Recombinant lymphocytic choriomeningitis virus expressing vesicular stomatitis virus glycoprotein

Abstract

A recombinant S segment RNA (Sr) of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) where the glycoprotein of vesicular stomatitis virus (VSVG) was substituted for the glycoprotein of LCMV (LCMV-GP) was produced intracellularly from cDNA under the control of a polymerase I promoter. Coexpression of the LCMV proteins NP and L allowed expression of VSVG from Sr. Infection of transfected cells with WT LCMV (LCMVwt) resulted in reassortment of the L segment of LCMVwt with the Sr at low frequency. Isolation of recombinant LCMV (rLCMV) expressing VSVG (rLCMV/VSVG) was achieved by selection against LCMVwt by using a cell line deficient in the cellular protease S1P. This approach was based on the finding that processing of LCMV-GP by S1P was required for virus infectivity. Characterization of protein and RNA expression of rLCMV/VSVG in infected cells confirmed the expected virus genome organization. rLCMV/VSVG caused syncytium formation in cultured cells and grew to approximately 100-fold lower titers than WT virus but, like the parent virus, it persisted in neonatally infected mice without clinical signs of disease.

Fig. 1.

Rescue of infectious LCMV VLPs by VSVG and intracellular reconstitution, reassortment, and passage of a rLCMV S segment. (A and B) BHK-21 cells were transfected with pMG-ARM/S, pC-NP, pC-Z, pC-L, pC-GP, and pC-VSVG for intracellular expression of MG RNA and proteins, as indicated. (B) Cell extracts were prepared 48 h later and assayed for CAT activity. (A) Infectious VLPs in the SN were assayed after passage of SN as described in Materials and Methods. Origin of sample application (O), nonacetylated (NAc), monoacetylated (MAc), and diacetylated chloramphenicol (DAc). (C, D, and G–J) BHK-21 cells on coverslips (C and D) or in M6 tissue culture wells (G and H) were transfected with pSr(-), pC-L, and pC-NP (C, G, and H) or with pSr(-) only (D). Forty-eight hours later, the cells were processed for VSVG-specific IF(C and D) or were superinfected with LCMV helper virus at a moi of 2(G and H). After 12 h, SN containing a mixture of LCMVwt and rLCMV/VSVG was passaged onto BHK-21 cells on coverslips (A and B). (I and J) Mock-infected control cells. Viral LCMV-NP (G and I) and VSVG (H and J) expression was monitored 24 h later by IF. (E) Comparison of the LCMV S segment with pSr(-). (F) Predicted RNA transcription and replication steps by the recombinant S RNP. Short straight arrows, transcription; bent arrows, replication; long straight arrows above RNA, ORF in sense orientation; long straight arrow below RNA, ORF in antisense orientation.

Fig. 2.

S1P-dependent LCMV propagation and GP processing. (A) CHO-K1 control cells and SRD-12B cells were infected with LCMV or VSV at a moi of 0.1. SNs harvested at the indicated time points were assayed for infectious virus. (B) CHO-K1 and SRD-12B cells were infected with LCMV at a moi of 0.1 or were left uninfected (Mock). Twenty-four hours later, infected cells (LCMV-NP⁺) were visualized by IF, and equal cell density was ascertained by bright-light microscopy (not shown). (C) CHO-K1 control cells and SRD-12B cells were infected with LCMV at moi of 5, and total cellular RNA was harvested at the indicated h p.i. S RNA and NP mRNA species were detected by Northern blot by using a NP cDNA probe. (D) Nucleotide (Upper) and amino acid sequence (Lower) in the predicted recognition site for S1P-mediated proteolytic cleavage (arrow) in GPC of LCMV-ARM. Mutations introduced in plasmid pC-GP_RRAA and resulting amino acid changes are indicated in red. (E) CHO-K1 control cells and SRD-12B cells were transfected with pC-GP or pC-GP_RRAA as indicated. Total cell lysates harvested 48 h later and purified LCMV virions as control were processed for SDS/10% PAGE. Western blot analysis using monoclonal antibody WE33.6 recognized unprocessed GPC (75 kDa) and GP-2 processing product (35 kDa). Crossreactivity of GP-2-specific antibody WE33.6 with a cellular protein of similar size as GP-C (lanes 1 and 4) has previously been observed (22). (F) For VSVG pseudotyping of LCMV, CHO-K1 control or SRD-12B cells (1.5 × 10⁶/M6 tissue culture well) were transfected with 2μg of pC-VSVG or with control vector (pC-“empty”), and 24 h later they were infected with LCMV at a moi of 1. Infectious virus in the SN was assessed 24 h later. Means of duplicate samples are shown. Variation between samples was ≤2-fold.

Fig. 3.

Isolation and characterization of rLCMV/VSVG. (A) BHK-21 cells were transfected with pSr(-), pC-L, and pC-NP 48 h before infection with LCMV helper virus (black). Twelve hours later, SN was harvested and contained a fraction of particles carrying a rLCMV/VSVG genome (white) mixed with LCMV helper virus at a ratio of ≈1:1,500. Note that both types of genomes may carry either surface GP (black or white). Two consecutive passages in SRD-12B cells (72 and 24 h, respectively) enriched rLCMV/VSVG to similar amounts as LCMVwt. Two consecutive rounds of treatment of this virus mixture with LCMV-neutralizing antibody and infection of BHK-21 cells under limiting dilution conditions allowed isolation of clonal rLCMV/VSVG populations. (B) RNA isolated 48 h after infection of BHK-21 cells with the indicated rLCMV/VSVG clonal populations (Tables 2 and 4) or with LCMVwt was analyzed by RT-PCR. LCMV-NP-, LCMV-GP-, and VSVG-specific amplification products obtained with or without RT polymerase were resolved by 1.2% agarose gel electrophoresis. (C–F) BHK-21 cells were infected with rLCMV/VSVG or with LCMVwt at a moi of 0.1 or were left uninfected (M). Total cellular RNA was harvested at the indicated h p.i. and was analyzed by Northern blot. Quadruplicate membranes were hybridized to LCMV-NP- (C), VSVG- (D), LCMV-GP- (E), or LCMV-Z- (F) specific cDNA probes for detection of corresponding genome segments and mRNA species. (G) BHK-21 cells were infected with rLCMV/VSVG or with LCMVwt at moi of 0.1 or were left uninfected (Mock). Thirty-six hours later, expression of VSVG, LCMV-GP, and LCMV-NP was determined by IF. (H) BHK-21 cells were infected with rLCMV-VSVG at moi of 0.1 for 24 h. LCMV-NP-specific IF revealed aggregated nuclei (exclusions) in fused cells. (I) Infectious rLCMV/VSVG (circles) and LCMV (diamonds) in the SN to the experiment described in C–F.(J) SRD-12B cells (open symbols) and CHO-K1 control cells (filled symbols) were infected with LCMV (squares) or rLCMV/VSVG (triangles) at a moi of 0.1. Viral titers in the SN were determined at the indicated time points. (K and L) BALB/c mice were injected intracerebrally within 24 h after birth with 2 × 10³ pfu of rLCMV/VSVG or 2 × 10³ pfu of LCMVwt or with the 30-μl diluent volume only (M). At weaning, all animals were killed, and brain RNA was extracted. S segment, NP-mRNA, and VSVG mRNA were detected by Northern blot hybridization by using LCMV-NP- (K) or VSVG- (L) specific cDNA probes. One representative of two experiments is shown.