Fig. 7 | Nature Communications

Fig. 7

From: Direct RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen

Fig. 7

Chimeric RL2–UL1 mRNA is expressed with late kinetics and by multiple HSV-1 strains. a The RL2–UL1 fusion transcript encodes an ICP0-gL fusion protein that lacks two phosphorylation sites (P2, P3) and the nuclear localization signal (NLS) domain present in ICP0. b Assessment of canonical RL2 exon2–exon3 and novel RL2 exon 2—UL1 internal splice junction usage at different times after infection by real-time RT-qPCR. Increased RNA abundance is reflected as lower crossover threshold (Ct) values and normalized to 18S rRNA. Three technical replicates were utilized per condition/time point. Representative data are shown from one of three biological replicates. c Detection of the unique RL2 exon 2—UL1 splice by RT-PCR using a primer scanning the splice junction. NHDFs were infected in parallel with either HSV-1 strain Patton (lanes 2–7) or with wild-type strain 17 syn+ (lane 9), KOS (lane 10), strain F (lane 11) viruses, or with n12, a KOS ICP4 null mutant (lane 12), and RNA was collected at either 6 h (lane 4) or 18 h (lanes 2–3, 5–7, and 8–12) post infection. Inhibitors of protein synthesis (cycloheximide, CHX) or the viral DNA polymerase (phosphonoacetic acid, PAA) were included as indicated (lanes 4–6). Amplification products were visualized with ethidium bromide. d Detection of the predicted ICP0-gL fusion protein. Lysates were prepared from mock (lane 3) or HSV-1 strain Patton-infected NHDFs collected at 6, 18, or 24 h post infection (lanes 1–2, 4–8) and analyzed by immunoblotting with (lanes 1–2 and 7–8) or without (lanes 3–6) prior immunoprecipitation using anti-ICP0 (lane 2 and 8) or control anti-flag- (lanes 1 and 7) loaded protein A beads. After fractionation by SDS-PAGE, membranes were probed using primary antibodies recognizing either the N terminus of ICP0 (lanes 1–2) or the C terminus of glycoprotein L (lanes 3–8). The ICP0-gL fusion peptide has a predicted mass of 32 kDa. Additional reactive species corresponding to ICP0, glycosylated and non-glycosylated gL, and antibody heavy (IgH) or light chains (IgL) are indicated. The image shown is representative of three independent replicates

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