G-quadruplexes formed by Varicella-Zoster virus reiteration sequences suppress expression of glycoprotein C and regulate viral cell-to-cell spread

Woo-Chang Chung; Subramaniyam Ravichandran; Daegyu Park; Gwang Myeong Lee; Young-Eui Kim; Youngju Choi; Moon Jung Song; Kyeong Kyu Kim; Jin-Hyun Ahn

doi:10.1371/journal.ppat.1011095

. 2023 Jan 11;19(1):e1011095. doi: 10.1371/journal.ppat.1011095

G-quadruplexes formed by Varicella-Zoster virus reiteration sequences suppress expression of glycoprotein C and regulate viral cell-to-cell spread

Woo-Chang Chung ^1,^#, Subramaniyam Ravichandran ^2,^¤,^#, Daegyu Park ¹, Gwang Myeong Lee ¹, Young-Eui Kim ¹, Youngju Choi ¹, Moon Jung Song ³, Kyeong Kyu Kim ^2,⁴, Jin-Hyun Ahn ^1,^4,^*

Editor: Robert F Kalejta⁵

PMCID: PMC9873165 PMID: 36630443

Abstract

G-quadruplex (G4) formed by repetitive guanosine-rich sequences plays important roles in diverse cellular processes; however, its roles in viral infection are not fully understood. In this study, we investigated the genome-wide distribution of G4-forming sequences (G4 motifs) in Varicella-Zoster virus (VZV) and found that G4 motifs are enriched in the internal repeat short and the terminal repeat short regions flanking the unique short region and also in some reiteration (R) sequence regions. A high density of G4 motifs in the R2 region was found on the template strand of ORF14, which encodes glycoprotein C (gC), a virulent factor for viral growth in skin. Analyses such as circular dichroism spectroscopy, thermal difference spectra, and native polyacrylamide gel electrophoresis with oligodeoxynucleotides demonstrated that several G4 motifs in ORF14 form stable G4 structures. In transfection assays, gC expression from the G4-disrupted ORF14 gene was increased at the transcriptional level and became more resistant to suppression by G4-ligand treatment. The recombinant virus containing the G4-disrupted ORF14 gene expressed a higher level of gC mRNA, while it showed a slightly reduced growth. This G4-disrupted ORF14 virus produced smaller plaques than the wild-type virus. Our results demonstrate that G4 formation via reiteration sequences suppresses gC expression during VZV infection and regulates viral cell-to-cell spread.

Author summary

G-quadruplexes (G4s) are found in herpesvirus genomes. However, whether G4 formation regulates the growth and pathogenesis of Varicella-Zoster virus (VZV), which causes chickenpox in children and shingles in older adults, is unclear. Here, we identified ~150 putative G4-forming sequences (G4 motifs) in the VZV genome and found that these sequences are enriched in the repeat regions in the genome. In particular, the G4 motifs found in the R2 reiteration region are located on the template strand of ORF14 that encodes glycoprotein C (gC). We demonstrate that several G4 motifs in R2 form anti-parallel G4 structures and that the formation of these G4s suppresses gC expression at the transcription level during virus infection. We also show that the G4-mediated attenuation of gC expression promotes viral spread to neighboring cells. Therefore, VZV has evolved to control gC expression through G4 formation by reiteration sequences for regulation of viral cell-to-cell spread.

Introduction

Repetitive guanosine-rich sequences connected by short stretches of nucleotides in single-stranded DNA or RNA can fold into a distinct type of tertiary structure known as a G-quadruplex (G4). In a G4 structure, four guanine bases are connected by Hoogsteen bonds to form a G-tetrad; multiple G-tetrads stack on top of each other, resulting in various types of G4 with different numbers of G-tetrads. G4s are stabilized by several monovalent and divalent cations including K⁺ and Na⁺. G4s can be produced within a single nucleic acid molecule to form intramolecular G4s or with several molecules to produce intermolecular G4s. G-runs in a G4 structure can be parallel or antiparallel [1,2]. G4s have been shown to play crucial roles in diverse biological processes, such as telomere protection from nuclease attack, regulation of gene transcription and translation, and regulation of DNA or RNA replication and genome stability [3,4].

The Herpesviridae family consists of enveloped viruses with a long linear double-stranded DNA genome. There are eight human herpesviruses and these are sub-classified as alpha-, beta-, and gamma-herpesviruses. Genome-wide bioinformatics analyses revealed that the herpesvirus genomes have high densities of G4-forming sequences (also called G4 motifs) [5,6]. Several studies have indicated the functional roles of G4s in the herpesvirus life cycle. G4 formation in the promoter regions of herpes simplex virus-1 (HSV-1), human cytomegalovirus (HCMV), and Kaposi’s sarcoma-associated herpesvirus (KSHV) has been shown to regulate viral gene expression [7–10]. G4 formation in Epstein-Barr virus (EBV) and KSHV mRNAs downregulates mRNA translation, suppressing antigen presentation [11–14]. In addition, several G4-binding ligands can inhibit herpesvirus growth [9,15,16]. Therefore, G4 formation in the herpesviral genomes is thought to affect diverse steps that are critical in viral lytic and latent infection.

Varicella-Zoster virus (VZV) belongs to the alpha-herpesvirinae subfamily and has an average 125-kb genome size with at least 71 open reading frames (ORFs). VZV can cause chickenpox (varicella), an early childhood disease [17]. After primary infection, VZV establishes lifelong latent infection in sensory trigeminal and dorsal root ganglia. In aged people, latent VZV can reactivate, often causing shingles (herpes zoster) in the skin [18,19]. The most common complication of herpes zoster is chronic nerve pain (called postherpetic neuralgia) [20] and about 25% of immunocompetent adults older than 50 years experiences herpes zoster-related complication [21]. Although VZV vaccines can reduce varicella and herpes zoster effectively [22], the number of VZV global incident case has increased, with more than 83 million new VZV infection cases in 2019 [23]. As elderly population worldwide has been increased, herpes zoster is a new global health burden.

Like HSV-1, the VZV genome consists of unique and repeat regions. The ~100-kb unique long (U_L) region is flanked by the terminal repeat long (TR_L) region and the internal repeat long (IR_L) region and is followed by the 5.2-kb unique short (U_S) region flanked by the internal repeat short (IR_S) region and the terminal repeat short (TR_S) region [17,24]. The VZV genome shows a unique feature having five GC-rich reiteration (R) sequence regions that have tandemly repeated units (called elements) that vary among strains [25,26]. A previous study on the distribution of G4 motifs in the VZV genome demonstrated enrichment of putative G4 motifs in the regulatory regions and the repeat regions [5]. Another study reported that the VZV immediate early (IE) promoters contain multiple G4 sequences that may have regulatory roles for VZV lytic replication [7]. However, studies on how G4 formation in the VZV genome regulates the viral life cycle are limited, especially in the context of virus infection.

In this study, we searched for G4 motifs in the entire VZV genome using in silico analysis and identified ~150 putative G4 motifs. These sequences were enriched in the IR_S and TR_S regions and were also found in some of the R regions. We found a high density of G4 motifs in R2. These sequences are located on the template strand of ORF14 encoding glycoprotein C (gC), which acts as a virulence factor [27,28]. We performed biophysical analyses to characterize G4 formation from the predicted sequences in R2. We investigated whether G4 formation in R2 regulates gC expression by generating the mutant ORF14 genes in which the G4-forming potential was disrupted. The role of G4s was also investigated by generating the recombinant viruses containing the G4-disrupted mutant ORF14 gene and its revertant gene. Finally, we investigated whether alteration of gC expression by G4s can regulate viral cell-to-cell spread.

Results

Genome-wide analysis of G4 motifs in the VZV genome

The 124 kb genome of the VZV Dumas strain (NC_001348.1), a reference strain of VZV, was mined for putative G4 motifs using the Quadparser program. The prediction identified 148 putative G4 motifs (denoted as GQ1 to GQ148) in the Dumas genome (S1 Appendix). We divided the predicted sequences into three main categories based on the loop architecture: conventional, long-looped, and bulged G4s. Conventional G4s were characterized by loops of 1–7-nt length and four complete G-tracts (3 G-runs). We predicted 14 conventional GQs in the entire genome. In the case of long-looped G4s, we restricted the loop length of two loops to 1–7 nt, whereas the length of the third loop was allowed to be 8–30 nt. We predicted 84 long-looped GQs in Dumas.

