Functional modulation of a peroxygenase cytochrome P450: novel insight into the mechanisms of peroxygenase and peroxidase enzymes

AbstractCytochrome P450BSβ is a peroxygenase that catalyzes the α- or β-hydroxylation of myristic acid by utilizing H2O2. The wild-type enzyme not only hydroxylated myristic acid, but oxidized 3,5,3′,5′-tetramethylbenzidine (TMB), a peroxidase substrate, in a myristic acid-dependent reaction. Study of inhibition of hydroxylation of myristic acid by TMB indicates these two substrates compete for the same highly reactive intermediate during the course of their respective reactions. When deuterated myristic acid was used as a substrate to decrease hydroxylation activity, the rate of TMB oxidation increased. This increased rate of TMB oxidation was greatly enhanced when the R242K mutant enzyme bound with deuterated myristic acid was used. These results suggest that there are critical structural elements at the distal active site which determine whether this enzyme acts as a peroxygenase or a peroxidase

高温下における麻疹ウイルスM蛋白合成阻害機講の解析

金沢大学医学部麻疹ウイルス感染細胞の培養温度上昇によりM蛋白合成が選択的かつ即座にペプチド鎖伸長のレベルで停止する現象を解析する目的で遺伝子導入実検を行ない、以下のような結果が得られた。ブル-スクリプトベクタ-でin vitro RNA合成・翻訳実験可能な麻疹ウイルス各遺伝子の完全長cDNAを4種類の動物細胞発現ベクタ-に組み込み、リン酸カルシウム法にて培養細胞内に導入し、それらの発現を検討した。その結果、Edmonston株のH蛋白およびM蛋白cDNAをSV40初期プロモ-タ-を含む動物細胞発現ベクタ-pSG5のクロ-ニングサイトに挿入したpSG5EHおよびpSG5EMと、同様にしてCAM株のM蛋白cDNAを挿入したpSG5CMが発現可能であった。各プラスミドDNAがトランスフェクションされたCOSー1細胞を35℃および39℃にて^Sーメチオニンで6あるいは18時間ラべルし、抗Hおよび抗Mモノクロ-ナル抗体を用いた免疫沈降とSDSーPAGE法にてウイルス蛋白合成を調ベた。その結果、H蛋白は両温度で同様に合成されていたが、M蛋白はウイルス感染細胞におけると同じく、35℃で検出され、39℃ではまったく検出されなかった。また、HおよびM特異的なriboprobeを用いたノ-ザンブロット解析によりトランスフェクションされた細胞内のHおよびmRNAは35℃と39℃において同様に存在していた。これらのことから単独のM遺伝子導入細胞においてもウイルス感染細胞におけると同様、高温下にて翻訳レベルでのM蛋白合成阻害が甫現されることが明らかとなった。今後、欠失変異M遺伝子等を用い、さらにこの阻害現象の解析を進める予定である。一方、Hackettらの方法に従い、培養細胞由来無細胞蛋白合成系の確立をこころみたが、ウイルス特異蛋白の合成は検出できなかった。今後さらに検計を加えたい。We analyzed a phenomenon that selective translational inhibition of measles virus Membrane (M) protein synthesis in the infected cells at elevated temperatures by transfection of measles virus fullーlength cDNA clones. We used four kinds of eukaryotic expression vectors for transient expression of measles virus Hemagglutinin (H) and M proteins. Among them, only pSG5 vector under the control of the SV40 early promoter was expressible. H protein was well expressed in the H cDNA-transfected COS-1 cells at both 35^ﾟC and 39^ﾟC. On the other hand, although M protein was expressed in the M cDNA-transfected cells at 35^ﾟC, its synthesis became undetected immediately after shiftーup to 39^ﾟC in spite of the presence of the M mRNA. Thus, it was demonstrated that the selective translational inhibition of M protein synthesis is reprosuced in the M cDNA-transfected cells by temperature elevation in the absence of the other MV specific RNAs and proteins.研究課題/領域番号:01570253, 研究期間(年度):1989 – 1990出典：研究課題「高温下における麻疹ウイルスM蛋白合成阻害機講の解析」課題番号01570253（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-01570253/015702531990kenkyu_seika_hokoku_gaiyo/）を加工して作

