Molecular characterization of a reassortant virus derived from Lassa and Mopeia viruses

Abstract

In this article we describe two new complete genomic sequences of Old World Arenaviruses: the Mopeia (MOP) virus and the reassortant MOP/LAS virus, clone 29, or ML29. This reassortant has the large (L) RNA from MOP virus and the small (S) RNA from Lassa (LAS) virus, Josiah strain. Recent studies showed that the ML29 virus is not pathogenic for mice, guinea pigs, or macaques, can completely protect guinea pigs from Lassa virus, and elicit vigorous cell-mediated immunity in immunized monkeys (Lukashevich, I. S., Patterson, J., Carrion, R., Moshkoff, D., Ticer, A., Zapata, J., Brasky, K., Geiger, R., Hubbard, G. B., Bryant, J., and Salvato, M. S., J Virol 79, 13934-13942, 2005). This is a molecular characterization of a reassortant virus, which has been put forward as a live attenuated vaccine candidate against Lassa Fever. Sequence analysis of this reassortant virus revealed 5 non-conservative amino acid substitutions that distinguished it from the parental LAS and MOP viruses. Three substitutions were found outside the conserved RNA-dependent RNA polymerase (RdRp) motifs. A fourth substitution was located between the glycoprotein (GPC)-cleavage site and the putative fusion peptide of GP2. The nucleocapsid protein (NP) contained a fifth substitution in the carboxyl-terminal region of the protein. Two mutations were found within each non-coding terminus of the L segment and one mutation was located in the 3' non-coding region of the S segment of the MOP/LAS virus. ML29 mutations in its genomic termini may have implications for the genetic stability and replication efficiency of ML29 reassortant.

Fig. 1

Genotyping of the ML29 virus. RNA from cells infected with ML29 or with parental viruses, MOP, or LAS was reverse transcribed and amplified with MOP L- or with LAS S-specific primers; m, markers, 100 nt DNA ladder

Fig. 2

Gel electrophoresis of amplified and purified L and S segments. A, 5′ portion (5.3 kilobases) of L segments; B, 3′ portion (2.1 kilobases) of L segments; C, full length S segments

Fig. 3

Location of amino acid substitutions detected in RdRp (L protein) of ML29 reassortant. Mutations in the coding region of the L RNA resulted in 3 non-conservative amino acid substitutions located outside the highly conserved motifs of at positions: A, 851 (Tyr → Asn); B, 1233 (Arg → Gly); C 2136 (Asp → Asn). GenBank accession numbers: MOP AN20410, NC_006574; ML29 reassortant, NC_006572; LAS-Josiah, NC_004297; LCMV-ARM, DQ361066; Guanarito, NC_005082; Junin, NC_005080; Machupo, NC_005079

Fig. 4

Location of amino acids substitutions in GPC and NP proteins of ML29 reassortant. A non-conservative substitution, K272E, is located in the amino-terminal region of GP2 glycoprotein between GPC cleavage motif (RRLL, underlined) and the putative fusion peptide of arenaviruses (GGYCLTRWMLIEAELKCFGNTAV, underlined). A conservative mutation, N173S, is mapped to the central region of NP protein. A non-conservative substitution, A485D, is located in the carboxyl-terminal region of NP protein. GenBank accession numbers: ML29 reassortant, NC_006573; LAS-Josiah, NC_004296; LAS-GA391, X52400; LAS-CSF, AF333969; LASAV, AF246121; LCMV-ARM, NC_004294

Fig. 5

The terminal RNA secondary structure predicted by a genetic algorithm (http://wwwmgs.bionet.nsc.ru/mgs/programs/2dstructrna). The fusion area is marked by a red star. Total energy for terminal structures of ML29 RNA and terminal structures formed by MOP L and LAS S segments are boxed