Baird et al., Page1

Comparison of VZV RNA sequences in human neurons and fibroblasts 1

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Nicholas L. Baird,a Jacqueline L. Bowlin,a Randall J. Cohrs,a,b Don Gildena,b,# and 3

Kenneth L. Jonesc 4

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Departments of Neurology,a Microbiology,b and Biochemistry & Molecular Geneticsc 6

University of Colorado School of Medicine, Aurora, CO 7

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Running Title: VZV transcriptome in human neurons and fibroblasts 9

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Word Count / Abstract: 76 11

Word Count / Text: 1,064 12

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#Corresponding author: Don Gilden, Department of Neurology, University of Colorado 14

Denver School of Medicine, 12700 E. 19th Avenue, Mail Stop B182, Aurora, CO 80045, 15

USA. Phone: (303) 724-4326. Fax: (303) 724-4329. don.gilden@ucdenver.edu16

JVI Accepts, published online ahead of print on 5 March 2014J. Virol. doi:10.1128/JVI.00476-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 18

Varicella zoster virus (VZV) infection causes varicella, after which virus becomes latent 19

in ganglionic neurons. In tissue culture, VZV-infected human neurons remain viable at 20

two weeks, whereas fibroblasts develop cytopathology. Next-generation RNA 21

sequencing was used to compare VZV transcriptomes in neurons and fibroblasts and 22

identified only 12 differentially transcribed genes of the 70 annotated VZV ORFs, 23

suggesting that defective virus transcription does not account for the lack of cell death in 24

VZV-infected neurons in vitro. 25

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TEXT 29

Varicella zoster virus (VZV) is an exclusively human neurotropic 30

alphaherpesvirus that usually produces varicella (chickenpox) upon primary infection, 31

after which virus becomes latent in ganglionic neurons along the entire neuraxis. In 32

tissue culture, VZV-infected neurons remain viable two weeks after infection, while all 33

non-neuronal cells develop a cytopathic effect (CPE). Our earlier studies of VZV-34

infected neurons derived from induced pluripotent stem (iPS) cells revealed that 35

neurons appeared healthy two weeks later, with no detectable infectious virus in the 36

tissue culture medium; analysis of the neurons revealed VZV DNA, transcripts and 37

proteins corresponding to VZV immediate-early, early and late kinetic phases of 38

replication (1). Furthermore, ultrastructural examination revealed few complete virions 39

and numerous aberrant viral particles (2). Together, these findings indicate VZV is not 40

latent in these neurons, despite the lack of CPE, and suggest a deficiency in replication 41

and viral assembly in neurons during the productive infection. Thus, next-generation 42

RNA sequencing (NextGen RNAseq) was used to examine all annotated VZV 43

transcripts in both cell types. 44

Terminally differentiated human neurons derived from human induced pluripotent 45

stem cells (iCell; Cellular Dynamics International, Madison, WI) and human fetal lung 46

(HFL) fibroblasts were infected with an MOI of 1 X 10-3 of cell-free vOka VZV (Zostavax; 47

Merck, Whitehouse Station, NJ) as described (2). Total RNA was extracted from VZV-48

infected fibroblasts at ~80% CPE and from VZV-infected neurons 14 days post-infection 49

(dpi) at which time 5-10% of the neurons were infected. cDNA libraries were 50

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constructed for each sample (3 neuron, 3 fibroblast) using the Illumina TruSeq Stranded 51

mRNA sample preparation kit (Illumina, San Diego, CA). The six independently and 52

uniquely indexed libraries were pooled and loaded onto a single lane of a HiSeq2000 53

flowcell for paired-end, 100-bp DNA sequencing using an Illumina HiSeq2000. 54

NextGen RNAseq of six cDNA libraries yielded 29.2 to 76.8 million total reads 55

per sample (Fig. 1A). Removal of low-quality bases [Phred score <15 (3, 4)] using a 56

custom Python script reduced total sequence data by ≤4%. The remaining sequences 57

were mapped to the annotated VZV genome (Dumas; NCBI NC_001348) using GSNAP 58

(5). Viral sequences comprised 3-7% of all reads from VZV-infected neurons and 17-59

29% from VZV-infected fibroblasts. The bi-directional, strand-specific cDNA library 60

construction protocol permitted alignment of sequences to either the annotated Dumas 61

strain (top strand) or the complementary (bottom strand) (Fig. 1A) of the VZV genome 62

using CUFFLINKS (6). After strand alignment of VZV sequences, the fragments per 63

kilobase of exon per million mapped reads (FPKM; a relative expression level 64

normalized to the sum of all viral sequence divided by specific ORF length) was 65

determined. FPKMs from all six libraries were analyzed using the statistical 66

transformation technique of principal components analysis (PCA) to visualize the 67

differences between samples (Fig. 1B). Samples were first separated by the largest 68

component of variance (principal component 1, PC1), followed by separation of the next 69

largest and independent component of variance (PC2). Based on PCA results and the 70

low number of reads from neuron-3, it was considered to be an outlier and removed 71

from further analysis. 72

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After mapping the five remaining libraries (two neuron, three fibroblast) the 73

average read depth per nucleotide was normalized to the total number of reads across 74

the entire virus genome for each sample, averaged for all neuron (Fig. 2, blue) or all 75

fibroblast (Fig. 2, red) samples, and plotted for the top (Fig. 2A) and bottom strands 76

