Residues K465 and G467 within the Cytoplasmic Domain of GP2 Play a Critical Role in the Persistence of Lymphocytic Choriomeningitis Virus in Mice

Abstract

Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever disease in humans and pose serious public health concerns in their regions of endemicity. Moreover, mounting evidence indicates that the worldwide-distributed prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. We have documented that a recombinant LCMV containing the glycoprotein (GPC) gene of LASV within the backbone of the immunosuppressive clone 13 (Cl-13) variant of the Armstrong strain of LCMV (rCl-13/LASV-GPC) exhibited Cl-13-like growth properties in cultured cells, but in contrast to Cl-13, rCl-13/LASV-GPC was unable to establish persistence in immunocompetent adult mice, which prevented its use for some in vivo experiments. Recently, V459K and K461G mutations within the GP2 cytoplasmic domain (CD) of rCl-13/LASV-GPC were shown to increase rCl-13/LASV-GPC infectivity in mice. Here, we generated rCl-13(GPC/VGKS) by introducing the corresponding revertant mutations K465V and G467K within GP2 of rCl-13 and we show that rCl-13(GPC/VGKS) was unable to persist in mice. K465V and G467K mutations did not affect GPC processing, virus RNA replication, or gene expression. In addition, rCl-13(GPC/VGKS) grew to high titers in cultured cell lines and in immunodeficient mice. Further analysis revealed that rCl-13(GPC/VGKS) infected fewer splenic plasmacytoid dendritic cells than rCl-13, yet the two viruses induced similar type I interferon responses in mice. Our findings have identified novel viral determinants of Cl-13 persistence and also revealed that virus GPC-host interactions yet to be elucidated critically contribute to Cl-13 persistence.

Importance: The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), provides investigators with a superb experimental model system to investigate virus-host interactions. The Armstrong strain (ARM) of LCMV causes an acute infection, whereas its derivative, clone 13 (Cl-13), causes a persistent infection. Mutations F260L and K1079Q within GP1 and L polymerase, respectively, have been shown to play critical roles in Cl-13's ability to persist in mice. However, there is an overall lack of knowledge about other viral determinants required for Cl-13's persistence. Here, we report that mutations K465V and G467K within the cytoplasmic domain of Cl-13 GP2 resulted in a virus, rCl-13(GPC/VGKS), that failed to persist in mice despite exhibiting Cl-13 wild-type-like fitness in cultured cells and immunocompromised mice. This finding has uncovered novel viral determinants of viral persistence, and a detailed characterization of rCl-13(GPC/VGKS) can provide novel insights into the mechanisms underlying persistent viral infection.

FIG 1

Generation and characterization in mice of rCl-13 containing mutations K465V and G467K within the CD of GP2. (A) Schematic diagram of LCMV GPC. LCMV surface glycoproteins are expressed as a single polypeptide, GPC, which is co- and posttranslationally processed into SSP and the mature GP1 and GP2. The TMD of GP2 is shaded. The asterisks indicate the positions where the mutations were introduced. (B) Amino acid sequence alignment of the C-terminal ends of GP2 from Cl-13, LASV, and Cl-13 containing mutations K465V and G467K (GPC/VGKS). (C) rCl-13(GPC/VGKS) does not persist in adult immunocompetent mice. Six-week-old B6 mice (n = 6 per group) were injected (2 × 10⁶ FFU i.v.) with either rCl-13 WT or rCl-13(GPC/VGKS). Blood (4 and 10 days [d] p.i.) and tissues (10 days p.i.), samples were collected from infected mice, and virus titers were determined. The data represent means ± standard deviations (SD) of six mice per group from two independent experiments.

FIG 2

Effects of K465V and G467K mutations on GPC processing and virus RNA replication and gene transcription. (A) Effects of K465V and G467K mutations on GPC processing. 293T cells (3.5 × 10⁵ cells/well) seeded in a 12-well plate were cultured overnight and transfected with 1 μg of pCAGGS-Empty (Empty), pC-GPC Cl-13 (GPC WT), or pC-GPC/VGKS Cl-13 (GPC/VGKS). At 48 h posttransfection, total cell lysates were prepared, and GPC processing was analyzed by Western blotting (WB) using an antibody to GP2. (B) Effects of K465V and G467K mutations on RNA replication and gene transcription. BHK-21 cells (3.5 × 10⁵ cells/well) seeded in a 12-well plate and cultured overnight were infected (MOI = 1.0) with either rCl-13 WT or rCl-13(GPC/VGKS). At the indicated time points, total cellular RNA was isolated and analyzed by Northern blotting (N) using a GPC antisense probe that detected the S genome and GPC mRNA species. 28S rRNA (28S) was detected by methylene blue staining.

FIG 3

Characterization of rCl-13(GPC/VGKS) growth properties in cultured cells. (A) Growth properties of rCl-13(GPC/VGKS) in different cell lines. L929 (1.25 × 10⁵ cells/well), BHK-21 (1.75 × 10⁵ cells/well), Vero (1.25 × 10⁵ cells/well), or A549 (1.25 × 10⁵ cells/well) cells were seeded in 24-well plates and cultured overnight. The cells were infected (MOI = 0.1) with either rCl-13 WT or rCl-13(GPC/VGKS). At the indicated times, TCSs were collected, and virus titers were determined by an immunofocus assay. The data represent means ± SD of the results of three independent experiments. (B) Comparison of the growth kinetics of rCl-13(GPC/VGKS) and rCl-13(LASV-GPC/KGGS). BHK-21 cells (1.75 × 10⁵ cells/well) were seeded in 24-well plates and cultured overnight, followed by infection (MOI = 0.01) with rCl-13 WT, rCl-13(GPC/VGKS), or rCl-13(LASV-GPC/KGGS). At the indicated times p.i., TCSs were collected and virus titers were determined. The data represent means ± SD of the results of three independent experiments.

