Hideo Fukuhara

Nectin4 Is an Epithelial Cell Receptor for Canine Distemper Virus and Involved in Neurovirulence

ABSTRACT Canine distemper virus (CDV) uses signaling lymphocyte activation molecule (SLAM), expressed on immune cells, as a receptor. However, epithelial and neural cells are also affected by CDV in vivo. Wild-type CDV strains showed efficient replication with syncytia in Vero cells expressing dog nectin4, and the infection was blocked by an anti-nectin4 antibody. In dogs with distemper, CDV antigen was preferentially detected in nectin4-positive neurons and epithelial cells, suggesting that nectin4 is an epithelial cell receptor for CDV and also involved in its neurovirulence.

Functional and Structural Characterization of Neutralizing Epitopes of Measles Virus Hemagglutinin Protein

ABSTRACT Effective vaccination programs have dramatically reduced the number of measles-related deaths globally. Although all the available data suggest that measles eradication is biologically feasible, a structural and biochemical basis for the single serotype nature of measles virus (MV) remains to be provided. The hemagglutinin (H) protein, which binds to two discrete proteinaceous receptors, is the major neutralizing target. Monoclonal antibodies (MAbs) recognizing distinct epitopes on the H protein were characterized using recombinant MVs encoding the H gene from different MV genotypes. The effects of various mutations on neutralization by MAbs and virus fitness were also analyzed, identifying the location of five epitopes on the H protein structure. Our data in the present study demonstrated that the H protein of MV possesses at least two conserved effective neutralizing epitopes. One, which is a previously recognized epitope, is located near the receptor-binding site (RBS), and thus MAbs that recognize this epitope blocked the receptor binding of the H protein, whereas the other epitope is located at the position distant from the RBS. Thus, a MAb that recognizes this epitope did not inhibit the receptor binding of the H protein, rather interfered with the hemagglutinin-fusion (H-F) interaction. This epitope was suggested to play a key role for formation of a higher order of an H-F protein oligomeric structure. Our data also identified one nonconserved effective neutralizing epitope. The epitope has been masked by an N-linked sugar modification in some genotype MV strains. These data would contribute to our understanding of the antigenicity of MV and support the global elimination program of measles.

Molecular Basis for LLT1 Protein Recognition by Human CD161 Protein (NKRP1A/KLRB1)

Human Th17 cells express high levels of CD161, a member of the killer cell lectin-like receptor (KLR) family (also referred to as NK receptor-P1A (NKRP1A) or KLRB1), as a representative marker. CD161 is also expressed on natural killer (NK) cells and NKT cells. Lectin-like transcript 1 (LLT1), another KLR family member, was recently identified as a ligand for CD161. This interaction may play pivotal roles in the immunomodulatory functions of Th17 cells as well as those of NK and NKT cells. However, the molecular basis for the interaction is poorly understood. Here we show that the extracellular domain of CD161 bound directly to LLT1 with a Kd of 48 μm and with the fast kinetics typical of cell-cell recognition receptors. Mutagenesis revealed that the similar membrane-distal β-sheet and loop regions of both CD161 and LLT1 were utilized for the binding, and notably, these regions correspond to the ligand-binding sites for major histocompatibility complex (MHC)-recognizing KLRs. Furthermore, we found a pair of detrimental mutations for both molecules that restored the binding. These results reveal a new template model for the recognition mode between the KLR family members and provide insights into the molecular mechanism underlying Th17/NK/NKT-mediated immune responses.

The Receptor-Binding Site of the Measles Virus Hemagglutinin Protein Itself Constitutes a Conserved Neutralizing Epitope

ABSTRACT Here, we provide direct evidence that the receptor-binding site of measles virus (MV) hemagglutinin protein itself forms an effective conserved neutralizing epitope (CNE). Several receptor-interacting residues constitute the CNE. Thus, viral escape from neutralization has to be associated with loss of receptor-binding activity. Since interactions with both the signaling lymphocyte activation molecule (SLAM) and nectin4 are critical for MV pathogenesis, its escape, which results from loss of receptor-binding activity, should not occur in nature.

TMPRSS2 Is an Activating Protease for Respiratory Parainfluenza Viruses

ABSTRACT Here, we show that human parainfluenza viruses and Sendai virus (SeV), like other respiratory viruses, use TMPRSS2 for their activation. The membrane fusion proteins of respiratory viruses often possess serine and glutamine residues at the P2 and P3 positions, respectively, but these residues were not critical for cleavage by TMPRSS2. However, mutations of these residues affected SeV growth in specific epithelial cell lines, suggesting the importance of these residues for SeV replication in epithelia.

Canine distemper virus with the intact C protein has the potential to replicate in human epithelial cells by using human nectin4 as a receptor.

