VZV-32 is a low passage laboratory strain; its genome has been completely sequenced and falls within European clade 1 of VZV genotypes (Peters et al., 2006). MRC-5 human fibroblast cells or HeLa cells were grown in six well tissue culture plates with and without 12 mm round or 22 mm square coverslips in Minimum Essential Medium (MEM; Gibco, Life Technologies) supplemented with 7% fetal bovine serum (FBS), L-glutamine, non-essential amino acids, and penicillin/streptomycin. When monolayers were nearly confluent, MRC-5 cells were inoculated with VZV-infected cells at a ratio of one infected cell to eight uninfected cells by previously described methods (Grose and Brunel, 1978).

HeLa cells were transfected with plasmids containing VZV gE (pTargeT_gE) or VZV ORF62 (pCMV_IE62) under the CMV promoter as described previously (Carpenter et al., 2011). The plasmids were transfected into HeLa cells using ExtremeGene HP (Roche) transfection reagent (Jacobsen et al., 2004) at 10 μl/ml and plasmid DNA at a concentration of 1.0 μg/ml. After 6 h, the culture medium was replaced with plasmid/transfection reagent free medium. At 24 h post-transfection, RNA was extracted from all wells in a culture plate and cells incubated on coverslips were fixed and processed for microscopy.

Total RNA was extracted from uninfected, tunicamycin treated and VZV infected fibroblast cells in six well plates at the given time points using the RNEasy mini kit (Qiagen). RNA quality and quantity was assayed by UV spectroscopy using a NanoDrop spectrometer. Both A260/A280 and A260/A230 ratios were within 20% of 2.0 and infected cells from a six well plate well (6.5 sq cm) yielded approximately 3 μg of RNA in 60 μl. Further, the RNA was electrophoresed in an Agilent Bioanalyzer 2100 (Agilent) and yielded RIN values within 20% of 10. Polyadenylated RNA was converted to cDNA using anchored Oligo(dT) primers and the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) to yield approximately 20 ng of cDNA. The entire cDNA sample from one well of cells was mixed into 1 ml of 1× diluted Power SYBR Green Master Mix (ABI) and split into all wells of a SA Biosciences UPR PCR array (Life Technologies) with a multichannel pipettor (25 μl per well). The measurements were carried out in triplicate using cDNA from three of the original six wells in the plate for all types of samples using a Model 7000 real time PCR instrument (ABI). The resulting PCR results were processed using the SDS 1.2.3 software (Applied Biosystems). C_T values of each measurement were normalized to an average of 16.0 for housekeeping genes (wells H1–H5 of the UPR array) to form ΔC_T values which were then used to calculate averages and standard deviations between triplicate measurements. Subsequent ΔΔC_T values were calculated by differences between averages of VZV infected ΔC_T values or tunicamycin treated ΔC_T values with the average uninfected ΔC_T values. Uncertainties correspond to propagation of errors using standard deviations between the uninfected and infected or tunicamycin averages.

To confirm measurements from the UPR specific PCR array, several RT-PCR measurements were carried out using the following primers: BiP: forward 5′-CCC CAA CTG GTG AAG AGG AT-3′ and reverse 5′-GCA GTA AAC AGC CGC TTA GG-3′; DNAJB9/ERDj4: forward 5′-ACA TCT GTG ACT TGC GTT GC-3′ and reverse 5′-TGG GCA ATA AAA CCA TTT CC-3′; CREBH: forward 5′-GGG AGA CGA GCT GTG AGC-3′ and reverse 5′-TGT CTG AGT GTC GGT TCC TG-3′; PERK: forward 5′-GCC TAA GGA GGT AGC AGC AA-3′ and reverse 5′-GGG ACA AAA ATG GAG TCA GC-3′.

Murine MAb antibodies to VZV gE (3B3) and IE62 (5C6) produced in our laboratory were used in addition to a rabbit polyclonal antibody to LC3B (Santa Cruz Biotech sc-28266).

Samples of infected and uninfected cells were prepared for confocal microscopy by methods described previously (Carpenter et al., 2008). Briefly, the samples were fixed with paraformaldehyde and permabilized with 0.05% Triton-X-100 in PBS and then blocked in 5% non-fat milk with 2.5% normal goat serum for 2 h at RT. The primary antibody (1:2000) was added for 2 h at RT and overnight at 4°C. After washing (3 × 5 min with PBS) the samples were incubated with the secondary antibody (1:1250) and the Hoechst 33342 dsDNA stain (1:500) for 2 h at RT then washed before mounting on slides for viewing. Following preparation, the samples were viewed on a Zeiss 710 confocal fluorescent microscope (Duus et al., 1995).

Conditions for treatment of cultured cells with tunicamycin (2.5 μg/ml; Calbiochem, #654380) have been described in earlier papers in which we were investigating VZV glycoprotein biosynthesis (Montalvo et al., 1985; Carpenter et al., 2010). For experiments in uninfected cells, tunicamycin (2.5 μg/ml) was added 24 h after subculturing and the monolayer was fixed after another 24 h.

DiOC₆ (3-3-dihexyloxa-carbocyanine iodide) was obtained in powder form from Molecular Probes (D-273) and dissolved (0.7 mg/ml) in ethanol (Sabnis et al., 1997). An aliquot of the DiOC₆ stock (2.8 μl/ml yielded a final concentration of DiOC₆ of 2 μg/ml) was added to warm cell culture medium; this medium was applied to live cells for 30 min, then rinsed 2× with PBS and processed for fluorescent microscopy as described above.

Within cell culture, VZV is entirely cell associated with no release of cell-free virus (Grose and Brunel, 1978; Weller, 1983). Monolayers are inoculated with VZV-infected cells. The susceptibly of cells to VZV determines how long it takes to infect the whole monolayer but spread typically requires 3–5 days. Within infected cell monolayers, we observe a range of fused cells. For example, VZV induces massive syncytia involving hundreds of nuclei in melanoma cells while VZV infection of less fusogenic cells such as lung fibroblasts or skin keratinocytes induces syncytia involving tens of nuclei.

Recently, we observed that VZV induces increased LC3-positive puncta formation indicative of autophagosomes within cultured cells as well as from cells removed from varicella and zoster vesicles (e.g., Figures 1A1,A2) (Takahashi et al., 2009). Unlike the closely related herpes simplex virus, VZV does not encode any known inhibitors of autophagy, such as ICP 34.5. Later we observed that VZV infected cells also exhibited signs of ER stress, namely XBP-1 splicing and a greatly enlarged ER (see, e.g., Figures 1B1,B2). The latter results led to the hypothesis that VZV glycoprotein synthesis induces ER stress that is partially relieved by an enlarging ER and increased autophagy (Carpenter et al., 2011).

Based on the observations in the previous section, we sought to further document the induction of the UPR within VZV infected cells via a UPR-specific PCR array manufactured by SA Biosciences (now part of Qiagen). This 96 well plate consists of 84 wells containing primers to the 3′ Untranslated Region (UTR) of transcripts associated with the UPR and the remaining 12 wells containing primers to housekeeping genes and PCR and cDNA quality control wells. Table 1 lists the UPR specific primers or wells where the wells are grouped by association with a given UPR function: ANTI or PRO (anti or pro-apoptotic), ERAD (ER associated degradation), FOLD (primarily folding chaperones), LIPID (transcripts associated with lipid synthesis and metabolism), SENSOR (transcripts associated with ER membrane resident proteins known to “sense” and signal ER stress conditions), TF (other transcription factors like C/EBPβ) and finally TRANS for two components associated with protein translation. Each group will be described more fully in the next sections.

Gene transcripts were measured in uninfected human fibroblasts, tunicamycin (TM) treated fibroblasts and VZV infected fibroblasts. Each measurement was done in triplicate. The measured C_T values were normalized so that in each case the housekeeping gene transcripts measured C_T average was 16 and then the triplicate measurements were averaged and standard deviations computed to generate ΔC_T. Differences between the uninfected ΔC_T and those associated with TM treated and VZV infected cell transcript measurements were then calculated to form the final measurements ΔΔC_T listed in Table 1. Graphs of the resulting values (Figure 2) showed that tunicamycin treatment, a classical ER stressor by inhibition of N glycosylation, upregulated 66 of the 84 UPR genes, with known folding chaperones, e.g., BiP (in blue), particularly upregulated. Also upregulated is the pro-apoptotic factor CHOP (pink). By contrast, only 43 of the UPR genes are upregulated in VZV infected cells. In particular, those genes most upregulated such as CREB3L3/CREBH (light blue) are more upregulated than after TM treatment. VZV infected cells also upregulated the LIPID transcripts AMFR/gp78 and INSIG (green) while downregulating a number of ERAD components such as UBXN4/erasin and EDEM3 (red). These differences will be considered by group in the subsequent sections.