Several studies have indicated that the stability of the G4 is inversely proportional to the loop length of the G4s [29,30]. The optimal loop length for stable G4 formation was predicted to be 10 nt long, but subsequent studies showed that G4s with longer loop lengths are also biologically relevant and should be considered for functional studies [31–34]. We also considered including bulged G4s in our study, since previous studies indicated that sequences could form stable G4s even in the presence of an incomplete G-tract [35]. The nucleotide between contiguous Gs in a G-tract can bulge outwards and accommodate the adjacent guanine to complete the G-tract with a bulge [35]. For consideration of G4 stability, only G4 motifs containing a single bulge were considered. In our computational study, we predicted 50 bulged GQs in the Dumas genome.

In addition to the conventional, long-looped and bulged G4s, G4 formation is also observed in sequences that do not contain loops between the G-tracts. The majority of G4s conform to the conventional, bulge, and long-loop G4 categories, but examples of G4s without loops have been observed in VZV ORF62/63 [7]. Additionally, in the case of the bulged G4s, the position of the bulge can alternate between the first or second G in a G-tract and the second and third G. In the case of Dumas, we observed that the sequence prediction was same in both bulge positions; therefore, we retained the sequences observed from one schema.

Our in silico analysis revealed that G4 motifs are highly enriched in two long repeated sequences, the IR_S and the TR_S regions (Fig 1A). The VZV genome contains five reiteration sequences (designated to R1, R2, R3, R4A, R4B, and R5). We found that several G4 motifs are located within the R2, R3, R4A, and R4B regions (Fig 1A). We also predicted the putative G4 motifs in the pOka strain of VZV (AB097933.1) to compare with the Dumas strain. pOka is a parental strain of vOka, a licensed vaccine strain [36], and the pOka-derived bacmid is wildly used for analysis of VZV gene functions [37]. We predicted 159 G4 motifs in the pOka genome. The number of conventional G4 in pOka was the same as that in Dumas, although the numbers for long-loop and bulged G4s were slightly different (94 and 51 in pOka, respectively) due to the sequence differences between these two strains (Fig 1B and S1 Appendix). The pOka genome also showed similar enrichment of G4 motifs in the IR_S and the TR_S regions and in R2, R3, R4A, and R4B (Fig 1C and S1 Appendix).

Distribution of G4 motifs in IR_S

IR_S contains ORF62, OriS, ORF63, and ORF64. TR_S, which contains ORF69, ORF70, OriS, and ORF71, is an inverted sequence of IR_S. The G4 motifs in IR_S are found upstream of ORF61, in the sense- and non-sense strands of ORF62, and in OriS, which overlaps with the bi-directional promoter for ORF62 and ORF63. The positions of G4 motifs in IR_S are indicated in S1 Fig. We found that GQ65 lies in the minimal ORF61 promoter activated by the ORF62 transactivator, with overlapping with two non-canonical Sp1-binding sites, which are required for effective ORF61 expression [38]. The ORF62/63 intergenic region containing OriS acts as a bi-directional promoter for ORF62 and ORF63 transcription [39]. Several G4 motifs are found in this intergenic region. GQ94 overlaps with the promoter region of ORF62 [40], while GQ103 and GQ104 are located in the promoter of ORF63 [41]. Besides the R4A reiteration sequence, the OriS region contains three consensus binding sites (Boxes A, B, and C) for the VZV origin-binding ORF51 protein and a neighboring 46-bp AT-rich palindrome sequence [42–44]. Notably, GQ95 and GQ96 are found in R4A, and GQ97 and several G4 motifs overlapping with GQ97 are localized between ORF51-binding sites [45] (S2 Fig).

Distribution of G4 motifs in R2

We focused on the G4 motifs in the R2 reiteration region since R2 shows a relatively high level of nucleotide variation among different strains [25,26] and is located within ORF14 encoding the glycoprotein C (gC). Most of the nucleotide variations observed in pOka and the other two clinical isolates YC01 and YC02, which were previously isolated from the patients of herpes zoster and chickenpox, respectively [46], were mapped in R2 (Fig 2A). The predicted G4 motifs in ORF14 (denoted as GQ12 to GQ41 in Dumas) were long-loop or bulge types and found on the template strand of ORF14 (Fig 2A). R2 consists of seven consecutive 42-bp elements and one final 32-bp element in these VZV strains. Based on the locations of G4 motifs, the R2 region could be divided into four segments, with each segment containing separated G4 motifs and G4 motifs overlapping them (Fig 2B). When sequences in pOka, YC01, and YC02 were aligned with Dumas, nucleotide variations found in R2 appeared to occur adjacent to G-runs in G4 motifs (Fig 2B). We performed a conservation analysis on 141 R2 sequences available in GenBank. Consistent with the result in Fig 2B, nucleotide variations in R2 largely occurred just downstream of G-runs in G4 motifs (S3 Fig and S2 Appendix).

Fig 2 — (A) The position of the ORF14 coding sequence is indicated as an open arrow on the bottom strand of the VZV Dumas genome and the region containing putative G4 motifs in R2 is indicated as a black box on the top strand. The variations found in ORF14 of pOka, YC01, or YC02 compared with Dumas are indicated as black vertical arrows. (B) The G4 motifs (GQ12 to GQ41) found in R2 are indicated below the genome sequence of Dumas (from 20681 to 21017). The variations found in pOka, YC01, and YC02 are indicated above the Dumas sequences. All G4 motifs of R2 are predicted on the anti-sense strand of ORF14. Black arrows, GQs for long-looped G4; red arrows, GQs for bulged G4. Seven 42-bp elements and one 32-bp element in R2 are indicated as light and dark grey bars, respectively, above the sequences.

Biophysical analysis of G4 formation by R2 G4 motifs

To investigate the G4 formation in R2 and its role in ORF14 gene expression, we used sequences predicted from pOka since the pOka bacmid was available for mutational analysis. The same numbers of G4 motifs (also named as GQ12 to GQ41) were predicted at the same position in R2 of pOka compared with those in Dumas (Fig 2 and S1 Appendix). We synthesized the oligodeoxynucleotides (ODNs) corresponding to the identified sequences and their mutant sequences, in which three or five G-to-T or G-to-A changes are introduced in G-runs to disrupt G4 formation without affecting coding potential (S1 Table). The sequences of GQ15, GQ16, GQ18, GQ19, GQ23, and GQ24 are identical. However, we tested two different mutant sequences, GQ15m/16m/18m/19m and GQ23m/24m, not to disrupt the coding potential for ORF14. We also used these two mutant sequences to make the mutant ORF14 gene for later gene expression studies. Likewise, two different mutant sequences, GQ17m/20m and GQ25m, were analyzed for identical GQ17, GQ20, and GQ25 G4 motifs. Circular dichroism (CD) spectroscopy analysis of ODNs suggested that nine GQ motifs, except GQ21 and GQ22, might form anti-parallel G4s, which displayed a prominent peak at ~290 nm and a trough at ~260 nm in the presence of 100 mM KCl as reported previously [47–49]. The GQ21 and GQ22 ODNs appeared to fold into an unusual structure, with a ~280 nm peak and ~245 nm trough, which is different from that of a single-stranded 24mer poly(T) [9]. The CD spectra of all GQ mutants, except GQ22m, were significantly changed from those of wild-type sequences, suggesting sequence-specific formation of G4s by most G4 motifs predicted in R2, except GQ21 and GQ22 (Fig 3).

Fig 3 — CD spectra of wild-type and mutant ORF14 GQ ODNs (15 μM) from pOka annealed in 10 mM Tris-HCl pH 7.5 and 100 mM KCl are shown. Each spectrum was an average of three accumulations in the wavelength range between 230–320 nm. The spectra were blanked with buffer only. GQ15/16/18/19 and GQ23/24 are same sequences, but their mutant sequences are different. Similarly, GQ17/20 and GQ25 are the same sequences, but their mutant sequences are different. The data were normalized using the maximum ellipticity and smoothed using GraphPad Prism 5.

The formation of G4s was also investigated by measuring the thermal difference spectra (TDS) that are calculated as the difference between the UV spectra at the maximum unfolding temperature (90°C) and the folding temperature (20°C). G4s are known to show the characteristic TDS spectra with a negative peak at ~295 nm and positive peaks at ~240 and ~270 nm [50]. We found that among the nine GQ motifs that showed the CD spectra of G4s, eight G4 motifs, except GQ17, showed the TDS spectra of G4 (Fig 4A), while their mutant sequences only showed the major peak at ~270 nm, indicating the disruption of the G4 structure (Fig 4B). The CD and TDS results suggest that GQ12, 13, 14, 15, 26, 28, 29, and 30 motifs form a G4 structure.

Fig 4 — TDS spectra of wild-type (A) and mutant (B) ORF14 GQ ODNs (15 μM) from pOka annealed in 10 mM Tris-HCl pH 7.5 and 100 mM KCl are shown. Each spectrum was an average of three accumulations in the wavelength range between 220–320 nm. The spectra were blanked with buffer only before TDS calculation.

