Elevated Expression of the Human Parainfluenza Virus Type 1 F Gene Downregulates HN Expression

AbstractInteractions involved in the expression of parainfluenza glycoproteins were examined by expressing cDNA clones of theHNandFgenes from human parainfluenza virus type-1 (hPIV1) or Sendai virus (SV) in recombinant Semliki Forest virus (recSFV) or vaccinia-T7 expression vectors. We found that expression of a cloned F protein gene of hPIV1 resulted in downregulation of the HN proteins of hPIV1 or SV. Compared to the amount of HN expressed in the absence of F, coexpression of HN and F led to about 70% reduction in HN. This reduction of HN was observed in both total cell lysates and in protein localized on the cell surface. In contrast to hPIV1 F, SV F did not suppress the expression of HN. Northern blot analysis indicated that similar levels of HN mRNA accumulated in the absence or presence of hPIV1 F. The reduction of HN protein expression by hPIV1 F was detectable after as little as a 10-min labeling period, suggesting that downregulation occurred at the level of translation or at an early stage of protein folding. In hPIV1-infected cells, the amount of F protein synthesized was only about 15% of that of HN, whereas SV F is expressed at high levels. When the level of F in hPIV1-infected cells was artificially increased by recSFV, HN expression was suppressed. The reduction of F protein production in hPIV1-infected cells was regulated at the level of transcription. Characterization of mRNAs produced in hPIV1-infected cells showed that only 20% of the hPIV1 F mRNAs were monocistronic transcripts; 80% were bicistronic M-F readthrough mRNAs. Because proteins are suggested to be synthesized from only the first cistron of bicistronic mRNA in paramyxovirus (T. C. Wong and A. Hirano (1987)J. Virol.61, 584–589), production of F protein is likely suppressed by transcriptional regulation in hPIV1-infected cells. These results suggest that F is capable of downregulating the synthesis of HN, but that this is normally prevented in hPIV1-infected cells by suppression of F protein synthesis by transcriptional regulation

上咽頭癌由来細胞(NPC-KT)に対するインターフェロンの効果

金沢大学医学部研究課題/領域番号59771195, 研究期間(年度):1984出典：研究課題「上咽頭癌由来細胞(NPC-KT)に対するインターフェロンの効果」課題番号59771195（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/grant/KAKENHI-PROJECT-59771195/）を加工して作

EBウイルスによる上皮細胞の発癌性について

金沢大学医学部附属病院細胞融合法を用いてEBウイルスゲノム陽性の上皮細胞を樹立化に成功した。その後、この細胞をクロ-ニングすることにより、EBウイルスゲノム陽性及び陰性の細胞を選びだした。この細胞を長期培養することにより、腫瘍原性の相違がみられた。その結果は以下のとおりであった。1.EBウイルスゲノム陽性及び陰性細胞とも短期間の培養では、ヌ-ドマウスへの腫瘍原性がみられなかった。2.長期培養を続けることにより、EBウイルスゲノム陽性細胞はヌ-ドマウスへの腫瘍原性がみられ、その生着した腫瘍の病理組織像は上咽頭癌と類似の低分化型偏平上皮癌であった。一方EBウイルスゲノム陰性細胞は、長期間培養にもかかわらず、何ら腫瘍原性がみられなかった。3.EBウイルス陽性細胞は腫瘍プロモ-タ-を作用させることにより、EBウイルスの活性化がみとめられた。以上の結果から、EBウイルスは上皮細胞にゲノムが定着、長期安定化することにより腫瘍原性を発揮するものと考えられた。このことは、EBウイルスと上咽頭癌は、密接な関連性を有していると考えられる。We have succeeded in establishing an Epstein-Barr virus (EBV) genome- positive and epithelial cell line by the fusion of EBV-transformed lymphoid cells with an epithelial cells. However, the epithelial hybrid cells (A2L/AH) were nontumorigenic in nude mice during early passage levels. Thereafter, an EBV genome-negative clone and two EBV genome-positive clones of the A2L/AH cells have been successfully cloned. We have continued to culture these three clones, as it is possible that EBV may disturb mechanisms that regulate tumorigenicity when cells are grown in vitro for long periods of time. Two EBV genome-positive A2L/AH clones have become tumorigenic in nude mice after long period of time. The data suggest that EBV may be associated with tumorigenicity in nude mice, at least in part.研究課題/領域番号:63570806, 研究期間(年度):1988 – 1989出典：研究課題「EBウイルスによる上皮細胞の発癌性について」課題番号63570806（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-63570806/635708061989kenkyu_seika_hokoku_gaiyo/）を加工して作

Biochemical Characterization of Enzyme Fidelity of Influenza A Virus RNA Polymerase Complex

