Background
Adenovirus infection is ubiquitous in the human population, and the species C subgroup (AdV-C1, 2, 5, and 6) is the most widespread of the viruses. Species C AdVs cause acute infection in the respiratory and gastrointestinal tracts [
1‐
4]. In addition to causing lytic infections in epithelial cells, adenoviruses have the ability to persist in a non-lytic state in mucosal lymphocytes [
2,
5‐
11]. AdV-C infections occur predominantly in the very young, and consequently nearly 80% of children contain viral DNA in the lymphocytes of their tonsils and adenoids [
1‐
4]. These infections can be life-threatening for immunocompromised pediatric transplant patients, and those receiving allogeneic hematopoietic stem cell transplants (allo-HSCT) are at significant risk for developing disseminated adenovirus disease. Although these infections and resulting disease can be initiated through de novo exposure to the virus, the predominant cause in severely immunocompromised patients is endogenous reactivation of AdV-C, types 1, 2, and 5 [
3]. The AdV-related post-transplantation mortality for these patients is estimated to be between 3.2 and 6.0%, potentially affecting more than 100 children per year in the U.S. [
3,
12,
13]. There is currently no medical intervention to protect against AdV reactivation, or FDA-approved treatment for AdV disease, and the mechanisms that allow the virus to persist and induce reactivation are almost entirely unknown [
14,
15].
Persistent AdV infections last for long periods of time following resolution of the initial lytic infection, and the virus can be intermittently detected in fecal samples for months to years after symptoms have abated [
16]. Persistent infections in lymphocytes have been reported to exhibit a range of repressed states, from truly latent (with no production of infectious particles) to a “smoldering” infection with low viral yield [
2,
8]. Immunoactivation of tonsillar lymphocytes has been shown to reactivate latent AdV, but the cell-type specific mechanisms behind this de-repression have not been studied [
2]. B and T lymphocytic cell line models of persistent infection have been established that exhibit long-term persistent AdV infections marked by retention of high levels of viral genomes and very low viral protein expression [
17,
18]. Interestingly, the persistent phase in these models has been shown to be regulated, in part, by transcriptional controls not seen in lytic infections. Several viral genes have been reported to display alternative patterns of expression when compared to lytic infections, suggesting specific programs of repression are present in persistent infections of lymphocytes [
19‐
21].
As B and T lymphocytes transition from a resting to an activated state, they undergo dramatic shifts in gene expression and metabolism to accommodate robust proliferation and differentiation into effector cells. Programs of gene expression during both resting and activated states have been shown to be regulated in part by chromatin remodelers and co-repressors, including DNA methyltransferases (DNMTs), Class I and II histone deacetylases (HDACs), Class III HDACs (sirtuins), ten-eleven translocation (TET) family proteins, and the C-terminal Binding Protein family [
22]. Because the adenovirus genome is chromatinized through rapid association with cellular histones upon entry into the host cell nucleus, viral gene expression is likely regulated by these cellular chromatin-modifying mechanisms and responsive to immunoactivation of the host lymphocyte [
23‐
25].
The C-terminal Binding Protein (CtBP) family of transcriptional corepressors was discovered through their high affinity binding to AdV E1A proteins [
26,
27]. Mammalian cells express both CtBP1 and its homolog CtBP2 (collectively known as CtBP), which both share a 2D-hydroxyacid dehydrogenase domain, RRT-binding domain, and the PxDLS-binding domain responsible for the interaction with E1A (reviewed in [
28]). CtBP homo- and hetero-dimers also likely form tetramers with the capacity to recruit many different chromatin modulators including Class I and II HDACs, histone methyltransferases, E3 ligases and other transcriptional regulators into large transcriptionally repressive complexes at the promoters of genes [
28‐
31]. The assembly and stability of these complexes are dependent on nicotinamide adenine dinucleotide (NAD
+ and its reduced form NADH) binding, and CtBP has been reported to function as an NAD(H) sensor and therefore a link between metabolic state and transcriptional regulation [
30,
32‐
36].