(Fig. 2B). Figure 2C shows the relative location of all annotated VZV ORFs and the 77

direction of transcription. 78

VZV ORFs differentially transcribed in neurons compared to fibroblasts were 79

identified based on the calculated fold-change of the FPKMs followed by ANOVA using 80

a 2 x 3 (neuron x fibroblast) design (Fig. 3A). Alignment of the fold change in transcript 81

levels from highest (greater transcription in neurons) to lowest (greater transcription in 82

fibroblasts) revealed a break at ±1.70-fold change (Fig. 3B, horizontal lines). Based on 83

a p-value ≤ 0.05 and ±1.70-fold change, transcription of eight VZV ORFs was increased 84

in neurons compared to fibroblasts, and four VZV ORFs were transcribed less in 85

neurons than in fibroblasts (Fig. 3A, asterisks). 86

To validate NextGen RNAseq results, reverse transcription quantitative PCR 87

(RT-qPCR) was performed on five differentially transcribed VZV ORFs as described (7) 88

using the same RNA used for RNAseq. VZV ORF 53, 54 and 64/69 transcripts were 89

more abundant in neurons than fibroblasts, while VZV ORF 23 and 50 transcripts were 90

more abundant in fibroblasts (Fig. 3B). Since RNAseq data were normalized to total 91

VZV transcript levels (FPKM), each RT-qPCR also required normalization. For this, 92

VZV ORF 29 was used since the ratio of this transcript in virus-infected neurons and 93

fibroblasts was one [i.e., the transcript abundance did not differ between cell types (Fig. 94

3)]. After normalization, the fold change for each VZV ORF from infected neurons 95

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compared to infected fibroblasts was determined (Fig. 4A, white bars). RT-qPCR 96

results were the same as found using NextGen RNAseq technology (Fig. 4A, black 97

bars). 98

Herein, next generation RNA sequencing was used to better understand the 99

absence of CPE during productive VZV-infection of human neurons compared to 100

fibroblasts by determining the complete virus transcriptome in each cell type. 101

Surprisingly, only 12 of the 70 VZV ORFs showed differences in transcript abundance 102

between the two cell types. 103

Unlike the limited viral transcripts present in human and monkey ganglia latently 104

infected with varicella virus (8-10), we found that VZV-infected human neurons in cell 105

culture transcribed every annotated ORF. This confirmed a significant difference in 106

virus gene transcription in vitro compared to human neurons latently infected with VZV. 107

A comparison of the 12 differentially transcribed VZV ORFs to orthologous HSV-1 108

genes revealed that they did not belong to a unique class of virus genes: one was 109

immediate-early (ORF 4), three were early (ORFs 8, 28 and 36), and eight were late 110

(ORFs 23, 33.5, 39, 50, 53, 54, 64/69 and 65); six were essential (ORFs 4, 28, 33.5, 39, 111

53 and 54) and six were non-essential (ORFs 8, 23, 36, 50, 64/69 and 65); and nine 112

mapped to the bottom strand (ORFs 4, 8, 23, 28, 33.5, 50, 53, 54 and 65), two to the 113

top strand (ORFs 36 and 39) and one to both strands (ORF 64/69). Overall, viral 114

transcription in neurons that survive two weeks after VZV infection does not appear to 115

be defective compared to that in fibroblasts. Further studies are needed to compare the 116

rate of VZV DNA replication in neurons versus fibroblasts. 117

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ACKNOWLEDGMENTS 119

This work was supported by Public Health Service grants AG006127 (D.G.), AG032958 120

(D.G. and R.J.C.) and NS082228 (R.J.C.) from the National Institutes of Health. Dr. 121

Baird was supported by Training Grant NS007321 to Dr. Gilden from the National 122

Institutes of Health. Dr. Jones and RNA sequencing were supported in part by the 123

Genomics and Biostatistics/Bioinformatics Shared Resources of Colorado’s NIH/NCI 124

Cancer Center Support Grant P30CA046934. We thank Marina Hoffman for editorial 125

review and Lori DePriest for manuscript preparation.126

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REFERENCES 128

1. Yu X, Seitz S, Pointon T, Bowlin JL, Cohrs RJ, Jonjic S, Haas J, Wellish M, 129

Gilden D. 2013. Varicella zoster virus infection of highly pure terminally 130

differentiated human neurons. J. Neurovirol. 19:75-81. 131

2. Grose C, Yu X, Cohrs RJ, Carpenter JE, Bowlin JL, Gilden D. 2013. Aberrant 132

Virion Assembly and Limited Glycoprotein C Production in Varicella-Zoster Virus-133

Infected Neurons. J. Virol. 87:9643-9648. 134

3. Ewing B, Green P. 1998. Base-calling of automated sequencer traces using phred. 135

II. Error probabilities. Genome Res. 8:186-194. 136

4. Ewing B, Hillier L, Wendl MC, Green P. 1998. Base-calling of automated 137

sequencer traces using phred. I. Accuracy assessment. Genome Res. 8:175-185. 138

5. Wu TD, Nacu S. 2010. Fast and SNP-tolerant detection of complex variants and 139

splicing in short reads. Bioinformatics 26:873-881. 140

6. Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL, Pachter L. 2013. 141

Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat. 142

Biotechnol. 31:46-53. 143

7. Cohrs RJ, Gilden DH. 2007. Prevalence and abundance of latently transcribed 144

varicella-zoster virus genes in human ganglia. J. Virol. 81:2950-2956. 145

8. Meyer C, Kerns A, Barron A, Kreklywich C, Streblow DN, Messaoudi I. 2011. 146

Simian varicella virus gene expression during acute and latent infection of rhesus 147

macaques. J. Neurovirol. 17:600-612. 148

on February 16, 2018 by guest

http://jvi.asm.org/

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nloaded from

Baird et al., Page9

9. Nagel MA, Choe A, Traktinskiy I, Cordery-Cotter R, Gilden D, Cohrs RJ. 2011. 149

Varicella-zoster virus transcriptome in latently infected human ganglia. J. Virol. 150

85:2276-2287. 151

10. Ouwendijk WJ, Choe A, Nagel MA, Gilden D, Osterhaus AD, Cohrs RJ, 152

Verjans GM. 2012. Restricted varicella-zoster virus transcription in human trigeminal 153

ganglia obtained soon after death. J. Virol. 86:10203-10206. 154

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FIGURE LEGENDS 158

FIG 1. Analysis of NextGen RNAseq data quality. (A) Quantification of viral 159

transcripts identified by RNAseq from VZV-infected fibroblasts and human neurons. (B) 160

Variance among all six samples was analyzed. FPKMs from all libraries were 161

separated using principal component analysis (PCA). Principal component 1 (PC1) 162

separated samples by their largest variance and resulted in separation between cell 163

types. PC2 separated samples by the next largest variance, independent of the first, 164

and resulted in separation of samples within cell types. PCA analysis indicated a lack of 165

variation between all three fibroblast samples whereas neuron-3 was different from 166

neuron-1 and -2. PCA analysis showed that neuron-3 was an outlier. 167

FIG 2. Normalized average read depths per base. Total number of times that each 168

nucleotide was sequenced (raw DNA read depth) was normalized to the sum of all VZV 169

transcripts in neurons (blue) and fibroblasts (red). Sequence reads were plotted for the 170

top (A) or bottom (B) strand of the VZV genome. (C) Positions of both viral ORFs and 171

physical features of VZV DNA. UL & US, unique long and short, respectively; TRL & 172

TRS, terminal repeat of UL and US, respectively; IRL & IRS, internal repeat of UL and US, 173

respectively. 174

FIG 3. Fold changes of VZV transcripts in human neurons compared to 175

fibroblasts. (A) ANOVA of VZV transcripts from VZV-infected fibroblasts and human 176

neurons. (B) Fold change of VZV transcripts for each ORF in infected human neurons 177

compared to fibroblasts arranged from highest (greater in neurons) to lowest (greater in 178

fibroblasts). Horizontal line denotes ±1.70-fold change in transcript levels between cell 179

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types. ORFs 53, 36, 64/69, 65, 39, 28, 54 and 4 were transcribed more in neurons, and 180

ORFs 50, 23, 33.5 and 8 were transcribed more in fibroblasts. 181

FIG 4. Validation of RNAseq by RT-qPCR. RNA used in RNAseq analysis was 182

reverse-transcribed with oligo[dT] primers and cDNA was analyzed by RT-qPCR. 183

Primer/probe sets were designed for five VZV ORFS that exceeded the ±1.70-fold 184

cutoff; three genes (ORFs 53, 64/69 and 54) were transcribed more in neurons and two 185

(ORFs 23 and 50) were transcribed less in neurons. Each RT-qPCR reaction contained 186

a primer/probe set for ORF 29 for normalization. (A) Fold change (neurons to fibroblast) 187

of the six ORFs from RNAseq analysis (black bars) or by RT-qPCR after normalization 188

to ORF 29 (white bars). (B) Raw data from RNAseq (FPKM) and RT-qPCR (copy 189

number) used to construct graph in (A). 190

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