Reverse genetics recovery of Lujo virus and role of virus RNA secondary structures in efficient virus growth

Abstract

Arenaviruses are rodent-borne viruses with a bisegmented RNA genome. A genetically unique arenavirus, Lujo virus, was recently discovered as the causal agent of a nosocomial outbreak of acute febrile illness with hemorrhagic manifestations in Zambia and South Africa. The outbreak had a case fatality rate of 80%. A reverse genetics system to rescue infectious Lujo virus from cDNA was established to investigate the biological properties of this virus. Sequencing the genomic termini showed unique nucleotides at the 3' terminus of the S segment promoter element. While developing this system, we discovered that reconstructing infectious Lujo virus using the previously reported L segment intergenic region (IGR), comprising the arenaviral transcription termination signal, yielded an attenuated Lujo virus. Resequencing revealed that the correct L segment IGR was 36 nucleotides longer, and incorporating it into the reconstructed Lujo virus restored the growth rate to that of the authentic clinical virus isolate. These additional nucleotides were predicted to more than double the free energy of the IGR main stem-loop structure. In addition, incorporating the newly determined L-IGR into a replicon reporter system enhanced the expression of a luciferase reporter L segment. Overall, these results imply that an extremely stable secondary structure within the L-IGR is critical for Lujo virus propagation and viral protein production. The technology for producing recombinant Lujo virus now provides a method to precisely investigate the molecular determinants of virulence of this newly identified pathogen.

Fig 1

Sequences of Lujo virus genomic termini. (A) 5′ and 3′ sequence analyses were performed as described in Materials and Methods. Chromatograms of the rapid amplification of cDNA ends (RACE) terminus products comprise a homopolymeric A tail incorporated after first-strand cDNA synthesis (5′ tailing) or directly on the 3′ end of the viral genome (3′ tailing). The arenavirus terminus consensus sequence is aligned above the Lujo virus sequences; red arrows indicate the first and last nucleotides of each segment. Note that the last S segment nucleotide (3′) is heterogeneous. Internal nucleotides diverging from the arenavirus consensus are boxed in red. WT, wild type. (B) Predicted base pairing of the 5′ and 3′ termini. Nucleotides not compatible with the current consensus are shown in red and possible nontemplated nucleotides in bold.

Fig 2

Rescue of recombinant Lujo virus. (A) Schematic representation of the primary antigenomic templates produced by the pLJL and pLJS constructs. (B) Strategy for recovering recombinant Lujo virus from cDNA. Transfecting pLJL and pLJS into T7 RNA polymerase-expressing cells generated antigenomic transcripts that were translated, respectively, into RNA-dependent RNA polymerase (L-RdRp) and nucleoprotein (NP), the minimal proteins required for the replication of T7-derived antigenomic templates and the production of recombinant Lujo virus (rLJV). (C) Immunostaining of Lujo virus antigens (green) from BSRT7/5 cells transfected with the indicated combination of plasmids; nuclei were counterstained with propidium iodide (red). (D) Propagation of rLJV compared to the parental strain isolated from a patient (LJV). Vero-E6 cells were initially infected with an MOI of 0.5 50% tissue culture infective dose (TCID₅₀)/cell.

Fig 3

Sequence and structure prediction of L segment intergenic regions. (A) Comparison of the newly derived 140-nt L-IGR with the 104-nt L-IGR. (B) The previous (L-IGR 104) and new (L-IGR 140) intergenic region (L-IGR) structures and free energy (ΔG) were predicted using the CLC Main Workbench 6 RNA prediction package (CLC Bio, Aarhus, Denmark). The predicted hairpin structures (I, II, and III) are annotated according to their locations with respect to the 5′ end. Additional nucleotides from L-IGR 140 not seen in L-IGR 104 are shown in green. ΔG of hairpin II and ΔG of hairpin III are indicated under the corresponding structures.

Fig 4

Effect of L-IGR on reporter gene expression. (A) Schematic representation of antigenomic T7 RNA polymerase transcripts derived by transfecting the indicated plasmids. The L segment Z protein and S segment glycoprotein precursor (GPC) genes were substituted with Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc) reporter genes, respectively. Subconfluent BSRT7/5 cell monolayers plated in 2-cm² wells were transfected using 1.5 μl of Mirus LT1 (Mirus Bio), 0.2 μg of pLJL-SDD, pLJL-GLuc IGR-104, or pLJL-GLuc IGR-140, 0.2 μg of pLJS-CLuc, 0.05 μg of pGL3-control luciferase (Promega, Fitchburg, WI), and 0.040 μg of pCAGGS-RVFV-NSs to suppress any cell background transcription of the reporter genes (21). At 48 h posttransfection, GLuc and CLuc activities were quantified by processing 25 μl of cell supernatant with BioLux Gaussia and Cypridina luciferase assay kits. Light emitted by the luciferase reactions was measured with a Synergy 4 plate reader (Biotek, Winooski, VT). GLuc and CLuc activities were normalized to firefly luciferase activity in the cell lysates by the use of a Bright-Glo luciferase assay system (Promega) per the manufacturer's instructions. Levels of CLuc (B) and GLuc (C) luciferase activity were measured in arbitrary units (AU). Error bars indicate standard deviations (n = 3).

Fig 5

Effect of L-IGR on viral propagation. (A) Schematic representation of the antigenomic T7 RNA polymerase transcripts derived from plasmids used to recover recombinant Lujo virus containing a 104-nt or 140-nt L-IGR. (B) Vero-E6 cells were initially infected at an MOI = 0.1 TCID₅₀/cell, and propagation of recombinant Lujo virus variants with the long L-IGR (rLJV-L-IGR140) or short L-IGR (rLJV-L-IGR104) was compared to propagation of the wild-type parental LJV isolate.