Preparation and biochemical characterization of Lassa virus subvirion fractions.

14C-labelled Lassa virus was purified by isopycnic centrifugation and split to subvirion fractions. The purified virus was treated with nonionic detergents Nonidet P-40 (NP-40) and with octylglycoside. After ultracentrifugation in urographin density gradient, two subvirion fractions with buoyant density of 1.24-1.26 and 1.08-1.10 g/cm3 were obtained. The first fraction corresponded to the nucleocapsid of Lassa virus: it contained a protein with molecular mass of 60 kDa, the L and S segments of the genomic RNA. The second one contained a protein with molecular mass of 48 kDa and represented, apparently, envelope fraction of virus particles.

[Identification of the mRNA of the nucleocapsid proteins of pathogenic arenaviruses]. / Identifikatsiia mRNK belkov nukleokapsida patogennykh arenavirusov.

Total RNA from cells infected with Machupo and Lassa viruses as well as individual sedimentation classes of these RNAs were translated in the cell-free protein-synthesizing system from rabbit reticulocytes. The translation products were precipitated either with anti-Machupo immune gamma-globulin or monoclonal antibodies to nucleocapsid protein (NP) of Lassa virus. Both total RNA and RNA fraction with the sedimentation coefficient 15-16S promoted the synthesis of protein which comigrated in gel with NP protein of purified virions. It is concluded that monocistron mRNA for NP protein of arenaviruses has the sedimentation coefficient 15-16 S.

[Pathogenic Machupo and Lassa arenaviruses: the biochemical properties of virion RNA and proteins]. / Patogennye arenavirusy Machupo i Lassa: nekotorye biokhimicheskie kharakteristiki virionnykh RNK i belkov.

Lassa virus purified in the isodensity sucrose concentration gradient had the following buoyant densities: 1.17 g/cm3 (sucrose), 1.19 g/cm3 (cesium chloride), 1.16 g/cm3 (urografin). Similar parameters were obtained for Machupo virus. Virion RNAs of these viruses contained 5 sedimentation classes of molecules: 30-31S, 28S, 22-24S, 18S, and 4-6S. Experiments on hybridization of individual sedimentation classes of RNA with an excess of poly(A)-containing RNA from the infected cells as well as inhibition of synthesis of 28S and 18S virion RNAs with low concentrations (0.005-0.5 micrograms/ml) of actinomycin D showed the genetic information for virus proteins to be coded for in two segments: 30-31S and 22-24S. The method of self-annealing demonstrated molecules with complementary sequence ("plus" and "minus" strands) in genome RNAs. In addition to previously described major proteins (78K, 64K, 37K), high performance liquid gel-penetrating chromatography of Machupo virus structural proteins revealed a minor protein with molecular weight of 50 kilodaltons. Pulse-chase experiments demonstrated in the infected cells a precursor-product metabolic bond between glycosylated proteins 78K and 37K. Lassa virus contained 3 structural major proteins with molecular weights 60, 48, and 34 kilodaltons (K). The 60K protein was detected in the nucleocapsid fraction, and 48 K protein in the soluble subvirion fraction. Proteins 60K and 34K were immunoprecipitated in greatest amounts in the infected cells.

Ribonucleic acids of Machupo and Lassa viruses.

Sucrose gradient velocity centrifugation, polyacrylamide gel electrophoresis and RNA-RNA hybridization were used to characterize Lassa and Machupo virion RNAs as well as virus-specific RNAs from cells infected with Pichinde and Machupo viruses. Five RNA species: 30-31S, 28S, 22-24S, 18S and 4-6S have been detected in Lassa, Machupo, and Pichinde virion RNAs. Among them 28S, 18S and 4-6S RNAs cosediment and comigrate with respectively cell RNAs. RNase resistance analyses suggest the presence of extensive secondary structures and complementary RNAs in Lassa, Machupo, and Pichinde virion RNAs. Annealing with poly(A)-containing RNA from infected cells has revealed that the bulk of "minus" strands of Machupo virion RNA is located in 22-24S and 28-31S fractions of sucrose gradient. Thus Machupo and Lassa viruses as well as Pichinde virus contain two genomic RNA fragments: "large" (molecular weight of about 2.2 X 10(6] and "small" (molecular weight of about 1.3 X 10(6]. In the cells infected with Pichinde virus and treated with actinomycin D (1.0 microgram/ml) synthesis of 18S, 22-24S and 30-31S RNAs has been registered. At least 22-24S and 30-31S classes comprise "plus" and "minus" strands. In cells infected with Machupo virus in the presence of actinomycin D the synthesis of similar sedimentation classes of RNAs and certain amounts of 28S RNA have been detected.

Structural and cell-associated proteins of Lassa virus.

Lassa virus was purified from culture fluids of infected CV-1 monkey kidney cells and its structural proteins analysed by polyacrylamide gel electrophoresis. Stained gels showed a typical arenavirus profile, with a prominent protein of molecular weight 60000, corresponding to the nucleocapsid protein N, and two faint broad bands with molecular weights of 45000 and 38000, the envelope glycoproteins G1 and G2. G1 and G2 were shown to be glycosylated by their ability to bind concanavalin A to nitrocellulose transfers of the separated proteins ('Western blots'). N and G2 bound antibody from guinea-pig or human convalescent sera but G1 was inactive, presumably as a result of denaturation. This technique also revealed other apparently virus-specific minor bands with molecular weights of 76000 and 68000. When Western blots of proteins of infected cells which had been lysed in SDS were probed with anti-Lassa virus serum or stained for glycoproteins, four virus-specific bands were apparent: the N, G1 and G2 proteins seen in purified virus, and a glycoprotein of molecular weight 72000 which probably corresponds to the envelope protein precursor (GPC) seen in other arenavirus systems. Immunoprecipitates from infected CV-1 cells labelled with [35S]methionine contained three major virus-specific proteins: the nucleocapsid protein N and proteins of 36000 and 24000 molecular weight (designated fN1 and fN2). Similar immunoprecipitates from Vero cells contained fN1 and fN2 and only very low levels of N. The polypeptides fN1 and fN2 are most probably fragments of N, since Western blots probed with anti-Lassa virus serum showed that lysis of cells in non-ionic detergent rather than SDS results in the appearance of fN2 with concomitant reduction or disappearance of N. These fragments do not exist in the intact cell, but are found as a consequence of rather specific proteolysis upon disruption under non-denaturing conditions. The proteolytic activity responsible was refractory to inhibition by phenylmethylsulphonyl fluoride, aprotinin, pepstatin A or sodium bisulphite, and was more active in Vero than in CV-1 cells.