Fig 5. Validation of V(D)J recombination in KSHV infected cultures.

(A) Primary naïve B lymphocytes were magnetically sorted from total tonsil lymphocytes, then subsequently flow sorted based on light chain expression and infected with KSHV or mock-infected. At 4 days post-infection total genomic DNA was harvested and BIOMED2 primers were used to amplify functional V-J rearrangements by PCR. (B) Mock Igλ+ or KSHV Igκ+ cultures were flow sorted at 7dpi for Igλ+ (Mock Control) or GFP+Igλ+ (KSHV-modified) and single cells were collected in 96-well PCR plates. RT-PCR was performed from single cell cDNA for Igλ transcripts. Data represents the percent of single cells expressing Igλ transcripts from three individual tonsil donors in three independent experiments. n≥96 for each sample. (C) KSHV Igκ+ cultures were flow sorted at 7dpi and GFP+Igκ+Igλ+ single cells were collected in 96-well PCR plates. RT-PCR was performed from single cell cDNA for both Igλ and Igκ transcripts. Data represents the number of single cells amplifying the indicated light chains or with no amplification (NA), overall n = 480 from three individual tonsil donors in two independent experiments. (D) Primary naïve B lymphocytes were KSHV or mock-infected. Total mRNA was harvested at 4 hours post-infection and RAG1 and RAG2 transcripts were amplified by nested RT-PCR. RT-PCR results were verified in 6 individual tonsil samples. (E) KSHV-modified Igλ transcripts (from Fig 4B, representing single infections from three individual tonsil donors) were sequenced and immunoglobulin lambda variable (IGLV) gene families were determined using IgBLAST (NCBI). Data displays the histogram actual frequency and Gaussian kernel density distribution for each IGLV gene family for Control (Mock Igλ+) and KSHV-modified (Igκ+ at infection, GFP+Igλ+ at sorting) sequences. Kolmogorov-Smirnov test indicates a statistically significant difference between the distributions (p<0.0001).