Much has been reported about CtBP and its interaction with the viral E1A proteins. Initiation of the lytic AdV infection is marked by expression of the immediate early gene
E1A, which has two main protein isoforms - large (13S E1A, 289R) and small (12S E1A, 243R) - responsible for transactivating other viral early genes and driving expression of cellular S-phase genes, respectively [
37]. Both E1A isoforms interact with high affinity with both CtBP1 and CtBP2 through a PLDLS-motif located in the shared conserved region 4 (CR4) at the C-terminal end of the E1A proteins. Large E1A has an additional CtBP interaction domain located in the CR3 region unique to this isoform [
38]. Of note, NADH was found to facilitate binding of CtBP to E1A at 1000-fold lower concentration than NAD
+, suggesting that the NAD
+/NADH ratio in the cell may affect the formation of CtBP-E1A protein complexes [
32].
The role of the CtBP-E1A interaction in the lytic AdV life cycle is complex and has been reported to be either repressive or faciliatory, depending on the context. Mutation of the CtBP-binding site in CR4 of E1A drastically reduces virus replication, but stable knock-down of CtBP2 increases viral yield [
39,
40]. CtBP1 and CtBP2 suppress the
ras-cooperative transformative activity of the E1A proteins, but are required for E1B-55 K cooperative transformation [
26,
39,
41‐
43]. At the level of transcriptional regulation, CtBP has been found to both repress and enhance E1A transactivation of viral and cellular genes [
38,
44]. In a reciprocal relationship, E1A can exert influence over CtBP function as well, such as by altering acetylation and repressor-complex composition [
44] and enhancing nuclear localization [
45,
46]. These findings suggest that the high affinity binding between the E1A proteins and the CtBP proteins could form different context-specific complexes with finely-tuned functions. Given the complex nature of CtBP function during lytic infections of epithelial cells, it seems plausible that the CtBP proteins function in yet a different capacity within the unique cellular backdrop of persistent infection in lymphocytes.
The present study focuses on the mechanisms of viral reactivation in lymphocytes infected with AdV-C and provides experimental evidence for metabolically-linked mechanisms that could contribute to viral reactivation following cell activation. We show that viral transcription in lymphocyte models of AdV persistence is repressed compared to lytically-infected cells, but that relative amounts across viral transcripts are similar between the two infection types. Our data reveal that activation of lymphocytes shifts the NAD+/NADH ratio and that viral transcription is linked to alterations in this ratio. We also report differential expression of the NAD-dependent CtBP protein homologs between lymphocytes and epithelial cells. Last, our data reveal that inhibition of CtBP interaction with PxDLS-motif binding partners upregulates AdV E1A expression in T lymphocytes but not epithelial cells. Together, our results provide novel insight into metabolic factors that can regulate adenoviral reactivation in human lymphocytes.
Material and methods
Cell lines
The human lung carcinoma cell line A549 was purchased from the American Type Culture Collection (ATCC, Manassas, VA). BJAB (EBV-negative Burkitt’s lymphoma, [
47]) and Jurkat (T cell Acute Lymphoblastic Leukemia [ALL]) were also obtained from the ATCC. KE37 (immature T cell ALL) cells were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Me-180 (HPV-positive cervical carcinoma) and CaLu1 (lung carcinoma) were obtained from Linda R. Gooding (Emory University, Atlanta, GA). A549 cells were grown in Dulbecco’s modified Eagle medium (DMEM) with 4.5 μg of glucose per ml, 10% fetal calf serum (FCS), and 10 mM glutamine. BJAB, Jurkat, and KE37 cells were grown in RPMI medium supplemented with 10% FCS and 10 mM glutamine. Me-180 and CaLu1 were grown in McCoy’s medium, 10% FCS, and 10 mM glutamine. Cells were routinely evaluated to ensure the absence of mycoplasma and lymphocyte cell lines were authenticated by Genetica Cell Line Testing (Burlington, NC).
Adenoviruses
The AdVC-5 mutant virus strain Ad5dl309 is phenotypically wild-type in cell culture and was obtained from Tom Shenk (Princeton University, Princeton, NJ). Ad5dl309 lacks genes necessary for evading adaptive immune attack (E3 RIDα and RIDβ proteins as well as the 14,700-molecular-weight protein (14.7 K protein)) in infected hosts [
48].