It is widely accepted that the highly error prone replication process of influenza A virus (IAV), together with viral genome assortment, facilitates the efficient evolutionary capacity of IAV. Therefore, it has been logically assumed that the enzyme responsible for viral RNA replication process, influenza virus type A RNA polymerase (IAV Pol), is a highly error-prone polymerase which provides the genomic mutations necessary for viral evolution and host adaptation. Importantly, however, the actual enzyme fidelity of IAV RNA polymerase has never been characterized.Here we established new biochemical assay conditions that enabled us to assess both polymerase activity with physiological NTP pools and enzyme fidelity of IAV Pol. We report that IAV Pol displays highly active RNA-dependent RNA polymerase activity at unbiased physiological NTP substrate concentrations. With this robust enzyme activity, for the first time, we were able to compare the enzyme fidelity of IAV Pol complex with that of bacterial phage T7 RNA polymerase and the reverse transcriptases (RT) of human immunodeficiency virus (HIV-1) and murine leukemia virus (MuLV), which are known to be low and high fidelity enzymes, respectively. We observed that IAV Pol displayed significantly higher fidelity than HIV-1 RT and T7 RNA polymerase and equivalent or higher fidelity than MuLV RT. In addition, the IAV Pol complex showed increased fidelity at lower temperatures. Moreover, upon replacement of Mg(++) with Mn(++), IAV Pol displayed increased polymerase activity, but with significantly reduced processivity, and misincorporation was slightly elevated in the presence of Mn(++). Finally, when the IAV nucleoprotein (NP) was included in the reactions, the IAV Pol complex exhibited enhanced polymerase activity with increased fidelity.Our study indicates that IAV Pol is a high fidelity enzyme. We envision that the high fidelity nature of IAV Pol may be important to counter-balance the multiple rounds of IAV genome amplification per infection cycle, which provides IAV Pol with ample opportunities to generate and amplify genomic founder mutations, and thus achieve optimal viral mutagenesis for its evolution

Antagonism of Innate Immunity by Paramyxovirus Accessory Proteins

Paramyxovirinae, a subfamily of Paramyxoviridae, are negative strand RNA viruses comprised of many important human and animal pathogens, which share a high degree of genetic and structural homology. The accessory proteins expressed from the P/V/C gene are major factors in the pathogenicity of the viruses, because of their ability to abrogate various facets of type I interferon (IFN) induction and signaling. Most of the paramyxoviruses exhibit a commonality in their ability to antagonize innate immunity by blocking IFN induction and the Jak/STAT pathway. However, the manner in which the accessory proteins inhibit the pathway differs among viruses. Similarly, there are variations in the capability of the viruses to counteract intracellular detectors (RNA helicases, mda-5 and RIG-I). Furthermore, a functional specificity in the antagonism of the IFN response has been reported, suggesting that specificity in the circumvention of innate immunity restricts viral host range. Available evidence indicates that paramyxoviruses employ specific strategies to antagonize the IFN response of their specific hosts, which is one of the major factors that determine viral pathogenicity and host range

Epstein-Barr virus with transforming and early antigen-inducing ability originating from nasopharyngeal carcinoma: Mapping of the viral genome

Epstein-Barr virus (EBV) from a human hybrid epithelial cell line (NPC-KT), derived from the fusion of human adenoidal cells and EBV genome-containing primary nasopharyngeal carcinoma cells (NPC) has both transforming and early antigen (EA)-inducing abilities. EBV DNA from NPC-KT cells was partially digested with TaqI and ligated to the cloning vector pJB8 at the ClaI site. This cosmid library encompassed the whole genomic DNA of the virus except for several kb of the terminal fragments. The identification and location of each of the cloned DNA fragments have been defined by hybridization to blots prepared with B95-8 and NPC-KT virion DNA. Defective heterogeneous restriction enzyme fragments of the viral DNA were not identified in any of the cosmid clones nor detected in hybridizations to virion DNA, which indicates that a single virus population derived from the NPC tissue has both transforming and EA-inducing activities

IL-10 Inhibits Transforming Growth Factor-ß-Induction of Type I Collagen mRNA Expression via Both JNK and p38 Pathways in Human Lung Fibroblasts

Transforming growth factor-ß (TGF-ß) is a key factor for understanding the pathogenesis of fibrotic disorders such as idiopathic pulmonary fibrosis (IPF). We have demonstrated that interleukin-10 (IL-10) suppresses TGF-ß-induced expression of type I collagen (COL1) mRNA in a human lung fibroblast cell line (WI-38). However, the inhibitory mechanism has not yet been clearly elucidated. Thus, in the current study, we investigate the effects of IL-10 blockade of TGF-ß signaling which regulates COL1 mRNA expression. In WI-38 cells, IL-10 inhibits TGF-ß-mediated phosphorylation of both, c-Jun HN2-terminal kinase (JNK) and p38, but does not suppress TGF-ß- mediated phosphorylation of Smad2 or affect TGF-ß-upregulation of Smad7 mRNA expression. In addition, SP600125 and SB203580, specific inhibitors of JNK and p38, respectively, attenuate TGF-ß-induced COL1 mRNA expression in WI-38 cells. These results suggest that IL-10 inhibits TGF-ß-induced COL1 mRNA expression via both JNK and p38 pathways but not Smad pathways in WI-38 cells. This inhibitory mechanism may provide a novel insight into therapeutic strategies for fibrotic disorders such as IPF

