Role of activating 3 in the synthesis PNAS PLUS of latency-associated transcript and maintenance of herpes simplex virus 1 in latent state in ganglia

Minfeng Shu, Te Du, Grace Zhou1, and Bernard Roizman2

Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, Chicago IL 60637

Contributed by Bernard Roizman, August 4, 2015 (sent for review July 17, 2015) A key property of herpes simplex viruses (HSVs) is their ability to rapidly deplete NGF or in medium containing both NGF and establish latent infection in sensory or autonomic ganglia and to epithelial growth factor (EGF) (9). In the former medium, ex- reactivate on physical, hormonal, or emotional stress. In latently pression of viral begins at ∼5 h after excision of the gan- infected ganglia, HSVs express a long noncoding RNA, a latency- glia. In medium containing NGF and EGF, the virus remains associated transcript (LAT), which plays a key role in maintaining silent for ∼18–24 h. Studies on this model revealed that in latently infected neurons, but not viral . To investigate the ganglia maintained in medium containing NGF plus EGF, the events leading to reactivation, we examined the use of ganglionic virus is maintained in a dynamic equilibrium that is disrupted organ cultures that enable rapid reactivation in medium contain- by inhibitors of HDAC-1 and -4, the PI3K-AKT-mTOR and STAT3 ing antibody to nerve growth factor (NGF) or delayed reactivation pathways, or proapoptotic drugs (10, 11). Reactivation in medium in medium containing NGF and epidermal growth factor (EGF). containing anti-NGF antibody is inhibited by the expression of Here we report the discovery that activating transcription factor 3 REST or by a p300/CBP inhibitor (11, 12). Perhaps more sig- (ATF3), a stress response , profoundly affects the interac- nificant for reactivation, in neurons all genes are expressed at once tion of HSV with its host. Specifically, (i) ATF3 is induced by stress, rather than sequentially in a coordinated fashion as is seen in in- such as inhibition of protein synthesis or infection; (ii) in infected fected cells at the portal of entry. cells, ATF3 enhances the accumulation of LAT by acting on the The present study emanated from an attempt to identify the MICROBIOLOGY response elements in the promoter of the LAT precursor RNA; neuronal genes whose expression maintains the virus in a latent (iii) ATF3 is induced nearly 100-fold in ganglionic organ cultures; state. In this study, we compared by microarray analyses RNA and (iv) ATF3 plays a key role in the maintenance of the latent transcripts in freshly excised ganglia with those of ganglia in- state, inasmuch as expression of ATF3 bereft of the C-terminal cubated for 5 h or 11 h in medium containing anti-NGF antibody activation domain acts as a dominant negative factor, inducing and medium containing NGF plus EGF. The cellular that HSV in ganglionic organ cultures harboring latent attracted our attention was that of ATF3, because its mRNA virus and incubated in medium containing NGF and EGF. Thus, ATF3 is increased nearly 100-fold both in ganglia incubated in medium a component of a cluster of cellular proteins that together with LAT containing anti-NGF and in medium containing NGF plus EGF. maintain the integrity of the neurons harboring latent virus. We found that ATF3 enhances the accumulation of LAT in in- fected cells in culture and plays a role in the maintenance of HSV-1 stress response | adenylate-uridylate–rich mRNA | reactivation in a latent state. ATF3 is a member of the ATF/CREB family of transcriptional ollowing infection at a portal of entry, usually the mouth or factors (13, 14). The current consensus holds that ATF3 is an Fgenitals, herpes simplex virus (HSV)-1 or HSV-2 infect sen- immediate early-response gene, a regulator of stress response, sory nerve endings and are transported retrograde to the neu- and a factor in determining cell fate (15). ATF3 binds to a cAMP ronal nucleus, where they establish a silent or “latent” infection response element (CRE) with the canonical sequence 5′-TGAC- (1). Characteristically, during the latent state, neurons accumu- GTCA-3′ in the form of homodimers or heterodimers with other late a 1.5- or 2-kb noncoding RNA known as latency-associated members of the ATF/CREB family. HSV-1 DNA contains eight transcript (LAT) (2–5). LAT is a stable intron derived from a much longer precursor RNA (pLAT) (3, 6). In a fraction of Significance neurons infected with HSV-1, the virus reactivates and is trans- ported anterograde to a site at or near the portal of entry, where A major unresolved issue confronting infectious diseases is the it replicates and becomes available for transmission to another mechanism by which virulent, potentially lethal viruses remain individual by physical tissue contact (7, 8). Knowledge is scant silent (latent) in selected cells in the human body until induced regarding the mechanisms by which HSV-1, a potentially virulent to replicate and spread in response to stress. The establishment virus, can remain silent in neurons for the life of the host or of latent infection by herpes simplex viruses in sensory or au- reactivate from latency. The model organism closest to the hu- tonomic neurons is a common event in a large fraction of the man is the mouse, a model system that does not lend itself human population. Here we report that activating transcription readily to molecular analyses of events occurring in the sensory factor 3 (ATF3) is induced by stress both in infected cells in ganglia. At the other extreme, neuronal cultures harboring HSV-1 culture and in ganglia harboring latent virus. The function silenced by drugs might not reflect the natural mechanisms by which of ATF3 is to block the reactivation of virus induced by neuro- the virus is silenced in ganglia. Moreover, in ganglia, sensory nal stress. neurons are coated by satellite cells that affect the physiology of