Infection of lymphocytes with adenovirus
Infection of lymphocyte cell lines with adenovirus was performed as described previously [
49] with minor modifications. Lymphocytes were collected and washed in serum-free (SF) RPMI medium, and cell density was adjusted to 10
7 cells per mL in SF-RPMI medium. Virus was added to the cell suspension at 50 PFU/cell, spun for 45 min at 1000 x g at 25 °C, and resuspended by agitation. Cells were then incubated at 37 °C for 1.5 h with gently flicking every 30 min. The infected cells were washed three times with complete RPMI medium and then resuspended in complete RPMI medium at 5 × 10
5 cells per mL for culture. Cell concentration and viability were monitored throughout the infection. Replicates for experiments were obtained from independent infections.
Stimulation of immune cell activation
Lymphocytes were treated for 24 h with 81 nM PMA + 1.35 μM Ionomycin (1X EZCell™ Cell Stimulation Cocktail, BioVision, Milpitas, CA). Following Fc block treatment (BD Pharmingen, San Jose, CA), cells were stained with fluorophore-conjugated antibodies against CD69 (PE, Biolegend, clone FN50) and CD25 (FITC, BioLegend, clone BC96), or stained with isotype control, and assessed by flow cytometry using LSR Fortessa (Becton Dickinson) and FlowJo Software (Becton Dickinson).
Drug treatments
Drug treatment concentration and time of exposure were optimized for all cell lines. For lymphocytic and epithelial cell lines, cells were seeded at a density of 3 × 105 and 1 × 105 cells per mL, respectively, in complete medium supplemented with treatment doses of drugs. Treatment drugs and doses tested include nicotinamide (NAM, Sigma-Aldrich, [2, 5, 10 mM]) and NSC95397 (CtBP inhibitor, Tocris, Bristol, UK, [0.5, 1, 5, 10, 20 μM]). Cell growth and viability were assessed by Trypan blue exclusion at 12 (NSC95397 only), 24, and 48 h. Experiments utilized the following doses which maintained the viability indicated: NAM-10 mM, > 80% for 48 h; NSC95397–10 μM for 24 h, which maintained > 40% viability in lymphocytes and > 70% viability for epithelial cells.
Reverse transcription and quantitative PCR analysis of viral and cellular mRNA levels
RT-qPCR was performed as described previously with minor modifications [
50]. Briefly, total RNA was isolated from 1 × 10
6 cells using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA) with RNase-free DNase treatment (Qiagen). After spectrophotometric quantification, 200 ng of RNA was reverse transcribed into cDNA in 20 μL reactions (Maxima First Strand cDNA Synthesis Kit, Thermo Fisher Scientific, Waltham, MA). RT-enzyme negative controls were included for each reaction. Primers and probes were obtained from Integrated DNA Technologies (Coralville, IA), with sequences specified below. Each cDNA sample was run in duplicate qPCR reactions using the Maxima Probe/ROX qPCR Master Mix (Thermo Fisher Scientific) with cycling conditions as described.
For all experiments in which changes to viral gene transcription were assessed and expression of our housekeeping gene (eukaryotic translation initiation factor 1, [
EIF1]) was unchanged by treatment, we quantified relative amounts of target (fold-change over untreated) as
\( {2}^{-\left(\Delta {C}_{T, treated}-\Delta {C}_{T, untreated}\right)}={2}^{-\Delta \Delta {C}_T} \) as described in [
51]. In experiments using NSC95397, four different housekeeping genes (
GAPDH,
HPRT1,
ACTB, and
EIF1) were all negatively impacted by treatment. Because our primer amplification efficiencies are similar, and cDNA was prepared using equal amounts of RNA for all treatments, we used
\( {2}^{-\Delta {C}_T^{\prime }}={2}^{-\left({C}_{T, treated}-{C}_{T, untreated}\right)} \) [
51] for each gene separately, and present the down-regulated housekeeping gene for reference. This formula was also used for comparing relative amounts across different viral transcripts of untreated samples. We approximate the constant
K = 1 (represents the ratio between the target gene and the housekeeping gene of the number of molecules present at threshold cycle given an initial number of molecules, defined in Eq. 4 [
51]). For this,
\( {2}^{-\Delta {C}_T^{\ast }}={2}^{-\left({C}_{T, target\ gene}-{C}_{T, housekeeping\ gene}\right)} \) was used to yield an approximate relative amount of target compared to the housekeeping gene for each viral gene.
