Replicon System for Lassa Virus

Meike Hass

Uta Gölnitz

Stefanie Müller

Beate Becker-Ziaja

Stephan Günther

Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany

^{Corresponding author. Mailing address: Bernhard-Nocht-Institute of Tropical Medicine, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany. Phone: 49 40 42818 421. Fax: 49 40 42818 378. E-mail:}ed.grubmah-inu.inb@rehtneug.

^{Present address: Department of Parasitology, Institute of Biomedical Science II, University of São Paulo, Cidade Universitária, São Paulo SP, 05508-900 Brazil.}

Received 2004 May 20; Accepted 2004 Aug 12.

Abstract

Lassa virus is endemic to West Africa and causes hemorrhagic fever in humans. To facilitate the functional analysis of this virus, a replicon system was developed based on Lassa virus strain AV. Genomic and antigenomic minigenomes (MG) were constructed consisting of the intergenic region of S RNA and a reporter gene (Renilla luciferase) in antisense orientation, flanked by the 5′ and 3′ untranslated regions of S RNA. MGs were expressed under the control of the T7 promoter. Nucleoprotein (NP), L protein, and Z protein were expressed from plasmids containing the T7 promoter and internal ribosomal entry site. Transfection of cells stably expressing T7 RNA polymerase (BSR T7/5) with MG in the form of DNA or RNA and plasmids for the expression of NP and L protein resulted in high levels of Renilla luciferase expression. The replicon system was optimized with respect to the ratio of the transfected constructs and by modifying the 5′ end of the MG. Maximum activity was observed 24 to 36 h after transfection with a signal-to-noise ratio of 2 to 3 log units. Northern blot analysis provided evidence for replication and transcription of the MG. Z protein downregulated replicon activity close to background levels. Treatment with ribavirin and alpha interferon inhibited replicon activity, suggesting that both act on the level of RNA replication, transcription, or ribonucleoprotein assembly. In conclusion, this study describes the first replicon system for a highly pathogenic arenavirus. It is a tool for investigating the mechanisms of replication and transcription of Lassa virus and may facilitate the testing of antivirals outside a biosafety level 4 laboratory.

Abstract

Lassa virus is a member of the family of Arenaviridae. This family comprises further human pathogens such as Junin virus, Guanarito virus, Machupo virus, and the prototype arenavirus lymphocytic choriomeningitis virus (LCMV).

The natural hosts of Lassa virus are rodents of the genus Mastomys (43). Transmission of the virus from its reservoir to humans causes Lassa fever, an acute febrile illness associated with bleeding. The disease is endemic to the West African countries of Sierra Leone, Guinea, Liberia, and Nigeria (5, 9, 42, 44), but the virus is probably endemic to larger areas of West Africa (24). Lassa fever is characterized initially by flu-like and gastrointestinal symptoms. Bleeding, organ failure, and shock may occur in the late phase of the disease (36). Death occurs after a mean period of 2 weeks after the onset of illness. The virus can also be transmitted from human to human, giving rise to nosocomial Lassa fever epidemics with fatality rates of up to 65% (17). There is no vaccination available for use in humans. The only drug with a proven therapeutic efficacy in humans with Lassa fever is the broad-spectrum nucleoside analogue ribavirin (35). However, the drug is only effective if given early during the course of disease. Cases of Lassa fever recently imported into Europe show that even state-of-the-art intensive care cannot prevent a fatal outcome (54). Due to the high pathogenicity of Lassa virus and the limitations to the prevention or treatment of infections, the virus is classified as a level 4 pathogen and must be handled under biosafety level 4 (BSL-4) conditions.

Arenaviruses belong to the segmented negative-strand RNA viruses. The genome of Lassa virus, like that of other arenaviruses, consists of two single-stranded RNA segments (33). The small (S) segment is 3.4 kb in length, and the large (L) segment is 7 kb in length. Each segment contains two genes, one in sense orientation and one in antisense orientation, a coding strategy that is called ambisense (4). The S RNA encodes the 75-kDa glycoprotein precursor (GPC) and the 63-kDa nucleoprotein (NP). GPC is posttranslationally cleaved into GP1 and GP2 (8). The L RNA encodes the 11-kDa Z protein, which binds zinc and acts as a matrix protein (47, 55), and the 200-kDa L protein, which is likely to function as the viral polymerase (34, 58). The genes are separated on each RNA segment by an intergenic region (IGR) that predictably folds into a stable secondary structure.

The terminal 19 nucleotides at the 3′ and 5′ ends of the RNA segments are complementary to each other and are highly conserved among all arenaviruses. The termini are essential for replication and transcription (46) and are believed to function as a binding site of the viral polymerase. Replication and transcription of the genome occur in the cytoplasm of an infected cell and both take place within ribonucleoprotein complexes (20). During genome replication, a full-length copy of genomic S and L RNAs is synthesized, yielding the corresponding antigenomic S and L RNAs. Due to the ambisense coding strategy, both genomic and antigenomic RNA serve as templates for the transcription of viral mRNA. The transcripts contain a cap but are not polyadenylated. They terminate within the IGR, suggesting that this element plays a role in transcription termination (37).

Recently, reverse genetics techniques were established for LCMV and Tacaribe virus (31, 32). While these minireplicon systems are still restricted in terms of rescue of genetically modified infectious viruses, they opened the possibility of studying the function of proteins and regulatory elements involved in RNA replication and transcription (30, 46, 48). In particular, the function of Z protein has been studied in detail (12, 13, 27, 47). A sophisticated selection strategy made it possible to generate the first infectious recombinant LCMV with the GPC gene replaced by the glycoprotein gene of vesicular stomatitis virus (49). A reverse genetics system for a highly pathogenic arenavirus like Lassa virus has not yet been published.

In this article, we report the establishment of a minireplicon system for Lassa virus. The system is analogous to the minireplicon systems published for LCMV and Tacaribe virus in gross terms, but it differs in several technical aspects from the other systems. It is also demonstrated that the system is suitable to test antivirals against Lassa virus outside BSL-4 laboratories.

Acknowledgments

We thank Ursula Buchholz and Karl-Klaus Conzelmann for providing vector pX12ΔT and BSR T7/5 cells, Gerd Sutter for providing MVA-T7, Simon Vieth for help with sequencing the S RNA termini of Lassa virus AV, Stephan Becker and Friedemann Weber for many helpful discussions, and Herbert Schmitz for continuous support of the work.

This work was supported by grant E/B41G/1G309/1A403 from the Bundesamt für Wehrtechnik und Beschaffung. The Bernhard-Nocht-Institut is supported by the Bundesministerium für Gesundheit and the Freie und Hansestadt Hamburg.

Acknowledgments

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