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1.
J Virol ; 98(5): e0041624, 2024 May 14.
Article in English | MEDLINE | ID: mdl-38624232

ABSTRACT

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.


Subject(s)
Antibodies, Neutralizing , Antibodies, Viral , COVID-19 , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/chemistry , SARS-CoV-2/immunology , SARS-CoV-2/genetics , Humans , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/virology , Epitopes/immunology , Cryoelectron Microscopy , Protein Domains , COVID-19 Vaccines/immunology
2.
J Infect Public Health ; 16(7): 1064-1072, 2023 Jul.
Article in English | MEDLINE | ID: mdl-37196370

ABSTRACT

BACKGROUND: Omicron variants with immune evasion have emerged, and they continue to mutate rapidly, raising concerns about the weakening of vaccine efficacy, and the very elderly populations are vulnerable to Coronavirus Disease 2019 (COVID-19). Therefore, to investigate the effect of multiple doses of mRNA vaccine for the newly emerged variants on these populations, cross-neutralizing antibody titers were examined against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants, including BQ.1.1 and XBB. METHODS: Blood samples were taken from residents at four long-term care facilities in Hyogo prefecture, Japan (median age, 91 years), after 3rd (n = 67) and 4th (n = 48) mRNA vaccinations, from April to October 2022. A live virus microneutralization assay was performed to determine the neutralizing antibody titers in participants' sera. RESULTS: After 3rd vaccination, cross-neutralizing antibody prevalence against conventional (D614G) virus, Delta, Omicron BA.2, BA.5, BA.2.75, BQ.1.1, and XBB were 100%, 97%, 81%, 51%, 67%, 4%, and 21%, respectively. After 4th vaccination, the antibody positivity rates increased to 100%, 100%, 98%, 79%, 92%, 31%, and 52%, respectively. The 4th vaccination significantly increased cross-neutralizing antibody titers against all tested variants. CONCLUSION: The positivity rates for BQ.1.1 and XBB increased after 4th vaccination, although the titer value was lower than those of BA.5 and BA.2.75. Considering the rapid mutation of viruses and the efficacy of vaccines, it may be necessary to create a system that can develop vaccines suitable for each epidemic in consideration of the epidemic of the virus.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Aged , Aged, 80 and over , SARS-CoV-2/genetics , COVID-19/prevention & control , Broadly Neutralizing Antibodies , Vaccination , RNA, Messenger , Antibodies, Viral
3.
J Virol ; 97(6): e0028623, 2023 06 29.
Article in English | MEDLINE | ID: mdl-37191569

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal , Antibodies, Viral , COVID-19 , Epitopes , Animals , Cricetinae , Humans , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/virology , Epitopes/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Male , Female , Middle Aged , mRNA Vaccines
5.
Cell Commun Signal ; 20(1): 95, 2022 06 21.
Article in English | MEDLINE | ID: mdl-35729616

ABSTRACT

BACKGROUND: Viruses must adapt to the environment of their host cells to establish infection and persist. Diverse mammalian cells, including virus-infected cells, release extracellular vesicles such as exosomes containing proteins and miRNAs, and use these vesicles to mediate intercellular communication. However, the roles of exosomes in viral infection remain unclear. RESULTS: We screened viral proteins to identify those responsible for the exosome-mediated enhancement of Epstein-Barr virus (EBV) infection. We identified BGLF2 protein encapsulated in exosomes, which were released by EBV-infected cells. BGLF2 protein is a tegument protein that exists in the space between the envelope and nucleocapsid, and it is released into the cytoplasm shortly after infection. BGLF2 protein-containing exosomes enhanced viral gene expression and repressed innate immunity, thereby supporting the EBV infection. CONCLUSIONS: The EBV tegument protein BGLF2 is encapsulated in exosomes and released by infected cells to facilitate the establishment of EBV infection. These findings suggest that tegument proteins support viral infection not only between the envelope and nucleocapsid, as well as in extraviral particles such as exosomes. Video abstract.


