RESUMO
The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued, enabling the virus to escape from host immunity by changing its spike antigen, while biased toward the receptor-binding domain and N-terminal domain. Here, we isolated a novel pan-SARS-CoV-2 neutralizing antibody (which we named MO11) for even the recent dominators XBB.1.16 and EG.5.1, from a convalescent patient who had received three doses of an original mRNA COVID-19 vaccination. A cryo-electron microscopy analysis of the spike-MO11 complex at 2.3 Å atomic resolution revealed that it recognizes a conserved epitope hidden behind a glycan shield at N331 on subdomain 1 (SD1), holding both the N- and C-terminal segments comprising SD1. Our identification of MO11 unveiled the functional importance of SD1 for the spike's function, and we discuss the potential availability of a novel common epitope among the SARS-CoV-2 variants.IMPORTANCENovel severe acute respiratory syndrome coronavirus 2 variants with immune evasion ability are still repeatedly emerging, nonetheless, a part of immunity developed in responding to the antigen of earlier variants retains efficacy against recent variants irrespective of the numerous mutations. In exploration for the broadly effective antibodies, we identified a cross-neutralizing antibody, named MO11, from the B cells of the convalescent patient. MO11 targets a novel epitope in subdomain 1 (SD1) and was effective against all emerging variants including XBB.1.16 and EG.5.1. The neutralizing activity covering from D614G to EG.5.1 variants was explained by the conservation of the epitope, and it revealed the importance of the subdomain on regulating the function of the antigen for viral infection. Demonstrated identification of the neutralizing antibody that recognizes a conserved epitope implies basal contribution of such group of antibodies for prophylaxis against COVID-19.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Humanos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , Epitopos/imunologia , Microscopia Crioeletrônica , Domínios Proteicos , Vacinas contra COVID-19/imunologiaRESUMO
Nascent nucleocapsids of herpesviruses acquire a primary envelope during their nuclear export by budding through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes. This process is mediated by a conserved viral heterodimeric complex designated the nuclear egress complex, which consists of the nuclear matrix protein and the nuclear membrane protein. In addition to its essential roles during nuclear egress, the nuclear matrix protein has been shown to interact with intracellular signaling pathway molecules including NF-κB and IFN-ß to affect viral or cellular gene expression. The human herpesvirus 6A (HHV-6A) U37 gene encodes a nuclear matrix protein, the role of which has not been analyzed. Here, we show that HHV-6A U37 activates the heat shock element promoter and induces the accumulation of the molecular chaperone Hsp90. Mechanistically, HHV-6A U37 interacts with heat shock transcription factor 1 (HSF1) and induces its phosphorylation at Ser-326. We report that pharmacological inhibition of HSF1, Hsp70, or Hsp90 decreases viral protein accumulation and viral replication. Taken together, our results lead us to propose a model in which HHV-6A U37 activates the heat shock response to support viral gene expression and replication. IMPORTANCE Human herpesvirus 6A (HHV-6A) is a dsDNA virus belonging to the Roseolovirus genus within the Betaherpesvirinae subfamily. It is frequently found in patients with neuroinflammatory disease, although its pathogenetic role, if any, awaits elucidation. The heat shock response is important for cell survival under stressful conditions that disrupt homeostasis. Our results indicate that HHV-6A U37 activates the heat shock element promoter and leads to the accumulation of heat shock proteins. Next, we show that the heat shock response is important for viral replication. Overall, our findings provide new insights into the function of HHV-6A U37 in host cell signaling and identify potential cellular targets involved in HHV-6A pathogenesis and replication.