the neuron. Author contributions: M.S., T.D., G.Z., and B.R. designed research; M.S. and T.D. per- In humans, mice, and rabbits, reactivation follows stimulation formed research; M.S., T.D., and B.R. analyzed data; and B.R. wrote the paper. or a reversible or irreversible injury (1). In an attempt to probe The authors declare no conflict of interest. the events occurring after injury, we developed a ganglionic or- 1Present address: Sino-French Hoffman Institute of Immunology, Guangzhou Medical gan culture model. In this model, trigeminal ganglia are har- University, Guangzhou 510182, China. vested at 30 d after mouse corneal inoculation and incubated in 2To whom correspondence should be addressed. Email: [email protected]. medium containing anti-nerve growth factor (NGF) antibody to edu.

www.pnas.org/cgi/doi/10.1073/pnas.1515369112 PNAS Early Edition | 1of7 Downloaded by guest on September 27, 2021 Fig. 1. HSV-1(F)–induced ATF3 mRNA is independent of new protein synthesis. (A) ATF3 mRNA is induced by HSV-1(F) infection. Cells (HEK293, Vero, and Hep2) were infected with HSV-1(F) at a multiplicity of infection (MOI) of 10, and then harvested at the indicated time points. ATF3 mRNA was detected by Northern blot analysis. (B) Effect of cycloheximide (CHX; 100 μg/mL) on HSV-1(F)–induced ATF3 mRNA level. Cells (HEK293, Vero, and Hep2) were infected with HSV-1(F) at an MOI of 10 in the absence or presence of CHX. 18s and 28s rRNAs served as loading controls.

