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. IMPORTANCEThe 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.A renaviruses are enveloped viruses with a bisegmented negative-strand RNA genome (1). Each genome segment, L and S, uses an ambisense coding strategy to direct the synthesis of two proteins in opposite orientations, separated by a noncoding intergenic region (IGR) (1). The S RNA encodes the viral nucleop...

An mRNA-LNP-based Lassa virus vaccine induces protective immunity in mice

“…The two different rLCMVs used in this study were generated by reverse genetics as described previously 34,67,68 . For the generation of WT rLCMV clone 13 strain (WT rLCMV), BHK-21 cells seeded at 7 × 10 5 cells per well (six-well plate) and cultured overnight were transfected with mPol1Sag Cl-13 (0.8 µg) and mPol1Lag Cl-13 (1.4 µg) that direct the RNA polymerase I (Pol-I)-mediated intracellular synthesis of S and L antigenome RNA species from the LCMV clone 13 strain 67, 69 , together with pC-NP (0.8 μg) and pC-L (1 μg) that supply the trans-acting factors LCMV NP and L 70 using 10 μL of Lipofectamine 2000 (Thermo Fisher Scientific) and incubated at 37°C and 5% CO2.…”

“…Cl-13(LASV-GPC/KGGS) 34 , where the GPC gene of mPol1Sag Cl-13 was replaced with the modified LASgpc gene containing nucleotide substitutions corresponding to the V459K and K461G mutations, instead of mPol1Sag Cl-13.…”

“…Further characterization of rLCMV/LASgpc mutants revealed that combining V459K and K461G mutations further increased the viral fitness of C57BL/6 mice. Conversely, rLCMV containing corresponding mutations from LCMV GPC toward LASgpc (K465V and G467K) showed poor replication in C57BL/6 mice, indicating that the K465 and G467 of the LCMV GPC are critical for the adaptation of LCMV in mice (Mus musculus) 34 .…”

An mRNA-LNP-based Lassa virus vaccine induces protective immunity in mice

“…Further characterization of rLCMV/LASgpc mutants revealed that rLCMV/LASgpc containing V459K and K461G mutations (rLCMV/LASgpc 2m ) further increased the viral fitness in C57BL/6 mice. Conversely, rLCMV containing corresponding mutations from LCMV GPC toward LASgpc (K465V and G467K) showed poor replication in C57BL/6 mice, indicating that the K465 and G467 of the LCMV GPC are critical for the adaptation of LCMV in mice ( Mus musculus ) ( 37 ).…”

Biological Characterization of Conserved Residues within the Cytoplasmic Tail of the Pichinde Arenaviral Glycoprotein Subunit 2 (GP2)

“…Accordingly, it has been shown that efficient virus replication and propagation require the presence of SSP and the GP2 TM and/or CT from a homologous virus (31). By use of recombinant LCMV (rLCMV) or rLCMV expressing LASV GP, two mutations in the GP2 CT have been shown to contribute to viral virulence in mice, suggesting an important role for the GP2 CT in viral fitness and virulence (32,33). However, the biological roles of the conserved GP2 CT region have not been comprehensively characterized in the context of an infectious arenavirus.…”