Yuichiro Nakatsu

The Matrix Protein of Measles Virus Regulates Viral RNA Synthesis and Assembly by Interacting with the Nucleocapsid Protein

ABSTRACT The genome of measles virus (MV) is encapsidated by the nucleocapsid (N) protein and associates with RNA-dependent RNA polymerase to form the ribonucleoprotein complex. The matrix (M) protein is believed to play an important role in MV assembly by linking the ribonucleoprotein complex with envelope glycoproteins. Analyses using a yeast two-hybrid system and coimmunoprecipitation in mammalian cells revealed that the M protein interacts with the N protein and that two leucine residues at the carboxyl terminus of the N protein (L523 and L524) are critical for the interaction. In MV minigenome reporter gene assays, the M protein inhibited viral RNA synthesis only when it was able to interact with the N protein. The N protein colocalized with the M protein at the plasma membrane when the proteins were coexpressed in plasmid-transfected or MV-infected cells. In contrast, the N protein formed small dots in the perinuclear area when it was expressed without the M protein, or it was incapable of interacting with the M protein. Furthermore, a recombinant MV possessing a mutant N protein incapable of interacting with the M protein grew much less efficiently than the parental virus. Since the M protein has an intrinsic ability to associate with the plasma membrane, it may retain the ribonucleoprotein complex at the plasma membrane by binding to the N protein, thereby stopping viral RNA synthesis and promoting viral particle production. Consequently, our results indicate that the M protein regulates MV RNA synthesis and assembly via its interaction with the N protein.

Long Untranslated Regions of the Measles Virus M and F Genes Control Virus Replication and Cytopathogenicity

ABSTRACT Measles is still a major cause of mortality mainly in developing countries. The causative agent, measles virus (MeV), is an enveloped virus having a nonsegmented negative-sense RNA genome, and belongs to the genus Morbillivirus of the family Paramyxoviridae. One feature of the moribillivirus genomes is that the M and F genes have long untranslated regions (UTRs). The M and F mRNAs of MeV have 426-nucleotide-long 3′ and 583-nucleotide-long 5′ UTRs, respectively. Though these long UTRs occupy as much as ∼6.4% of the virus genome, their function remains unknown. To elucidate the role of the long UTRs in the context of virus infection, we used the reverse genetics based on the virulent strain of MeV, and generated a series of recombinant viruses having alterations or deletions in the long UTRs. Our results showed that these long UTRs per se were not essential for MeV replication, but that they regulated MeV replication and cytopathogenicity by modulating the productions of the M and F proteins. The long 3′ UTR of the M mRNA was shown to have the ability to increase the M protein production, promoting virus replication. On the other hand, the long 5′ UTR of the F mRNA was found to possess the capacity to decrease the F protein production, inhibiting virus replication and yet greatly reducing cytopathogenicity. We speculate that the reduction in cytopathogenicity may be advantageous for MeV fitness and survival in nature.

Both RIG-I and MDA5 RNA Helicases Contribute to the Induction of Alpha/Beta Interferon in Measles Virus-Infected Human Cells

ABSTRACT Measles virus (MV), a member of the family Paramyxoviridae, is a nonsegmented negative-strand RNA virus. The RNA helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are differentially involved in the detection of cytoplasmic viral RNAs and induction of alpha/beta interferon (IFN-α/β). RIG-I is generally believed to play a major role in the recognition of paramyxoviruses, whereas many viruses of this family produce V proteins that can inhibit MDA5. To determine the individual roles of MDA5 and RIG-I in IFN induction after MV infection, small interfering RNA-mediated knockdown of MDA5 or RIG-I was performed in the human epithelial cell line H358, which is susceptible to wild-type MV isolates. The production of IFN-β mRNA in response to MV infection was greatly reduced in RIG-I knockdown clones compared to that in H358 cells, confirming the importance of RIG-I in the detection of MV. The IFN-β mRNA levels were also moderately reduced in MDA5 knockdown clones, even though these clones retained fully functional RIG-I. A V protein-deficient recombinant MV (MVΔV) induced higher amounts of IFN-β mRNA at the early stage of infection in H358 cells compared to the parental virus. The reductions in the IFN-β mRNA levels in RIG-I knockdown clones were less pronounced after infection with MVΔV than after infection with the parental virus. Taken together, the present results indicate that RIG-I and MDA5 both contribute to the recognition of MV and that the V protein promotes MV growth at least partly by inhibiting the MDA5-mediated IFN responses.