FIG 4

Effects of K465V and G467K mutations on virus replication in mice. (A) rCl-13(GPC/VGKS) persists in IFN-I-deficient mice. IFNAR^−/− B6 mice (n = 5 per group) were injected (2 × 10⁶ FFU i.v.) with either rCl-13 WT or rCl-13(GPC/VGKS). At the indicated times p.i., blood samples were collected, and serum virus titers were determined by an immunofocus assay. One mouse infected with rCl-13 died at 11 days p.i. The data represent means ± SD among five mice per group. (B) DNA sequence chromatogram of the region containing amino acid positions 465 and 467 of rCl-13(GPC/VGKS) present in serum at 4 days p.i. BHK-21 cells were infected with sera from rCl-13(GPC/VGKS)-infected IFNAR^−/− mice. At 72 h p.i., total cellular RNA was isolated from infected cells and used to amplify, by RT-PCR, DNA fragments containing the GPC ORF, and their sequences were analyzed with Sequencher DNA sequence analysis software.

FIG 5

Cell distribution of rCl-13(GPC/VGKS) in spleens of infected mice. (A) Comparison of virus antigen distributions in different immune cell populations in mice infected with either rCl-13 WT or rCl-13(GPC/VGKS). Six-week-old B6 mice (n = 5 per group) were injected (2 × 10⁶ FFU i.v.) with either rCl-13 WT or rCl-13(GPC/VGKS). At 24 h p.i., their spleens were collected, and NP-positive cells in different immune cell populations were detected by flow cytometry. The data represent means and SD among five mice per group. MMM, metallophilic marginal zone macrophages; Macs/monos, macrophages and monocytes; NK cells, natural killer cells; FRCs, fibroblastic reticular cells. (B) rCl-13(GPC/VGKS) does not prevent persistence of Cl-13. Six-week-old B6 mice (n = 4 per group) were injected (i.v.) with 2 × 10⁶ FFU of either rARM WT or rCl-13(GPC/VGKS), together with 2 × 10⁶ FFU of rCl-13 WT. At the indicated times p.i., blood samples were collected and virus serum titers were determined. The data represent means ± SD among four mice per group.

FIG 6

Effects of K465V and G467K mutations on Cl-13's ability to inhibit induction of IFN-I. (A) rCl-13(GPC/VGKS) prevents IFN-I induction. L929 cells seeded in 24-well plates (1.25 × 10⁵ cells/well) and cultured overnight were infected (MOI = 0.1) with rCl-13 WT, rCl-13(GPC/VGKS), or rCl-13(NP/D382A). Twenty-four hours later, LCMV-infected cells were infected with rVSV WT (MOI = 0.01) [rVSV(+)] or remained uninfected [rVSV(−)]. At 36 h p.i. with rVSV, the cells were fixed and stained with crystal violet to assess rVSV-induced CPE. (B) Detection of apoptosis in L929 cells infected with either rCl13 WT or rCl-13(GPC/VGKS). L929 cells (2.5 × 10⁵ cells/well) seeded in a 12-well plate and cultured overnight were infected with either rCl-13 WT or rCl-13(GPC/VGKS) (MOI = 0.1) or remained uninfected (mock). At 48 h p.i., cells were collected. As a positive control, mock-infected cells were incubated at 55°C for 20 min [Heat (+)]. The cells were then stained with fluorescently labeled annexin V and anti-NP antibody (VL-4) and analyzed by flow cytometry. (C) Sera from mice infected with rCl-13(GPC/VGKS) contain levels of bioactive IFN-I that are able to induce the antiviral state in L929 cells and protect against VSV-induced CPE. L929 cells (1.25 × 10⁵ cells/well) seeded in 24-well plates and cultured overnight were treated with sera (1:100 dilution) from mice infected (2 × 10⁶ FFU i.v.) with rCl-13 WT, rCl-13(GPC/VGKS), or rCl-13(NP/D382A) collected at 24 h p.i. After 24 h, the serum-treated cells were infected with rVSV WT (MOI = 0.01) [rVSV (+)] or remained uninfected [rVSV (−)], and at 36 h p.i. with rVSV, the cells were fixed and stained with crystal violet to assess rVSV-induced CPE. (D) K465V and G467K mutations do not affect Cl-13 susceptibility to an IFN-I-induced antiviral state. L929 (1.25 × 10⁵ cells/well) and Vero (1.25 × 10⁵ cells/well) cells were seeded in 24-well plates and cultured overnight, followed by infection (MOI = 0.1) with either rCl-13 WT or rCl-13(GPC/VGKS). After 90 min of virus adsorption, the cells were treated with universal IFN-α at a low (5-U/ml) or high (500-U/ml) concentration or remained untreated. At 24 or 48 h p.i., TCSs were collected and virus titers were determined by an immunofocus assay. The data represent means ± SD of the results of three independent experiments.