Primers and Probes:
E1A (Sense sequence, 5′- GTTAGATTATGTGGAGCASCCC-3′, anti-sense sequence, 5′-CAGGCTCAGGTTCAGACAC − 3′, probe sequence, 5′-6 FAM-ATGAGGACCTGTGGCATGTTTGTCT-3IABkFQ-3′).
E3GP19K (Sense sequence, 5′-TTTACTCACCCTTGCGTCAG-3′, anti-sense sequence, 5′-GCAGCTTTTCATGTTCTGTGG-3′, probe sequence, 5′-6 FAM-CTGGCTCCTTAAAATCCACCTTTTGGG-3IABkFQ-3′).
TLP HEXON (Sense sequence, 5′-AAAGGCGTCTAACCAGTCAC-3′, anti-sense sequence, 5′-CCCGAGATGTGCATGTAAGAC-3′, probe sequence, 5′-6 FAM-CGCTTTCCAAGATGGCTACCCCT-3IABkFQ-3′).
EIF1 (Sense sequence, 5′- GATATAATCCTCAGTGCCAGCA-3′, anti-sense sequence, 5′-GTATCGTATGTCCGCTATCCAG-3′, probe sequence, 5′-6 FAM-CTCCACTCTTTCGACCCCTTTGCT-3IABkFQ-3′).
Quantitative real time PCR analysis of viral DNA levels
Infected or uninfected control cells were washed in phosphate-buffered saline (PBS) and 5 × 10
5 cells for each sample were lysed in 100 μL of NP-40–Tween buffer containing proteinase K, as described in [
5]. Samples were tested by real-time PCR for a region of
hexon gene that is conserved among species C adenovirus serotypes. Samples were run in duplicate for each independent experiment, with cycling conditions as described. Viral genome numbers were quantified by comparison to an Ad2 DNA standard curve and normalized relative to
GAPDH expression to account for small differences in cell input [
5].
Immunoblots for protein detection
Protein lysates were prepared using RIPA buffer (Sigma-Aldrich) with protease/phosphatase inhibitors (Cell Signaling Technologies), and protein concentrations were quantified using a BCA protein assay (Thermo Scientific). 30μg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5 to 12% polyacrylamide gels (Mini-PROTEAN TGX gels, BioRad, Hercules, CA). Proteins were transferred onto nitrocellulose membranes (Thermo Scientific) overnight at 30 mV at 4 °C. Following confirmation of protein transfer with Ponceau S staining (Aqua Solutions, Deer Park, TX), membranes were blocked at room temperature (RT) with 5% bovine serum albumin (BSA) for 1 h, washed three times with Tris-Buffered-Saline with 1% Tween (TBST), and incubated with primary antibodies on a rocker overnight at 4 °C. Following three washes with TBST, membranes were incubated with secondary HRP-conjugated antibodies for 1 h at RT. Membranes were washed three times with TBST, the HyGLO HRP chemiluminescent reagent (Denville, Quebec, CA) used as substrate, and signal detected using x-ray film (MTC Bio). Primary antibodies include CtBP1 (mouse, 612,042, BD Transduction Lab, San Jose, CA), CtBP2 (mouse, 612,044, BD Transduction Lab), and β-actin (rabbit, D6A8, Cell Signaling, Danvers, MA). Secondary antibodies used were also from Cell Signaling: HRP-linked anti-rabbit IgG (7074) and HRP-linked anti-mouse IgG (7076S).
Quantification of total cellular NAD+ and NADH concentrations
NAD+ and NADH concentrations were determined using the bioluminescent NAD/NADH-Glo Assay from Promega (Madison, WI). Cells were plated at a density of 1.5–3 × 104 cells per well in 250 μL complete media on 96-well plates. For determining the effects of treatments on NAD+/NADH ratios, cells were left untreated or drugs added, and all cells were incubated for times specified in figures. Nanomolar concentrations of NAD+ and NADH were determined following manufacturer’s instructions by comparison to a standard curve consisting of dilutions of β-Nicotinamide adenine dinucleotide (N8285, Sigma).
