Reverse genetic mapping of LCMV Cl13 persistence. (A) Schematic of the LCMV genome consisting of the S (GP and NP genes) and L segments (Z and L genes), with intergenic regions (IGR) separating the respective two genes. Three coding mutations (N176D and F260L in GP and K1079Q in L) as well as the noncoding mutation in the IGR of the L segment (g416a) are indicated, differentiating the Cl13 and ARM strains (red). (B and C) C57BL/6 mice were infected i.v. with 2 × 10⁶ pfu of the viruses indicated in the chart. The individual virus nomenclature (e.g., C/C/C/C) and denomination of virus groups (e.g., C/C/X/X) according to the four analyzed genome positions reflect the combination of Cl13 and ARM mutations and are described in detail in Results. At the indicated time points after infection, viral titers and viral RNA loads were determined in blood (B), spleen, and liver (C) as indicated. Each symbol and bar represents the mean ± SEM of three to five mice. In B, representative results from one of three experiments are shown.

Cl13 polymerase mutation enhances replication in pDCs and increases early viremia. (A) C57BL/6 mice were infected with 2 × 10⁶ pfu of the indicated viruses i.v., and viremia was determined 4 d later. Symbols represent individual mice. Representative results from one of two independent experiments are shown. (B) Schematic representation of the replication-deficient rLCMV/Cre_L1079-ARM (lysine at position 1079 of L) and rLCMV/Cre-L1079-Cl13 vector genomes (glutamine at position 1079 of L). Note that substitution of GP for Cre recombinase renders the resulting vector replication-deficient. (C and D) STOP-GFP reporter mice were inoculated with 10⁷ pfu of rLCMV/Cre_L1079-Cl13 pseudotyped with GP of Cl13 from producer cells; 3 d later, pDC and mDC subsets (discriminated according to mPDCA-1 and CD11c expression) were analyzed for GFP expression. rLCMV/OVA (vector expressing ovalbumin instead of Cre recombinase) served as GFP background control. Numbers indicate the percentage of GFP-positive cells. (D) STOP-GFP reporter mice were inoculated with 10⁷ pfu of rLCMV/Cre_L1079-Cl13 or rLCMV/Cre_L1079-ARM. Both vectors were pseudotyped with the GP of Cl13 from producer cells to assure identical cell targeting; 3 d later, GFP-positive pDCs were sorted by FACS, total cellular RNA was extracted, and LCMV S segment RNA was determined by quantitative RT-PCR. Each symbol represents a pool of sorted cells prepared from splenocytes obtained from three mice.

Genetic determinants of LCMV-induced CD8⁺ T-cell exhaustion and generalized immunosuppression. (A–D) C57BL/6 mice were infected i.v. with 2 × 10⁶ pfu of the indicated LCMV; 22 d later, IFN-γ/TNF-α coproducing CD8⁺ T cells specific for NP396 (A) and GP33 (B) were enumerated by intracellular cytokine staining from blood. On day 13 after infection, analogous analyses were performed in spleen (C and D). (E and F) C57BL/6 mice were infected i.v. with 2 × 10⁶ pfu of the indicated LCM virus and 18 d later, were given 2 × 10⁶ pfu VSV i.p.; 8 d later (day 26 after LCMV infection), VSV-specific IgG was measured in serum (E), and VSV-NP52–specific IFN-γ–producing CD8⁺ T cells were enumerated in peripheral blood (F). Symbols in A, B, E, and F represent individual mice. Bars in C and D indicate the mean ± SEM of three to five mice. A, B, E, and F show representative results from two independent experiments.