Bin Gotoh

Sendai Virus C Protein Plays a Role in Restricting PKR Activation by Limiting the Generation of Intracellular Double-Stranded RNA

ABSTRACT Sendai virus (SeV) C protein is a multifunctional protein that plays important roles in regulating viral genome replication and transcription, antagonizing the host interferon system, suppressing virus-induced apoptosis, and facilitating virus assembly and budding. We here report a novel role of SeV C protein, the limitation of double-stranded RNA (dsRNA) generation for maintaining the rate of protein synthesis in infected cells. It was found that the intracellular protein synthesis rate was maintained even after wild-type (wt) SeV infection, but markedly suppressed following C-knockout SeV infection. This indicates the requirement of C protein for maintaining protein synthesis after infection. In contrast to wt SeV infection, C-knockout SeV infection caused phosphorylation of both the translation initiation factor eIF2α and dsRNA-dependent protein kinase (PKR). Phosphorylation of eIF2α occurred mainly due to the action of PKR, since knockdown of PKR by small interfering RNA limited eIF2α phosphorylation. C protein, however, could inhibit neither poly(I):poly(C)-activated nor Newcastle disease virus-induced phosphorylation of PKR and eIF2α, suggesting that C protein does not target common pathways leading to PKR activation. Immunofluorescent staining experiments with a monoclonal antibody specifically recognizing dsRNA revealed generation of a large amount of dsRNA in cells infected with C-knockout SeV but not wt SeV. The dsRNA generation as well as phosphorylation of PKR and eIF2α induced by C-knockout SeV was markedly suppressed in cells constitutively expressing C protein. Taken together, these results demonstrate that the SeV C protein limits generation of dsRNA, thereby keeping PKR inactive to maintain intracellular protein synthesis.

Induction of necroptotic cell death by viral activation of the RIG-I or STING pathway

Necroptosis is a form of necrotic cell death that requires the activity of the death domain-containing kinase RIP1 and its family member RIP3. Necroptosis occurs when RIP1 is deubiquitinated to form a complex with RIP3 in cells deficient in the death receptor adapter molecule FADD or caspase-8. Necroptosis may play a role in host defense during viral infection as viruses like vaccinia can induce necroptosis while murine cytomegalovirus encodes a viral inhibitor of necroptosis. To see how general the interplay between viruses and necroptosis is, we surveyed seven different viruses. We found that two of the viruses tested, Sendai virus (SeV) and murine gammaherpesvirus-68 (MHV68), are capable of inducing dramatic necroptosis in the fibrosarcoma L929 cell line. We show that MHV68-induced cell death occurs through the cytosolic STING sensor pathway in a TNF-dependent manner. In contrast, SeV-induced death is mostly independent of TNF. Knockdown of the RNA sensing molecule RIG-I or the RIP1 deubiquitin protein, CYLD, but not STING, rescued cells from SeV-induced necroptosis. Accompanying necroptosis, we also find that wild type but not mutant SeV lacking the viral proteins Y1 and Y2 result in the non-ubiquitinated form of RIP1. Expression of Y1 or Y2 alone can suppress RIP1 ubiquitination but CYLD is dispensable for this process. Instead, we found that Y1 and Y2 can inhibit cIAP1-mediated RIP1 ubiquitination. Interestingly, we also found that SeV infection of B6 RIP3−/− mice results in increased inflammation in the lung and elevated SeV-specific T cells. Collectively, these data identify viruses and pathways that can trigger necroptosis and highlight the dynamic interplay between pathogen-recognition receptors and cell death induction.

A Tryptophan-Rich Motif in the Human Parainfluenza Virus Type 2 V Protein Is Critical for the Blockade of Toll-Like Receptor 7 (TLR7)- and TLR9-Dependent Signaling

ABSTRACT Plasmacytoid dendritic cells (pDCs) do not produce alpha interferon (IFN-α) unless viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs and macrophages. We show here that even paramyxoviruses, whose infections are restricted to the respiratory tract, have a V protein able to prevent Toll-like receptor 7 (TLR7)- and TLR9-dependent IFN-α induction specific to pDCs. Mutational analysis of human parainfluenza virus type 2 demonstrates that the second Trp residue of the Trp-rich motif (Trp-X3-Trp-X9-Trp) in the C-terminal domain unique to V, a determinant for IRF7 binding, is critical for the blockade of TLR7/9-dependent signaling.

An anti-interferon activity shared by paramyxovirus C proteins: inhibition of Toll-like receptor 7/9-dependent alpha interferon induction.

