. The P protein-dynein LC8 interaction was confirmed by colocalization with laser confocal microscopy in infected cells and by coimmunoprecipitation. The dynein-interacting P protein domain was mapped to the 186 amino acid residues of the N-terminal half of the protein. Dynein LC8 is a component of both cytoplasmic dynein and myosin V, which are involved in a wide range of intracellular motile events, such as microtubule minus-end directed organelle transport in axon "retrograde transport" and actin-based vesicle transport, respectively. Our results provide support for a model of viral nucleocapsid axoplasmic transport. Furthermore, the role of LC8 in cellular mechanisms other than transport, e.g., inhibition of neuronal nitric oxide synthase, suggests that the P protein interactions could be involved in physiopathological mechanisms of rabies virus-induced pathogenesis.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila , Lyssavirus/metabolismo , Fosfoproteínas/metabolismo , Proteínas Virais/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular , Citoplasma/fisiologia , DNA Complementar , Dineínas , Biblioteca Gênica , Humanos , Lyssavirus/genética , Microscopia Confocal , Chaperonas Moleculares , Fosfoproteínas/genética , Testes de Precipitina , Vírus da Raiva/genética , Vírus da Raiva/metabolismo , Infecções por Rhabdoviridae/virologia , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismoRESUMO
Diagnosis of rabies is routinely confirmed by detection of rabies virus antigens in acetone-fixed frozen brain tissues or imprint smears using an immunofluorescence method with commercial antirabies virus antibodies. Since recent molecular analyses disclosed wide heterogeneity in the genome sequences of rabies virus strains and related lyssaviruses, it is necessary to confirm the presence of common epitopes in these lyssaviruses. In this study we confirmed the presence of cross-reactive antigens of various lyssaviruses in paraffin sections of formalin-fixed tissue using a monospecific rabbit antiserum prepared by immunization with a recombinant nucleoprotein of rabies virus. By immunohistochemical application, the antigen was detected predominantly in the cytoplasm of neurons in the brains of mice infected with rabies virus, Duvenhage virus, Mokola virus and European bat lyssavirus-1, while no cross-reaction was observed in uninfected humans and animals including dogs, bats, and raccoons. In addition, we examined one autopsy case that was infected in a rabies-endemic nation and developed the clinical manifestation of rabies after returning to Japan in 1970, and found that the antigen was well preserved in paraffin sections of formalin-fixed tissues. Thus, this suggests that the lyssavirus-specific antigen is recognized by the monospecific antibody against rabies virus nucleoprotein, and that this cross-reactive antigen is detectable on formalin-fixed paraffin-embedded tissues by immunohistochemical analysis.