Optical Control of Mononegavirus Gene Expression and Replication.

Mononegaviruses are promising tools as oncolytic and transgene vectors for gene therapy and regenerative medicine. However, when mononegaviruses are used for therapeutic applications, the viral activity must be strictly controlled due to concerns about toxicity and severe side effects. With this technology, mononegavirus vectors can be grown where they are intended and can be easily removed when they are no longer needed. In particular, a photoswitch protein called Magnet (consisting of two magnet domains) is incorporated into the hinge region between the connector and methyltransferase domains of the mononegavirus polymerase protein (L protein) to disrupt the L protein functions. Blue light (470 ± 20 nm) irradiation causes the dimerization of the two magnet domains, and the L protein is restored to activity, allowing viral gene expression and virus replication. Since the magnet domains' dimerization is reversible, viral gene expression and replication cease when blue light irradiation is stopped.

Rapid quantitative detection system for measles virus-neutralizing antibodies using HiBiT-tagged virus-like particles.

Immunological testing to detect neutralizing antibodies (NAbs) is important in measles (MV) infection control. Currently, the plaque reduction neutralization test is the only credible method for measuring actual virus NAbs; however, its feasibility is hampered by drawbacks, such as long turnaround times, low throughput, and the need for laboratory biosafety equipment. To solve these problems, we developed a simple and rapid MV-NAb detection system using lentivirus-based virus-like particles incorporated with the NanoLuc fragment peptide HiBiT comprising the MV fusion protein and hemagglutinin on their exterior surface. Overall, this simple, safe, and rapid method could be used to detect MV NAbs.

Generation of a photocontrollable recombinant bovine parainfluenza virus type 3.

Bovine parainfluenza virus type 3 (BPIV3) is a promising vaccine vector against various respiratory virus infections, including the human PIV3, respiratory syncytial virus, and severe acute respiratory syndrome-coronavirus 2 infections. In this study, we combined the Magnet system and reverse genetic approach to generate photocontrollable BPIV3. An optically controllable Magnet gene was inserted into the H2 region of the BPIV3 large protein gene, which encodes an RNA-dependent RNA polymerase. The generated photocontrollable BPIV3 grew in specific regions of the cell sheet only when illuminated with blue light, suggesting that spatiotemporal control can aid in safe clinical applications of BPIV3.

Essential role of TMPRSS2 in SARS-CoV-2 infection in murine airways.

In cultured cells, SARS-CoV-2 infects cells via multiple pathways using different host proteases. Recent studies have shown that the furin and TMPRSS2 (furin/TMPRSS2)-dependent pathway plays a minor role in infection of the Omicron variant. Here, we confirm that Omicron uses the furin/TMPRSS2-dependent pathway inefficiently and enters cells mainly using the cathepsin-dependent endocytosis pathway in TMPRSS2-expressing VeroE6/TMPRSS2 and Calu-3 cells. This is the case despite efficient cleavage of the spike protein of Omicron. However, in the airways of TMPRSS2-knockout mice, Omicron infection is significantly reduced. We furthermore show that propagation of the mouse-adapted SARS-CoV-2 QHmusX strain and human clinical isolates of Beta and Gamma is reduced in TMPRSS2-knockout mice. Therefore, the Omicron variant isn't an exception in using TMPRSS2 in vivo, and analysis with TMPRSS2-knockout mice is important when evaluating SARS-CoV-2 variants. In conclusion, this study shows that TMPRSS2 is critically important for SARS-CoV-2 infection of murine airways, including the Omicron variant.

Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells.

A novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused a large respiratory outbreak in Wuhan, China in December 2019, is currently spreading across many countries globally. Here, we show that a TMPRSS2-expressing VeroE6 cell line is highly susceptible to SARS-CoV-2 infection, making it useful for isolating and propagating SARS-CoV-2. Our results reveal that, in common with SARS- and Middle East respiratory syndrome-CoV, SARS-CoV-2 infection is enhanced by TMPRSS2.

Non-transmissible MV Vector with Segmented RNA Genome Establishes Different Types of iPSCs from Hematopoietic Cells.