To further analyze G4 structures formed by GQ12, 13, 14, 15, 26, 28, 29, and 30 motifs, we used a well-known G4-binding ligand Pyridostatin (PDS) (Fig 5A), which has previously been shown to have high affinity for the anti-parallel G4 structure [51–53]. We tested a minimum 1:2 stoichiometry of the ligand to consider the binding of the ligand on the G4 structure. Treatment with 2× molar ratio of PDS did not show any shifts in the ~290 nm peak for all G4s in the CD spectra, indicating that the topology of these G4s is not affected by PDS. We next investigated the stability of G4s by analyzing their CD thermal melting curves. The ellipticity at 290 nm, a wavelength attributed to the formation of the anti-parallel G4, was monitored in the temperature range of 20–90°C. All eight G4 motifs displayed conventional sigmoid curves with Tm values of 52.2–58.0°C (Fig 5B and Table 1). The Tm values of the ORF14 G4s were increased by 0.5 to 7.5°C in the presence of PDS (Fig 5B and Table 1). The thermal stability appeared to be determined by sequences rather than G4 structures. Among the eight G4 motifs that showed formation of a G4 structure in both CD and TDS analyses, the sequences of GQ12, 13, 15, 26, 28, and 29 appear to form a G4 structure whose Tm can be substantially increased (>3.0°C) by PDS (Table 1).

Table 1. Effect of PDS on the Tm values of ORF14 G4s.

G4 motif	Tm (°C) ± PDS^a
G4 motif	DW^b	PDS	ΔTm
GQ12	55.89±0.7	60.73±0.88	4.83
GQ13	56.83±0.64	62.38±0.54	5.55
GQ14	58.02±0.1	59.86±0.33	1.84
GQ15	53.98±0.49	57.53±0.09	3.54
GQ26	56.02±0.49	60.04±0.91	4.02
GQ28	56.64±0.99	62.02±0.88	5.38
GQ30	57.29±0.25	57.79±0.57	0.49

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^aTm represents the mean and ±SD of 3 independent replicates.

^bDW; distilled water

ODNs with an intramolecular G4 structure are known to be more compact and migrate faster than unstructured mutants in a native polyacrylamide gel electrophoresis (PAGE) [54–56]. Therefore, we tested whether the G4 motifs identified to form a G4 structure can form a compact intramolecular structure in a native PAGE. When the wild-type and mutant GQ12, 13, 14, 15, 26, 28, 29, and 30 motifs were annealed in 100 mM KCl buffer and subjected to a native PAGE, most of wild-type ODNs, except GQ26 and GQ28 motifs, migrated faster than their mutant ODNs, while the difference of mobility between wild-type and mutants became diminished under denaturing conditions (with 7M Urea) (Fig 6). These results suggest that the GQ12, 13, 14, 15, 29, and 30 motifs can form a compact intramolecular structure.

Fig 6 — Wild-type and mutant ORF14 GQ ODNs (1 μM) were subjected to native PAGE analyses after annealing in 100 mM K⁺ buffer (top) or denaturing PAGE with 7M urea (bottom). Scrambled (Sc DNA; 5′-TAACCGATGATATGAGTCAGATATAT-3′) and poly-T ODNs (Poly-T; 26mer) used as controls are shown. Double-stranded DNA (ds DNA) size makers are shown.

G4-mediated suppression of ORF14 gene expression in transfection assays

Having confirmed the G4 formation by several G4 motifs, which are distributed throughout the R2, we next asked whether G4s regulate ORF14 gene expression. To address this question, the G4-disrupted ORF14 mutant DNA (denoted as ORF14-G4m), in which all G4 motifs were replaced with the mutant sequences used for in vitro analysis (in S1 Table), was synthesized (S4 Fig). This ORF14-G4m DNA contains the same coding potential as the wild-type ORF14 (S5 Fig). The wild-type ORF14 and ORF14-G4m DNAs were cloned into the pSG5 vector containing the simian virus 40 early promoter [57] to express gC with a C-terminal HA-tag. gC is expressed as a ~64-kDa unmodified form and larger glycosylated forms [58]. In immunoblotting of lysates of 293T cells transfected with wild-type ORF14-HA and ORF14-G4m-HA plasmids, we observed the unmodified form of gC-HA largely accumulated in transfected 293T cells, although some modified forms (probably, glycosylated forms) were also detectable (Fig 7A). The unmodified form of gC-HA showed a slightly larger size than expected due to the addition of extra-amino acids between gC and the HA tag, which was introduced during cloning using LR clonase. We found that gC-HA expression was higher with the G4m plasmid than with the wild-type plasmid, suggesting that the G4 formation in ORF14 inhibits gC expression.

Fig 7 — (A) 293T cells (1x10⁶ cells per well) in six-well plates were transfected with 2 μg of empty vector (EV) or plasmids expressing pOka ORF14-HA or its G4-disrupted form (G4m). Total cell lysates were prepared in RIPA buffer at 72 h after transfection. The total cell lysates were subjected to immunoblotting with anti-HA antibody. Non-specific bands are indicated by the open circle. The levels of β-actin are shown as a loading control. (B) 293T cells were transfected as in (A) and treated with DW or increasing amounts (2.5 and 5 μM) of PDS at 16 h after transfection. At 72 h after transfection, total RNA was prepared and the ORF14 mRNA levels were measured by qRT-PCR and normalized with those of β-actin. Results are shown as mean values and standard errors of triplicates. (C) 293T cells (1x10⁶ cells per well) in six-well plates were transfected with 2 μg of empty vector (EV) or plasmids expressing wild-type ORF14-HA (Wt) or its different G4-mutated forms (G4m-1, G4m-2, or G4m) and treated with DW (D) or 2.5 μM of PDS (P) at 16 h after transfection. At 72 h after transfection, total RNA was prepared and the ORF14 mRNA levels were measured by qRT-PCR as in (B). (D) 293T cells were transfected as in (A) and treated with actinomycin D for the indicated times. The gC mRNA levels were determined as in (B and C). (E) 293T cells were transfected with 2 μg of empty vector (EV) or plasmids expressing pOka ORF14 (Wt) or its G4-disrupted form (G4m). The gC mRNA levels were determined as in (B) and (C).

G4 formation on the template strand is known to inhibit gene transcription [59–61]. Therefore, we investigated whether the observed effect of G4 formation on gC expression occurs at the transcriptional level. We transfected 293T cells with wild-type and G4m ORF14 plasmids and treated cells with PDS, and gC mRNA levels were measured by qRT-PCR. The level of gC mRNA in PDS-untreated cells was higher with ORF14-G4m than that with wild-type ORF14, and the gC mRNA levels from wild-type ORF14 were dose-dependently reduced by PDS treatment, while those from ORF14-G4m were not affected (Fig 7B). These indicate that the ORF14 gene expression is suppressed by G4 formation at the transcriptional level.

To further support the G4-mediated suppression of gC expression, we constructed two more ORF14 mutant genes (G4m-1 and G4m-2). Compared to G4m, which contains all GQ mutant sequences (from GQ12m to GQ41m), G4m-1 contains only GQ34m to GQ41m sequences, whereas G4m-2 contains only GQ18m to GQ41m sequences. When the gC mRNA levels were compared among G4m, G4m-1, and G4m-2 ORF14 genes by qRT-PCR in transfected cells, both G4m-1 and G4m-2 also expressed higher levels of gC mRNA than wide-type, but the increase of gC mRNA by these mutants was much less than that by G4m (Fig 7C). Furthermore, PDS treatment repressed gC transcription from G4m-1 and G4m-2 but not from G4m (Fig 7C). The increased gC-HA mRNA transcription in the G4-disrupted ORF14 was not due to the enhanced stability of gC-HA mRNA since the stability of mRNAs produced from wild-type and G4m ORF14 was similar (Fig 7D). We observed that the difference in gC mRNA levels between wild-type and G4m was less than that in gC protein levels in transfected cells. Although the mutations introduced did not affect mRNA stability, it cannot be excluded that they also affected protein expression at the post-transcriptional level in transfection assays. The production of gC mRNA from the untagged ORF14 plasmid was similarly increased by G4-disrupting mutations, indicating that the HA tag did not affect ORF14 transcription (Fig 7E). Together, these results support the G4-mediated suppression of ORF14 expression and also demonstrate that the more G4 is formed, the more ORF14 is suppressed.

G4 formation suppresses gC expression at the transcriptional level during virus infection

We further investigated the suppressive effect of G4 formation on gC expression using recombinant viruses. We first produced a recombinant pOka ORF14-HA virus that expresses C-terminal HA-tagged gC using bacmid mutagenesis (S6A Fig). This recombinant virus is designed to express gC-HA without inserting additional amino acids between gC and HA. The pOka bacmid contains a green fluorescent protein (GFP) expression cassette between ORF60 and ORF61 and a luciferase reporter gene driven by the simian 40 virus promoter between ORF66 and ORF67 [37]. The recombinant viruses were grown in MeWo cells after transfection with bacmid DNAs. Human fibroblast (HF) cells were infected with wild-type pOka (prepared from the pOka bacmid) and pOka-ORF14-HA viruses and their growth was measured by luciferase activity. The growth of the ORF14-HA virus was similar to that of the wild-type virus, indicating that the C-terminal HA tag on gC did not affect viral replication (Fig 8A). In immunoblotting with anti-HA antibody, the gC-HA proteins in ORF14-HA virus-infected cells were expressed as highly glycosylated forms with a major form between 100 and 140 kDa (Fig 8B).

Fig 8 — (A) HF cells were infected with recombinant pOka viruses containing the wild-type ORF14 or ORF14-HA gene at an MOI of 0.01. Cells were harvested at 24, 48, 72 and 96 h after infection and luciferase activities in cell lysates were measured. Results are shown as mean values and standard errors of three independent experiments. (B) HF cells were infected with recombinant viruses (wild-type or ORF14-HA) at an MOI of 0.01 Total cell lysates were prepared at 96 h after infection and subjected to SDS-PAGE and immunoblotting with ant-HA and anti-gE (ORF68) antibodies. Levels of β-actin are shown as a loading control. (C) HF cells in 12-well plates were infected with recombinant pOka viruses [wild-type, G4-disrupted mutant (G4m), and its revertant (R)] at an MOI of 0.001. Cells were harvested at 24, 48, 72, 96, and 120 h after infection. The cells were trypsinized and the virus titers were determined by plaque assay. Results are shown as mean values and standard errors of three independent experiments. p-values <0.01 (**) are indicated when significance was observed both between G4m and Wt and between G4m and R. (D and E) HF cells in 12-well plates were infected with recombinant viruses as in (C). Total cell lysates were also prepared at 48, 72, 96, and 120 h after infection and the expression levels of gC-HA and gE were determined by immunoblotting with anti-HA and anti-gE antibodies, respectively. Levels of β-actin are shown as a loading control (panel D, left). The gC-HA and gE bands in three independent experiment were quantitated with ImageJ and shown as graphs (panel D, right). Total mRNA was also prepared at 72 and 96 h after infection and the mRNA levels of gC (ORF14) and gE (ORF68) were determined by qRT-PCR. Results are shown as mean values of viral mRNA normalized with β-actin and standard errors of three independent experiments (E). (F) HF cells in 12-well plates were infected with recombinant viruses as in (C). At 72 h after infection, cells were treated with PDS (5 μM) for 48 h before qRT-PCR assays. The gC and gE mRNA levels and the gC/gE ratio are shown as in (E).

We then produced the recombinant virus containing the G4 formation-defective ORF14 gene. The G4 motifs-containing region in the pOka ORF14-HA bacmid was replaced with the corresponding region from the ORF14-G4m plasmid, resulting in the ORF14-G4m-HA bacmid. Its revertant bacmid, ORF14-G4m-HA-(R), was also produced from the ORF14-G4m-HA bacmid (S6B Fig). Recombinant viruses were grown in MeWo cells transfected with the bacmid DNAs. HF cells were then infected with recombinant viruses [ORF14-HA, ORF14-G4m-HA, or ORF14-G4m-HA-(R)] at an MOI of 0.001 and their growth was compared by determining cell-associated virus titers by performing plaque assays in MeWo cells. The ORF14-G4m-HA virus showed slightly reduced growth compared with that of the wild-type and revertant viruses (Fig 8C).

We next compared the gC levels in HF cells infected with recombinant viruses at an MOI of 0.001 by immunoblot analysis. During ORF14-G4m-HA virus infection, gC was detected as early as 72 h at a significantly higher level than those in wild-type and revertant viruses (Fig 8D). As a control, gE (encoded by ORF68) was detected from 48 h during ORF14-G4m-HA virus infection and its levels were comparable at 48 h and lower at 96 and 120 h compared to those in wild-type and revertant viruses (Fig 8D). The delayed expression of gC compared with that of gE during wild-type virus infection was consistent with the previous reports [62–64]. We also compared the gC and gE mRNA levels in recombinant virus-infected HF cells using qRT-PCR. The gC mRNA levels in G4m virus were much higher at 72 h and 96 h than those in wild-type and revertant viruses (Fig 8E, left), while gE mRNA levels in G4m virus similar or reduced compared to those in wild-type and revertant viruses (Fig 8E, right), demonstrating that G4 formation in ORF14 indeed inhibits gC expression at the transcriptional level during virus infection. We also investigated the effect of PDS on gC transcription in virus-infected cells. Since the G4 ligands could affect the expression of several viral and cellular genes from the early stage of infection, PDS treatment reduced gC and gE transcription from both wild-type and G4-disrupted (G4m) viruses (Fig 8F, left and center); however, the gC/gE mRNA ratio was higher by 3-fold in G4m virus than in wild-type and revertant viruses (Fig 8F, right). Furthermore, the gC/gE mRNA ratio was decreased by PDS treatment in wild-type and revertant viruses, while it was increased in G4m virus (Fig 8F, right). These results demonstrate that gC transcription was less severely suppressed by PDS during G4m virus infection than in wild-type virus infection.

Enhanced gC expression in the ORF14-G4m virus attenuates viral cell-to-cell spread

Previous studies suggested that enhanced gC expression may result in the reduction of viral cell-to-cell spread in VZV and Marek’s disease virus (MDV), an alphaherpesvirus of chickens [62,65]. Therefore, we also investigated whether the ORF14-G4m virus displays a similar growth property. MeWo cells were transfected with the VZV bacmids containing the ORF14-HA, ORF14-G4m-HA, or its revertant gene and GFP⁺ plaque size was measured at 8 days after transfection. The plaque size of the ORF14-G4m virus was significantly smaller than those of the wild-type and revertant viruses (Figs 9A and S7A). The similar small plaque property of the ORF14-G4m virus was observed when MeWo cells were infected with cell-associated recombinant viruses (Figs 9B and S7B). A similar small plaque phenotype of the ORF14-G4m virus was observed in HF cells (S8 Fig). These results demonstrate that the enhanced gC expression in the ORF14-G4m virus is inhibitory for viral cell-to-cell spread.

Fig 9 — (A) MeWo cells in six-well plates were transfected with the VZV bacmids (1 μg) containing the ORF14-HA gene [wild-type, G4m, or revertant (R)]. At 8 days after transfection, images of GFP+ plaques were taken. A similar plaque size reduction pattern was found with the G4m virus in two independent experiments. Since the efficiency of plaque generation in bacmid-transfected cell is low, we combined all plaques (n = 11 for Wt, n = 17 for G4m, and n = 8 for G4m-R) in two experiments to make the graph. Plaque area size was measured with ImageJ and shown as a box and whisker graph. Box plots show the first and third quartiles as a box. Horizontal lines are samples median. X corresponds to the mean. Whiskers correspond to 1.5 times the inter-quartile distance (IQR) from the difference between first and third quartiles. Images showing plaques are shown in S7A Fig. (B) MeWo cells in six-well plates were infected with cell-associated recombinant viruses. At 5 days after infection, cells were stained with crystal violet and plaque size was measured (n = 20) and shown as a graph as in (A). The experiments were performed more than three times and showed similar results. The data from a representative experiment are shown. Plaques images are shown in S7B Fig.

Discussion

Our genome-wide analysis of G4 motifs in the VZV genome revealed that they are enriched in the IR_S and the TR_S regions and in some of the R regions (R2, R3, R4A, and R4B). Each R region has its unique repeat sequences (elements) and the copy number of elements varies among the different VZV strains [26]. The G4 motifs found in R2 have interesting features. First, nucleotide variations found in R2 mostly occur in relation to G4 motifs. Comparative analysis of nine glycoprotein genes in different strains indicated a higher level of single nucleotide polymorphisms in ORF14 [64]. It is intriguing to speculate that G4 formation during gene transcription or DNA replication is responsible for this high level variation in ORF14. Second, the G4 motifs are located at the junction between two elements, suggesting the involvement of G4s in determining the copy numbers of elements. G4s may cause RNA polymerase pausing and DNA nicks, promoting recombination. Notably, in the absence of BLM helicase, of which mutation can cause Bloom syndrome, a cancer predisposition disorder, sister chromatid exchange events are enriched at sites with G4 motifs in transcribed genes [66]. Furthermore, the recombination breakpoints found in the HSV-1 genome are associated with G4 motifs, suggesting a role of G4s in recombination during HSV-1 infection [67]. The numbers of repeated units in R2 vary in strains, although they seem to be relatively stable during viral replication [68]. Whether the copy numbers of elements in the R regions affect the VZV life cycle is not clear. However, it is conceivable that recombination at the R regions maintains the genome length and G4 formation is involved in this process by promoting recombination.

In this study, we provided biophysical evidences that several G4 motifs found in R2 within ORF14 form a G4 structure. Analyses of ODNs showed that nine motifs out of 11 predicted motifs, except GQ21 and GQ22, formed an anti-parallel G4s in CD. Among these nine G4 motifs, eight G4 motifs such as GQ12, 13, 14, 15, 26, 28, 29, and 30, except GQ17, showed the G4 characteristic in TDS. Therefore, we considered that these eight G4 motifs can form a G4 structure. In the thermostability assays using PDS, the stability of G4s formed by these G4 motifs could be increased by PDS. The native PAGE analysis demonstrated that GQ12, 13, 14, 15, 29, and 30 can form a compact intramolecular structure. These sequences for these six G4 motifs are distributed throughout the ORF14 R2, indicating that G4 formation is a prominent structural characteristic in the R2 region.

We showed that the G4 formation in R2 (on the template strand of ORF14) negatively regulates gC transcription. This finding is consistent with the notion that G4 formation on the template strand can inhibit transcriptional elongation by RNA polymerases. VZV expresses 11 glycoproteins that play important roles in virus attachment, membrane fusion, and pathogenesis [69]. Unlike gC proteins in HSV-1 and PRV [70,71], gC in VZV is not required for virion binding to cell surface receptors for virus entry, since the mutant viruses that lack gC expression normally grow in cell culture [27,72]. However, gC appears to be involved in tissue tropism, since the gC-defective mutant viruses show poor growth in human skin tissue [27,28]. Previous studies showed that expression level of gC varies among strains, with a lower level of expression in pOka than Scott [58]. gC is a true late protein, as its transcription occurs at very late during VZV infection probably due to the lack of the factors required for gC synthesis during early phases of infection [62–64]. We also observed delayed expression of gC compared with that of gE. Furthermore, our analysis of the G4 formation in R2 revealed that gC transcription is suppressed by G4s. Whether delayed expression of gC is solely attributed to the G4-mediated suppression of gC transcription is not clear. However, our results provide a clue for this long-standing question of gC expression.

We also found that the enhanced gC expression in the mutant virus containing the G4-disrupted ORF14 gene led to a small plaque phenotype. Previous studies reported that higher gC expression of VZV or MDV led to a reduction of plaque size [62,65]. We also verified that higher gC expression by the ORF14-G4m virus results in a decreased plaque size in both bacmid DNA-transfected or recombinant virus-infected cells. The ORF14-G4m virus also showed a slightly reduced growth. This is consistent with findings in MDV showing that the overexpression of gC reduced viral growth [62,65]. Our results support the notion that higher expression of gC has a negative impact on the cytopathology and viral growth rate during VZV growth. Since the copy number varies in some strains, it is speculated that strains with more 42-bp elements in ORF14 will express less gC due to G4 formation and show better cell-to-cell spread.

González-Motos et al. recently showed that gC of VZV binds to chemokines with high affinity and facilitates the migration of leukocytes [73]. VZV infects dendritic cells and T lymphocytes, and this may be important for systemic dissemination of the virus [74,75]. Since gC is expressed at true late stages of the virus life cycle [63,76], the delayed gC expression by G4s may be a viral strategy to optimize the timing of virus dissemination, unless the virus is eradicated by immune responses before maturation processes for infectious virions. Further studies are necessary to elucidate how G4-mediated regulation of gC expression affects VZV replication in vivo.

Materials and methods

Cells and chemicals

Immortalized human melanoma MeWo cells, human embryonic kidney (HEK) 293T cells, and human foreskin fibroblast (HF) cells were grown in Dulbecco’s modified Eagle medium (GIBCO) supplemented with 10% fetal bovine serum and 100 U of penicillin-streptomycin at 37°C and 5% CO₂. PDS was purchased from Cayman.

In silico G4 prediction

The Dumas VZV genome (NC_001348.1) and pOka VZV genome (AB097933.1) were mined for potential non-overlapping G4 motifs using the Quadparser program [77]. We divided the G4s into conventional, long-loop, and bulged categories based on the schema used for prediction of the G4s. Conventional G4s were predicted using the schema G_3-6N_1-7G_3-6N_1-7G_3-6N_1-7G_3-6 and represent the conventional 3-stack G4s. In our case, long-loop G4s were restricted to a single long loop and predicted using the schema G_3-6N_8-30G_3-6N_1-7G_3-6N_1-7G_3-6 (and subsequent loops). Bulged G4s were predicted using the schema G_3-6N_1-7G_3-6N_1-7G_3-6N_1-7G₂(A/T/C)G and remaining loop permutations. Duplicates were removed if the same sequence was predicted under the same category (long-loop, conventional, or bulged). Sequences were retained if predicted under different categories.

Circular dichroism (CD) spectroscopy and thermal melting curve analysis

ODNs used for the CD spectroscopy are described in S1 Table. CD spectroscopy was performed on a Jasco J-810 spectroscopy fitted with a Peltier temperature controller (CDF426S, Jasco, Japan). The ODNs were dissolved at a concentration of 15 μM in a buffer containing 10 mM Tris-HCl (pH 7.5) and 100 mM KCl, followed by denaturation at 95°C for 5 min and annealing at room temperature for 2 h. For studies with G4-binding ligands, preformed G4s were treated with 30 μM PDS for a DNA-to-ligand ratio of 1:2. CD spectra were measured at 25°C using a 1 mm path length quartz cuvette (Hellma, Germany) as the average of three accumulations between 230–320 nm, with a response time of 2 sec, scanning speed of 100 nm/min, and data pitch of 1 nm. CD melting curves were recorded between 20–90°C at a wavelength of 290 nm for all nucleotides with 2°C/min heating rate. After subtracting the spectrum of buffer only from all samples, the data were normalized to the maximum ellipticity. The first derivative of the melting curve was plotted and fitted and smoothed using inbuilt functions in GraphPad Prism 5.

Thermal Difference Spectra (TDS)

The UV spectra was measured on a Jasco V-750 UV spectrophotometer fitted with a Peltier temperature controller (ETCS-761, Jasco, Japan). The ODNs were dissolved at a concentration of 15 μM in a buffer containing 10 mM Tris-HCl (pH 7.5) and 100 mM KCl, followed by denaturation at 95°C for 5 min and annealing at room temperature for 2 h. The UV spectra was measured using a 1 mm path length quartz cuvette (Hellma, Germany) at 20°C and 90°C following which the spectra were blanked using buffer only spectra. Finally, the TDS was measured as the difference between absorbance at 20°C and 90°C. The data was normalized to the maximum TDS value to obtain the normalized TDS.

Plasmids

The ORF14 of pOka was amplified by PCR as a SalI-NotI fragment from the pOKa bacmid with specific primers (LMV2825/2847 for HA-tagged plasmid or LMV2825/2826 for untagged plasmid) (S2 Table). The PCR products were cloned into the SalI and NotI sites of the pENTR-1A vector (Invitrogen). The G4-defective ORF14 (G4m) DNA was chemically synthesized (Synbio Technologies and Cosmogentech Inc.) and cloned into HindIII-BamH1 sites in pUC57. The G4-defective ORF14 was then PCR amplified with primers (LMV2825/2847) and cloned into the SalI and NotI sites of pENTR-1A. Expression plasmids encoding C-terminal HA-tagged ORF14 were generated by transferring the DNA in pENTR-1A to the pSG5-based destination vector using LR clonases (Invitrogen).

Antibodies

Anti-HA mouse monoclonal antibodies (MAbs) 3F10 conjugated with horseradish peroxidase (HRP) and 12CA5 were purchased from Roche. Anti-gE mouse MAb 9C8 was purchased from Santa Cruz Biotechnology. Anti-β-actin mouse MAb GT5512 was purchased from Gene Tex, Inc.

Immunoblot analysis

Cells were washed with PBS and total cell lysates were prepared in RIPA buffer. Equal amounts of clarified cell extracts were separated on an SDS-polyacrylamide gel and electroblotted onto nitrocellulose membranes. Blots were blocked with PBS plus 0.1% Tween 20 (PBST) containing 5% nonfat dry milk for 1 h at room temperature. After three washes with PBST, blots were incubated with the appropriate antibodies in PBST for 1 h at room temperature or overnight at 4°C. After three 5-min washes with PBST, blots were incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (Amersham) for 1 h at room temperature. For HA-tagged proteins, HRP-conjugated anti-HA antibody was used. Blots were then washed three times with PBST and the protein bands were visualized with an enhanced chemiluminescence system (Amersham).

