麻疹ウイルスＮ蛋白質及びＰ蛋白質におけるリン酸化修飾の機能解析

学位の種別: 論文博士審査委員会委員 : （主査）東京大学教授 甲斐 知惠子, 東京大学教授 辻本 元, 東京大学准教授 芳賀 猛, 東京大学准教授 堀本 泰介, 東京大学准教授 米田 美佐子University of Tokyo(東京大学

Determination of a phosphorylation site in Nipah virus nucleoprotein and its involvement in virus transcription

Many viruses use their host’s cellular machinery to regulate the functions of viral proteins. The phosphorylation of viral proteins is known to play a role in genome transcription and replication in paramyxoviruses. The paramyxovirus nucleoprotein (N), the most abundant protein in infected cells, is a component of the N–RNA complex and supports the transcription and replication of virus mRNA and genomic RNA. Recently, we reported that the phosphorylation of measles virus N is involved in the regulation of viral RNA synthesis. In this study, we report a rapid turnover of phosphorylation in the Nipah virus N (NiV-N). The phosphorylated NiV-N was hardly detectable in steady-state cells, but was detected after inhibition of cellular protein phosphatases. We identified a phosphorylated serine residue at Ser451 of NiV-N by peptide mass fingerprinting by electrospray ionization–quadrupole time-of-flight mass spectrometry. In the NiV minigenome assay, using luciferase as a reporter gene, the substitution of Ser451 for alanine in NiV-N resulted in a reduction in luciferase activity of approximately 45 % compared with the wild-type protein. Furthermore, the substitution of Ser451 for glutamic acid, which mimics a phosphoserine, led to a more significant decrease in luciferase activity – approximately 81 %. Northern blot analysis showed that both virus transcription and replication were reduced by these mutations. These results suggest that a rapid turnover of the phosphorylation of NiV-N plays an important role in virus transcription and replication

Peroxiredoxin 1 Is Required for Efficient Transcription and Replication of Measles Virus▿

Measles is a highly contagious human disease caused by the measles virus (MeV). In this study, by proteomic analysis, we identified peroxiredoxin 1 (Prdx1) as a host factor that binds to the C-terminal region of the nucleoprotein (N; NTAIL) of MeV. Glutathione S-transferase (GST) pulldown experiments showed that the Prdx1-binding site overlapped with the MeV phosphoprotein (P)-binding site on NTAIL and that Prdx1 competed for the binding to NTAIL with the P protein, which is a component of RNA-dependent RNA polymerase (RdRp). Furthermore, RNA interference for Prdx1 resulted in a significant reduction in MeV growth in HEK293-SLAM cells. A minigenome assay indicated that Prdx1 suppression affected the viral RNA transcription and/or replication step. Relative quantification of viral RNA by real-time PCR (RT-PCR) showed that Prdx1 suppression not only reduced viral RNA transcription and replication but also enhanced polar attenuation in viral mRNA transcription. Surface plasmon resonance analysis showed that the binding affinity of Prdx1 to MeV-N was 40-fold lower than that of MeV-P to MeV-N, which suggested that Prdx1 might be involved in the early stage of MeV infection, when the expression level of Prdx1 was much higher than that of MeV-P. Since Prdx1 was expressed abundantly and constitutively in various cells, the results in this study indicate that Prdx1 is one of the inherent host factors implicated in MeV RNA synthesis

Robustness and Vulnerability of the Autoregulatory System That Maintains Nuclear TDP-43 Levels: A Trade-off Hypothesis for ALS Pathology Based on in Silico Data

Abnormal accumulation of TAR DNA-binding protein 43 (TDP-43) in the cytoplasm and its disappearance from the nucleus are pathological features of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) and are directly involved in the pathogenesis of these conditions. TDP-43 is an essential nuclear protein that readily aggregates in a concentration-dependent manner. Therefore, cells must strictly maintain an appropriate amount of nuclear TDP-43. In one relevant maintenance mechanism, TDP-43 binds to its pre-mRNA and promotes alternative splicing, resulting in mRNA degradation via nonsense-mediated mRNA decay. The level of nuclear TDP-43 is tightly regulated by these mechanisms, which control the amount of mRNA that may be translated. Based on the results of previous experiments, we developed an in silico model that mimics the intracellular dynamics of TDP-43 and examined TDP-43 metabolism under various conditions. We discovered an inherent trade-off in this mechanism between transcriptional redundancy, which maintains the robustness of TDP-43 metabolism, and vulnerability to specific interfering factors. These factors include an increased tendency of TDP-43 to aggregate, impaired nuclear-cytoplasmic TDP-43 transport, and a decreased efficiency of degrading abnormal proteins, all of which are functional abnormalities related to the gene that causes familial ALS/FTD. When these conditions continue at a certain intensity, the vulnerability of the autoregulatory machinery becomes apparent over time, and transcriptional redundancy enters a vicious cycle that ultimately results in TDP-43 pathology. The results obtained using this in silico model reveal the difference in TDP-43 metabolism between normal and disease states. Furthermore, using this model, we simulated the effect of a decrease in TDP-43 transcription and found that this decrease improved TDP-43 pathology and suppressed the abnormal propagation of TDP-43. Therefore, we propose a potential therapeutic strategy to suppress transcriptional redundancy, which is the driving force of the pathological condition caused by the specific factors described above, in patients with ALS presenting with TDP-43 pathology. An ALS animal model exhibiting TDP-43 pathology without overexpression of exogenous TDP-43 should be developed to investigate the effect of alleviating the transcriptional redundancy of TARDBP

Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs.

Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV-LACK, rCDV-TSA, and rCDV-LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV-LACK showed markedly smaller nodules without ulceration. Although the rCDV-TSA- and rCDV-LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV-LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs

Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs

Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV–LACK, rCDV–TSA, and rCDV–LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV–LACK showed markedly smaller nodules without ulceration. Although the rCDV–TSA- and rCDV–LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV–LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs