Targeted Genome Sequencing Reveals Varicella-Zoster Virus Open Reading Frame 12 Deletion

Abstract

The neurotropic herpesvirus varicella-zoster virus (VZV) establishes a lifelong latent infection in humans following primary infection. The low abundance of VZV nucleic acids in human neurons has hindered an understanding of the mechanisms that regulate viral gene transcription during latency. To overcome this critical barrier, we optimized a targeted capture protocol to enrich VZV DNA and cDNA prior to whole-genome/transcriptome sequence analysis. Since the VZV genome is remarkably stable, it was surprising to detect that VZV32, a VZV laboratory strain with no discernible growth defect in tissue culture, contained a 2,158-bp deletion in open reading frame (ORF) 12. Consequently, ORF 12 and 13 protein expression was abolished and Akt phosphorylation was inhibited. The discovery of the ORF 12 deletion, revealed through targeted genome sequencing analysis, points to the need to authenticate the VZV genome when the virus is propagated in tissue culture.IMPORTANCE Viruses isolated from clinical samples often undergo genetic modifications when cultured in the laboratory. Historically, VZV is among the most genetically stable herpesviruses, a notion supported by more than 60 complete genome sequences from multiple isolates and following multiple in vitro passages. However, application of enrichment protocols to targeted genome sequencing revealed the unexpected deletion of a significant portion of VZV ORF 12 following propagation in cultured human fibroblast cells. While the enrichment protocol did not introduce bias in either the virus genome or transcriptome, the findings indicate the need for authentication of VZV by sequencing when the virus is propagated in tissue culture.

Keywords: ORF 12; VZV; deletion; genome; transcriptome.

FIG 1

VZV nucleic acid enrichment dependency on initial target abundance. The fold target enrichment (y axis) in VZV DNA (open circles) or cDNA sequences (closed circles) was plotted as a function of percent input VZV nucleic acid in each sample. The efficiency of virus DNA or cDNA enrichment spanned almost 2 orders of magnitude.

FIG 2

Alignment of DNA sequence from VZV-DEN09 to VZV32 passage 72. The VZV genome contains a long unique (U_L) and short unique (U_S) segment joined through the internal repeat of the long (IR_L) and short (IR_S) segments, which are inverted at the genome end as terminal repeats of the long (TR_L) and short (TR_S) segments, respectively. The 51-nucleotide VZV-DEN09 reads, mapped to the archived reference strain (VZV32 passage 72) genome, showed overrepresented DNA segments at A, C, and C′ along with a deletion at region B. The average read depth per nucleotide was 24,000 (red line).

FIG 3

VZV DNA region A sequence determination. (Left) Two independent samples of VZV-DEN09 DNA (sample 1, lanes 1 and 2; sample 2, lanes 5 and 6) along with duplicate samples of VZV BAC (lanes 3 and 4) were PCR amplified with primers flanking region A. All PCR products showed similar 2.8-kbp bands. M, 500-bp marker. (Right) The complete DNA sequence of the 2.8-kbp PCR product was assembled in two steps. Seq 1 obtained 702 to 976 nucleotides beginning at approximately nucleotide 565 (yellow) and 936 to 998 nucleotides ending at approximately nucleotide 3275 (green). Seq 2 completed the 738 to 1070 internal sequence (orange), with 1842 to 1869 nucleotides extending from nucleotides 712 to 738 to nucleotides 2557 to 2582 (blue).

FIG 4

Deletion of 2,160 bp in VZV-DEN09 ORF 12. (A) Agarose gel of PCR products generated using primers spanning ORF 12 and VZV DNA from VZV-DG after 21 and 48 passages in MeWo cells (lanes 1 and 2, respectively), VZV-NASA after <10 passages in HFL cells (lane 3), VZV Web A (RIT) after >50 passages in BSC-1 cells (lane 4), VZV-DEN09 after 7 passages in HFL cells (lane 5), three independent DNA extractions of VZV-DEN09 in HFL cells after >20 passages (lanes 6 to 8), and two independent DNA extractions of VZV-DEN13 after 2 passages in MRC-5 cells (lanes 9 and 10). M, 100-bp marker (left) and 500-bp marker (right). (B) Sequence of the ORF 12 deletion junction determined from analysis of the PCR product from lane 6. Red nucleotides indicate newly formed ORF 12 termination codons, underlined nucleotides indicate ORF 13 initiation codons, and numbers indicate nucleotide location of deletion relative to archive VZV32 passage 72.

FIG 5

VZV-DEN09 does not express ORFs 12 or 13 and does not induce Akt phosphorylation. (A to C) Flow cytometry analyses of VZV gE expression in mock (blue)- and VZV (red)-infected cells. Human brain vascular adventitia fibroblasts (HBVAFs) (A), HFL (B), or MeWo (C) cultures were left uninfected or infected with VZV-DEN09 or VZV-DEN13 for 72 h. (D) Graphical representation of percent VZV gE⁺ cells from panels A to C. HBVAFs, gray; HFLs, red; MeWos, green. Error bars represent average percent VZV gE⁺ cells ± standard deviations from duplicates for HBVAFs. An asterisk denotes 0% of mock-infected cells expressed VZV gE⁺. (E) Cell lysates were prepared for immunoblotting and resolved on 12.5% SDS-PAGE gels. Membranes were probed for ORFs 12 and 13 and total and phospho-Akt. gE was included as an infection control, and β-actin was included as a loading control.

FIG 6

Growth analysis of VZV-DEN09 and VZV-DEN13 in MeWo and HFL cells. DNA was isolated from infected cells (VZV-DEN09 [dashed lines] and VZV-DEN13 [solid lines]) and quantified by TaqMan-based qPCR at the indicated days postinfection. Fold increase in VZV DNA was determined by the delta C_T method, normalized to day 0 (2^{[CT(day 0) − CT(day X)]}). Data represent duplicate growth curves. *, P < 0.05.

FIG 7

Bias value plot for VZV-DEN09 DNA. The bias value, an index of enrichment efficiency, was determined for each nucleotide of VZV-DEN09. Bias values of <0 indicate less-than-expected enrichment, while a bias value of >0 indicates more-than-expected enrichment. A single region of VZV-DEN09 DNA that was deleted from VZV32 was outside a 2-fold deviation (blue lines) from the average (red line).

FIG 8

Bias value plot for VZV-DEN09 transcripts. Bias values were determined from transcriptome analysis for all nucleotide locations on both top and bottom DNA strands of VZV-DEN09. Bias values of >1 (red dots) indicate nucleotide locations with >2-fold the expected counts per nucleotide (cpn) in enriched samples. Bias values of <−1 (green dots) indicate nucleotide locations with <2-fold less than the expected cpn in enriched samples. Bias values between −1 and 1 (black dots) indicate nucleotide locations with cpn within 2-fold of the expected value. Percentages of total cpn that reside within each area are listed on the right. Total numbers and locations of cpn from 51-nucleotide reads residing outside the 2-fold expected value are listed on the right, with relative location shown by dots within blue circles.

FIG 9

VZV-DEN09 annotated genome. Open reading frames as annotated in reference genome VZV32 passage 72 were mapped to 1st, 2nd, or 3rd reading frames of top (T) or bottom (B) DNA strands of VZV-DEN09.

FIG 10

VZV-DEN09 transcriptome. cDNA reads were converted to counts per nucleotide (cpn) and plotted against the top or bottom strand of the annotated VZV-DEN09 genome. Direction and location of ORFs (red arrows) are as shown in Fig. 8.

FIG 11

Location of candidate novel VZV transcripts. Enlargement of the 1.4-kbp ORF 60/61 intergenic segment of VZV-DEN09 shown in Fig. 9 revealed three locations of increased cpn (arrows) on the DNA strand (black) opposite that encoding ORFs 60 and 61 (red).