Recent outbreaks in monkeys have proven that canine distemper virus (CDV) causes diseases in a wide range of mammals. CDV uses SLAM and nectin4 as receptors to replicate in susceptible animals. Here, we show that human nectin4, but not human SLAM, is fully functional as a CDV receptor. The CDV Ac96I strain hardly replicated in nectin4-expressing human epithelial NCI-H358 cells, but readily adapted to grow in them. Unsurprisingly, no amino acid change in the H protein was required for the adaptation. The original Ac96I strain possessed a truncated C protein, and a subpopulation possessing the intact C protein was selected after growth in NCI-H358 cells. Other CDV strains possessing the intact C protein showed significantly higher growth abilities in NCI-H358 cells than the Ac96I strain with the truncated C protein. These findings suggest that the C protein is functional in human epithelial cells and critical for CDV replication in them.

Crystal structure of extracellular domain of human lectin‐like transcript 1 (LLT1), the ligand for natural killer receptor‐P1A

Emerging evidence has revealed the pivotal roles of C‐type lectin‐like receptors (CTLRs) in the regulation of a wide range of immune responses. Human natural killer cell receptor‐P1A (NKRP1A) is one of the CTLRs and recognizes another CTLR, lectin‐like transcript 1 (LLT1) on target cells to control NK, NKT and Th17 cells. The structural basis for the NKRP1A‐LLT1 interaction was limitedly understood. Here, we report the crystal structure of the ectodomain of LLT1. The plausible receptor‐binding face of the C‐type lectin‐like domain is flat, and forms an extended β‐sheet. The residues of this face are relatively conserved with another CTLR, keratinocyte‐associated C‐type lectin, which binds to the CTLR member, NKp65. A LLT1‐NKRP1A complex model, prepared using the crystal structures of LLT1 and the keratinocyte‐associated C‐type lectin‐NKp65 complex, reasonably satisfies the charge consistency and the conformational complementarity to explain a previous mutagenesis study. Furthermore, crystal packing and analytical ultracentrifugation revealed dimer formation, which supports a complex model. Our results provide structural insights for understanding the binding modes and signal transduction mechanisms, which are likely to be conserved in the CTLR family, and for further rational drug design towards regulating the LLT1 function.

Silkworm Baculovirus Expression System for Molecular Medicine

The characterization of immunologically or biologically relevant proteins is prerequisite for the development of effective drugs or therapies. Since large amounts of proteins are often required for this purpose, more effective expression systems for recombinant human proteins must be developed. Among the various expression systems, the baculovirus expression system (BEVS) is an attractive tool with certain advantages for high-level expression and post-translational modifications. The silkworm BEVS utilizes silkworm larvae or pupae as bioreactors programmed by recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) or bacmid DNA. Recombinant human protein production is generally higher in silkworm BEVS than insect cell line BEVS. In addition, transgenic silkworm technology has been developed for stable recombinant protein expression. In this review, we introduce the use of this silkworm BEVS for two human proteins that are potential targets for drug development, the ectodomains of immunoreceptors (i.e. IL-4Ra, IL13Ra, KIR2DL1 and Fas) and guanine nucleotide-binding protein (G-protein) coupled receptors (GPCR) (i.e. μ-opioid receptor and nociceptin receptor). These proteins were successfully expressed in the silkworm BEVS, showing the advantages in both the expression level and simpler manipulation, as compared to other systems. The silkworm BEVS is expected to be convenient for recombinant protein production for molecular medical studies.

Establishment of the BacMam system using silkworm baculovirus.

The BacMam system uses modified insect viruses (baculoviruses) as vehicles to efficiently deliver genes for expression in mammalian cells. The technique can be widely applied to large-scale recombinant protein production with appropriate modifications, high-throughput screening platforms for cell-based assays, and the delivery of large genes. The silkworm system is often employed as a rapid and cost-effective approach for recombinant baculovirus generation. Here we have developed the novel BacMam system using silkworm baculovirus, and shown the successful expression of EGFP in mammalian cells. The transduction to mammalian cells via the BacMam system was improved by adding phosphate-buffered saline and sodium butyrate to the culture medium and lowering the temperature after viral infection. This study provides an alternative gene delivery system for mammalian cells, which has various potential applications, including efficient native protein production and gene therapy.

The Role of Heparan Sulfate Proteoglycans as an Attachment Factor for Rabies Virus Entry and Infection

Rabies virus (RABV) is the causative agent of fatal neurological disease. Cellular attachment is the initial and essential step for viral infections. Although extensive studies have demonstrated that RABV uses various target cell molecules to mediate infection, no specific molecule has been identified as an attachment factor for RABV infection. Here we demonstrate that cellular heparan sulfate (HS) supports RABV adhesion and subsequent entry into target cells. Enzymatic removal of HS reduced cellular susceptibility to RABV infection, and heparin, a highly sulfated form of HS, blocked viral adhesion and infection. The direct binding between RABV glycoprotein and heparin was demonstrated, and this interaction was shown to require HS N- and 6-O-sulfation. We also revealed that basic amino acids in the ectodomain of RABV glycoprotein serve as major determinants for the RABV-HS interaction. Collectively, our study highlights a previously undescribed role of HS as an attachment factor for RABV infection.