Recent advances in gene therapy technologies have enabled the treatment of congenital disorders and cancers and facilitated the development of innovative methods, including induced pluripotent stem cell (iPSC) production and genome editing. We recently developed a novel non-transmissible and non-integrating measles virus (MV) vector capable of transferring multiple genes simultaneously into a wide range of cells through the CD46 and CD150 receptors. The MV vector expresses four genes for iPSC generation and the GFP gene for a period of time sufficient to establish iPSCs from human fibroblasts as well as peripheral blood T cells. The transgenes were expressed differentially depending on their gene order in the vector. Human hematopoietic stem/progenitor cells were directly and efficiently reprogrammed to naive-like cells that could proliferate and differentiate into primed iPSCs by the same method used to establish primed iPSCs from other cell types. The novel MV vector has several advantages for establishing iPSCs and potential future applications in gene therapy.

Biophysical characterization and single-chain Fv construction of a neutralizing antibody to measles virus.

The measles virus (MV) is a major cause of childhood morbidity and mortality worldwide. We previously established a mouse monoclonal antibody, 2F4, which shows high neutralizing titers against eight different genotypes of MV. However, the molecular basis for the neutralizing activity of the 2F4 antibody remains incompletely understood. Here, we have evaluated the binding characteristics of a Fab fragment of the 2F4 antibody. Using the MV infectious assay, we demonstrated that 2F4 Fab inhibits viral entry via either of two cellular receptors, SLAM and Nectin4. Surface plasmon resonance (SPR) analysis of recombinant proteins indicated that 2F4 Fab interacts with MV hemagglutinin (MV-H) with a KD value at the nm level. Furthermore, we designed a single-chain Fv fragment of 2F4 antibody as another potential biopharmaceutical to target measles. The stable 2F4 scFv was successfully prepared by the refolding method and shown to interact with MV-H at the µm level. Like 2F4 Fab, scFv inhibited receptor binding and viral entry. This indicates that 2F4 mAb uses the receptor-binding site and/or a neighboring region as an epitope with high affinity. These results provide insight into the neutralizing activity and potential therapeutic use of antibody fragments for MV infection.

Predominant Detection of the Subgroup A2b Human Metapneumovirus Strain with a 111-Nucleotide Duplication in the G gene in Yokohama City, Japan in 2018.

Human metapneumovirus (HMPV) has been a major causative agent of acute respiratory infections in humans. Recently, two types of variant A2b subtype HMPV strains possessing a 111- or 180-nucleotide duplication (nt-dup) in the G gene (HMPV A2b180nt-dup and HMPV A2b111nt-dup, respectively) were detected in Japan, Spain, Vietnam, and China. Our surveillance for infectious agents in Yokohama City, Japan revealed that the HMPV A2b111nt-dup strain became predominant in Yokohama City in 2018. In contrast, no classic HMPV A2b strain was detected after 2017. These data indicate a beneficial role of the 111nt-dup in the G gene for the transmission of HMPV.

Photocontrollable mononegaviruses.

Mononegaviruses are promising tools as oncolytic vectors and transgene delivery vectors for gene therapy and regenerative medicine. By using the Magnet proteins, which reversibly heterodimerize upon blue light illumination, photocontrollable mononegaviruses (measles and rabies viruses) were generated. The Magnet proteins were inserted into the flexible domain of viral polymerase, and viruses showed strong replication and oncolytic activities only when the viral polymerases were activated by blue light illumination.

Consensus and variations in cell line specificity among human metapneumovirus strains.