CRE sites, but only two of these are in a promoter, specifically in ATF3 mutants, shown schematically in Fig. 2A. As noted in the the promoter 1 of LAT (LP1) (16). Mutagenesis of the CRE1 site introductory section, ATF3 consists of an N-terminal activation (5′-CTGCGTCA-3′) proximal to the pLAT TATA box results in domain (A), followed by a repressor domain (R), a basic domain diminished levels of LAT and reduced reactivation rates in a rabbit (B), a ZIP domain (Z), and a second C-terminal activation do- model (17, 18). Those results are consistent with results of other main (A) (Fig. 2A). In these experiments, replicate cultures of studies showing that LAT protects neurons from apoptosis (19, 20). HEK293T cells were transfected with 1 μg of pcDNA or plasmids Finally, the 181-residue protein has been shown to consist of encoding intact or mutant ATF3 tagged at the N terminus with five domains: an N-terminal activation domain, a repressor do- Flag. The cells were exposed to 1 PFU of HSV-1(F) per cell at main, a basic domain, a ZIP domain essential for dimerization, 40 h after transfection and harvested at 20 h after infection. and a C-terminal activation domain (21, 22). Like many stress Northern blots of extracted LAT are shown in Fig. 2B. LAT was response genes, its mRNA contains adenylate-uridylate (AU)-rich quantified by phosphoimaging (Fig. 2E) or by quantitative RT- elements in the 3′ UTR (23). Curiously, ATF3 is not subject to PCR (qRT-PCR) (Fig. 2F). The amounts of wild type (WT) and degradation by the viral host shutoff RNase. The other AU-rich mutant ATF3 accumulating in transfected cells are shown in Fig. RNAs spared from degradation play significant roles in the biology 2D. The results show that the accumulation of LAT is enhanced of HSV (24, 25). by ATF3, and that the C-terminal activation domain is essential for the observed effect. Results ATF3 Is Induced in Untreated HSV-1–Infected Cells and in Infected Cells ATF3 Binds to CRE1 and CRE2 Sites in the Promoter of LAT Precursor Treated with Cycloheximide (100 μg/mL) at the Time of Infection. RNA. As noted above, only two of the eight CRE sites in HSV-1 Replicate cultures of HEK293T, Vero, and HEp-2 cells were ex- DNA are contained in a promoter, specifically in the promoter posed to 10 PFU of HSV-1(F) per cell alone (Fig. 1A)orinthe of the LAT precursor RNA (LP1). The sequences of CRE1 presence of cycloheximide (Fig. 1B). The cells were harvested at (5′-CTGCGTCA-3′)andCRE2(5′-TTACATCA-3′)differfromthe the time of infection (0 h) and at the times shown in Fig. 1. The sequence of canonical CRE (5′-TGACGTCA-3′). Previous studies salient features of the results can be summarized as follows. All have shown that mutagenesis of the CRE1 site results in decreased infected cells showed accumulation of ATF3 mRNA. The patterns accumulation of LAT concurrent with decreased reactivation of of accumulation varied. We noted a huge increase in ATF3 mRNA HSV-1 in the rabbit model (17). To determine whether ATF3 in infected HEK 293T and HEp-2 cells, but a relatively modest binds to CRE1 or CRE2, 32P-labeled oligonucleotides expressing accumulation in infected Vero cells. The presence of high molec- CRE1 or CRE2 were reacted with nuclear extracts from cells ular weight bands in HEK 293T and HEp-2 cells late in the course transfected with plasmids encoding WT N-terminal Flag-tagged of infection suggested accumulation of unspliced ATF3 RNAs. ATF3, M2, or M3. As described in detail in Materials and Methods, In addition, the amounts of ATF3 mRNA were higher in cells the mixtures were subjected to electrophoresis in nondenaturing exposed to cycloheximide late in infection. These data are consis- gels, transferred to a cellulose sheet, and subjected to autoradiog- tent with the conclusion that activation of ATF3 in infected cells raphy. To ensure that the CRE probes bound WT or mutant ATF3, does not require previous viral protein synthesis. The apparent the reaction mixture contained a competitor oligonucleotide or increase in the amount of ATF3 mRNAs could reflect either a antibody to Flag. In mixtures in which the oligonucleotide con- more intense stress response to the presence of cycloheximide or taining the CRE1 or CRE2 sequence, ATF3, M2, or M3 anti- inhibition of ATF3 mRNA accumulation by a viral gene product body bound to the Flag tag retarded the electrophoretic mobility produced relatively late after infection in untreated cells. of the complex. Fig. 3C shows that transfected cells produced N-terminal Flag-tagged ATF3, M2, or M3 proteins. Fig. 3 A and B Accumulation of LAT Is Enhanced in Cells Expressing Wild Type ATF3 show the results obtained on CRE1 and CRE2, respectively. The or ATF3 Mutants Containing the C-Terminal Activation Domain. To results indicate that WT ATF3 bound both CRE1 (Fig. 3A,lane5) follow up preliminary studies showing that ATF3 enhances the and CRE2 (Fig. 3B, lane 5), but neither M2 nor M3 bound CRE1 expression of LAT (26), we constructed a series of truncated or CRE2.

2of7 | www.pnas.org/cgi/doi/10.1073/pnas.1515369112 Shu et al. Downloaded by guest on September 27, 2021 AD were harvested at 2, 12, 24, or 48 h after exposure to 0.1 PFU of PNAS PLUS virus per cell. The accumulation of ATF3 in the transfected cells at the time of infection is shown in Fig. 5C. The results of ti- trations shown in Fig. 5B indicate that WT ATF3, M2, or M3 had no effect on viral replication.