Assuntos
Fatores de Transcrição de Choque Térmico , Resposta ao Choque Térmico , Herpesvirus Humano 6 , Proteínas da Matriz Viral , Humanos , Fatores de Transcrição de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , Herpesvirus Humano 6/metabolismo , Herpesvirus Humano 6/patogenicidade , Proteínas da Matriz Viral/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Regiões Promotoras Genéticas , Replicação Viral , Fosforilação , Regulação Viral da Expressão Gênica , Transdução de SinaisRESUMO
We identified neutralizing monoclonal antibodies against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) variants (including Omicron variants BA.5 and BA.2.75) from individuals who received two doses of mRNA vaccination after they had been infected with the D614G virus. We named them MO1, MO2, and MO3. Among them, MO1 showed particularly high neutralizing activity against authentic variants: D614G, Delta, BA.1, BA.1.1, BA.2, BA.2.75, and BA.5. Furthermore, MO1 suppressed BA.5 infection in hamsters. A structural analysis revealed that MO1 binds to the conserved epitope of seven variants, including Omicron variants BA.5 and BA.2.75, in the receptor-binding domain of the spike protein. MO1 targets an epitope conserved among Omicron variants BA.1, BA.2, and BA.5 in a unique binding mode. Our findings confirm that D614G-derived vaccination can induce neutralizing antibodies that recognize the epitopes conserved among the SARS-CoV-2 variants. IMPORTANCE Omicron variants of SARS-CoV-2 acquired escape ability from host immunity and authorized antibody therapeutics and thereby have been spreading worldwide. We reported that patients infected with an early SARS-CoV-2 variant, D614G, and who received subsequent two-dose mRNA vaccination have high neutralizing antibody titer against Omicron lineages. It was speculated that the patients have neutralizing antibodies broadly effective against SARS-CoV-2 variants by targeting common epitopes. Here, we explored human monoclonal antibodies from B cells of the patients. One of the monoclonal antibodies, named MO1, showed high potency against broad SARS-CoV-2 variants including BA.2.75 and BA.5 variants. The results prove that monoclonal antibodies that have common neutralizing epitopes among several Omicrons were produced in patients infected with D614G and who received mRNA vaccination.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , COVID-19 , Epitopos , Animais , Cricetinae , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , Epitopos/imunologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Masculino , Feminino , Pessoa de Meia-Idade , Vacinas de mRNARESUMO
BACKGROUND: Although pure GAA expansion is considered pathogenic in SCA27B, non-GAA repeat motif is mostly mixed into longer repeat sequences. This study aimed to unravel the complete sequencing of FGF14 repeat expansion to elucidate its repeat motifs and pathogenicity. METHODS: We screened FGF14 repeat expansion in a Japanese cohort of 460 molecularly undiagnosed adult-onset cerebellar ataxia patients and 1022 controls, together with 92 non-Japanese controls, and performed nanopore sequencing of FGF14 repeat expansion. RESULTS: In the Japanese population, the GCA motif was predominantly observed as the non-GAA motif, whereas the GGA motif was frequently detected in non-Japanese controls. The 5'-common flanking variant was observed in all Japanese GAA repeat alleles within normal length, demonstrating its meiotic stability against repeat expansion. In both patients and controls, pure GAA repeat was up to 400 units in length, whereas non-pathogenic GAA-GCA repeat was larger, up to 900 units, but they evolved from different haplotypes, as rs534066520, located just upstream of the repeat sequence, completely discriminated them. Both (GAA)≥250 and (GAA)≥200 were enriched in patients, whereas (GAA-GCA)≥200 was similarly observed in patients and controls, suggesting the pathogenic threshold of (GAA)≥200 for cerebellar ataxia. We identified 14 patients with SCA27B (3.0%), but their single-nucleotide polymorphism genotype indicated different founder alleles between Japanese and Caucasians. The low prevalence of SCA27B in Japanese may be due to the lower allele frequency of (GAA)≥250 in the Japanese population than in Caucasians (0.15% vs 0.32%-1.26%). CONCLUSIONS: FGF14 repeat expansion has unique features of pathogenicity and allelic origin, as revealed by a single ethnic study.
RESUMO
During the nuclear export of nascent nucleocapsids of herpesviruses, the nucleocapsids bud through the inner nuclear membrane (INM) by acquiring the INM as a primary envelope (primary envelopment). We recently reported that herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), which consists of UL34 and UL31, interacts with an endosomal sorting complex required for transport III (ESCRT-III) adaptor ALIX and recruits ESCRT-III machinery to the INM for efficient primary envelopment. In this study, we identified a cluster of six arginine residues in the disordered domain of UL34 as a minimal region required for the interaction with ALIX, as well as the recruitment of ALIX and an ESCRT-III protein CHMP4B to the INM in HSV-1-infected cells. Mutations in the arginine cluster exhibited phenotypes similar to those with ESCRT-III inhibition reported previously, including the mislocalization of NEC, induction of membranous invagination structures containing enveloped virions, aberrant accumulation of enveloped virions in the invaginations and perinuclear space, and reduction of viral replication. We also showed that the effect of the arginine cluster in UL34 on HSV-1 replication was dependent primarily on ALIX. These results indicated that the arginine cluster in the disordered domain of UL34 was required for the interaction with ALIX and the recruitment of ESCRT-III machinery to the INM to promote primary envelopment. IMPORTANCE Herpesvirus UL34 homologs contain conserved amino-terminal domains that mediate vesicle formation through interactions with UL31 homologs during primary envelopment. UL34 homologs also comprise other domains adjacent to their membrane-anchoring regions, which differ in length, are variable in herpesviruses, and do not form distinguished secondary structures. However, the role of these disordered domains in infected cells remains to be elucidated. In this study, we present data suggesting that the arginine cluster in the disordered domain of HSV-1 UL34 mediates the interaction with ALIX, thereby leading to the recruitment of ESCRT-III machinery to the INM for efficient primary envelopment. This is the first study to report the role of the disordered domain of a UL34 homolog in herpesvirus infections.
Assuntos
Arginina , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Herpesvirus Humano 1/fisiologia , Proteínas Virais/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HeLa , Humanos , Morfogênese , Mutação , Membrana Nuclear/metabolismo , Nucleocapsídeo/metabolismo , Fosforilação , Proteínas Virais/química , Proteínas Virais/genética , Vírion/crescimento & desenvolvimento , Liberação de Vírus , Replicação ViralRESUMO
The stimulus-induced cAMP response element (CRE)-binding protein (CREB) family of transcription factors bind to CREs to regulate diverse cellular responses, including proliferation, survival, and differentiation. Human herpesvirus 6A (HHV-6A), which belongs to the Betaherpesvirinae subfamily, is a lymphotropic herpesvirus frequently found in patients with neuroinflammatory diseases. Previous reports implicated the importance of CREs in the HHV-6A life cycle, although the effects of the binding of transcription factors to CREs in viral replication have not been fully elucidated. In this study, we analyzed the role of the CREB family of transcription factors during HHV-6A replication. We found that HHV-6A infection enhanced phosphorylation of the CREB family members CREB1 and activating transcription factor 1 (ATF1). Knockout (KO) of CREB1 or ATF1 enhanced viral gene expression and viral replication. The increase in viral yields in supernatants from ATF1-KO cells was greater than that in supernatants from CREB1-KO cells. Transcriptome sequencing (RNA-seq) analysis showed that sensors of the innate immune system were downregulated in ATF1-KO cells, and mRNAs of beta interferon (IFN-ß) and IFN-regulated genes were reduced in these cells infected with HHV-6A. IFN-ß treatment of ATF1-KO cells reduced progeny viral yields significantly, suggesting that the enhancement of viral replication was caused by a reduction of IFN-ß. Taken together, our results suggest that ATF1 is activated during HHV-6A infection and restricts viral replication via IFN-ß induction. IMPORTANCE Human herpesvirus 6A (HHV-6A) is a ubiquitous herpesvirus implicated in Alzheimer's disease, although its role in its pathogenesis has not been confirmed. Here, we showed that the transcription factor ATF1 restricts HHV-6A replication, mediated by IFN-ß induction. Our study provides new insights into the role of ATF1 in innate viral immunity and reveals the importance of IFN-ß for regulation of HHV-6A replication, which possibly impairs HHV-6A pathogenesis.
Assuntos
Fator 1 Ativador da Transcrição , Herpesvirus Humano 6 , Interferon beta , Replicação Viral , Fator 1 Ativador da Transcrição/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Técnicas de Inativação de Genes , Herpesvirus Humano 6/fisiologia , Humanos , Interferon beta/genéticaRESUMO
Varicella-zoster virus-specific cell-mediated immunity has been associated with the onset and severity of herpes zoster (HZ), and the administration of the HZ vaccine enhanced the immunity. However, limited data is available on the duration of cell-mediated immunity enhancement by soluble antigen of varicella-zoster virus (VZV) skin test. A prospective, community-based cohort study was conducted in Shozu County, Kagawa Prefecture, Japan. Repeated VZV skin tests containing inactivated VZV antigen and blood tests were performed on 365 subjects aged 60 years and older at baseline, 1, 2, and 3 years later. The differential immunity indices of VZV over time for cell-mediated and humoral immunity were evaluated. VZV skin test reaction and ELISpot counts increased significantly at 1, 2, and 3 years later compared to the baseline. However, humoral immunity indices did not change materially over time. Soluble antigen by VZV skin test enhanced VZV-specific cell-mediated immunity, and it persisted for at least 1 year. In addition, the inoculation with inactivated antigens every year by VZV skin test continued to enhance VZV-specific cell-mediated immunity after 2 and 3 years.
