The Association of Virus-specified Proteins with Membrane Structures in Vero Cells Infected with Japanese Encephalitis or Murray Valley Encephalitis Virus

日本脳炎ウイルス(JE)またはマレー渓谷脳炎ウイルス(MEV)に感染したVero細胞から3つの分画(E1, E2, E3)を抽出し,それぞれを不連続庶糖密度勾配超遠心法で分画したところ,E1から3つのバンド(E1/F1, E1/F2, E1/F3), E2からも3つのバンド(E2/F4, E2/F5,E2/F6)が得られたが,E3からは1つのバンド(E3/F7)だけが得られた.超遠心の後,E1とE2から管底に得られたpelletを各々P1とP2となづけた.各分画について放射線性同位元素で標識したウイルス特異的タンパクと糖タンパクを解析し電子顕微鏡による観察結果と比較した.MVE特異的タンパクは殆ど全ての分画に存在したが,JE特異的タンパクは平滑膜を含むE1/F2, E1/F3, E2/F4分画に存在した.JE及びMVC両者共,感染細胞のE1分画から得られたPdllet(P1)に多量のウイルス特異的タンパクが存在し,ウイルス感染によって生じた膜構造とリボソームが観察された.P1分画にはウイウルス特異的タンパクの全ての種類(E, ES1,PrM)が存在したが,E1/F2, E1/F3及びP2分画にはEとNS1のみ認められた.すなわち,平滑膜及びP1分画の膜構造がJE特異的タンパクと強く関係しており多分それらの翻訳過程に関すると推測されるが,核に極めて近接した膜構造にJE特異的なタンパクは集積していなかった.これに対して,MBE特異的タンパクは核周辺膜を含む殆ど全ての膜構造に存在した.従って,平滑膜,及び粗面膜共にJE及びMVEに特異的なタンパクの翻訳と集積に重要であるとみられる.Vero cells infected with Japanese encephalitis (JE) or Murray Valley encephalitis (MVE) virus were fractionated into three extracts (E1, E2 and E3). Each extract was centrifuged through 25-60% discontinuous sucrose gradients. Three distinct bands were separated from Extract 1 (E1/F1, E1/F2, E1/F3) and Extract 2 (E2/F4, E2/F5, E2/F6) in contrast to a single band obtained from Extract 3 (E3/F7). In addition, pellets were also obtained from Extracts 1 and 2, and were termed P1 and P2 pellets respectively. Analyses of the labelled viral proteins and glycoproteins in the various fractions were carried out in conjunction with electron microscopy. MVE virus-specified proteins were present in virtually all fractions. In contrast, JE virus-speficied proteins were observed almost exclusively in the smooth membranes of E1/F2, E1/F3 and E2/F4 fractions. Intense virus protein profiles were seen in the E1 pellet (P1) from both JE virus-and MVE virus-infected cells, and contained most of the virus-induced membranous structures and nbosomes. All the virus-specified glycoproteins (E, NS1 and PrM) were found in the P1 fraction but only E and NS1 were found in the E1/F2, E1/F3 and P2 fractions. In summary, the smooth membranes and membranes of the P1 fraction have strong association with the JE virus-specified proteins and probably have a role in their translation. However, the membranes very close to the nuclei had no accumulation of the JE viral proteins. The association of the MVE virus-specified proteins was found with almost all the membrane fractions including the membranes of the perinuclear region. Thus it seems that both smooth and rough membranes are important for the translation and accumulation of both the JE and viral proteins in Vero cells