M2, but Not M3 or WT ATF3, Induced the Reactivation of Recombinant E Virus in Ganglia That Were Excised and Incubated in Medium Containing NGFandEGFat30dAfterInfection.We constructed a series of recombinant viruses carrying WT ATF3, M2, or M3 driven by the cytomegalovirus (CMV) immediate early promoter and inserted between UL3andUL4 using procedures described elsewhere (27). B Trigeminal ganglia of mice infected by corneal scarification were harvested at 30 d after infection and either analyzed immediately for viral gene expression or incubated for 24 h in medium con- taining NGF plus EGF or anti-NGF antibody. The harvested F ganglia in groups of six were analyzed for the amounts of mRNAs encoding representative α (ICP27), β (thymidine kinase), γ1 (VP16), or γ2 (UL41) genes. The results, shown in Fig. 6, indicate that the C recombinant virus encoding the M2 ATF3 mutant induced the expression of all representative viral gene groups to levels in- distinguishable from those of ganglia incubated in medium containing anti-NGF antibody. The increases in the amounts of viral mRNAs in ganglia harboring latent WT virus or recombi- nant viruses carrying WT ATF3 or M3 were significantly lower.

WT and Recombinant Viruses Induce the Expression of ATF3 on Excision and Incubation in Medium Containing Anti-NGF Antibody MICROBIOLOGY Fig. 2. Effect of different ATF3 truncations on LAT expression. (A) Sche- matic diagram of different ATF3 truncations. A, activation domain; B, basic and Medium Containing NGF and EGF. In these experiments, we domain; R, repression domain; Z, ZIP domain. Both the basic domain and used qRT-PCR to quantify ATF3 mRNA in the extracts of the ZIP domain are required for binding to DNA. The truncations were named ganglia described above, that is, ganglia harboring latent WT or serially from M1 to M6. (B) Northern blot analysis showing the effect of recombinant viruses encoding ATF3, M2, or M3 and prepared transfection of ATF3 truncations on LAT (2.0-kb intron) expression. (C) The either immediately after excision or after incubation in medium 18s and 28s rRNAs served as loading controls. (D) Protein expression of dif- containing anti-NGF antibody or medium containing both NGF ferent ATF3 truncations. (E) Intensities of the bands of various truncations and EGF. The results, shown in Fig. 7, indicate that ATF3 was transfection in B were quantified by phosphoimaging analysis. The value induced and increased ∼100-fold after excision and incubation obtained was normalized with respect to 18s rRNA and plotted relative to the RNA levels present in the pcDNA-transfected cells. (F) Total RNA was in medium containing anti-NGF antibody as well as in medium extracted, reverse-transcribed, and used for quantification of LAT expression containing NGF plus EGF. by qRT-PCR. The results are expressed as fold change compared with the RNA level present in the cells of pcDNA transfection. Data represent mean ± Discussion SD. **P < 0.01; ns, not significant. The finding that prompted the studies described in this report was the observation that ATF3 is induced by nearly 100-fold in murine ganglia harboring latent HSV-1 and maintained in either WT ATF3 Interacts with ATF3, but Not with M2 or M3 Mutants. As medium containing anti-NGF antibody or medium containing noted above, ATF3 interacts with its response elements by forming NGF plus EGF (Fig. 7). The salient features of the results pre- homodimers or heterodimers with itself or other ATF/CREB sented in this report are as follows. proteins. The central question that we posed is whether WT ATF3 ATF3 is induced in cells productively infected with HSV-1. forms heterodimers with M2 lacking only the C-terminal activation The HSV-1 DNA sequence contains eight CREs, of which only domain or with M3, which lacks both the ZIP domain and the two are located in a promoter region, namely in LP1. Our results C-terminal activation domain. In these experiments, HEK 293T cells show that ATF3 binds to both CRE sites in LP1, forms a homodimer, were transfected with plasmids encoding ATF3, M2, or M3 tagged and fosters increased accumulation of LAT. at the N terminus with Flag. Soluble cell extracts prepared at 48 h As noted above, ATF3 consists of several discrete domains. after transfection (Fig. 4 A and B, lanes 1–4) were reacted with ATF3 mutant M2 bereft of the C-terminal activation domain and GST or GST-ATF3 bound to agarose beads (Fig. 4C). The pro- M3 mutant lacking both the ZIP and the C-terminal activation teins bound to the beads were solubilized, subjected to electro- domains did not enhance LAT synthesis, bind to the CRE sites in phoresis in denaturing gels, transferred to a nitrocellulose sheet, and LP1, or interact with WT ATF3. Neither WT ATF3 nor the reacted with Ponceau stain (Fig. 4B) and then with antibody to Flag mutants tested in this study affected the replication of HSV-1 in (Fig. 4A). The presence of ATF3 on the agarose beads is shown in productively infected cells. Fig. 4C, lanes 3 and 4. The pull-down assays shows that the tagged A very different picture emerged from the studies on ganglia ATF3 pulls down itself, but not M2 or M3 (Fig. 4A, lanes 8–13). harboring latent viruses expressing WT ATF3, M2, or M3. The viruses carrying WT ATF3 or M3 could not be differentiated Overexpression of ATF3, M2, or M3 Has No Effect on the Replication of from HSV-1(F) with respect to viral gene expression on excision HSV-1(F). We next asked whether overexpression of WT ATF3, of the ganglia, on incubation in medium containing anti-NGF M2, or M3 acting as a dominant negative factor affects virus antibody, or in medium containing both NGF and EGF. In replication. As illustrated in Fig. 5A, at 24 h before infection, contrast, viral gene expression was activated in ganglia harboring replicate cultures of HEK 293T cells were transfected with 1 μg the mutant virus M2 in medium containing either anti-NGF of plasmids encoding pcDNA, WT-ATF3, M2, or M3. The cells antibody or both NGF and EGF.

