Glycosylation site conservation and recombinant LCMV genetic stability.

Specific mutations of N-glycosylation sites were engineered to delete or add N-glycan on the LCM virus glycoprotein.aConservation of the glycosylation site from alignment of arenavirus GP sequences performed using CLC Sequence Viewer software. The 8 Old Word arenaviruses are: Lassa virus (LASV), X52400; Ippy virus (IPPYV), DQ328877; Mobala virus (MOBV), AY342390; Mopeia virus (MOPV), DQ328874; Morogoro virus (MORV), EU914103; Dandenong virus (DANV), EU136038; Lymphocytic choriomeningitis virus (LCMV), AY847350; Lujo virus (LUJV), FJ952384; while the 20 New World arenaviruses are: Pirital virus (PIRV), AF277659; Allpahuayo virus (ALLV), AY012687; Pichinde virus (PICV), NC_006447, Parana virus (PARV), AF512829; Flexal virus (FLEV), AF512831; Oliveros virus (OLVV), U34248; Latino virus (LATV), AF512830; Machupo virus (MACV), NC_005078; Junin virus (JUNV), D10072; Tacaribe virus (TCRV), NC_004293; Chapare virus (CHAV), EU260463; Sabia virus (SABV), NC_006317; Guanarito virus (GTOV), NC_005077; Amapari virus (AMAV), AF512834; Cupixi virus (CPXV), AF512832; Skinner Tank virus (SKTV), EU123328; Whitewater Arroyo virus (WWAV), AF228063; Catarina virus (CATV), DQ865245; Tamiami virus (TAMV), AF512828; Bear Canyon virus (BCNV), AF512833.bStability controlled by sequencing of GP for each rescued virus mutant 72 h post infection in (i) epithelial cell lines: Vero E6 and HEK293T, (ii) neurons: cell line OBL21A and primary mouse cortical neuron, (iii) macrophages: cell line RAW264.7 and bone marrow derived macrophages. Stability was measured on a scale from 0–10 and represents the stability of the mutants during 10 passages, on epithelial cells, or during a 72 h infection, on primary cells. ‘0′ means no rLCMV rescued and ‘10′ mutation stable for more than 10 passages on epithelial cells and more than 72 h on primary cells.