Bacmid mutagenesis

The pOka bacmid [37], which was kindly provided by Dr. Hua Zhu (Rutgers-New Jersey Medical School, NJ, USA), was used as a template for mutagenesis. The pOka bacmid containing the ORF14-HA gene was generated using a counter-selection bacterial artificial chromosome (BAC) modification kit (Gene Bridges, Germany). Briefly, the rpsL-neo cassettes flanked by a 50-nucleotide homology arm targeting the C-terminal region of ORF14 were PCR amplified (with LMV2877 and LMV2878) and introduced into E. coli DH10B containing wild-type pOka bacmid for recombination by electroporation using a Gene Pulser II (Bio-Rad). The primer sequences used for ORF14 bacmid mutagenesis are shown in S2 Table. The rpsL-neo containing intermediate bacmid was selected on Luria Broth (LB) agar plates containing kanamycin. In second round of homologous recombination, the rpsL-neo marker cassette was replaced by the annealed 130 nucleotide homologous arms containing an HA-tag (LMV2879 and LMV2880), resulting in recombinant bacmid containing the ORF14-HA gene. The mutation in the bacmid was confirmed by direct sequencing.

The G4-disrupted mutant (G4m) ORF14 gene was generated in the ORF14-HA bacmid background. The rpsL-neo cassettes flanked by 50 nucleotide homology arms upstream and downstream of the target region (i.e., the G4-forming region) were PCR amplified using specific primer sets (LMV2881 and LMV2882). The amplified rpsL-neo fragments were purified and introduced via electroporation into E. coli DH10B containing the G4m sequences for recombination. The intermediate pOka bacmid construct containing the rpsL-neo cassette was selected on LB agar plates containing kanamycin. Next, to replace the rpsL-neo cassette, the G4-disrupted ORF14 DNA fragments were PCR amplified with specific primers (LMV2883 and LMV2884) and were recombined into the pOka bacmid containing the rpsL-neo cassette. E. coli cells containing the pOka-ORF14-G4m bacmid were selected on LB plates containing streptomycin. Mutated regions were amplified and sequenced to verify the desired mutations. To generate the revertant pOka bacmid from ORF14-G4m, the intermediate pOka bacmid construct containing the rpsL-neo cassette was selected and the rpsL-neo cassette was replaced with DNA fragments containing the wild-type ORF14 sequences, which were PCR amplified with specific primers (LMV2883 and LMV2884), by homologous recombination as described above.

To generate recombinant viruses, pOka bacmids were introduced into MeWo cells by transfection using the OmicsFect reagents (Omics Bio) according to the manufacturer’s protocol. After 10 days post-transfection, the cells were transferred to new MeWo cell culture to propagate the viruses. The titers of propagated virus stocks were determined using plaque assay with MeWo cells.

Plaque assays

Plaque assays were performed to determine titers of recombinant viruses. MeWo cells were infected with serial 1 to 10 dilutions of viruses. At 5 days post-infection, the medium was discarded, and cells were stained with crystal violet for 6 h prior to plaque counting.

Quantitation of viral mRNAs

Total RNA was extracted from virus-infected cells (5×10⁵) using TRI reagents (Molecular Research Center). RNA quality was checked by determining the integrity of rRNAs in agarose gel electrophoresis. RNA to cDNA EcoDry Premix (TaKaRa) was used to generate cDNAs. qRT-PCR was performed using the Power SYBR Green PCR Master Mix and QuantStudio Real-Time PCR System. The primers used for ORF14 and ORF68 were 5′-GGATGCATAGGGTTGCGATAA-3′ (ORF14 forward), 5′-TGCATCTACCTACGCCACTA-3′ (ORF14 reverse), 5′-GTACATTTGGAACATGCGCG-3′ (ORF68 forward), and 5′-TCCACATATGAAACTCAGCCC-3′ (ORF68 reverse) [76]. The primers for β-actin were 5′-AGCGGGAAATCGTGCGTG-3′ (forward), and 5′-CAGGGTACAT GGTGGTGCC-3′ (reverse).

Luciferase reporter assays

Cells were collected and lysed by three freeze-thaw steps in 100 μl of 0.25 M Tris-HCl (pH 7.9) with 1 mM dithiothreitol. Cells extracts were clarified in a centrifuge and 20 μl of extracts were incubated with 350 μl of reaction buffer A (25 mM glycylglycine [pH 7.8], 15 mM MgSO₄, 5 mM ATP, and 4 mM EGTA) and then mixed with 100 μl of 0.25 mM luciferin (Sigma-Aldrich) in reaction buffer A. The TD-20/20 luminometer (Turner Designs) was used for a 10-s assay of the photons produced (measured in relative light units).

Statistical analysis

Statistical significance are determined using Student’s t-test and indicated by p-values <0.05 (*), <0.01 (**), and <0.001 (***) are indicated.

Supporting information

S1 Appendix. Putative G4 motifs predicted in the VZV genomes (Dumas and pOka).

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S2 Appendix. Nucleotide variations found in the R2 reiteration sequence regions of VZV.

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S1 Fig. Putative G4 motifs in IR_S.

(A) The positions of ORFs and OriS in the IR_L, IR_S, U_S and TR_S regions in the VZV genome are shown. (B) The G4 motifs (GQ64 to GQ104) found in IR_L and IR_S are indicated on the top and bottom strands of the genome. Similar G4 motifs with overlapping sequences are clustered in a box. The types of G4 (conventional, long-loop, and bulged) are indicated with different colors.

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S2 Fig. Indication of putative G4 motifs in IR_S on the VZV genome sequence.

The G4 motifs (GQ65 to GQ104) found in IR_S are indicated below the genome sequence of Dumas. The G4 motifs predicted for conventional, long-loop, and bulged G4s are indicated as blue, black, and red lines, respectively. The positions of ORF61, ORF62, OriS, and ORF63 are indicated. In OriS, the locations of the R4A reiteration sequence, Boxes A, B, and C, and palindrome sequence are indicated.

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S3 Fig. Conservation of G-runs and nucleotide variations in R2 reiteration sequences.

Multiple nucleotide sequence alignment of the R2 reiteration regions in ORF14 of 141 VZV strains was performed using MAFFT [78,79]. R2 of the DUMAS strain (from 20,681 to 21,017 in the genome) was used as the reference sequence. Graphical representation of R2 variation indicates preserved G-runs (3 Gs) in orange, other preserved sequences in gray, and sequences with variations in black letters. Nucleotide sequences with <1% variations are represented by a single black letter. The degree of variation in nucleotides is indicated as a percentage.

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S4 Fig. Comparison of wild-type and G4-disrupted mutant (G4m) ORF14 gene sequences.

Alignment of the top strand sequences for part of ORF14 from 20602 to 21033 (in pOka) with its corresponding G4-disrupted mutant (G4m) sequences is shown. The identical sequences are denoted as *. The positions of G4 motifs (GQ12 to GQ41) in pOka are indicated below the sequences. Black arrows, long-looped G4; red arrows, bulged G4. The sequences corresponding to the translation initiation codon for gC are indicated in the box.

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S5 Fig. Complete sequences of the G4-disrupted ORF14 gene and its translation product.

(A) The complete sequences on the template strand of the wild-type ORF14 gene of pOka and its G4-disrupted mutant (G4m) are shown. The mutated sequences in G4m are highlighted. (B) The gC amino acid sequences produced from the wild-type and G4m ORF14 genes are compared and show 100% identity. The repeated sequences of 14 amino acids encoded from the R2 region are indicated as arrows.

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S6 Fig. Schemes for the production of recombinant viruses containing the ORF14-HA and ORF14-G4m-HA gene.

The scheme for the production of pOka bacmid containing the ORF14-HA gene (A) and pOka bacmids containing the ORF14-G4m-HA gene and its revertant gene (B). See the Materials and Methods for the detailed procedure.

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S7 Fig. Images showing plaque sizes of recombinant viruses in MeWo cells.

Images of MeWo cells used for measuring plaque sizes in Fig 9 are shown. The boundary of green fluorescence was used as a plaque boundary for measuring plaque regions in VZV-bacmid infected MeWo cells. (A) GFP images of cells showing plaques taken at 8 days after bacmid transfection. (B) Cell images showing plaques taken at 5 days after virus infection. To determine the plaque region in VZV-infected MeWo cells, empty regions for the confluent cell monolayer are measured.

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S8 Fig. Comparison of plaque sizes of ORF14 recombinant viruses in HF cells.