Statistical analysis
Experiments were repeated at least three times unless otherwise indicated. The experimental data were analyzed using a student’s t-test in GraphPad Prism software. P-values less than 0.05 were considered statistically significant. Independent infections of lymphocytes exhibit a high degree of variability in gene expression preventing the ability to average observations across infections, thus for some experiments we have shown the results of independent replicate experiments.
Discussion
Most of what is known about adenovirus is from studies of lytically-infected cells, and much about adenovirus latency and reactivation is not well characterized. The virus can be life-threatening for immunocompromised individuals as well as pediatric transplant patients, however, the mechanisms that allow the virus to persist, or those that induce reactivation, are almost entirely unknown. Patient samples have shown that lymphocytes of the tonsils, adenoids [
5], and gastrointestinal tract [
8] contain AdV DNA and are presumably the sites of reactivation. The lack of small-animal models of persistent adenovirus infection has been an obstacle to studying infection dynamics in vivo, but a study of AdV infection using humanized mice has recently shown that persistently-infected cells could also be found in the bone marrow [
71].
Our previous studies of AdV-infected lymphocytes from tonsils or adenoids suggest that replicating virus is more common among younger donors, however high genome copy number did not appear to correlate with active replication [
2]. Replicating virus could be detected from cells containing a range of genome copy numbers, from as few as 10
4 to as many as 10
6 AdV genomes per 10
7 cells [
2]. Our cell line models of persistent lymphocyte infection carry AdV DNA levels in a range between 1 × 10
5–1 × 10
9 copies per 10
7 cells (Fig.
1a). Within these persistently-infected models, many viral transcripts can be detected in low amounts with fewer than 1% of the cells expressing detectable levels of viral proteins or producing virus [
20,
21].
The persistent phase of infection has been shown to be regulated, in part, by transcriptional controls not seen in lytic infections. Murali et al. (2014) determined that the
E3-Adenovirus Death Protein (
ADP) gene is repressed both transcriptionally and post-transcriptionally in cells which harbor persistent AdV infection [
21]. Krzywkowski et al. (2017) showed that in persistently-infected BJAB, very few individual cells express E1A mRNA or Major Late Transcription Unit mRNA at levels comparable to lytically-infected HeLa cells, even when the cells harbored large amounts of viral DNA [
19]. In contrast, Furuse et al. (2013) determined that persistently-infected BJAB expressed amounts of VA RNAI and VA RNAII that were comparable to those expressed in lytic infections. However, the relative proportion of the two transcripts differed when compared to lytic infection [
20]. In our current study, we report low expression of both early (
E1A and
E3) and late genes (
hexon) in infected lymphocytes as compared to lytically-infected cells (Fig.
1b). Indeed, the level of viral transcripts are all relatively lower than the expression level of the representative housekeeping gene. In contrast, AdV transcript levels are relatively higher than housekeeping gene expression in both the lytically-infected T cells (Jurkat) and lytically-infected epithelial cells (A549). However, we found reduced levels of viral transcripts in lytically-infected T cells as compared to lytically-infected epithelial cells revealing that lymphocytes in general have lower levels of AdV gene expression. We attempted to confirm differences in viral gene expression at the protein level but were unable to detect viral proteins which are in low abundance during viral persistence (data not shown). Despite some degree of transcriptional repression in the lymphocytes, viral mRNA ratios were surprisingly similar between persistently-infected and lytically-infected cells (Fig.
1c and d, respectively). These findings in lymphocytes are in line with amounts of E1A, E3, and hexon mRNAs (~ 4, 35, and 90%, respectively), quantified as a percent of GAPDH, at 36 h post-infection in normal lung fibroblasts recently reported by Crisostomo et al. (2019) [
54].