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Fig. 3. ATF3 directly binds to the LAT promoter at both CRE1 and CRE2 sites. (A and B) EMSA and supershift assay showing that ATF3 interacts directly with both CRE1 (A) and CRE2 (B) sites. HEK 293T cells were transfected with 1 μg of pN-Flag-ATF3, pN-Flag-M2, or pN-Flag-M3 plasmid. The cells were collected at 48 h after transfection and fractionated into nuclear, membrane, and cytoplasmic fractions as described in Materials and Methods. Then 20 μg of nuclear protein extract was reacted with 32P end-labeled DNA with the CRE, CRE1, or CRE2 consensus binding site. The gel supershift bands showing the specific binding of ATF3 to CRE1 or CRE2 were manifested in the presence of anti-Flag antibody and indicated by a solid arrow. (C) Nuclear extracted ATF3, M2, and M3 protein expression from transfected cells.

The results presented in this report suggest the following regarding the role of STAT3 is based on studies showing that in- conclusions. ATF3 is expressed in response to stress and carries hibition of STAT3, PI3K, Akt, or mTOR, or interference with the AU-rich elements in its 3′ UTR characteristic of mRNAs encoding phosphorylation of tyrosine 705, results in activation of virus (44). stress response proteins (23). Activation of stress response genes is a ATF3 is the sole stress-inducible factor currently associated common consequence of infection (28). Furthermore, the mRNAs with suppression of activation of latent virus. Depending on the encoding stress response proteins are a primary target of VHS- system, ATF3 acts to promote viability or death of the cell in which RNase encoded by the UL41 gene of HSV-1 (24, 25, 28, 29). The it is induced. In recorded instances, ATF3 acts with STAT3 to in- presence of two CREs in LP1 suggests that these sites have evolved duce gene expression (36). The effect on LAT expression and the to respond to cellular stress and raises the question of whether the synthesis of LAT is a stress response. Moreover, in natural infection, HSV-1 reactivates as a con- sequence of stress induced by physical injury to neurons [e.g., AC UV light exposure (30, 31)] or cytokines [e.g., IL6 (32, 33)] in- duced by hormonal or emotional stress (34, 35). In the model system used in these studies, the primary stress is excision of ganglia, and the induction of ATF3 appears to be at least one of the induced responses to stress. The observation that M2 induces viral gene expression in ganglia harboring latent virus and maintained in medium containing NGF plus EGF suggests that M2 acts as a dominant negative factor, possibly by interacting with a partner other than ATF3. As noted in the introductory B section, ATF3 can form heterodimers with other members of ATF/CREB proteins (36, 37). M2 contains the ZIP domain re- quired for the formation of homodimers or heterodimers, but it does not interact with WT ATF3. A relevant conclusion based on our results is that ATF3 plays a role in maintaining the integrity of the affected neuron and concurrently maintains viral DNA in a latent state. Fig. 4. ATF3 forms homodimerization, but M2 and M3 fail to dimerize with It is appropriate to place ATF3 in the context of other factors ATF3. (A) Dimerization was detected by GST pull-down assay. HEK-293T cells were transiently transfected with the indicated plasmids encoding pN-Flag–tag- that play roles in maintaining HSV in a latent state. These known ged full-length ATF3 (residues 1–181), M2 (residues 1–135), or M3 (residues factors fall into one of three categories: constitutively active, 1–118). Soluble cell extracts were prepared at 48 h after transfection and reacted activated on stress, or inducible on stress (Fig. 8). All share a com- with 10 μL of glutathione-agarose beads bound to GST (lanes 6, 8, 10, and 12) mon goal: maintenance of neuronal integrity. or 80 μL of GST-ATF3 (lanes 7, 9, 11, and 13). (B) The proteins bound to the The constitutively active factors have been known for some beads were solubilized and subjected to electrophoresis on a denaturing poly- time. Current consensus holds that the function of LAT and, acrylamide gel, transferred to a nitrocellulose membrane, and stained with most likely, of the miRNAs is to maintain the integrity of the Ponceau stain, followed by reaction with antibody against Flag. Immunoreac- neuron to maintain the virus in a silent state (38, 39). In the absence tivities of intact ATF3, M2, and M3 are shown in lanes 2, 3, and 4 indicated by of LAT, neurons harboring latent virus succumb to apoptosis (31). arrows. The amounts loaded in each lane represent 10% (vol/vol) of the whole- cell lysates. pcDNA, pcDNA 3.1(+). (C) Ponceau staining showing the GST and NGF is similarly required for the maintenance of neurons; with- GST-ATF3 proteins bound to the beads used in the pull-down assay. Note that the drawal of NGF results in apoptosis (40, 41). amounts of beads in the reaction mixtures were determined on the basis of GST- In neurons, STAT3 resides in the cytoplasm and is trans- ATF3 proteins bound to the beads. Shown are the amounts of GST and GST-ATF3 located to the nucleus on neuronal stress (42). The function of proteins solubilized from the beads subjected to electrophoresis in a denaturing STAT3 is to block neuronal death (43). Much of the information gel and stained with Coomassie blue.