(A) HF cells in six-well plates were infected with cell-associated recombinant viruses. At 14 days after infection, cells were stained with crystal violet and plaque size was measured (n = 18). Plaque area size was measured with ImageJ in triplicate experiments and shown as a box and whisker graph. p-values <0.001 (***) are indicated. (B) Plaques images are shown as in S7 Fig.

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S1 Table. G4 motifs found in pOka R2 and their mutant sequences used in this study.

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S2 Table. Primers used for ORF14 cloning and bacmid mutagenesis.

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Acknowledgments

We thank Dr. Hua Zhu for providing the pOka bacmid.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This work was supported by grants from the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2020R1A4A1018019, 2021M3A9I2080488, and 2022R1A2C1006748) to JHA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Pathog. doi: 10.1371/journal.ppat.1011095.r001

Decision Letter 0

Blossom Damania, Robert F Kalejta

11 Oct 2022

Dear Dr. Ahn,

Thank you very much for submitting your manuscript "G-quadruplexes formed by Varicella-Zoster virus reiteration sequences suppress expression of glycoprotein C and regulate viral cell-to-cell spread" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

There is a major concern from one reviewer (#2) that the observed phenotype may be the result of the HA tag on gC interrupting proper processing, as opposed to the mutations in the G4 quadruplex. This issue, as well as the multiple comments from reviewer #1, need to be addressed.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Robert F. Kalejta

Associate Editor

PLOS Pathogens

Blossom Damania

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Chung and co-authors submitted a manuscript dealing with the presence of G-quadruplexes (G4s) in the genome of Varicella Zoster virus (VZV). In particular, the research work intends to shed light on the downregulating role of a cluster of repeated short regions, able to fold into G4s, on the expression of the viral glycoprotein C.

The work is clearly written, and the topic is very interesting. Nonetheless, there are issues in the experimental design and in the described results that need to be clarified.

Reviewer #2: In this manuscript, Chung et al report evidence to suggest that there high G/C regions of VZV include structures called G Quadruplexes. G Quad elements are known to form in single stranded DNA and RNA under some considtions to form different structures that can affect expression of RNA at the transcriptional level. They particularly focus on the VZV gC protein, which in most strains has 7/2/3 or a 42bp high G+C repeating element as part of its coding region. The work claims there is the formation of G Quadruplexes by the repeat element that regulates the transcription of gC so that, when such elements are mutated, there is a higher level of gC expression.

The work first involves scanning the genome for G Quadruplex formation elements using a program. They report the finding of about 150 potential elements. Including several found in the gC coding repeating elements. The then do a series of biophysical studies on the specific elements assessed outside of the context of their VZV environment using oligonucleotides and mutant oligonucleotides. They show subtle differences in circular Dichroism spectroscopy, Thermal difference spectra and the consequences of a known binding drug to G4 Quad pyridostatin. Which inhibits the actions ( it is not clear why the drug also affects the mutant oligonucleotides despite the text indicating it does not, See Figure 7b. Whiles this shows effects on the G4 Q oligonucleotides, it is of course separated from the context of the virus, so the intepretation of these data and if it occurs in the context of the virus is difficult. While interesting, I have several major concerns. I do not think this work is of sufficient significance and asdvancement for the field for publication in the Journal Plos Pathogens

Reviewer #3: The manuscript by Chung et al explores the potential role of G-quadruplexes for regulating expression of glycoprotein C in Varicella Zoster virus. This is a nicely presented story, beginning with a bioinformatic evaluation of potential G quadruplexes within the VZV genome and then biophysical analyses of G-quadruplexes found within the coding region of ORF 14 (gC). From there, the investigators compared WT gC to a gC engineered with disrupted G-quadruplexes both in transient transfection experiments and then further via recombinant viruses. Overall, the evidence suggests that G4 formation within the gC coding sequences suppresses gC expression and that disruption of these motifs can impart an observable phenotype on virus growth in culture.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: -It is not clear how the authors chose the viral strains and bacmids used in the study. The strains pOKA and YC01 and YC02 were not introduced and a better description would help the reader.

-The performed research on Putative G-quadruplex-forming sequences (PQS) on the genome and the choice to consider also bulged sequences and PQS with no loops is interesting: please add a summary table that categorizes the PQS.

-How many bulges per sequence were considered, were there sequences with more than one bulge?

-The authors report a partial conservation analysis on VZV genomes, taking into consideration another strain. A conservation analysis performed on all VZV genomes in GenBank needs to be performed. Add also a detailed description on PQSs location in the genome (i.e. promoter, coding, UTR etc) (partial information is contained in the S1 Appendix file).

-The authors noticed that many PQSs are located in the internal and terminal repeats. Which genomic features are contained in those regions? Promoters? Enhancers? Origins of replication?

-The final sentence of “Distribution of G4 motifs in R2” (page 7, lines 165-166) is not clear. Figure 2b shows that mutations are on non-G nucleotides but the sentence states the opposite. Can the authors clarify what was the conveyed message?

-The authors mutated the G residues (Page 7, lines 174) to T o A, but it is not explained why these nucleotides were chosen. Were all mutations aligned with the wt coding sequence (as shown in Fig S2 C), to state that the coding potential was not disrupted? Can the authors add new supplementary material on these analyses?

-Page 8, line 184, the authors define the sequence with a positive peak around 280 nm and a negative peak around 245 nm as an unusual structure. Are there differences between the recorded spectra and those provided by a random nucleotide unstructured sequence?

-Figure 4 is very difficult to interpret (too many spectra on the same graph), a better one should be provided.

-Page 8, lines 205-210, may the authors comment on the thermal stability and further G4-ligand stabilization of sequences taking into consideration the presence of bulges, short and long loops? Is there a trend in PDS binding and stabilization?

-pSG5 vector is used for different studies, it is not clear which promoter drives gC expression? Are there PQS in the expressing promoter?

-Please add a scheme showing the mutations inserted in the gC sequence

-Figure 7 A, why do the authors perform an IP prior to WB? What are the bands around 140 KDa? If they were specific HA targets, why were they not concentrated by the IP passage? There is a band corresponding to gC-HA also in the first lane of EV (empty vector): can the authors comment on that? Panels B and C, was a normalization formula used (B-actin as reference is stated in the methods section)?

-Figure 8 panel b, gC protein is indicated at a different height with respect to the band shown in Fig. 7.

-In the last result section, (G4 formation suppresses gC expression at the transcriptional level during virus infection) the infection assays are described in a very unclear way (page 10, lines 268-271), please carefully revise all this part.

-The authors state that the virus expressing the mutated gC, which is expressed earlier and more abundantly than the wild type counterpart, attenuates the cell to cell spread in plaque reduction assays. The plaque size may depend on various factors. Please analyze and show the overall replication kinetics of the mutated virus, to understand whether the gC G4-related mutation hampers or exacerbates VZV infection. Moreover, the viral titres generated by the two viruses (wild type and mutated) would give information of the viral infectivity in human cells.

-Are there circulating strains bearing the studied mutation?

Reviewer #2: They then assess the effect of mutating the g Quad elements on gene expression from a strong transfection based plasmid promoter and from a recombinant virus This raises my Major concern. In both transiently infected and recombinant infected cells, they report the expression of gC using a tagged protein. The gC protein detected in infected cells and from transiently expressed protein is far too small for fully mature gC. Multiple authors have shown the major predominant form of gC is 100-110KD, and is bigger than gE. gC contains 40KD of additional post translational modifications. However the gC in the blots in figure 8b and d, as well as in 7A, all show a protein at less than 75Kd. I do not know why, but one reason is that the C terminal HA tag is affecting gC processing; or that the mutations made are affecting translation of gC. Whatever the basis, all the subtle virus phenotypes ( and they are subtle, as gC is not required for VZV growth; could all be a result of the errant processing of the protein. As such, this must be resolved for the data to be of meaning. Blots are not quantified in anyway and need to be

2.The blots for gC expression are poor and not easily visible. In contrast blots for gE are way overexposed in figure b and d. these need to be repeated and fully quantified

3. The RNA for gE is different for the mutant and the parent/repaired virus (and apparently significant) (8E) no explanation is provided.

Reviewer #3: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: -In the introduction, the infection linked to VZV was classified as an early childhood, but nothing is said about the worldwide distribution of VZV-positive persons and about the results of the worldwide vaccination campaign (so the actual VZV circulation in the population). The social health burden brought about Varicella and its reactivation (shingles) should be more emphasised.