Immunoactivation of tonsillar lymphocytes has been shown to reactivate latent AdV causing increases in viral gene expression and productive infection [
2]. In previous studies, a cocktail of immune cell stimulators was used including PMA, Ionomycin, IL-2, anti-CD3 and anti-CD28, however, no specific mechanisms for viral gene de-repression were determined. In addition, these prior studies on activation of naturally infected lymphocytes were done using samples that contained both T cells and B cells together. In the current study, we report that PMA/Iono alone is sufficient to induce AdV gene expression in B and T cell models of persistent infection, as well as in lytically-infected Jurkat cells (Fig.
2b). In addition, we found that the magnitude of change in viral expression mirrors the change observed in the NAD
+/NADH ratio (Fig.
3a). PMA/Iono treatment increased total cellular NAD
+ and NADH concentrations (data not shown) and significantly increased the NAD
+/NADH ratio in BJAB and Jurkat cells; large increases in AdV early gene expression were readily observable in these cells by 24 h. Stimulation, including PMA/Iono treatment, of resting lymphocytes has been well-documented to shift the metabolic program from primarily oxidative phosphorylation to glycolysis, which increases lactate production, increases synthesis of biosynthetic intermediates, and shifts the NAD
+/NADH ratio [
63,
72,
73]. Thus, our data support the notion that changes in the metabolic status of lymphocytes can promote reactivation of AdV gene expression. In the current study, PMA/Iono had the least impact on AdV gene expression in KE37 cells which corresponded with the non-significant change detected in the NAD
+/NADH ratio in these cells. Whether the addition of other T cell stimulating agents (IL-2, anti-CD3 and anti-CD28) can induce a significant change in this ratio, as well as more robust changes in AdV gene expression, is still under investigation.
Interestingly, when comparing the basal NAD
+/NADH ratios in the two persistently-infected cell lines, KE37 and BJAB, a trend toward viral infection reducing the NAD
+/NADH ratio relative to their uninfected counterparts could be seen, though significance was not reached (Fig.
3b). These samples were evaluated at different times post-infection, and it is intriguing to speculate that AdV may significantly impact the NAD
+/NADH ratio of the cells it persistently infects at some point during the course of the infection. How the virus would modulate cell metabolism mechanistically is unclear. Persistent adenovirus infection of B-lymphocytes has been shown to significantly down-regulate several cellular genes (
BBS9,
BNIP3,
BTG3,
CXADR,
SLFN11, and
SPARCL - [
50]), however, none are reported to obviously function in the regulation of metabolism. Nonetheless, it is possible that some of the other genes identified as altered by AdV infection could play a role in this effect ([
50], supplemental data).
Nicotinamide (NAM), which is recycled by the cellular NAD
+-salvage pathway and converted into NAD
+, can be used to manipulate the NAD
+/NADH ratio of cells [
74]. NAM treatment of persistently-infected cell lines significantly increased the NAD
+/NADH ratio in KE37 while a much smaller change was induced in BJAB cells (Fig.
4a). Nonetheless, increased viral gene expression could be detected in both cell lines (Fig.
4b) suggesting that alterations in this metabolic ratio can induce viral gene expression in lymphocytes. Interestingly, in contrast to the robust PMA/Iono-induced upregulation of
E1A and large increase in NAD
+/NADH ratio seen in BJAB (3.3-fold, Fig.
2b), there was no apparent change in
E1A expression when the ratio was only increased 1.3-fold with NAM (Fig.
4b). A similar relationship is seen between
E1A expression and the shift in the metabolic ratio in KE37, where more
E1A expression is seen following larger increases in the NAD
+/NADH ratio (Figs.
4,
2b). These findings support a link between metabolic shifts in lymphocytes and the magnitude of AdV de-repression induced.
The link between the metabolic state of cells and gene expression contributes to lymphocyte functional responses following immune stimulation [
64,
75,
76]. This transcriptional regulation involves chromatin remodelers dependent upon specific concentrations of metabolites that serve as co-substrates or co-factors [
64]. CtBP is well-known repressor of gene expression that was discovered through its interaction with E1A [
26,
27,
77]. CtBP tetramers associate with epigenetic enzymes forming complexes that modify the chromatin environment through coordinated histone modifications, allowing for the effective repression of genes targeted by DNA binding proteins associated with the complex [
30‐
36,
78‐
80]. The stability of CtBP tetramers is dependent upon NAD(H) binding. Because AdV gene expression in lymphocytes is responsive to shifts in the NAD
+/NADH ratio, we investigated whether CtBP, as a reported metabolic sensor, could be contributing to the transcriptional repression evident in persistent infection. When comparing CtBP protein levels, we found that our three lymphocyte cell lines only expressed CtBP1 and that CtBP2 protein could not be detected (Fig.