Subject(s)
Epstein-Barr Virus Infections , Exosomes , Animals , Epstein-Barr Virus Infections/genetics , Epstein-Barr Virus Infections/metabolism , Exosomes/metabolism , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/metabolism , Mammals/metabolism , Viral Fusion Proteins , Viral Proteins
6.
Sci Rep ; 11(1): 22919, 2021 11 25.
Article in English | MEDLINE | ID: mdl-34824296

ABSTRACT

CD81 is an integral membrane protein of the tetraspanin family and forms complexes with a variety of other cell surface membrane proteins. CD81 is involved in cell migration and B cell activation. However, the mechanism of the transcriptional regulation of the CD81 gene remains unclear. Here, we revealed that CD81 transcriptional activation was required for binding of the transcription factor Pax5 at the Pax5-binding sequence (-54)GCGGGAC(-48) located upstream of the transcriptional start site (TSS) of the CD81 gene. The reporter assay showed that the DNA sequence between - 130 and - 39 bp upstream of the TSS of the CD81 gene had promoter activity for CD81 transcription. The DNA sequence between - 130 and - 39 bp upstream of TSS of CD81 harbors two potential Pax5-binding sequences (-87)GCGTGAG(-81) and (-54)GCGGGAC(-48). Reporter, electrophoresis mobility shift, and chromatin immunoprecipitation (ChIP) assays disclosed that Pax5 bound to the (-54)GCGGGAC(-48) in the promoter region of the CD81 gene in order to activate CD81 transcription. Pax5 overexpression increased the expression level of CD81 protein, while the Pax5-knockdown by shRNA decreased CD81 expression. Moreover, we found that the expression level of CD81 was positively correlated with Pax5 expression in human tumor cell lines. Because CD81 was reported to be involved in cell migration, we evaluated the effects of Pax5 overexpression by wound healing and transwell assays. The data showed that overexpression of either Pax5 or CD81 promoted the epithelial cell migration. Thus, our findings provide insights into the transcriptional mechanism of the CD81 gene through transcription factor Pax5.


Subject(s)
Neoplasms/metabolism , PAX5 Transcription Factor/metabolism , Promoter Regions, Genetic , Tetraspanin 28/metabolism , Transcriptional Activation , A549 Cells , Binding Sites , Cell Movement , Gene Expression Regulation, Neoplastic , HEK293 Cells , HeLa Cells , Humans , MCF-7 Cells , Neoplasm Invasiveness , Neoplasms/genetics , Neoplasms/pathology , PAX5 Transcription Factor/genetics , Protein Binding , Tetraspanin 28/genetics
7.
Sci Rep ; 10(1): 6671, 2020 04 21.
Article in English | MEDLINE | ID: mdl-32317666

ABSTRACT

Herpes simplex virus 1 (HSV-1) causes a number of clinical manifestations including cold sores, keratitis, meningitis and encephalitis. Although current drugs are available to treat HSV-1 infection, they can cause side effects such as nephrotoxicity. Moreover, owing to the emergence of drug-resistant HSV-1 strains, new anti-HSV-1 compounds are needed. Because many viruses exploit cellular host proteases and encode their own viral proteases for survival, we investigated the inhibitory effects of a panel of protease inhibitors (TLCK, TPCK, E64, bortezomib, or MG132) on HSV-1 replication and several host cell signaling pathways. We found that HSV-1 infection suppressed c-Raf-MEK1/2-ERK1/2-p90RSK signaling in host cells, which facilitated viral replication. The mechanism by which HSV-1 inhibited ERK signaling was mediated through the polyubiquitination and proteasomal degradation of Ras-guanine nucleotide-releasing factor 2 (Ras-GRF2). Importantly, the proteasome inhibitor MG132 inhibited HSV-1 replication by reversing ERK suppression in infected cells, inhibiting lytic genes (ICP5, ICP27 and UL42) expression, and overcoming the downregulation of Ras-GRF2. These results indicate that the suppression of ERK signaling via proteasomal degradation of Ras-GRF2 is necessary for HSV-1 infection and replication. Given that ERK activation by MG132 exhibits anti-HSV-1 activity, these results suggest that the proteasome inhibitor could serve as a novel therapeutic agent against HSV-1 infection.


Subject(s)
Antiviral Agents/pharmacology , Herpesvirus 1, Human/drug effects , Leupeptins/pharmacology , MAP Kinase Signaling System/drug effects , Animals , Bortezomib/pharmacology , Caspases/metabolism , Chlorocebus aethiops , DNA Replication/drug effects , Enzyme Activation/drug effects , Gene Expression Regulation, Viral/drug effects , Hep G2 Cells , Humans , Leucine/analogs & derivatives , Leucine/pharmacology , Models, Biological , NF-KappaB Inhibitor alpha/metabolism , NF-kappa B/metabolism , Phosphorylation/drug effects , Polyubiquitin/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Protein Stability/drug effects , Proteolysis/drug effects , Tosyllysine Chloromethyl Ketone/pharmacology , Tosylphenylalanyl Chloromethyl Ketone/pharmacology , Vero Cells , Virus Replication/drug effects
8.
Biol Pharm Bull ; 43(3): 540-545, 2020 Mar 01.
Article in English | MEDLINE | ID: mdl-31902824