Inhibitory effect of papaverine on HVJ (Sendai virus) replication in rat glioma C6 cells

The replication of HVJ (Sendai virus) in C6 rat glial cells was found to be inhibited by treatment of the cells with papaverine, an inhibitor of cAMP phosphodiesterase, but not with cAMP or dibutyryl cAMP. In addition, cyclic GMP which often manifests a reciprocal relationship to cAMP did not counteract the inhibition of HVJ yield by papaverine. Both viral genome replication and transcription were suppressed slightly by treatment of the cells with papaverine. In the cells cultured in the presence of papaverine, the synthesis of viral proteins and their phosphorylation occurred at normal rates. Membrane immunofluorescence and cell surface immunoprecipitation showed that the viral glycoproteins HN and F0 were expressed on the cell surface of the papaverine-treated cells. Moreover, all the viral structural proteins were associated with plasma membrane isolated from the treated cells. These results indicate that papaverine treatment suppresses some part of the process of virus budding at the plasma membrane

Temperature-sensitive HVJ (Sendai virus) with altered P polypeptide derived from persistently infected cell lines

HVJ isolated from culture fluids of G2, THEL and GM2 cells persistently infected with HVJ (G2-HVJ, THEL-HVJ and GM2-HVJ) were characterized in comparison with wild-type HVJ (HVJo). Viral structural proteins were analysed by 10% SDS-polyacrylamide gel electrophoresis and it was found that only the P polypeptides of all the HVJ clones isolated from G2-HVJ cell shad a smaller size mol. wt. 77000 (77K), than that of HVJo with a mol. wt. of 79000 (79K). One of six clones from THEL-HVJ cells and one of ten clones from GM2-HVJ cells exhibited the same migration pattern of P polypeptide as that of the clones from G2-HVJ cells. However, the other structural proteins were not different from those of the wild-type virions. All the clones from these carrier cultures were temperature-sensitive and were blocked in early step(s) required for RNA synthesis. These results indicate that some mutation(s) associated with P polypeptide could occur during the course of HVJ persistent infection in cell cultures

An intracellular interaction between a temperature-sensitive mutant and the original wild-type HVJ (Sendai virus) is responsible for the establishment and maintenance of HVJ persistent infection

In order to understand the selective survival of temperature-sensitive (ts) mutants in persistent infection by HVJ (Sendai virus), an intracellular interaction between a ts clone (HVJ cl. 14) isolated from HVJ carrier G2 cells and the original wild-type virus (HVJo) was studied. HVJ cl.14 differed from HVJo mainly in its ts property at 39°C, weak cytopathogenicity and faster electrophoretic mobility of P protein (P(77K)), but showed similar trypsin-activated growth to that of HVJo. When LLCMK2 cells were simultaneously infected with HVJo and HVJ cl.14 at 32°C, synthesis of HVJo-derived P protein (P(79K)) was inhibited with concomitant reduction of cytopathic effect (c.p.e.) and more dominant growth of HVJ cl.14 was observed. For the analysis of progeny viruses in these mixed infections, another mutant of HVJo designated HVJe which formed plaques activated only by elastase was isolated and employed instead of HVJo. At 39°C, HVJ cl.14 was rescued by coinfected HVJe at about 900- to 13000-fold over single infection. This recovery was also shown by sequential synthesis of HVJ cl. 14-derived P protein (P(77K)) following the earlier synthesis of HVJo-derived P polypeptide (P(79K)) in the mixed infection at 39°C. However, the u.v. inactivation of HVJe or HVJ cl. 14 resulted in a loss of their activity on rescue or on c.p.e., reduction, suggesting the necessity of protein synthesis by opposite viruses for these interactions. The mechanisms involved in the predominant growth of the ts mutant and concomitant reduction of c.p.e. seemed to provide a general explanation for the preferable persistence of the ts mutant in the HVJ carrier cells

Human cytomegalovirus persistent infection in a human central nervous system cell line: Production of a variant virus with different growth characteristics