5a). CtBP2 expression has previously been reported to be in low abundance or undetectable in leukocytes, immune tissues, and lymphocyte cell lines [
29]. In contrast to the lymphocytes evaluated in our study, A549 cells expressed high levels of CtBP2 with lower levels of CtBP1 (Fig.
5b). This finding suggested that the composition of CtBP complexes in lymphocytes is different than in epithelial cells, and therefore CtBP may interact differently with viral proteins in lymphocytes than what has been reported for epithelial cells.
NSC95397 is a small-molecule inhibitor of CtBP which acts through the disruption of CtBP binding to PxDLS-containing partners, including E1A [
70]. Interestingly, treatment with NSC95397 resulted in mixed changes in expression of AdV genes (Fig.
6b-e).
E1A expression was increased in the T cells lines (KE37 and Jurkat) but minimally impacted in the B cell line (BJAB). In sharp contrast to
E1A,
hexon expression was consistently downregulated across all the lymphocyte cell lines. The ability of NSC95397 to impact
E1A expression in both a lytically-infected T cell line as well as a persistently-infected T cell line could indicate a T lymphocyte specific role for the disrupted interaction. Unlike the impact seen in T lymphocytes, none of the epithelial cell lines showed an increase in
E1A expression with NSC95397 treatment (Fig.
6f). Among the epithelial cell lines, A549 showed negligible changes in AdV expression following treatment with NSC95397 while Me-180 and CaLu exhibited moderate downregulation of both
hexon and
E3 (Fig.
6e). Whether this downregulation is attributable to the higher amount of CtBP1 present in these two epithelial cell lines as compared to A549 (Fig.
5b) is still unclear.
Of note, cell viability, especially that of transformed cell lines, can be negatively impacted following treatment with NSC95397 [
70]. In our experiments, we optimized treatment timing to maintain cell viability at or above roughly 50% (data not shown). NSC95397 also induced substantial downregulation of multiple housekeeping genes (Fig.
6b-d, and unpublished data), although this effect did not directly relate to the viability of the cells. For example, among the epithelial cell lines, Me-180 cells exhibited the highest reduction in viability with treatment (data not shown), however the housekeeping gene remained unchanged. One limitation to our study is the inherent variability between individual infections of lymphocytes which does not allow for averaging of data across independent infections. Nonetheless, our primary observations remain consistent between multiple infections, which are shown individually.
In addition to the use of small-molecule inhibitor NSC95397, another potential experimental strategy for understanding the impact of CtBP1 on persistent infection in lymphocytes is transient knock-down of CtBP1 expression using shRNA or siRNA. Primary lymphocytes and lymphocytic cell lines are notoriously challenging to transfect using lipid-based approaches [
81], but electroporation has been used successfully to deliver regulatory RNA with high efficiency [
82]. In our current study, we attempted to transfect our persistently-infected lymphocytic cell lines with knock-down siRNA through electroporation and found that electroporation alone was sufficient to upregulate viral gene expression (data not shown). Future attempts to use a CtBP1 knock-down approach may include stable transduction with an inducible shRNA expression vector prior to infection of the lymphocytes, which would allow controlled expression of the regulatory RNA and resulting CtBP1 knock-down only after the persistent phase of infection has been established.