The SENSOR grouping includes the best known ER stress sensors: PERK, IRE1α and ATF6 but also two CREB proteins (CREB3/LUMAN and CREBH) as well as primers to the Golgi resident proteases S1P and S2P that activate AT6 and the CREB proteins by cleavage (Ye et al., 2000; Asada et al., 2011). Included in the group are lesser known transcripts including IRE1β, ATF6β, and NUCB1.

CREBH, the cAMP responsive element binding protein H, is an ER anchored transcription factor implicated in nutrient metabolism and the proinflammatory response. VZV infected cells displayed more transcripts of CREBH and fewer of ATF6β and PERK (all with p < 0.001) than in TM treated cells (Figure 3A). TM treatment generally upregulated all ER sensor transcripts with CREBH the most upregulated. CREBH transcription has previously been described as upregulated in hepatocytes and has been associated with lipid synthesis and acute phase transcription in T-cells (Zhang et al., 2006, 2012). More recently, CREBH as a transcription factor has been described as increasing the capacity of the secretory pathway (Barbosa et al., 2013). ATF6β and ATF6α share similar structures but differ in function. In particular, ATF6β has been reported to inhibit transcription of ATF6α one of the primary ER stress sensors (Thuerauf et al., 2007).

In order to confirm the results of the UPR specific PCR array, we carried out qPCR measurements using primers to CREBH and PERK (Figure 3B) in TM treated cells and at several timepoints in VZV infected cells. Those measurements confirm the upregulation of CREBH by TM treatment but particularly in VZV infected cells (p < 0.01). However, the downregulation of PERK in VZV infected cells was not confirmed.

Within the FOLD group, the largest, there are 32 wells with primers to eight HSP-70 homologs including HSPA5/BiP and SIL1/BaP; five DNAJ HSP-40 homologs including DNAJB9/ERdj4 and DNAJC10/ERdj5; twelve wells contain primers to transcripts encoding ER lumen folding components including CALR and CANX; three components of the folding chaperonin complex and finally four components in the ER membrane including RPN1 and SEC63. Many of these transcripts encode proteins which assist secretory protein folding but also sense misfolded proteins in the ER (Schroder, 2008). For example, DNAJC10/ERdj5 is a disulfide reductase that associates with ERAD component EDEM (Hagiwara et al., 2011).

VZV infected cells exhibited very uneven transcription of folding chaperones (FOLD) while TM treatment robustly upregulated transcription of these chaperones particularly BiP (Figure 4A). Measurements with the UPR specific PCR array showed VZV infected cells upregulated BiP while downregulating DNAJB9/ERdj4 and HSPA1L. In order to reassess these observations, we carried out qPCR measurements of BiP and DNAJB9/ERDj4 using primers specific to those transcripts (Figure 4B) and found that neither the upregulation of BiP nor downregulation of DNAJB9/ERDj4 was confirmed. Rather the qPCR results found BiP to be moderately downregulated as the infection progressed to more cells (p < 0.05). However, we also reassessed the regulation of the ER-co-chaperone DNAJC10/ERdj5 and found a correlation with the UPR-specific array (data not shown).

There are 21 ERAD associated wells that amplify a number of known transcripts that code for proteins that are involved in the degradation of misfolded proteins in the ER through a number of steps: recognition of misfolding (OS9, PPIA and SELS along with a number of FOLD transcripts), trimming of mannose residues prior to recognition by E3 ubiquitin ligases (EDEM1 and EDEM3), recognition of misfolded proteins by E3 ubiquition ligases (DERL3/HRD1, DERL2, DERL1, HERP, RNF5, and associated factors SEL1L and FBX06), exportation to the cytosolic side of the ER membrane (SEC62) where the VCP/p97 complex poly-ubiquitinates protein substrates before extracting/clipping the protein from the membrane to be ultimately degraded in the cytosol by the proteasome (Schroder, 2008; Merulla et al., 2013). The VCP/p97 complex includes its cofactors UFD1L and NPLOC4 and regulators ATAXIN3 and ARMET/erasin as well as the E2 ubiquitin-conjugators UBE2J2 and UBE2G2 (Ballar et al., 2011). Finally, there is a cytosolic protease, USP14, included in this grouping.

