Replicon System for Lassa Virus

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.

Structure of the Lassa Virus Nucleoprotein Revealed by X-ray Crystallography, Small-angle X-ray Scattering, and Electron Microscopy

“…LASV Replicon Assay-Constructs pCITE-Las-NP, pCITELas-L, and pLAS-MG for T7 RNA polymerase-driven expression in mammalian cells of NP, L protein, and minigenome, respectively, of LASV strain AV have been described (12,42). For transfection, the functional cassette of pCITE-Las-L and pLAS-MG was PCR-amplified with Phusion DNA polymerase (Finnzymes).…”

“…The S segment encodes the glycoprotein precursor and the nucleoprotein (NP). The virus genome, NP, and L protein represent the minimum components required for replication and transcription (12)(13)(14). NP is the major component of the viral ribonucleoprotein complex.…”

Generation of recombinant lymphocytic choriomeningitis viruses with trisegmented genomes stably expressing two additional genes of interest

“…The L segment encodes the viral RNA-dependent RNA polymerase (or L polymerase), and a small (Ϸ12 kDa) RING finger protein Z that is functionally the counterpart of the matrix protein found in many enveloped NS RNA viruses. Reverse genetics systems have been now developed for LCMV (11), LASV (12), and Tacaribe virus (13), which are allowing investigators to examine the cis-acting sequences and trans-acting factors that control arenavirus replication and gene expression, as well as assembly and budding (14). Moreover, infectious LCMV has been rescued from cloned cDNAs (15,16), which has opened the possibility of examining the phenotypes of recombinant LCMV (rLCMV) with predetermined mutations in cultured cells and whole organisms.…”

Assembly of Arenavirus Envelope Glycoprotein GPC in Detergent-Soluble Membrane Microdomains

“…The nucleoprotein (N) and the multidomain RNAdependent RNA polymerase (L) make up the ribonucleoprotein core of the virion and together are sufficient for the replication and transcription of the RNA genome (30,35,40). The small matrix protein (Z) is myristoylated and associates with the inner leaflet of the plasma membrane to drive the formation and budding of virion particles (16,51,65).…”