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

Abstract

Several arenaviruses cause hemorrhagic fever disease in humans for which no licensed vaccines are available and current therapeutic intervention is limited to the off-label use of the wide-spectrum antiviral ribavirin. However, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) has proven to be a Rosetta stone for the investigation of virus-host interactions. Arenaviruses have a bisegmented negative-strand RNA genome. The S segment encodes for the virus nucleoprotein and glycoprotein, whereas the L segment encodes for the virus polymerase (L) and Z protein. The ability to generate recombinant LCMV (rLCMV) expressing additional foreign genes of interest would open novel avenues for the study of virus-host interactions and the development of novel vaccine strategies and high-throughput screens to identify antiarenaviral molecules. To this end, we have developed a trisegmented (1L + 2S) rLCMV-based approach (r3LCMV). Each of the two S segments in r3LCMV was altered to replace one of the viral genes by a gene of interest. All r3LCMVs examined expressing different reported genes were stable both genetically and phenotypically and exhibited wild-type growth properties in cultured cells. Reporter gene expression in r3LCMV-infected cells provided an accurate surrogate of levels of virus multiplication. Notably, some r3LCMVs displayed highly attenuated virulence in mice but induced protective immunity against a subsequent lethal challenge with wild-type LCMV, supporting the potential development of r3LCMV-based vaccines.

Fig. 1.

Schematic representation of LCMV WT and r3LCMV GFP/CAT genomes. Genes in solid (NP, CAT and L) and hatched (GPC, GFP and Z) gray are transcribed from genome and antigenome templates, respectively.

Fig. 2.

Growth properties and foreign gene expression of r3LCMV GFP/CAT in cultured cells. BHK-21 cells were infected (moi of 0.1) either with LCMV WT or r3LCMV GFP/CAT. At the indicated time points, virus titers in supernatants were determined (A), and cells were either harvested for a CAT assay (B) or fixed and examined for NP (α-NP) and GFP expression (C).

Fig. 3.

Phenotypic stability of r3LCMV GFP/CAT during serial passages. LCMV WT and r3LCMV GFP/CAT were serially passed on BHK-21 cells. At each passage, virus titers in supernatants were determined (A), and r3LCMV GFP/CAT-infected cells were collected for CAT assay (B).

Fig. 4.

Effect of genome location on foreign gene expression. (A) Two r3LCMVs (r3LCMV GFP/CAT and r3LCMV CAT/GFP) were rescued that differed only on the location within the S segment of the two foreign genes, which were inverted. (B and C) Viral titers (B) and CAT activity (C) were measured in infected (moi = 0.01) BHK-21 cells. (D and E) In an independent experiment, viral titers (D) and GFP expression (E) were determined in infected (moi = 0.01) BHK-21. (F) The ratio of expression levels for the same foreign gene (GFP or CAT) expressed from the NP over GPC loci was determined and normalized by the corresponding virus titers.

Fig. 5.

Morphological comparison of LCMV WT and r3LCMV virions. Purified LCMV WT and r3LCMV GFP/GFP were analyzed by electron microscopy. (A) The diameter of particles was determined in the same horizontal axis. (B) Virion morphology was examined by negative staining.

Fig. 6.

Use of r3LCMV CAT/FLuc to identify antiarenaviral compounds. (A) BHK-21 cells (96-well plate) were infected (moi = 0.1) with either LCMV WT or r3LCMV CAT/FLuc and at different time points, virus titers and FLuc activity were determined. (B) The correlation between virus replication and FLuc expression was estimated by plotting together virus titer and FLuc activity values. (C and D) In an independent experiment, BHK-21 cells were infected at low (0.05) or high (2) moi and treated with Rib or 2-OHM or left untreated. At the indicated time, virus production (C) and FLuc activity (D) were measured.

Fig. 7.

Growth properties of r3LCMV in mice. (A) Growth properties in cultured cells. BHK-21 (IFN−) and L929 (IFN+) cells were infected with the indicated viruses (moi = 0.01), and at the indicated hour p.i., virus titers in supernatants were determined. (B) r3LCMV GFP/GFP was compared with LCMV WT in the mouse model of LCMV-induced lethal meningitis. Mice (n = 8) were injected i.c. with either 10³ or 10⁵ ffu of LCMV WT or r3LCMV GFP/GFP and checked daily for clinical symptoms and survival. (C) The tropism and GFP expression of r3LCMV GFP/GFP were determined in brains of mice injected (i.c.) with 10⁵ ffu at day 5 p.i.

Fig. 8.

Amino acid diversity among arenavirus proteins. The amino acid alignments used to infer phylogenetic relationships among arenavirus species (34) were used to measure the amino acid diversity by pairwise distance in different arenavirus groups. Because GP1 was too divergent to be aligned among groups, we combined the signal peptide and GP2 sequences (GP*) instead of the full GPC for this analysis.