Alejo A, Andrés G, Viñuela E, Salas M L
Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
J Biol Chem. 1999 Jun 18;274(25):18033-9. doi: 10.1074/jbc.274.25.18033.
In a previous study, it was shown that the protein encoded by the gene B318L of African swine fever virus (ASFV) is a trans-prenyltransferase that catalyzes in vitro the condensation of farnesyl diphosphate and isopentenyl diphosphate to synthesize geranylgeranyl diphosphate and longer chain prenyl diphosphates (Alejo, A., Yáñez, R. J., Rodríguez, J. M., Viñuela, E., and Salas, M. L. (1997) J. Biol. Chem. 272, 9417-9423). To investigate the in vivo function of the viral enzyme, we have determined, in this work, its subcellular localization and activity in cell extracts. Two systems were used in these studies: cells infected with ASFV and cells infected with a recombinant pseudo-Sindbis virus carrying the complete B318L gene. In this latter system, the trans-prenyltransferase was found to colocalize with the endoplasmic reticulum marker protein-disulfide isomerase, whereas in cells infected with ASFV, the viral enzyme was present in cytoplasmic viral assembly sites, associated with precursor viral membranes derived from the endoplasmic reticulum. In addition, after subcellular fractionation, the viral enzyme partitioned into the membrane fraction. Extraction of membrane proteins with alkaline carbonate and Triton X-114 indicated that the ASFV enzyme behaved as an integral membrane protein. The membrane enzyme synthesized predominantly all-trans-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate. These results indicate that the viral B318L protein is a trans-geranylgeranyl-diphosphate synthase, being the only enzyme of this type that is known to have a membrane localization.
在先前的一项研究中，已表明非洲猪瘟病毒（ASFV）基因B318L编码的蛋白质是一种反式异戊二烯基转移酶，它在体外催化法呢基二磷酸和异戊烯基二磷酸的缩合反应，以合成香叶基香叶基二磷酸和更长链的异戊二烯基二磷酸（阿莱霍，A.，亚涅斯，R. J.，罗德里格斯，J. M.，维纽埃拉，E.，和萨拉斯，M. L.（1997年）《生物化学杂志》272卷，9417 - 9423页）。为了研究这种病毒酶在体内的功能，我们在这项工作中确定了它在细胞提取物中的亚细胞定位和活性。这些研究中使用了两种系统：感染ASFV的细胞和感染携带完整B318L基因的重组伪辛德毕斯病毒的细胞。在后一种系统中发现，反式异戊二烯基转移酶与内质网标记蛋白二硫键异构酶共定位，而在感染ASFV的细胞中，这种病毒酶存在于细胞质中的病毒装配位点，与源自内质网的前体病毒膜相关。此外，在进行亚细胞分级分离后，这种病毒酶分配到膜部分。用碱性碳酸盐和Triton X - 114提取膜蛋白表明，ASFV酶表现为一种整合膜蛋白。这种膜酶主要从法呢基二磷酸和异戊烯基二磷酸合成全反式香叶基香叶基二磷酸。这些结果表明，病毒B318L蛋白是一种反式香叶基香叶基二磷酸合酶，是已知的唯一一种具有膜定位的此类酶。