Assuntos
Herpes Zoster , Herpesvirus Humano 3 , Humanos , Pessoa de Meia-Idade , Idoso , Estudos de Coortes , Estudos Prospectivos , Imunidade Celular , Testes CutâneosRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant omicron is now under investigation. We evaluated cross-neutralizing activity against omicron in coronavirus disease 2019 (COVID-19) convalescent patients (n = 23) who had received 2 doses of an mRNA vaccination (BNT162b2 or mRNA-1273). Intriguingly, after the second vaccination, the neutralizing antibody titers of subjects against SARS-CoV-2 variants, including omicron, all became seropositive, and significant fold-increases (21.1-52.0) were seen regardless of the disease severity. Our findings thus demonstrate that 2 doses of mRNA vaccination to SARS-CoV-2 convalescent patients can induce cross-neutralizing activity against omicron.
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacina BNT162 , COVID-19/prevenção & controle , Humanos , Testes de Neutralização , RNA Mensageiro , VacinaçãoRESUMO
Sialic acid immunoglobulin-like lectin (Siglec) family molecules are immune regulatory receptors that bind to specific molecules containing sialic acids. Varicella-zoster virus (VZV), a member of the herpesvirus family, infects hematopoietic cells and spreads throughout the body, causing chickenpox, shingles, and, sometimes fatal encephalomyelitis. However, the cellular entry receptors that are required for VZV to infect hematopoietic cells have remained unclear. Here, we found that Siglec-7, mainly expressed on hematopoietic cells, binds to VZV envelope glycoprotein B in a sialic acid-dependent manner. Furthermore, Siglec-7 mediated VZV infection by inducing membrane fusion. Our findings provide the first evidence for a molecular mechanism by which VZV infects hematopoietic cells.
Assuntos
Antígenos de Diferenciação Mielomonocítica , Varicela , Herpes Zoster , Lectinas , Antígenos de Diferenciação Mielomonocítica/metabolismo , Herpesvirus Humano 3 , Humanos , Lectinas/metabolismo , Ácido N-Acetilneuramínico , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Proteínas do Envelope ViralRESUMO
Varicella-zoster virus (VZV) first infects hematopoietic cells, with the infected cells then acting to distribute the virus throughout the body. Sialic acid-binding immunoglobulin-like lectin (Siglec) family molecules recognize sialic acid-containing molecules on the same cell surface, called cis-ligands, or molecules on other cells or soluble agents, called trans-ligands. Among the Siglec family molecules, Siglec-4 and Siglec-7 mediate VZV infection through association with glycoprotein B (gB). As Siglec-7, but not Siglec-4, is expressed on hematopoietic cells such as monocytes, the regulatory mechanism by which Siglec-7 associates with gB is important to our understanding of VZV infection of blood cells. Here, we found that Siglec-7 is required for VZV to infect human primary monocytes. Furthermore, treatment of primary monocytes with sialidase enhanced both VZV gB binding to monocytes and VZV infectivity. Calcium influx in primary monocytes decreased the expression of Siglec-7 cis-ligands and increased VZV infectivity. These results demonstrate that the Siglec-7 cis-ligands present on primary monocytes play an important role in VZV infection through regulation of the interaction between gB and Siglec-7.
