0026-8933/01/3503- $25.00 © 2001 MAIK “Nauka / Interperiodica” 0417 Molecular Biology, Vol. 35, No. 3, 2001, pp. 417–422. Translated from Molekulyarnaya Biologiya, Vol. 35, No. 3, 2001, pp. 492–499. Original Russian Text Copyright © 2001 by Kachko, Cheusova, Sorokin, Kazachinskaya, Cheshenko, Belanov, Bukreev, Ivanova, Razumov, Ryabchikova, Netesov. INTRODUCTION The Marburg virus (MV) is classed with the family Filoviridae, order Mononegavirales, and causes severe hemorrhagic fever with high lethality in humans and primates. Little is still known on the role of individual MV proteins in assembly of the viral nucleocapsid and in transcription and replication of genomic RNA [1]. The MV genome is negatively- stranded RNA of 19,112 nt [2] coding for seven struc- tural proteins. Glycoprotein is the only glycosylated MV protein; its homotrimers form spikes on the virus surface [3]. Two proteins, VP40 and VP24, are in the matrix between the envelope and the nucleocapsid. The other four proteins (nucleoprotein (NP), VP35, VP30, and L) form the nucleocapsid complex [4]. In MV, NP of 695 amino acid residues (calc. 78 kDa) is the major nucleocapsid component, forming the pro- tein backbone of viral RNP. NP is present at 1062 molecules per virion and, by weight, accounts for 27% of the total virion protein [5]. In addition to its structural role, NP induces the immune response in filovirus infection [6, 8], but the antigenic structure of NP is still unknown. In the virus particle, NP forms hexagonal helical tubules [7]. The mechanisms and domains involved in NP self-assembly are also obscure. Hence it is necessary to study in detail the immune and morphological properties of NP. Such a study can be done with recombinant analogs of MV NP obtained in eukaryotic and prokaryotic expression systems. Expression of viral genes in Escherichia coli commonly yields recombinant proteins that are immunologically similar to their natural counterparts, as demonstrated by comparing their properties [8]. Here we study the antigenic properties and self- assembly of MV NP synthesized in various expression systems. EXPERIMENTAL Virus and cell cultures. MV strain Popp was iso- lated, purified, concentrated, and inactivated as described previously [9, 11]. Cell line 293 (human kidney cells transformed with genome region E1 of the adenovirus type 2) was maintained in DMEM (Vector) supplemented with 5% fetal bovine serum (Serva) and 80 mg/ml gentamicin. Antivirus sera (AS) and monoclonal antibodies (mAb). We used control AS of a patient with Marburg disease in record [10] and AS of BALB/c mice immu- nized with MV in order to obtain mAb. Immunization and mAb preparation, purification, and properties have been described elsewhere [11]. Morphology and Antigenic Properties of Recombinant Analogs of the Marburg Virus Nucleoprotein A. V. Kachko 1 , T. B. Cheusova 1 , A. V. Sorokin 1 , E. I. Kazachinskaya 1 , I. O. Cheshenko 1 , E. F. Belanov 1 , A. A. Bukreev 2 , A. V. Ivanova 1 , I. A. Razumov 1 , E. I. Ryabchikova 1 , and S. V. Netesov 1 1 Institute of Molecular Biology, VECTOR State Research Center of Virology and Biotechnology, Kol’tsovo, Novosibirsk Region, 630559 Russia; E-mail: a_kachko@chat.ru 2 National Institute of Allergy and Infectious Diseases, National Cancer Institute, Bethesda, Maryland, United States Received October 24, 2000 Abstract—The full-length gene for Marburg virus (MV) nucleoprotein (NP) was cloned in prokaryotic pQE32 under the control of the T5 promoter and in eukaryotic pTM1 under the control of the T7 RNA polymerase pro- moter. Recombinant NP was synthesized in Escherichia coli and in human kidney cell line 293 cotransfected with recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase. On evidence of electron microscopy with immune detection, recombinant NP formed tubules of two types in E. coli and of a single type in cell line 293. ELISA and immunoblotting with polyclonal and monoclonal antibodies revealed common antigenic deter- minants in recombinant NP and natural MV NP. Key words: filovirus, Marburg virus, nucleoprotein, recombinant protein, expression system, morphology UDC 616.988:576.858 MISCELLANEOUS