Measles Virus Circumvents the Host Interferon Response by Different Actions of the C and V Proteins

ABSTRACT Measles is an acute febrile infectious disease with high morbidity and mortality. The genome of measles virus (MV), the causative agent, encodes two accessory products, V and C proteins, that play important roles in MV virulence. The V but not the C protein of the IC-B strain (a well-characterized virulent strain of MV) has been shown to block the Jak/Stat signaling pathway and counteract the cellular interferon (IFN) response. We have recently shown that a recombinant IC-B strain that lacks C protein expression replicates poorly in certain cell lines, and its growth defect is related to translational inhibition and strong IFN induction. Here, we show that the V protein of the MV IC-B strain also blocks the IFN induction pathway mediated by the melanoma differentiation-associated gene 5 product, thus actively interfering with the host IFN response at two different steps. On the other hand, the C protein per se possesses no activity to block the IFN induction pathway. Our data indicate that the C protein acts as a regulator of viral RNA synthesis, thereby acting indirectly to suppress IFN induction. Since recombinant MVs with C protein defective in modulating viral RNA synthesis or lacking C protein expression strongly stimulate IFN production, in spite of V protein production, both the C and V proteins must be required for MV to fully circumvent the host IFN response.

Efficient Multiplication of Human Metapneumovirus in Vero Cells Expressing the Transmembrane Serine Protease TMPRSS2

ABSTRACT Human metapneumovirus (HMPV) is a major causative agent of severe bronchiolitis and pneumonia. Its fusion (F) protein must be cleaved by host proteases to cause membrane fusion, a critical step for virus infection. By generating Vero cells constitutively expressing the transmembrane serine protease TMPRSS2 and green fluorescent protein-expressing recombinant HMPV, we show that TMPRSS2, which is expressed in the human lung epithelium, cleaves the HMPV F protein efficiently and supports HMPV multiplication. The results indicate that TMPRSS2 is a possible candidate protease involved in the development of lower respiratory tract illness in HMPV-infected patients.

Translational Inhibition and Increased Interferon Induction in Cells Infected with C Protein-Deficient Measles Virus

ABSTRACT In addition to the phosphoprotein, the P gene of measles virus (MV) also encodes the V and C proteins by an RNA editing process and by alternative initiation of translation in a different reading frame, respectively. Although the MV C protein is required for efficient MV replication in vivo and in some cultured cells, its exact functions in virus infection are currently unclear. Here, we report that a recombinant MV lacking the C protein (MVΔC) grew poorly in a human cell line possessing the intact interferon (IFN) pathway and that this growth defect was associated with reduced viral translation and genome replication. The translational inhibition was correlated with phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2. Moreover, increased IFN induction was observed in MVΔC-infected cells. The NS1 protein of influenza virus, which binds to double-stranded RNA (dsRNA) and consequently inhibits IFN induction and dsRNA-dependent protein kinase activation, complemented the growth defect of MVΔC. These results indicate that the MV C protein inhibits IFN induction and modulates host antiviral responses, thereby ensuring MV growth in host cells.

The Host Protease TMPRSS2 Plays a Major Role in In Vivo Replication of Emerging H7N9 and Seasonal Influenza Viruses

ABSTRACT 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.