Assuntos
Antígenos de Diferenciação Mielomonocítica , Herpesvirus Humano 3 , Lectinas , Monócitos , Antígenos de Diferenciação Mielomonocítica/metabolismo , Herpesvirus Humano 3/fisiologia , Humanos , Lectinas/metabolismo , Ligantes , Monócitos/virologia , Ácido N-Acetilneuramínico , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Infecção pelo Vírus da Varicela-Zoster/metabolismo , Infecção pelo Vírus da Varicela-Zoster/virologiaRESUMO
Viral infection induces host cells to mount a variety of immune responses, which may either limit viral propagation or create conditions conducive to virus replication in some instances. In this regard, activation of the NF-κB transcription factor is known to modulate virus replication. Human herpesvirus 6A (HHV-6A), which belongs to the Betaherpesvirinae subfamily, is frequently found in patients with neuroinflammatory diseases, although its role in disease pathogenesis has not been elucidated. In this study, we found that the HHV-6A-encoded U14 protein activates NF-κB signaling following interaction with the NF-κB complex protein, p65. Through induction of nuclear translocation of p65, U14 increases the expression of interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein 1 transcripts. We also demonstrated that activation of NF-κB signaling is important for HHV-6A replication, since inhibition of this pathway reduced virus protein accumulation and viral genome copy number. Taken together, our results suggest that HHV-6A infection activates the NF-κB pathway and promotes viral gene expression via late gene products, including U14. IMPORTANCE Human herpesvirus 6A (HHV-6A) is frequently found in patients with neuro-inflammation, although its role in the pathogenesis of this disease has not been elucidated. Most viral infections activate the NF-κB pathway, which causes the transactivation of various genes, including those encoding proinflammatory cytokines. Our results indicate that HHV-6A U14 activates the NF-κB pathway, leading to upregulation of proinflammatory cytokines. We also found that activation of the NF-κB transcription factor is important for efficient viral replication. This study provides new insight into HHV-6A U14 function in host cell signaling and identifies potential cellular targets involved in HHV-6A pathogenesis and replication.
Assuntos
Herpesvirus Humano 6/genética , Herpesvirus Humano 6/metabolismo , NF-kappa B/metabolismo , Infecções por Roseolovirus/imunologia , Transdução de Sinais/imunologia , Proteínas Virais/imunologia , Linhagem Celular , Regulação da Expressão Gênica , Genes Virais , Genoma Viral , Humanos , NF-kappa B/genética , Doenças Neuroinflamatórias , Receptor EphB2 , Proteínas Virais/genética , Replicação ViralRESUMO
Like all herpesviruses, the roseoloviruses (HHV6A, -6B, and -7) establish lifelong infection within their host, requiring these viruses to evade host antiviral responses. One common host-evasion strategy is the downregulation of host-encoded, surface-expressed glycoproteins. Roseoloviruses have been shown to evade the host immune response by downregulating NK-activating ligands, class I MHC, and the TCR/CD3 complex. To more globally identify glycoproteins that are differentially expressed on the surface of HHV6A-infected cells, we performed cell surface capture of N-linked glycoproteins present on the surface of T cells infected with HHV6A, and compared these to proteins present on the surface of uninfected T cells. We found that the protein tyrosine phosphatase CD45 is downregulated in T cells infected with HHV6A. We also demonstrated that CD45 is similarly downregulated in cells infected with HHV7. CD45 is essential for signaling through the T cell receptor and, as such, is necessary for developing a fully functional immune response. Interestingly, the closely related betaherpesviruses human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) have also separately evolved unique mechanisms to target CD45. While HCMV and MCMV target CD45 signaling and trafficking, HHV6A acts to downregulate CD45 transcripts. IMPORTANCE Human herpesviruses-6 and -7 infect essentially 100% of the world's population before the age of 5 and then remain latent or persistent in their host throughout life. As such, these viruses are among the most pervasive and stealthy of all viruses. Host immune cells rely on the presence of surface-expressed proteins to identify and target virus-infected cells. Here, we investigated the changes that occur to proteins expressed on the cell surface of T cells after infection with human herpesvirus-6A. We discovered that HHV-6A infection results in a reduction of CD45 on the surface of infected T cells and impaired activation in response to T cell receptor stimulation.