4of7 | www.pnas.org/cgi/doi/10.1073/pnas.1515369112 Shu et al. Downloaded by guest on September 27, 2021 Materials and Methods PNAS PLUS Cells and Viruses. HEp-2, Vero, and HEK 293T cell lines were obtained from American Type Culture Collection and cultured. The limited-passage HSV-1(F) is the prototype HSV-1 strain used in this laboratory (45).

Antibodies. Mouse monoclonal antibodies against Flag and β-actin were pur- chased from Sigma-Aldrich. The antibodies were used at a dilution of 1:1,000.

Plasmid Construction. The WT ATF3 (1–181) and truncated ATF3 (1–148), ATF3 (1–135), ATF3 (1–118), ATF3 (40–181), ATF3 (40–148), and ATF3 (1–39, 85–181) fragments were obtained by PCR using the following primers: ATF3 (1–181)- N-Flag,forward:5′-CGCGGATCCATGGATTACAAGGATGACGATGACAAGATGC- TTCAACACCCAGGCCAGGTC-3′;ATF3(1–181), reverse: 5′-CCGGAATTCTTAGC- TCTGCAATGTTCCTTC-3′;ATF3(1–148), reverse: 5′-CCGGAATTCTTAAAGGTTGA- GCATGTATATCAAATGCTGC-3′; ATF3 (1–135), reverse: 5′-CCGGAATTCTTA- GAGCTCCTCAATCTGAGCCTTCAGTTC-3′;ATF3(1–118), reverse: 5′-CCGGAAT- TCTTACGACTCTTTCTGCAGGCACTCCGTCTTC-3′; ATF3 (40–181)-N-Flag, forward: MICROBIOLOGY

Fig. 5. ATF3, M2, and M3 transient transfections have no effect on HSV-1(F) growth in vitro. (A) Schematic diagram of the experimental design. In brief, HEK293 T cells were transfected with 1 μg of pcDNA, pN-Flag–tagged ATF3, M2, or M3 plasmids for 24 h. Cells were then exposed to HSV-1(F) at a multiplicity of 0.1 PFU per cell. The inocula were replaced at 2 h after ex- posure to virus. Cells were harvested at 2, 12, 24, and 48 h after infection. (B) Viral progeny were titrated on Vero cells. (C) Protein expression of transfected ATF3, M2, and M3. β-actin served as a loading control.

