FIG. 5.

Replication of two-component genome viruses having a helper genome with a deleted NS1 and the chimeric PIV2 genomes having either a mutated NS2A or mutations in both the NS2A and furin cleavage sites. (A) The schematic representation of chimeric PIV2 genomes having either mutation in YFV NS2A (C_YF/DEN/YFV*/GFP) or in both YFV NS2A and furin cleavage site of DEN2V-specific prM (C_YF/DEN*/YFV*/GFP), and helper genome YF/YFV/ΔNS1/Cherry. YFV-specific sequences are indicated by open boxes. DEN2V-specific sequences are indicated by filled, grey boxes. Arrows indicate the positions of mutations introduced into prM and NS2A. (B) Alignments of amino acid sequences of the protein fragments containing furin cleavage site and the internal cleavage site of NS2A. Identical amino acids are denoted by dashes. The introduced mutations are indicated by blue. Arrows indicate positions of cleavage. (C) BHK-21 cells were electroporated by indicated PIV2 and helper genomes and seeded into 100-mm dishes. Samples harvested at 96 h post electroporation or after starting passage 1 were used to perform the next passage, to infect naïve BHK-21 cells at an MOI of 1 inf.u/cell. At the indicated time points, media were replaced and titers of packaged PIV2 and helper genomes were measured by infecting BHK-21 cells containing VEErep/Pac-2A-NS1 replicon, by different dilutions of the samples and evaluating the numbers of GFP- and Cherry-positive cells. Dashed lines indicate the limits of detection.