Human metapneumovirus (HMPV) has been a notable etiological agent of acute respiratory infection in humans, but it was not discovered until 2001, because HMPV replicates only in a limited number of cell lines and the cytopathic effect (CPE) is often mild. To promote the study of HMPV, several groups have generated green fluorescent protein (GFP)-expressing recombinant HMPV strains (HMPVGFP). However, the growing evidence has complicated the understanding of cell line specificity of HMPV, because it seems to vary notably among HMPV strains. In addition, unique A2b clade HMPV strains with a 180-nucleotide duplication in the G gene (HMPV A2b180nt-dup strains) have recently been detected. In this study, we re-evaluated and compared the cell line specificity of clinical isolates of HMPV strains, including the novel HMPV A2b180nt-dup strains, and six recombinant HMPVGFP strains, including the newly generated recombinant HMPV A2b180nt-dup strain, MG0256-EGFP. Our data demonstrate that VeroE6 and LLC-MK2 cells generally showed the highest infectivity with any clinical isolates and recombinant HMPVGFP strains. Other human-derived cell lines (BEAS-2B, A549, HEK293, MNT-1, and HeLa cells) showed certain levels of infectivity with HMPV, but these were significantly lower than those of VeroE6 and LLC-MK2 cells. Also, the infectivity in these suboptimal cell lines varied greatly among HMPV strains. The variations were not directly related to HMPV genotypes, cell lines used for isolation and propagation, specific genome mutations, or nucleotide duplications in the G gene. Thus, these variations in suboptimal cell lines are likely intrinsic to particular HMPV strains.

Analysis of VSV pseudotype virus infection mediated by rubella virus envelope proteins.

Rubella virus (RV) generally causes a systemic infection in humans. Viral cell tropism is a key determinant of viral pathogenesis, but the tropism of RV is currently poorly understood. We analyzed various human cell lines and determined that RV only establishes an infection efficiently in particular non-immune cell lines. To establish an infection the host cells must be susceptible and permissible. To assess the susceptibility of individual cell lines, we generated a pseudotype vesicular stomatitis virus bearing RV envelope proteins (VSV-RV/CE2E1). VSV-RV/CE2E1 entered cells in an RV envelope protein-dependent manner, and thus the infection was neutralized completely by an RV-specific antibody. The infection was Ca2+-dependent and inhibited by endosomal acidification inhibitors, further confirming the dependency on RV envelope proteins for the VSV-RV/CE2E1 infection. Human non-immune cell lines were mostly susceptible to VSV-RV/CE2E1, while immune cell lines were much less susceptible than non-immune cell lines. However, susceptibility of immune cells to VSV-RV/CE2E1 was increased upon stimulation of these cells. Our data therefore suggest that immune cells are generally less susceptible to RV infection than non-immune cells, but the susceptibility of immune cells is enhanced upon stimulation.

Heat Shock Protein 90 Ensures Efficient Mumps Virus Replication by Assisting with Viral Polymerase Complex Formation.

Paramyxoviral RNAs are synthesized by a viral RNA-dependent RNA polymerase (RdRp) consisting of the large (L) protein and its cofactor phosphoprotein (P protein). The L protein is a multifunctional protein that catalyzes RNA synthesis, mRNA capping, and mRNA polyadenylation. Growing evidence shows that the stability of several paramyxovirus L proteins is regulated by heat shock protein 90 (Hsp90). In this study, we demonstrated that Hsp90 activity was important for mumps virus (MuV) replication. The Hsp90 activity was required for L-protein stability and activity because an Hsp90-specific inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), destabilized the MuV L protein and suppressed viral RNA synthesis. However, once the L protein formed a mature polymerase complex with the P protein, Hsp90 activity was no longer required for the stability and activity of the L protein. When the Hsp90 activity was inhibited, the MuV L protein was degraded through the CHIP (C terminus of Hsp70-interacting protein)-mediated proteasomal pathway. High concentrations of 17-AAG showed strong cytotoxicity to certain cell types, but combined use of an Hsp70 inhibitor, VER155008, potentiated degradation of the L protein, allowing a sufficient reduction of 17-AAG concentration to block MuV replication with minimum cytotoxicity. Regulation of the L protein by Hsp90 and Hsp70 chaperones was also demonstrated for another paramyxovirus, the measles virus. Collectively, our data show that the Hsp90/Hsp70 chaperone machinery assists in the maturation of the paramyxovirus L protein and thereby in the formation of a mature RdRp complex and efficient viral replication.IMPORTANCE Heat shock protein 90 (Hsp90) is nearly universally required for viral protein homeostasis. Here, we report that Hsp90 activity is required for efficient propagation of mumps virus (MuV). Hsp90 functions in the maintenance of the catalytic subunit of viral polymerase, the large (L) protein, prior to formation of a mature polymerase complex with the polymerase cofactor of L, phosphoprotein. Hsp70 collaborates with Hsp90 to regulate biogenesis of the MuV L protein. The functions of these chaperones on the viral polymerase may be common among paramyxoviruses because the L protein of measles virus is also similarly regulated. Our data provide important insights into the molecular mechanisms of paramyxovirus polymerase maturation as well as a basis for the development of novel antiviral drugs.