While TM treatment showed almost complete upregulation of ERAD transcripts particularly HERP (Figure 5A), VZV infection showed considerable downregulation of ERAD transcript (Figure 5A) where EDEM3, UBXN4/erasin and SEC62 were downregulated. However, ATAXIN3 was upregulated in VZV infected cells. Both erasin and ATAXIN3 are regulators, positive and negative, respectively, of VCP/p97 (Lim et al., 2009; Liu and Ye, 2012). As noted above, VCP/p97 forms the protein complex in the ER membrane on the cytosolic side that poly-ubiqininates ERAD substrates that are then released into the cytosol to be degraded by the proteasome (Ballar et al., 2011). Downregulation of VCP/p97 via its regulators appeared to reduce ERAD in VZV infected cells. All observed differences were significant with p < 0.001. Again, in order to reassess two of the more striking observations from the UPR specific PCR array, we carried out qPCR measurements using primers to UBXN4/erasin and ATAXIN-3 (Figure 5B). These measurements showed UBXN4/erasin to be modestly downregulated in VZV infected cells while ATAXIN-3 was essentially unchanged.

Transcripts associated with lipid synthesis and metabolism such as RAMP4 showed similar transcription in both VZV infected and TM treated cells but VZV infected cells, in particular, showed increased transcription of cholesterol synthesis regulators AMFR/gp78 and INSIG (Figure 6A). AMFR/gp78 is an E3 ubiquitin ligase and INSIG is an insulin signaling factor (Flury et al., 2005; Chen et al., 2012). Both are localized to the ER membrane and when activated function together to degrade HMG COA reductase, a cholesterol synthesis enzyme (Jo et al., 2011; Tsai et al., 2012).

In order to reassess the upregulation of AMFR/gp78 and INSIG, we carried out qPCR measurements using primers to each transcript (Figure 6B). Of note, INSIG was upregulated in VZV infected cells at early timepoints in agreement with the UPR specific array, while AMFR/gp78 was not increased. Of note, greater transcription of INSIG may lead to reduced cholesterol synthesis in VZV infected cells with the consequence of a more fluid ER in those cells. Cholesterol acts as a stabilizing agent in lipid membranes by supporting adjacent lipid head groups and reducing disorder of the lipid hydrocarbon chains internal to the bilayer (Mouritsen and Zuckermann, 2004). All observed differences were significant with p < 0.001.

Transcription of cellular transcription factor C/EBPβ was significantly downregulated (p < 0.001) in VZV infected cells as compared to the value in TM treated cells (Figure 7A) C/EBPβ is a transcription factor with a large effect on cellular proliferation (Tang and Lane, 2000). Downregulation of this factor may put VZV infected cells into a non-proliferative state. Transcription of apoptotic genes differed considerably between VZV infected cells and TM treated cells. TM treated cells exhibited much higher CHOP transcription than VZV infected cells while VZV infected cells showed a greater number of transcripts associated with cellular apoptosis such as HTRA4 and the MAP kinases JNK1 and JNK3 (Figure 7B). Finally there was no difference between the levels of two protein translation associated transcripts in VZV infected cells vs. TM treated cells (Figure 7C).

In 2011, we found that transfecting cells with VZV glycoprotein genes led to increased autophagosome production and inflation of the ER. Transfection with VZV IE62 led to neither increased autophagosomes nor a larger ER. Therefore, we measured by qPCR whether CREBH and BiP transcription was increased by transfection of a glycoprotein vs. a non-glycoprotein that is also the major transactivator encoded by VZV. Transfection with a plasmid encoding VZV gE under the CMV immediate early promoter led to approximately 10% of transfected cells (Figure 8A1) while transfection with VZV IE62 also under the CMV immediate early promoter led to a larger number, approximately 40%, of transfected cells (Figure 8A2). Even though a low fraction of cells were transfected with VZV gE, increased transcription of CREBH and BiP were observed in those cells (Figures 8B1,B2) whereas not in cells transfected with VZV IE62 even though many more cells were transfected in those samples (p < 0.01). We therefore conclude that expression of a single VZV glycoprotein gene in cells is sufficient to activate the CREBH arm of the UPR.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.