ABSTRACT

CD81 is a highly conserved four-transmembrane protein in mammals and widely expressed on many tissues. It belongs to the tetraspanin family and forms complexes with various cell surface membrane proteins. It also functions in cell migration and B-cell activation, which is induced by CD81 complexing with CD19, CD21 and the B-cell receptor. Thus, CD81 is thought to play a key role in regulating cell function and fate. However, little is known about the degradation mechanism of CD81. Here we found that CD81 on the plasma membrane is degraded by the lysosome pathway via endocytosis. The expression levels of CD81 in HEK293T cells treated with a proteasome inhibitor (lactacystin) and lysosome inhibitors (chloroquine and bafilomycin A1) were analyzed by flow cytometry. The expression of CD81 on the cell surface was increased by the lysosome inhibitors, but not lactacystin. A pulldown assay revealed that CD81 was conjugated with a K63- and K29-linked poly-ubiquitin chain before its degradation, and the poly-ubiquitination site was Lys8 at the N-terminal intracellular domain of CD81. Furthermore, mutant CD81, in which Lys8 was substituted with alanine (Ala), extended the CD81 half-life compared with wildtype. CD81 was mainly localized on the plasma membrane in normal cells, but also co-localized with lysosomal LAMP1 and early endosomal EEA1 in chloroquine-treated cells. Furthermore, a clathrin-mediated endocytosis inhibitor, chlorpromazine, stabilized CD81 expression on the cell surface. Hence, we demonstrated that CD81 is internalized by clathrin-mediated endocytosis and subsequently degraded via a lysosome pathway requiring the K63- and K29-linked poly-ubiquitination of CD81.


Subject(s)
Clathrin/metabolism , Lysosomes/metabolism , Polyubiquitin/metabolism , Tetraspanin 28/metabolism , Cell Membrane , Endocytosis , HEK293 Cells , Humans , Membrane Proteins/metabolism , Proteolysis , Ubiquitination
9.
Biol Pharm Bull ; 42(12): 2109-2112, 2019.
Article in English | MEDLINE | ID: mdl-31787726

ABSTRACT

Primary effusion lymphoma (PEL) is a rare subtype of non-Hodgkin's B-cell lymphoma and is caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. PEL is an aggressive lymphoma and is frequently resistant to conventional chemotherapies. Sulforaphane (SFN), a natural compound found in cruciferous vegetables and broccoli sprouts, modulates signaling pathways and epigenetic gene expression. However, the anti-proliferative effects of SFN on PEL cells and the underlying mechanisms have not been identified. Here, we found that SFN decreased the viability of KSHV-infected PEL cells compared to KSHV-uninfected B-lymphoma cells. The anti-proliferative effects of SFN on PEL cells were mediated by apoptosis with activating caspases. In addition, SFN inhibited the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and AKT in PEL cells. We also showed that p38MAPK and AKT inhibitors reduced PEL cell growth. The constitutive and/or transient activation of p38MAPK and AKT signaling are necessary for the survival and proliferation of PEL cells. Our data and previous literature indicate that SFN represses the phosphorylation of p38MAPK and AKT, which results in PEL cell apoptosis. Moreover, we investigated whether MG132 or sangivamycin (Sangi) in combination with SFN potentiated the cytotoxic effects of SFN on PEL cells. Compared to treatment with SFN alone, the addition of MG132 or Sangi enhanced the cytotoxic activity of SFN in a synergistic manner. In conclusion, the anti-proliferative effects of SFN indicate its potential as a new substance for the treatment of PEL.


Subject(s)
Antineoplastic Agents/pharmacology , Isothiocyanates/pharmacology , Leupeptins/pharmacology , Lymphoma, Primary Effusion/drug therapy , Pyrimidine Nucleosides/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Drug Synergism , Humans , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Sulfoxides , p38 Mitogen-Activated Protein Kinases/metabolism
10.
J Nat Med ; 72(2): 439-447, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29288328

ABSTRACT

Nigella species are rich source of dolabellane diterpenes. During our study of Nigella species, new dolabellane diterpenes, damasterpenes V-VIII were isolated. The structural determination of new compounds damasterpenes V-VIII is described with consideration of their absolute configurations. The antiviral activities against herpes simplex virus type-1 of the isolated compounds and their derivatives are also evaluated. Damasterpene V (inhibition 35.0%) and 2-phenylacetyl 13-benzoyl damasterpenol (32.0%) showed significant antiviral activity at 10 µM.


Subject(s)
Antiviral Agents/therapeutic use , Diterpenes/chemistry , Herpesvirus 1, Human/drug effects , Nigella damascena/chemistry , Seeds/chemistry , Antiviral Agents/pharmacology
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