[Measles Virus].

Measles virus (MeV) is exceptionally contagious and still a major cause of death in child.However, recently significant progress towards the elimination of measles has been made through increased vaccination coverage of measles-containing vaccines. The hemagglutinin (H) protein of MeV interacts with a cellular receptor, and this interaction is the first step of infection. MeV uses two different receptors, signaling lymphocyte activation molecule (SLAM) and nectin-4 expressed on immune cells and epithelial cells, respectively. The interactions of MeV with these receptors nicely explain the immune suppressive and high contagious properties of MeV. Binding of the H protein to a receptor triggers conformational changes in the fusion (F) protein, inducing fusion between viral and host plasma membranes for entry. The stalk region of the H protein plays a key role in the F protein-triggering. Recent studies of the H protein epitopes have revealed that the receptor binding site of the H protein constitutes a major neutralizing epitope. The interaction with two proteinaceous receptors probably imposes strong functional constraints on this epitope for amino acid changes. This would be a reason why measles vaccines, which are derived from MV strains isolated more than 60 years ago, are still highly effective against all MV strains currently circulating.

Erratum: Tahara, M., et al. Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability. Viruses 2016, 8, 216.

The authors wish to make the following change to their paper [1].[...].

Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability.

Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors.

Transmembrane serine protease TMPRSS2 activates hepatitis C virus infection.

UNLABELLED: The human liver reacts to hepatitis C virus (HCV) with a balanced response consisting of host anti- and proviral activities. To explore these subtle host responses, we used oligonucleotide microarrays to investigate the differential gene expression between two groups of liver samples with high and low HCV loads (>100-fold difference). We identified and validated 26 genes that were up-regulated in livers with high HCV loads, including transmembrane protease serine 2 (TMPRSS2). Trypsin inhibitors inhibited the infection of Huh7-25-CD81 cells with cell-culture-derived HCV (HCVcc) of Japanese fulminant hepatitis 1 isolate at the postbinding and entry step, and trypsin enhanced HCVcc infection at an early stage of infection. Several major transmembrane serine proteases, in particular, furin and hepsin, were detected in Huh7-25-CD81 cells, but TMPRSS2 was not. Huh7-25-CD81 cell clones stably expressing TMPRSS2- WT (wild type) and inactive TMPRSS2-mutant genes showed positive and negative enhancement of their susceptibility to HCVcc infection, respectively. The enhanced susceptibility of TMPRSS2-WT Huh7-25-CD81 cells was confirmed by knockdown of TMPRSS2 using small interfering RNA. The cell-surface protease activity of TMPRSS2-WT cells was markedly active in the cleavage of QAR and QGR, corresponding to amino acid residues at P3 to P1. CONCLUSION: The cell-surface activity of a trypsin-like serine protease, such as TMPRSS2, activates HCV infection at the postbinding and entry stage. Host transmembrane serine proteases may be involved in the sensitivity, persistence, and pathogenesis of HCV infection and be possible targets for antiviral therapy.

The host protease TMPRSS2 plays a major role in in vivo replication of emerging H7N9 and seasonal influenza viruses.