Anti-fibrotic Effects of ONO-EF-345, a Specific Phosphodiesterase IV inhibitor, on Lung Fibroblasts

Phosphodiesterase (PDE) IV inhibitors have been shown to inhibit various inflammatory reactions in pulmonary diseases such as bronchial asthma and chronic obstructive lung diseases (COPD). However, there have been no studies evaluating the effect of PDE IV inhibitors on airway fibrosis, which is a critical feature of airway remodeling in asthma and COPD. We therefore examined whether ONO-EF-345 (ONO), a PDE IV inhibitor, affected the function of lung fibroblasts. ONO suppressed TGF-ß-induced type I collagen (COL1) mRNA expression in lung fibroblasts and also inhibited TGF-ß-induced a- smooth muscle actin (SMA) protein expression. ONO did not affect Smad2 phosphorylation or Smad7 expression. However, ONO reduced JNK and p38 activation, which regulates TGF-ß-induced COL1 expression. These results indicate that PDE IV inhibitors exert anti-fibrotic effects through the JNK and/or p38 pathways

Heliocentric Distance Dependence of Zodiacal Light Observed by Hayabusa2#

Zodiacal light (ZL) is sunlight scattered by interplanetary dust particles (IDPs) at optical wavelengths. The spatial distribution of IDPs in the Solar System may hold an important key to understanding the evolution of the Solar System and material transportation within it. The number density of IDPs can be expressed as $n(r) \sim r^{-\alpha}$, and the exponent $\alpha \sim 1.3$ was obtained by previous observations from interplanetary space by Helios 1/2 and Pioneer 10/11 in the 1970s and 1980s. However, no direct measurements of $\alpha$ based on ZL observations from interplanetary space outside Earth's orbit have been performed since then. Here, we introduce initial results for the radial profile of the ZL at optical wavelengths observed over the range 0.76-1.06 au by ONC-T aboard the Hayabusa2# mission in 2021-2022. The ZL brightness we obtained is well reproduced by a model brightness, although there is a small excess of the observed ZL brightness over the model brightness at around 0.9 au. The radial power-law index we obtained is $\alpha = 1.30 \pm 0.08$, which is consistent with previous results based on ZL observations. The dominant source of uncertainty arises from the uncertainty in estimating the diffuse Galactic light (DGL).Comment: 22 pages, 19 figures, 4 tables, accepted for publication by Earth, Planets and Spac

Trafficking of Sendai Virus Nucleocapsids Is Mediated by Intracellular Vesicles

Paramyxoviruses are assembled at the plasma membrane budding sites after synthesis of all the structural components in the cytoplasm. Although viral ribonuclocapsid (vRNP) is an essential component of infectious virions, the process of vRNP translocation to assembly sites is poorly understood.To analyze real-time trafficking of vRNPs in live infected cells, we created a recombinant Sendai virus (SeV), rSeVLeGFP, which expresses L protein fused to enhanced green fluorescent protein (eGFP). The rSeVLeGFP showed similar growth kinetics compared to wt SeV, and newly synthesized LeGFP could be detected as early as 8 h postinfection. The majority of LeGFP co-localized with other components of vRNPs, NP and P proteins, suggesting the fluorescent signals of LeGFP represent the locations of vRNPs. Analysis of LeGFP movement using time-lapse digital video microscopy revealed directional and saltatory movement of LeGFP along microtubules. Treatment of the cells with nocodazole restricted vRNP movement and reduced progeny virion production without affecting viral protein synthesis, suggesting the role of microtubules in vRNP trafficking and virus assembly. Further study with an electron microscope showed close association of vRNPs with intracellular vesicles present in infected cells. In addition, the vRNPs co-localized with Rab11a protein, which is known to regulate the recycling endocytosis pathway and Golgi-to-plasma membrane trafficking. Simultaneous movement between LeGFP and Rab11a was also observed in infected cells, which constitutively express mRFP-tagged Rab11a. Involvement of recycling endosomes in vRNP translocation was also suggested by the fact that vRNPs move concomitantly with recycling transferrin labeled with Alexa 594.Collectively, our results strongly suggest a previously unrecognized involvement of the intracellular vesicular trafficking pathway in vRNP translocation and provide new insights into the transport of viral structural components to the assembly sites of enveloped viruses