CtBP gene regulation is complex with many paradoxical activities reported for its function. The differences in CtBP expression profile between our cell line models of lytic and persistent infection suggest that distinctions in known function, structure, and localization of the two CtBP homologs may be important for infection outcome in these cells. While CtBP1 is ubiquitously expressed, CtBP2 expression is more tissue and cell-type specific [
29]. Structurally, CtBP1 and CtBP2 differ slightly by a nuclear localization signal (NLS) only present in the N-terminal of CtBP2 and a PDZ-binding domain only present in the C-terminal of CtBP1 [
83]. The NLS present, and a key p300 acetylation site on lysine 10 within the NLS, are responsible for the nuclear localization of CtBP2 [
45]. On the other hand, the localization of CtBP1, which is found both in the cytoplasm and the nucleus, is subject to more complex regulation; sumoylation at lysine K428, in conjunction with the PDZ-binding domain regulate nuclear localization [
83]. CtBP1 can also be recruited to the nucleus by a CtBP2-dependent mechanism [
84]. Additionally, distribution of CtBP1 between the cytoplasm and the nucleus is also reported to be dependent upon the cell-type, further implicating other factors in localization regulation [
83‐
86]. How these reported differences in the complex regulation of CtBP impact the viral life cycle in these cells will require additional study.
This is the first investigation into a possible role for CtBP in persistent infection of lymphocytes, and we observed that NSC95397 treatment could release a CtBP-associated repression of
E1A in infected T cell lines. Although the Jurkat infections are lytic and KE37 infections persist for months, both show suppression of infection kinetics relative to epithelial cells [
17]. A549 cells produce high levels of viral late proteins within 24 h of infection, while Jurkat and KE37 do not achieve peak levels until 1–3 or 3–7 dpi, respectively, despite equivalent amounts of viral DNA (Fig.
1a and [
17,
21]). Transcription is also repressed in both cell lines relative to A549 (Fig.
1b). Whether these overall reduced levels of viral transcripts stem from a repressive mechanism at the
E1A promoter remains to be determined, but it seems likely that repression of the master regulator of AdV infection,
E1A, could have a profound influence on the infection dynamics. We were surprised to find that, under the same treatment conditions, we observed no de-repression of
E1A in BJAB cells. It is possible that the binding partners incorporated into CtBP complexes between our B and T cell lines may be different, and additionally, may be influenced by the differences in basal NAD
+/NADH ratios detected in our lymphocyte cell lines [
35]. These are all areas worthy of further investigation.
In one of the only other reports of a direct mechanism involved in establishment of persistent infection, Zheng et al. showed that repression of AdV transcription, resulting from interferon (IFN) α- and IFNγ-induced recruitment of E2F/Rb complexes to the
E1A enhancer, was able to induce persistent infection in primary and normal epithelial cells [
87]. While IFN-treatment allowed epithelial cells to survive infection for long periods of time with reduced viral gene expression in this study, production of infectious virus could be detected at all time points [
87]. Notably, upon cessation of IFN-treatment, viral replication rebounded dramatically [
87]. In contrast, in both naturally-infected lymphocytes extracted from tonsil and adenoid tissue and in lymphocyte cell lines, viral transcription is similarly repressed but infectious virus can be detected only in rare instances [
2,
17]. This suggests that, even without chronic IFN exposure, a more extensive repression of viral gene expression has occurred in lymphocytes than what was described for IFN-treated epithelial cells. Whether the IFN-E2F/Rb axis contributes to persistent infection in lymphocytes has not been determined, but different and/or additional mechanisms of transcriptional repression likely regulate persistence in lymphocytes.
Other mechanisms of viral transcriptional repression have been reported in AdV infection of epithelial cells that potentially link the metabolic state of the cell to regulation of persistent infection through NAD-dependent enzymes. Sirtuins (NAD
+-dependent Class III HDACs) have been implicated in regulation of AdV gene expression. Silencing RNA (siRNA) knockdown of all seven human sirtuins (SIRT1–7) has been shown to increase AdV-C5 titers by 1.5- to 3-fold [
88]. In the same vein, activation of sirtuins through resveratrol treatment inhibits adenovirus DNA replication [
89,
90]. Another NAD
+-dependent enzyme to have been studied in lytic infection is Poly (ADP-Ribose) Polymerase 1 (PARP1); the AdV E4orf4 protein has been found to increase production of viral progeny through inhibition of PARP1, which is activated by the infection-induced DNA damage response (DDR) [
91]. PARP-induced synthesis and attachment of long poly (ADP-ribose) chains to proteins has been shown to regulate cellular transcription through chromatin remodeling and modification of transcription factors [
92,
93]. Whether sirtuins or PARP1 contribute to the transcriptional repression of persistent infection needs further investigation.