UNLABELLED: Proteolytic cleavage of the hemagglutinin (HA) protein is essential for influenza A virus (IAV) to acquire infectivity. This process is mediated by a host cell protease(s) in vivo. The type II transmembrane serine protease TMPRSS2 is expressed in the respiratory tract and is capable of activating a variety of respiratory viruses, including low-pathogenic (LP) IAVs possessing a single arginine residue at the cleavage site. Here we show that TMPRSS2 plays an essential role in the proteolytic activation of LP IAVs, including a recently emerged H7N9 subtype, in vivo. We generated TMPRSS2 knockout (KO) mice. The TMPRSS2 KO mice showed normal reproduction, development, and growth phenotypes. In TMPRSS2 KO mice infected with LP IAVs, cleavage of HA was severely impaired, and consequently, the majority of LP IAV progeny particles failed to gain infectivity, while the viruses were fully activated proteolytically in TMPRSS2+/+ wild-type (WT) mice. Accordingly, in contrast to WT mice, TMPRSS2 KO mice were highly tolerant of challenge infection by LP IAVs (H1N1, H3N2, and H7N9) with ≥1,000 50% lethal doses (LD50) for WT mice. On the other hand, a high-pathogenic H5N1 subtype IAV possessing a multibasic cleavage site was successfully activated in the lungs of TMPRSS2 KO mice and killed these mice, as observed for WT mice. Our results demonstrate that recently emerged H7N9 as well as seasonal IAVs mainly use the specific protease TMPRSS2 for HA cleavage in vivo and, thus, that TMPRSS2 expression is essential for IAV replication in vivo. IMPORTANCE: Influenza A virus (IAV) is a leading pathogen that infects and kills many humans every year. We clarified that the infectivity and pathogenicity of IAVs, including a recently emerged H7N9 subtype, are determined primarily by a host protease, TMPRSS2. Our data showed that TMPRSS2 is the key host protease that activates IAVs in vivo through proteolytic cleavage of their HA proteins. Hence, TMPRSS2 is a good target for the development of anti-IAV drugs. Such drugs could also be effective for many other respiratory viruses, including the recently emerged Middle East respiratory syndrome (MERS) coronavirus, because they are also activated by TMPRSS2 in vitro. Consequently, the present paper could have a large impact on the battle against respiratory virus infections and contribute greatly to human health.

The MyD88 pathway in plasmacytoid and CD4+ dendritic cells primarily triggers type I IFN production against measles virus in a mouse infection model.

Infection by measles virus (MV) induces type I IFN via the retinoic acid-inducible gene I/melanoma differentiation-associated gene 5/mitochondrial antiviral signaling protein (MAVS) pathway in human cells. However, the in vivo role of the MAVS pathway in host defense against MV infection remains undetermined. CD150 transgenic (Tg) mice, which express human CD150, an entry receptor for MV, with the disrupting IFNR gene (Ifnar(-/-)), are susceptible to MV and serve as a model for MV infection. In this study, we generated CD150Tg/Mavs(-/-) mice and examined MV permissiveness compared with that in CD150Tg/Ifnar(-/-) mice. MV replicated mostly in the spleen of i.p.-infected CD150Tg/Ifnar(-/-) mice. Strikingly, CD150Tg/Mavs(-/-) mice were not permissive to MV in vivo because of substantial type I IFN induction. MV barely replicated in any other organs tested. When T cells, B cells, and dendritic cells (DCs) isolated from CD150Tg/Mavs(-/-) splenocytes were cultured with MV in vitro, only the DCs produced type I IFN. In vitro infection analysis using CD150Tg/Mavs(-/-) DC subsets revealed that CD4(+) and plasmacytoid DCs, but not CD8α(+) and CD8α(-)CD4(-) double negative DCs, were exclusively involved in type I IFN production in response to MV infection. Because CD150Tg/Mavs(-/-) mice turned permissive to MV by anti-IFNAR Ab, type I IFN produced by CD4(+) DCs and plasmacytoid DCs plays a critical role in antiviral protection for neighboring cells expressing IFNAR. Induction of type I IFN in these DC subsets was abolished by the MyD88 inhibitory peptide. Thus, production of type I IFN occurs via the MyD88-dependent and MAVS-independent signaling pathway during MV infection.

TMPRSS2 is an activating protease for respiratory parainfluenza viruses.

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 associated with a lethal outbreak in monkeys can readily adapt to use human receptors.

A canine distemper virus (CDV) strain, CYN07-dV, associated with a lethal outbreak in monkeys, used human signaling lymphocyte activation molecule as a receptor only poorly but readily adapted to use it following a P541S substitution in the hemagglutinin protein. Since CYN07-dV had an intrinsic ability to use human nectin-4, the adapted virus became able to use both human immune and epithelial cell receptors, as well as monkey and canine ones, suggesting that CDV can potentially infect humans.