Paramyxovirus C protein targets the host interferon (IFN) system for virus immune evasion. To identify its unknown anti‐IFN activity, we examined the effect of Sendai virus C protein on activation of the IFN‐α promoter via various signaling pathways. This study uncovers a novel ability of C protein to block Toll‐like receptor (TLR) 7‐ and TLR9‐dependent IFN‐α induction, which is specific to plasmacytoid dendritic cells. C protein interacts with a serine/threonine kinase IKKα and inhibits phosphorylation of IRF7. This anti‐IFN activity of C protein is shared across genera of the Paramyxovirinae, and thus appears to play an important role in paramyxovirus immune evasion.

Human Parainfluenza Virus Type 2 V Protein Inhibits TRAF6-Mediated Ubiquitination of IRF7 To Prevent TLR7- and TLR9-Dependent Interferon Induction

ABSTRACT Paramyxovirus V proteins block Toll-like receptor 7 (TLR7)- and TLR9-dependent signaling leading to alpha interferon production. Our recent study has provided evidence that interaction of the V proteins with IRF7 is important for the blockade. However, the detailed mechanisms still remain unclear. Here we reexamined the interaction of the human parainfluenza virus type 2 (HPIV2) V protein with signaling molecules involved in TLR7/9-dependent signaling. Immunoprecipitation experiments in HEK293T cells transfected with V protein and one of the signaling molecules revealed that the V protein interacted with not only IRF7 but also TRAF6, IKKα, and MyD88. Whereas overexpression of TRAF6 markedly enhanced the level of V protein associating with IRF7, IKKα, and MyD88 in HEK293T cells, the level of V protein associating with TRAF6 was little affected by overexpression of IRF7, IKKα, and MyD88. Moreover, knockdown or knockout of endogenous TRAF6 in HEK293T or mouse embryonic fibroblast cells resulted in dissociation of the V protein from IRF7, IKKα, and MyD88. These results demonstrate that binding of the V protein to IRF7, IKKα, and MyD88 is largely indirect and mediated by endogenous TRAF6. It was found that the V protein inhibited TRAF6-mediated lysine 63 (K63)-linked polyubiquitination of IRF7, which is prerequisite for IRF7 activation. Disruption of the tryptophan-rich motif of the V protein significantly affected its TRAF6-binding efficiency, which correlated well with the magnitude of inhibition of K63-linked polyubiquitination and the resultant activation of IRF7. Taken together, these results suggest that the HPIV2 V protein prevents TLR7/9-dependent interferon induction by inhibiting TRAF6-mediated K63-linked polyubiquitination of IRF7.

Human metapneumovirus M2-2 protein inhibits viral transcription and replication

M2-2 protein of human metapneumovirus (HMPV) is encoded by one of two overlapping open reading frames within M2 mRNA. The precise function of HMPV M2-2 protein remains unknown. We here examined effect of M2-2 protein on HMPV transcription and replication using a minigenome construct and monitoring luciferase reporter gene expression. The minigenome assays demonstrated that M2-2 protein inhibited both transcription and RNA replication. The inhibitory function of M2-2 protein was completely abrogated by removal of eight or four amino acids from its N- or C-terminus, respectively, demonstrating importance of both short terminal sequences for maintaining its functional structure. Immunoprecipitation experiments revealed interaction of M2-2 protein with L protein, which might be involved in inhibition of HMPV transcription and replication. Prior accumulation of intracellular M2-2 protein severely restrained HMPV from replicating. Thus inherent viral control of the M2-2 gene expression in infected cells seems to be essential for efficient HMPV replication.

F-Actin Modulates Measles Virus Cell-Cell Fusion and Assembly by Altering the Interaction between the Matrix Protein and the Cytoplasmic Tail of Hemagglutinin

ABSTRACT Actin filament (F-actin) is believed to be involved in measles virus (MV) assembly as a cellular factor, but the precise roles remain unknown. Here we show that Phe at position 50 of the MV matrix (M) protein is important for its association with F-actin, through which the function of the M protein is regulated. In plasmid-expressed or MV-infected cells, a coimmunoprecipitation study revealed that the wild-type M (M-WT) protein associated strongly with F-actin but only weakly with the cytoplasmic tail of the hemagglutinin (H) protein. Since the F50P mutation allowed the M protein the enhanced interaction with the H protein in return for the sharply declined association with F-actin, the mutant M (M-F50P) protein strongly inhibited MV cell-cell fusion and promoted the uptake of the H protein into virus particles. The abundantly incorporated H protein resulted in the increase in infectivity of the F50P virus, although the virus contained a level of genome RNA equal to that of the WT virus. When the structure of F-actin was disrupted with cytochalasin D, the M-WT protein liberated from F-actin interacted with the H protein as tightly as the M-F50P protein, suppressing cell-cell fusion and promoting virus assembly comparably efficiently as the M-F50P protein. The cell-cell fusion activity of the WT virus appeared to be upheld by F-actin, which prevents the M protein interaction with the H protein. Our results indicate that F-actin in association with the M protein alters the interaction between the M and H proteins, thereby modulating MV cell-cell fusion and assembly.

Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells

ABSTRACT Human metapneumovirus (HMPV) has the ability to inhibit Toll-like receptor 7 (TLR7)- and TLR9-dependent alpha interferon (IFN-α) production by plasmacytoid dendritic cells (pDCs). However, the inhibition mechanism remains largely unknown. To identify viral proteins responsible for this inhibition, we performed a screening of HMPV open reading frames (ORFs) for the ability to block TLR7/9-dependent signaling reconstituted in HEK293T cells by transfection with myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6), IKKα, and IFN regulatory factor 7 (IRF7). This screening demonstrated that the M2-2 protein was the most potent inhibitor of TLR7/9-dependent IFN-α induction. A recombinant HMPV in which the M2-2 ORF was silenced indeed induced greater IFN-α production by human pDCs than wild-type HMPV did. Immunoprecipitation experiments showed direct physical association of the M2-2 protein with the inhibitory domain (ID) of IRF7. As a natural consequence of this, transfection of IRF7 lacking the ID, a constitutively active mutant, resulted in activation of the IFN-α promoter even in the presence of M2-2. Bioluminescence resonance energy transfer assays and split Renilla luciferase complementation assays revealed that M2-2 inhibited MyD88/TRAF6/IKKα-induced homodimerization of IRF7. In contrast, expression of the M2-2 protein did not result in inhibition of IPS-1-induced homodimerization and resultant activation of IRF7. This indicates that inhibition of MyD88/TRAF6/IKKα-induced IRF7 homodimerization does not result from a steric effect of M2-2 binding. Instead, it was found that M2-2 inhibited MyD88/TRAF6/IKKα-induced phosphorylation of IRF7 on Ser477. These results suggest that M2-2 blocks TLR7/9-dependent IFN-α induction by preventing IRF7 homodimerization, possibly through its effects on the phosphorylation status of IRF7. IMPORTANCE The family Paramyxoviridae is divided into two subfamilies, the Paramyxovirinae and the Pneumovirinae. Members of the subfamily Paramyxovirinae have the ability to inhibit TLR7/9-dependent IFN-α production, and the underlying inhibition mechanism has been intensively studied. In contrast, little is known about how members of the subfamily Pneumovirinae regulate IFN-α production by pDCs. We identified the M2-2 protein of HMPV, a member of the subfamily Pneumovirinae, as a negative regulator of IFN-α production by pDCs and uncovered the underlying mechanism. This study explains in part why the M2-2 knockout recombinant HMPV is attenuated and further suggests that M2-2 is a potential target for HMPV therapy.

Expeditious neutralization assay for human metapneumovirus based on a recombinant virus expressing Renilla luciferase

BACKGROUNDnHuman metapneumovirus (HMPV) is a common cause of respiratory diseases in persons of all ages. Because of its slow replication and weak cytopathic effect in cultured cells, conventional neutralization assays for HMPV require around one week for completion.nnnOBJECTIVESnThe purpose of this study is to establish a rapid neutralization assay based on a recombinant virus expressing Renilla luciferase (Rluc).nnnSTUDY DESIGNnA recombinant HMPV expressing both Rluc and green fluorescent protein (GFP) was created by reverse genetics method. Two-fold serial dilutions of human 23 sera were made in a 96-well plate and incubated with 50 pfu/well of the recombinant virus at 4°C for 1 h. The mixtures were then transferred to LLC-MK2 cells in a 96-well plate, incubated for 2 h, and replaced with trypsin-free fresh media. After incubation at 32°C for 24 h, the cells were lysed and measured for Rluc activity. The neutralization titer was defined as the reciprocal of the highest serum dilution that resulted in 50% reduction of Rluc activity.nnnRESULTSnThe novel assay could be completed within 24 h and eliminated the requirement of trypsin supporting multistep replication in cultured cells, as well as laborious processes including the plaque assay with immunostaining. Neutralization titers correlated well with those determined by a GFP-based assay previously developed.nnnCONCLUSIONSnThe neutralization assay based on Rluc activity is the fastest and the most straightforward of all previous assays, and may be available for high throughput screening of neutralizing antibodies.

Intramolecular complementation of measles virus fusion protein stability confers cell–cell fusion activity at 37 °C

The fusion (F) protein of measles virus mediates membrane fusion. In this study, we investigated the molecular basis of the cell–cell fusion activity of the F protein. The N465H substitution in the heptad repeat B domain of the stalk region of the F protein eliminates this activity, but an additional mutation in the DIII domain of the head region – N183D, F217L, P219S, I225T or G240R – restores cell–cell fusion. Thermodynamically stabilized by the N465H substitution, the F protein required elevated temperature as high as 40 °C to promote cell–cell fusion, whereas all five DIII mutations caused destabilization of the F protein allowing the highest fusion activity at 30 °C. Stability complementation between the two domains conferred an efficient cell–cell fusion activity on the F protein at 37 °C.