The susceptibility of human central nervous system cell lines to human cytomegalovirus (HCMV) and the fate of infected cultures were studied. Significant amounts of infectious progeny virus were produced in 118MGC glioma and IMR-32 neuroblastoma, but not in KGC oligodendroglioma cells when the cultures were infected with wild-type virus (HCMVwt) at an m.o.i. of 10 p.f.u. per cell. Further passage of infected 118MGC cells resulted in the establishment of a long-term persistent infection. This infection, designated 118MGC/Towne, continuously produced infectious virus (HCMVpi) with titres ranging from 102 to 105 p.f.u./106 cells up to 360 days post-infection (corresponding to 50 subcultures). Since no temperature-sensitive mutants, defective interfering particles or interferon-like activity were found in the 118MGC/Towne cultures, maintenance of the persistent infection seemed to be due to a balance between the release of infectious virus and the growth of uninfected cells. The HCMVpi produced in long-term persistently infected cultures was shown to be different from the HCMVwt originally used to infect by the following characteristics: (i) HCMVpi replicated slowly and yielded lower amounts of progeny virus than HCMVwt; (ii) HCMVpi induced a 73000 mol. wt. immediate early protein that was not synthesized in HCMVwt-infected cells; (iii) HCMVpi had a different DNA structure from that of HCMVwt. These results suggest that HCMVpi is a slower growing variant of HCMVwt and probably plays an important role in the maintenance of the persistent infection

Development of protease activation mutants of HVJ (Sendai virus) in persistently infected cell cultures

HVJ wild-type virus, in which the F protein is activated by trypsin but not by elastase, was spontaneously converted to a mutant with an F protein characterized by being activated by elastase alone. This spontaneous mutation generally occurred during serial passages of cells persistently infected with HVJ, even though the cells were first established by infection with plaque-purifed wild-type virus. Multiple-cycle replication, plaque formation, haemolysis and SDS-polyacrylamide gel electrophoretic (SDS-PAGE) analysis showed that all the elastase-activated mutants isolated from HVJ carrier cells no longer required trypsin for F protein activation. At early passages, these protease activation mutants did not show temperature-sensitive (ts) growth, while at a later stage the mutants, together with the ts mutation, appeared dominant. The frequency of such a protease activation mutation during passage in the HVJ carrier cells seemed to depend on the cell species, but was increased when compared to lytic infections

Antigenic variation of HVJ (Sendai virus) HN glycoprotein detectable by monoclonal antibodies during persistent infection

Three newly established monoclonal hybridoma antibodies to the haemagglutinin molecule of HVJ, designated A7, B3 and F11, recognize operationally non-overlapping antigenic determinants and have neutralizing activity. Using these antibodies, the frequencies of occurrence of neutralization-resistant antigenic variants were analysed in virus populations released from four cell lines persistently infected with HVJ, namely GM2-HVJ, LLCMK2-HVJ, Vero-HVJ and GEsl-HVJ at various passage stages. Antigenic variants were selected from culture fluids of these HVJ carrier cells at a total frequency of 10-3.3, 10-3.8 and 10-3.6 by monoclonal antibodies A7, B3 and F11, respectively. These values were considerably higher than those of 10-4.7 to 10-5.2 detected in a stock preparation of wild-type virus with these antibodies. All the variant viruses isolated as above were negative in neutralization, haemagglutination inhibition and immunofluorescent staining tests with each monoclonal antibody used for their isolation, but were positive with the other antibodies

Temperature elevation enhances cell surface expression of measles virus fusion protein in infected cells

Cell fusion proceeded gradually in measles virus-infected cells incubated at 35°C. Shift-up of incubation temperature to 39°C induced rapidly increased cell fusion in spite of the cessation of de novo synthesis of the fusion (F) protein. Pulse-chase experiments showed that there was little difference in the acquisition of immunoreactivity by haemagglutinin (H) and F proteins between the two temperatures. H protein was detected on the cell surface 60 min after the chase at either temperature. However, appearance of F protein on the cell surface took less than 3 h at 39°C whereas it took 5 h at 35°C. These data indicate that temperature elevation induces more efficient expression of F protein on the cell surface accompanied by marked syncytium formation in measles virus-infected cells