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 6/genética , Herpesvirus Humano 7/genética , Antígenos Comuns de Leucócito/genética , Linfócitos T/virologia , Linhagem Celular , Regulação para Baixo , Células HEK293 , Herpesvirus Humano 6/metabolismo , Herpesvirus Humano 7/metabolismo , Humanos , Estabilidade Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Human herpesvirus 6A (HHV-6A) and HHV-6B use different cellular receptors, human CD46 and CD134, respectively and have different cell tropisms although they have 90% similarity at the nucleotide level. An important feature that characterizes HHV-6A/6B is the glycoprotein H (gH)/gL/gQ1/gQ2 complex (a tetramer) that each virus has specifically on its envelope. Here, to determine which molecules in the tetramer contribute to the specificity for each receptor, we developed a cell-cell fusion assay system for HHV-6A and HHV-6B that uses the cells expressing CD46 or CD134. With this system, when we replaced the gQ1 or gQ2 of HHV-6A with that of HHV-6B in the tetramer, the cell fusion activity mediated by glycoproteins via CD46 was lower than that done with the original-type tetramer. When we replaced the gQ1 or the gQ2 of HHV-6A with that of HHV-6B in the tetramer, the cell fusion mediated by glycoproteins via CD134 was not seen. In addition, we generated two types of C-terminal truncation mutants of HHV-6A gQ2 (AgQ2) to examine the interaction domains of HHV-6A gQ1 (AgQ1) and AgQ2. We found that amino acid residues 163 to 185 in AgQ2 are important for interaction of AgQ1 and AgQ2. Finally, to investigate whether HHV-6B gQ2 (BgQ2) can complement AgQ2, an HHV-6A genome harboring BgQ2 was constructed. The mutant could not produce an infectious virus, indicating that BgQ2 cannot work for the propagation of HHV-6A. These results suggest that gQ2 supports the tetramer's function, and the combination of gQ1 and gQ2 is critical for virus propagation.IMPORTANCE Glycoprotein Q2 (gQ2), an essential gene for virus propagation, forms a heterodimer with gQ1. The gQ1/gQ2 complex has a critical role in receptor recognition in the gH/gL/gQ1/gQ2 complex (a tetramer). We investigated whether gQ2 regulates the specific interaction between the HHV-6A or -6B tetramer and CD46 or CD134. We established a cell-cell fusion assay system for HHV-6A/6B and switched the gQ1 or gQ2 of HHV-6A with that of HHV-6B in the tetramer. Although cell fusion was induced via CD46 when gQ1 or gQ2 was switched between HHV-6A and -6B, the activity was lower than that of the original combination. When gQ1 or gQ2 was switched in HHV-6A and -6B, no cell fusion was observed via CD134. HHV-6B gQ2 could not complement the function of HHV-6A's gQ2 in HHV-6A propagation, suggesting that the combination of gQ1 and gQ2 is critical to regulate the specificity of the tetramer's function for virus entry.
RESUMO
Viral cell-to-cell spread, a method employed by several viral families for entrance via cell junctions, is highly relevant to the pathogenesis of various viral infections. Cell-to-cell spread of herpes simplex virus 1 (HSV-1) is known to depend greatly on envelope glycoprotein E (gE). However, the molecular mechanism by which gE acts in HSV-1 cell-to-cell spread and the mechanisms of cell-to-cell spread by other herpesviruses remain poorly understood. Here, we describe our identification of prohibitin-1 as a novel gE-interacting host cell protein. Ectopic expression of prohibitin-1 increased gE-dependent HSV-1 cell-to-cell spread. As observed with the gE-null mutation, decreased expression or pharmacological inhibition of prohibitin-1 reduced HSV-1 cell-to-cell spread without affecting the yield of virus progeny. Similar effects were produced by pharmacological inhibition of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, wherein prohibitin-1 acts as a protein scaffold and is required for induction of this pathway. Furthermore, artificial activation of the MAPK/ERK pathway restored HSV-1 cell-to-cell spread impaired by the gE-null mutation. Notably, pharmacological inhibition of prohibitins or the MAPK/ERK pathway reduced viral cell-to-cell spread of representative members in all herpesvirus subfamilies. Our results suggest that prohibitin-1 contributes to gE-dependent HSV-1 cell-to-cell spread via the MAPK/ERK pathway and that this mechanism is conserved throughout the Herpesviridae, whereas gE is conserved only in the Alphaherpesvirinae subfamily.IMPORTANCE Herpesviruses are ubiquitous pathogens of various animals, including humans. These viruses primarily pass through cell junctions to spread to uninfected cells. This method of cell-to-cell spread is an important pathogenic characteristic of these viruses. Here, we show that the host cell protein prohibitin-1 contributes to HSV-1 cell-to-cell spread via a downstream intracellular signaling cascade, the MAPK/ERK pathway. We also demonstrate that the role of the prohibitin-1-mediated MAPK/ERK pathway in viral cell-to-cell spread is conserved in representative members of every herpesvirus subfamily. This study has revealed a common molecular mechanism of the cell-to-cell spread of herpesviruses.
Assuntos
Comunicação Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Repressoras/metabolismo , Proteínas do Envelope Viral/metabolismo , Células A549 , MAP Quinases Reguladas por Sinal Extracelular/genética , Herpes Simples/genética , Herpes Simples/metabolismo , Humanos , Junções Intercelulares , Proteínas Quinases Ativadas por Mitógeno/genética , Proibitinas , Proteínas Repressoras/genética , Proteínas do Envelope Viral/genética , Replicação ViralRESUMO
A unique glycoprotein is expressed on the virus envelope of human herpesvirus 6B (HHV-6B): the complex gH/gL/gQ1/gQ2 (hereafter referred to as the HHV-6B tetramer). This tetramer recognizes a host receptor expressed on activated T cells: human CD134 (hCD134). This interaction is essential for HHV-6B entry into the susceptible cells and is a determinant for HHV-6B cell tropism. The structural mechanisms underlying this unique interaction were unknown. Herein we solved the interactions between the HHV-6B tetramer and the receptor by using their neutralizing antibodies in molecular and structural analyses. A surface plasmon resonance analysis revealed fast dissociation/association between the tetramer and hCD134, although the affinity was high (KD = 18 nM) and comparable to those for the neutralizing antibodies (anti-gQ1: 17 nM, anti-gH: 2.7 nM). A competition assay demonstrated that the anti-gQ1 antibody competed with hCD134 in the HHV-6B tetramer binding whereas the anti-gH antibody did not, indicating the direct interaction of gQ1 and hCD134. A single-particle analysis by negative-staining electron microscopy revealed the tetramer's elongated shape with a gH/gL part and extra density corresponding to gQ1/gQ2. The anti-gQ1 antibody bound to the tip of the extra density, and anti-gH antibody bound to the putative gH/gL part. These results highlight the interaction of gQ1/gQ2 in the HHV-6B tetramer with hCD134, and they demonstrate common features among viral ligands of the betaherpesvirus subfamily from a macroscopic viewpoint.
Assuntos
Herpesvirus Humano 6/metabolismo , Receptores OX40/metabolismo , Infecções por Roseolovirus/metabolismo , Proteínas do Envelope Viral/metabolismo , HumanosRESUMO
Primary infection of human herpesvirus 6B (HHV-6B) occurs in infants after the decline of maternal immunity and causes exanthema subitum accompanied by a high fever, and it occasionally develops into encephalitis resulting in neurological sequelae. There is no effective prophylaxis for HHV-6B, and its development is urgently needed. The glycoprotein complex gH/gL/gQ1/gQ2 (called 'tetramer of HHV-6B') on the virion surface is a viral ligand for its cellular receptor human CD134, and their interaction is thus essential for virus entry into the cells. Herein we examined the potency of the tetramer as a vaccine candidate against HHV-6B. We designed a soluble form of the tetramer by replacing the transmembrane domain of gH with a cleavable tag, and the tetramer was expressed by a mammalian cell expression system. The expressed recombinant tetramer is capable of binding to hCD134. The tetramer was purified to homogeneity and then administered to mice with aluminum hydrogel adjuvant and/or CpG oligodeoxynucleotide adjuvant. After several immunizations, humoral and cellular immunity for HHV-6B was induced in the mice. These results suggest that the tetramer together with an adjuvant could be a promising candidate HHV-6B vaccine.
Assuntos
Exantema Súbito/imunologia , Vacinas contra Herpesvirus/imunologia , Proteínas do Envelope Viral/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Exantema Súbito/virologia , Herpesvirus Humano 6 , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Coronavirus disease 2019 (COVID-19) is a global health problem that causes millions of deaths worldwide. The clinical manifestation of COVID-19 widely varies from asymptomatic infection to severe pneumonia and systemic inflammatory disease. It is thought that host genetic variability may affect the host's response to the virus infection and thus cause severity of the disease. The SARS-CoV-2 virus requires interaction with its receptor complex in the host cells before infection. The transmembrane protease serine 2 (TMPRSS2) has been identified as one of the key molecules involved in SARS-CoV-2 virus receptor binding and cell invasion. Therefore, in this study, we investigated the correlation between a genetic variant within the human TMPRSS2 gene and COVID-19 severity and viral load. RESULTS: We genotyped 95 patients with COVID-19 hospitalised in Dr Soetomo General Hospital and Indrapura Field Hospital (Surabaya, Indonesia) for the TMPRSS2 p.Val160Met polymorphism. Polymorphism was detected using a TaqMan assay. We then analysed the association between the presence of the genetic variant and disease severity and viral load. We did not observe any correlation between the presence of TMPRSS2 genetic variant and the severity of the disease. However, we identified a significant association between the p.Val160Met polymorphism and the SARS-CoV-2 viral load, as estimated by the Ct value of the diagnostic nucleic acid amplification test. Furthermore, we observed a trend of association between the presence of the C allele and the mortality rate in patients with severe COVID-19. CONCLUSION: Our data indicate a possible association between TMPRSS2 p.Val160Met polymorphism and SARS-CoV-2 infectivity and the outcome of COVID-19.