Generation of Measles Virus with a Segmented RNA Genome

ABSTRACT Viruses classified in the order Mononegavirales have a single nonsegmented RNA molecule as the genome and employ similar strategies for genome replication and gene expression. Infectious particles of Measles virus (MeV), a member of the family Paramyxoviridae in the order Mononegavirales, with two or three RNA genome segments (2 seg- or 3 seg-MeV) were generated using a highly efficient reverse genetics system. All RNA segments of the viruses were designed to have authentic 3′ and 5′ self-complementary termini, similar to those of negative-stranded RNA viruses that intrinsically have multiple RNA genome segments. The 2 seg- and 3 seg-MeV were viable and replicated well in cultured cells. 3 seg-MeV could accommodate up to six additional transcriptional units, five of which were shown to be capable of expressing foreign proteins efficiently. These data indicate that the MeV genome can be segmented, providing an experimental insight into the divergence of the negative-stranded RNA viruses with nonsegmented or segmented RNA genomes. They also illustrate a new strategy to develop mononegavirus-derived vectors harboring multiple additional transcriptional units.

The SI Strain of Measles Virus Derived from a Patient with Subacute Sclerosing Panencephalitis Possesses Typical Genome Alterations and Unique Amino Acid Changes That Modulate Receptor Specificity and Reduce Membrane Fusion Activity

ABSTRACT Subacute sclerosing panencephalitis (SSPE) is a fatal sequela associated with measles and is caused by persistent infection of the brain with measles virus (MV). The SI strain was isolated in 1976 from a patient with SSPE and shows neurovirulence in animals. Genome nucleotide sequence analyses showed that the SI strain genome possesses typical genome alterations for SSPE-derived strains, namely, accumulated amino acid substitutions in the M protein and cytoplasmic tail truncation of the F protein. Through the establishment of an efficient reverse genetics system, a recombinant SI strain expressing a green fluorescent protein (rSI-AcGFP) was generated. The infection of various cell types with rSI-AcGFP was evaluated by fluorescence microscopy. rSI-AcGFP exhibited limited syncytium-forming activity and spread poorly in cells. Analyses using a recombinant MV possessing a chimeric genome between those of the SI strain and a wild-type MV strain indicated that the membrane-associated protein genes (M, F, and H) were responsible for the altered growth phenotype of the SI strain. Functional analyses of viral glycoproteins showed that the F protein of the SI strain exhibited reduced fusion activity because of an E300G substitution and that the H protein of the SI strain used CD46 efficiently but used the original MV receptors on immune and epithelial cells poorly because of L482F, S546G, and F555L substitutions. The data obtained in the present study provide a new platform for analyses of SSPE-derived strains as well as a clear example of an SSPE-derived strain that exhibits altered receptor specificity and limited fusion activity.

Intracellular Transport of the Measles Virus Ribonucleoprotein Complex Is Mediated by Rab11A-Positive Recycling Endosomes and Drives Virus Release from the Apical Membrane of Polarized Epithelial Cells

ABSTRACT Many viruses use the host trafficking system at a variety of their replication steps. Measles virus (MV) possesses a nonsegmented negative-strand RNA genome that encodes three components of the ribonucleoprotein (RNP) complex (N, P, and L), two surface glycoproteins, a matrix protein, and two nonstructural proteins. A subset of immune cells and polarized epithelial cells are in vivo targets of MV, and MV is selectively released from the apical membrane of polarized epithelial cells. However, the molecular mechanisms for the apical release of MV remain largely unknown. In the present study, the localization and trafficking mechanisms of the RNP complex of MV were analyzed in detail using recombinant MVs expressing fluorescent protein-tagged L proteins. Live cell imaging analyses demonstrated that the MV RNP complex was transported in a manner dependent on the microtubule network and together with Rab11A-containing recycling endosomes. The RNP complex was accumulated at the apical membrane and the apical recycling compartment. The accumulation and shedding of infectious virions were severely impaired by expression of a dominant negative form of Rab11A. On the other hand, recycling endosome-mediated RNP transport was totally dispensable for virus production in nonpolarized cells. These data provide the first demonstration of the regulated intracellular trafficking events of the MV RNP complex that define the directional viral release from polarized epithelial cells.