-What is DW? Is it the vehicle used in controls? This is not clearly stated

Reviewer #2: 4.A few spellers including the lack of m last lane figure 6. Also “strand” versus “stand” in a couple of places Also the references lack capitalizations and occasionally some reference details

5.Methods lack how plaques were quantified, and measured. No details of the imaging process used. Studies were done in mewo cells in which plaques are invariably asymetric

Reviewer #3: Type page 5 line 112, should be “disrupted”

Fig. 7: The difference in RNA levels is about 2-fold between WT and G4m, yet protein levels seem to be substantially more than that. The investigators should provide additional explanations, addressing such issues as whether G4 might affect mRNA stability or translation efficiency to account for this. An additional consideration to address is whether WT versus G4m gC mRNA are processed and transported from the nucleus to the cytoplasm with equal efficiency.

Fig. 8: Within the context of the virus, mRNA levels for gC seem to be concordant with protein expression (in contrast to the transfection experiments). The investigators should provide some speculation to explain these apparent discrepancies.

Some VZV strains apparently have varying numbers of 42bp repeats within their gC coding regions. Additional commentary should be provided on whether the number of G4 repeats might have additional positive/negative effects on gC expression.

**********

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PLoS Pathog. 2023 Jan 11;19(1):e1011095. doi: 10.1371/journal.ppat.1011095.r002

Author response to Decision Letter 0

6 Dec 2022

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PLoS Pathog. doi: 10.1371/journal.ppat.1011095.r003

Decision Letter 1

Blossom Damania, Robert F Kalejta

29 Dec 2022

Dear Dr. Ahn,

Thank you very much for submitting your manuscript "G-quadruplexes formed by Varicella-Zoster virus reiteration sequences suppress expression of glycoprotein C and regulate viral cell-to-cell spread" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

The reviewers are impressed with the revised manuscript and ask only for some small changes to the text. We invite you to make the changes you see fit and resubmit the manuscript for quick, editorial review.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

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Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

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Blossom Damania

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Kasturi Haldar

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***********************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Chung and co-authors submitted a revised manuscript dealing with the presence of G-quadruplexes (G4s) in the genome of the Varicella Zoster virus (VZV).

We thank the authors for considering and addressing the major issues raised in the first revision.

The information added in the introduction and the prediction and distribution of PQSs were appreciated. The initial part of the manuscript is now clearer and more complete.

Reviewer #2: the authors have done a thorough and remarkable job and have made the work far more convincing than the first version. figures have been clarified and better explained.

indeed i am ready to almost reccomend it being accepted for publication.

the article needs a spellcheck, as quite a few of the revised comments contain spellers or incorrect grammatical wordings. for a couple of examples see iines 95 global incident case

ln 169 lin 195 and other places. in s1 fig should be figure S1

line 266 the unmodified from of

265 how do they know the other forms are glycosylated?

Reviewer #3: (No Response)

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Reviewer #1: none required

Reviewer #2: no mJOR ISSUES

Reviewer #3: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Still, there are minor issues with the new data:

- A summary table for all the analyzed PQS, reporting CD data on topology, and stability and the results of the TDS would help the reader to have an overview of all sequences and in particular of those reported with atypical CD spectra.

- The presence of a table/figure panel reporting the aminoacidic changes caused by the G mutations would help the reader. Moreover, the authors should comment on how the changed aminoacids may influence protein folding and may contribute to the phenotypic changes of the virus. Is this new protein degraded faster than wt ORF14? which protein domain is involved in aminoacidic changes? Does mORF14 accumulate in cell granules, changing the ability of VZV to replicate? Please complete and discuss

- The authors conclude that the lack of G4s changes the VZV phenotype: this information is not sustained by the experimental data. mORF14 has aminoacidic changes that may cause viral cycle changes, this aspect needs to be commented.

Reviewer #2: spellers and ways of saying things and Figures need to be corrected

Reviewer #3: (No Response)

**********

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References:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

PLoS Pathog. 2023 Jan 11;19(1):e1011095. doi: 10.1371/journal.ppat.1011095.r004

Author response to Decision Letter 1

30 Dec 2022

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PLoS Pathog. doi: 10.1371/journal.ppat.1011095.r005

Decision Letter 2

Blossom Damania, Robert F Kalejta

2 Jan 2023

Dear Dr. Ahn,

We are pleased to inform you that your manuscript 'G-quadruplexes formed by Varicella-Zoster virus reiteration sequences suppress expression of glycoprotein C and regulate viral cell-to-cell spread' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Robert F. Kalejta

Academic Editor

PLOS Pathogens

Blossom Damania

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

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Editor-in-Chief

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***********************************************************

Reviewer Comments (if any, and for reference):

PLoS Pathog. doi: 10.1371/journal.ppat.1011095.r006

Acceptance letter

Blossom Damania, Robert F Kalejta

9 Jan 2023

Dear Dr. Ahn,

We are delighted to inform you that your manuscript, "G-quadruplexes formed by Varicella-Zoster virus reiteration sequences suppress expression of glycoprotein C and regulate viral cell-to-cell spread," has been formally accepted for publication in PLOS Pathogens.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Appendix. Putative G4 motifs predicted in the VZV genomes (Dumas and pOka).

(XLSX)

Click here for additional data file.^{(28.9KB, xlsx)}

S2 Appendix. Nucleotide variations found in the R2 reiteration sequence regions of VZV.

(XLSX)

Click here for additional data file.^{(32.9KB, xlsx)}

S1 Fig. Putative G4 motifs in IR_S.

(TIF)

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S2 Fig. Indication of putative G4 motifs in IR_S on the VZV genome sequence.

(TIF)

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S3 Fig. Conservation of G-runs and nucleotide variations in R2 reiteration sequences.

(TIF)

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S4 Fig. Comparison of wild-type and G4-disrupted mutant (G4m) ORF14 gene sequences.

(TIF)

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S5 Fig. Complete sequences of the G4-disrupted ORF14 gene and its translation product.

(TIF)

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S6 Fig. Schemes for the production of recombinant viruses containing the ORF14-HA and ORF14-G4m-HA gene.

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S7 Fig. Images showing plaque sizes of recombinant viruses in MeWo cells.

(TIF)

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S8 Fig. Comparison of plaque sizes of ORF14 recombinant viruses in HF cells.

(TIF)

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S1 Table. G4 motifs found in pOka R2 and their mutant sequences used in this study.

(XLSX)

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S2 Table. Primers used for ORF14 cloning and bacmid mutagenesis.

(XLSX)

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Attachment

Submitted filename: Review (author reponse) 2022-12-04.pdf

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Attachment

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Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

G-quadruplexes formed by Varicella-Zoster virus reiteration sequences suppress expression of glycoprotein C and regulate viral cell-to-cell spread

Woo-Chang Chung

Subramaniyam Ravichandran

Daegyu Park

Gwang Myeong Lee

Young-Eui Kim

Youngju Choi

Moon Jung Song

Kyeong Kyu Kim

Jin-Hyun Ahn

Roles

Abstract

Author summary

Introduction

Results

Genome-wide analysis of G4 motifs in the VZV genome

Fig 1. Distribution of putative G4 motifs in the VZV genome.

Distribution of G4 motifs in IRS

Distribution of G4 motifs in R2

Fig 2. Putative G4 motifs in R2 within ORF14.

Biophysical analysis of G4 formation by R2 G4 motifs

Fig 3. CD spectroscopy analysis of ORF14 GQ ODNs from pOka.

Fig 4. Normalized TDS spectra of ORF14 GQ ODNs from pOka.

Fig 5. Effect of PDS on thermal melting curves of ORF14 G4s.

Table 1. Effect of PDS on the Tm values of ORF14 G4s.

Fig 6. Non-denaturing polyacrylamide gel electrophoresis (PAGE) analysis of GQ oligonucleotides.

G4-mediated suppression of ORF14 gene expression in transfection assays

Fig 7. Effect of G4 disruption in ORF14 on gC expression in transfected cells.

G4 formation suppresses gC expression at the transcriptional level during virus infection

Fig 8. Altered gC expression in the recombinant virus containing the G4-disrupted ORF14-HA gene.

Enhanced gC expression in the ORF14-G4m virus attenuates viral cell-to-cell spread

Fig 9. Comparison of plaque sizes of ORF14 recombinant viruses.

Discussion

Materials and methods

Cells and chemicals

In silico G4 prediction

Circular dichroism (CD) spectroscopy and thermal melting curve analysis

Thermal Difference Spectra (TDS)

Plasmids

Antibodies

Immunoblot analysis

Bacmid mutagenesis

Plaque assays

Quantitation of viral mRNAs

Luciferase reporter assays

Statistical analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Blossom Damania

Robert F Kalejta

Roles

Author response to Decision Letter 0

Decision Letter 1

Blossom Damania

Robert F Kalejta

Roles

Author response to Decision Letter 1

Decision Letter 2

Blossom Damania

Robert F Kalejta

Roles

Acceptance letter

Blossom Damania

Robert F Kalejta

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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Distribution of G4 motifs in IR_S