Assuntos
COVID-19/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único , SARS-CoV-2/isolamento & purificação , Serina Endopeptidases/genética , Adulto , Alelos , COVID-19/diagnóstico , COVID-19/virologia , Estudos Transversais , Feminino , Frequência do Gene , Genótipo , Humanos , Indonésia , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/fisiologia , Carga Viral/genéticaRESUMO
We followed 45 participants in Surabaya, Indonesia, for 10 months and compared their PCR and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) results. As much as 13 out of 45 participants were IgG seropositive at least once while the remaining 32 stayed IgG seronegative throughout the study. Among 13 seropositive participants, 9 were consecutively seropositive at least twice and were eligible for IgG longevity evaluation. The duration of IgG detection varied from 47 to ≥233 days. We observed intermittent re-positive PCR results suggestive of viral shedding in participants with a longer duration of IgG detection.
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Humanos , Imunoglobulina G , Imunoglobulina M , Eliminação de Partículas ViraisRESUMO
BACKGROUND: The impact of body mass index on incidence of herpes zoster is unclear. This study investigated whether body mass index was associated with a history of herpes zoster and incidence during a 3-year follow-up, using data from a prospective cohort study in Japan. METHODS: In total, 12,311 individuals were included in the cross-sectional analysis at baseline, of whom 1,818 with a history of herpes zoster were excluded from the incidence analysis, leaving 10,493 individuals. Body mass index (kg/m2) was classified into three categories (underweight: <18.5; normal: 18.5 to <25; and overweight: ≥25). To evaluate the risk of herpes zoster, we used a logistic regression model for prevalence and a Cox proportional hazard regression model for incidence. RESULTS: Being overweight or underweight was not associated with herpes zoster prevalence at baseline. The multivariate hazard ratios of herpes zoster incidence for overweight versus normal-weight groups were 0.67 (95% confidence interval, 0.51-0.90) in all participants, and 0.57 (95% confidence interval, 0.39-0.83) in women, with no significant difference for men. CONCLUSION: Being overweight was associated with a lower incidence of herpes zoster than being normal weight in older Japanese women.
Assuntos
Herpes Zoster , Sobrepeso , Idoso , Índice de Massa Corporal , Estudos Transversais , Feminino , Herpes Zoster/complicações , Herpes Zoster/epidemiologia , Humanos , Incidência , Japão/epidemiologia , Masculino , Sobrepeso/epidemiologia , Estudos Prospectivos , MagrezaRESUMO
BACKGROUND: Although COVID-19 severity in cancer patients is high, the safety and immunogenicity of the BNT162b2 mRNA COVID-19 vaccine in patients undergoing chemotherapy for solid cancers in Japan have not been reported. METHODS: We investigated the safety and immunogenicity of BNT162b2 in 41 patients undergoing chemotherapy for solid cancers and in healthy volunteers who received 2 doses of BNT162b2. We evaluated serum IgG antibody titers for S1 protein by ELISA at pre-vaccination, prior to the second dose and 14 days after the second vaccination in 24 cancer patients undergoing cytotoxic chemotherapy (CC group), 17 cancer patients undergoing immune checkpoint inhibitor therapy (ICI group) and 12 age-matched healthy volunteers (HV group). Additionally, inflammatory cytokine levels were compared between the HV and ICI groups at pre and the next day of each vaccination. RESULTS: Anti-S1 antibody levels were significantly lower in the ICI and CC groups than in the HV group after the second dose (median optimal density: 0.241 [0.063-1.205] and 0.161 [0.07-0.857] vs 0.644 [0.259-1.498], p = 0.0024 and p < 0.0001, respectively). Adverse effect profile did not differ among the three groups, and no serious adverse event occurred. There were no differences in vaccine-induced inflammatory cytokines between the HV and ICI groups. CONCLUSION: Although there were no significant differences in adverse events in three groups, antibody titers were significantly lower in the ICI and CC groups than in the HV group. Further protection strategies should be considered in cancer patients undergoing CC or ICI.