high levels of expression in excised ganglia suggest that in the HSV- 1–infected cells, ATF3 functions primarily to maintain the integrity of neurons. Finally, it is noteworthy that each of the factors associated with the maintenance of HSV in a latent state is associated with blocking neuronal death. Conversely, deprivation of NGF, ab- sence of LAT, or inhibition of STAT3 can cause virus reac- tivation and apoptosis. Is activation of apoptosis compatible with reactivation? There are no definite studies on this issue. The only available data are based on studies of ganglionic organ cultures reported elsewhere (10). In brief, exposure of ganglia harboring latent virus in medium containing NGF and EGF and two dif- ferent proapoptotic drugs resulted in viral gene expression and Fig. 6. Reactivation of HSV-1(F), recombinant viruses of HSV-1 expressing viral DNA synthesis at levels compatible with those attained in WT ATF3, M2, or M3. At 30 d postinoculation, TG excised from F, recombi- nant viruses expressing WT-ATF3, M2, or M3 were processed immediately medium containing anti-NGF antibody. In essence, the data in- (blue columns) or after 24 h incubation in medium containing anti-NGF dicate that proapoptotic stimuli could induce virus reactivation antibody (red columns), or NGF plus EGF (green columns). Copy numbers of

and may be the principal mechanism by which neuronal injury ICP27, TK, VP16, and UL41 normalized to cellular RNA were plotted. *P < causes reactivation of latent virus. 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Shu et al. PNAS Early Edition | 5of7 Downloaded by guest on September 27, 2021 previously (25). The membrane was probed for Flag tag and β-actin with the antibodies listed above.

Construction of Recombinant ATF3 and Mutant Viruses. The recombinant HSV-1 viruses expressing WT ATF3 or mutants were constructed using the BAC system, as described previously (27).Inbrief,ORFsofWThumanATF3(46) or two mutants (M2 and M3) tagged with Flag at the N terminus were

inserted between the genes encoding UL3andUL4 under CMV promoter, and the resulting viruses were designated R501 (WT-ATF3), R502 (M2), and R503 (M3).

Northern Blot Analysis. The Northern blot analyses were carried out as described previously (47). In brief, 10 μg of total RNA was loaded onto denaturing formaldehyde gel, transferred, and probed with random hexanucleotide- primed 32P-labeled fragments of the indicated genes (ATF3 and LAT).

