Comparison of varicella-zoster virus RNA sequences in human neurons and fibroblasts

Abstract

Varicella-zoster virus (VZV) infection causes varicella, after which the virus becomes latent in ganglionic neurons. In tissue culture, VZV-infected human neurons remain viable at 2 weeks, whereas fibroblasts develop cytopathology. Next-generation RNA sequencing was used to compare VZV transcriptomes in neurons and fibroblasts and identified only 12 differentially transcribed genes of the 70 annotated VZV open reading frames (ORFs), suggesting that defective virus transcription does not account for the lack of cell death in VZV-infected neurons in vitro.

FIG 1

Analysis of NextGen RNA-seq data quality. (A) Quantification of viral transcripts identified by RNA-seq from VZV-infected fibroblasts and human neurons. (B) Variance among all six samples was analyzed. FPKMs from all libraries were separated using principal-component analysis (PCA). Principal component 1 (PC1) separated samples by their largest variance and resulted in separation between cell types. PC2 separated samples by the next-largest variance, independent of the first, and resulted in separation of samples within cell types. PCA analysis indicated a lack of variation between all three fibroblast samples, whereas neuron 3 was different from neurons 1 and 2. PCA analysis showed that neuron 3 was an outlier.

FIG 2

Normalized average read depths per base. The total number of times that each nucleotide was sequenced (raw DNA read depth) was normalized to the sum of numbers of all VZV transcripts in neurons (blue) and fibroblasts (red). Sequence reads were plotted for the top (A) or bottom (B) strands of the VZV genome. (C) Positions of both viral ORFs and physical features of VZV DNA. U_L and U_S, unique long and short sequences, respectively; TR_L and TR_S, terminal repeats of U_L and U_S, respectively; IR_L and IR_S, internal repeats of U_L and U_S, respectively.

FIG 3

Fold changes in numbers of VZV transcripts in human neurons from their numbers in fibroblasts. (A) ANOVA of VZV transcript numbers from VZV-infected fibroblasts and human neurons. (B) Fold changes in the numbers of VZV transcripts for all ORFs in infected human neurons from those in fibroblasts, arranged from highest (greater in neurons) to lowest (greater in fibroblasts). Dotted horizontal lines denote a ±1.70-fold change in transcript levels between cell types. ORFs 53, 36, 64/69, 65, 39, 28, 54, and 4 were transcribed more in neurons, and ORFs 50, 23, 33.5, and 8 were transcribed more in fibroblasts.

FIG 4

Validation of RNA-seq by RT-qPCR. RNA used in RNA-seq analysis was reverse transcribed with oligo(dT), and primers and cDNA were analyzed by RT-qPCR. Primer/probe sets were designed for five VZV ORFS that exceeded the ±1.70-fold cutoff; three genes (ORFs 53, 64/69, and 54) were transcribed more in neurons, and two genes (ORFs 23 and 50) were transcribed less in neurons. Each RT-qPCR mixture contained a primer/probe set for ORF 29 for normalization. (A) Fold changes (from neurons to fibroblasts) in the levels of transcription of the six ORFs from RNA-seq analysis (black bars) or by RT-qPCR after normalization to ORF 29 (white bars). (B) Raw data from RNA-seq (FPKMs) and RT-qPCR (copy numbers) used to construct the graph in panel A.