Electrophoretic Mobility Shift and Supershift Assays. For this experiment, 20 μg of nuclear extracted protein was added to a binding reaction mixture con- taining 0.5 ng of 32 P-labeled oligonucleotide, followed by electrophoresis and autoradiography. The Oligonucleotide labeling, nuclear protein fraction extraction, and electrophoretic mobility shift assay (EMSA) were done as described previously (47–49). The canonical CRE and CRE-like (CRE1 and Fig. 7. ATF3 mRNA was stimulated by HSV-1(F), recombinant viruses of CRE2) oligonucleotides were designed to the LAT promoter sequence HSV-1(F) HSV-1 expressing WT ATF3, M2, or M3 infection. Copy numbers of ATF3 corresponding to ATF3-binding sites. The two complementary single- normalized to cellular RNA were plotted as described in Fig. 6. strain oligonucleotides were synthesized and annealed before labeling. The sequences were as follows: WT CRE1 (118621–118646), forward: 5′-TGT- TTTTGCTGCGTCATCTG AGCCTT-3′, reverse: 5′-AAGGCTCAGATGACGCAG- 5′-CGCGGATCCATGGATTACAAGGATGACGATGACAAGTTTGTCAAGGAAGAGC- ′ ′ ′ TGAGGTTTGCC-3′;ATF3(1–39), reverse: 5′-GCTCTAGAGGGCGTCAGGTTAGCA- CAAAAACA-3 ; mutant CRE1, forward: 5 -TGTTTTTGCTGCGCA ATCTGAGCCTT-3 , ′ ′ – AAATCCTCAAAC-3′; and ATF3 (85–181), forward: 5′-GCTCTAGAGAAGAT- reverse: 5 -AAGGCTCAGATTGCGC AGCAAAAACA-3 ; WT CRE2 (118581 118606), ′ ′ ′ GAAAGGAAAAAGAGGCGACGA-3′. The WT and mutant ATF3 ORFs tagged forward: 5 -AAAATAAAATTACATCACCTACCCAC-3 ,reverse:5-GTGGGTAGGT- ′ ′ with Flag at the N terminus were subcloned in the multiple cloning site of GATGTAATTTTATTTT-3 ; mutant CRE2, forward: 5 -AAAATAAAATTACACA- ′ ′ ′ pcDNA3.1(+).The resulting plasmids were named pN-Flag-ATF3 (WT, 1–181), ACCTACCCAC-3 , reverse: 5 -GTGGGTAGGTTGTGTAATTTTATTTT-3 ; canonical ′ ′ ′ M1 (1–148), M2 (1–135), M3 (1–118), M4 (40 181), M5 (40–148), and M6 (1–39, CRE, forward: 5 AGAGATTGCCTGACGTCAGAGAGC TAG-3 ,reverse:5-CTAG- ′ 85–181), respectively. Plasmids containing ATF3 ORFs were generously provided CTCTCTGACG TCAGGCAATCTCT-3 . by Shigetaka Kitajima (37) and Hsonwin Hai (22). Murine Model of Virus Infection and Reactivation from Latency. The 4-wk-old HEK 293T Cell Transient Transfection. The procedure for transfection of HEK 293T inbred female CBA/J mice (The Jackson Laboratory) received unrestricted cells has been described previously (24). The cells were harvested at 24 or 48 h access to food and water. All animal studies were done in accordance with after transfection and lysed according to the protocol for subsequent use. protocols approved by the Institutional Animal Care and Use Committee of the University of Chicago. Mice were infected by cornea scarification, and GST Pull-Down Assay. In brief, HEK 293T cells were transfected with 1 μgof virus in the trigeminal ganglia was reactivated by a ganglionic organ culture pN-Flag-ATF3 (1–181), M2 (1–135), or M3 (1–118). Extracted total proteins model as described previously (9). were pulled down by beads bound to GST fused to the ATF3 protein, and then subjected to immunoblot analysis with the mouse anti-Flag antibody. RNA Extraction and RT-qPCR Analysis. The procedures for RNA extraction from The pull-down procedures were performed as described previously (25). cells or murine tissues and RT-qPCR analysis have been described previously (9). Viral mRNA expression levels in ganglia immediately after excision, after Immunoblot Analysis. Cells were collected at the indicated time points after a 24-h incubation in anti-NGF antibody, and after a 24-h incubation in infection. The procedures for harvesting, solubilization, protein quantifica- NGF plus EGF were compared using the Student t test. A two-tailed P value tion, SDS/PAGE, and transfer to nitrocellulose membranes were as described of < 0.05 was considered statistically significant.

Fig. 8. Schematic representation of the known factors maintaining HSV in latent form. The transition from the silent (latent) to activated state, shown here by a long black arrow, is irreversible. As indicated above the black arrow, the transition is blocked by constitutively present LAT, NGF, and chromatin-modifying enzymes (e.g., HDACs, methyl transferases) (50–53), activated factors exemplified by STAT3, and inducible factors such as ATF3. Inhibitors, dominant negative mutants, and other entities that support the roles of constitutively present, activated, or inducible factors are shown below the black arrow.

6of7 | www.pnas.org/cgi/doi/10.1073/pnas.1515369112 Shu et al. Downloaded by guest on September 27, 2021 ACKNOWLEDGMENTS. We thank Lindsay Smith for technical assistance College of Medicine) for the generous gifts of plasmids encoding PNAS PLUS withtheanimalexperiments,andDr.Shigetaka Kitajima (Tokyo Medical ATF3. This study was supported by National Cancer Institute Grant and Dental University) and Dr. Hsonwin Hai (Ohio State University CA71933.

1. Roizman B, Knipe DM, Whitley RJ (2013) Herpes simplex viruses. Fields’ Virology, eds 27. Gu H, Roizman B (2007) Herpes simplex virus-infected cell protein 0 blocks the si- Knipe DM, et al. (Wolters Kluwer/Lippincott-Williams and Wilkins, New York), 6th Ed, lencing of viral DNA by dissociating histone deacetylases from the CoREST-REST pp 1823–1897. complex. Proc Natl Acad Sci USA 104(43):17134–17139. 2. Kang W, Mukerjee R, Fraser NW (2003) Establishment and maintenance of HSV latent 28. Taddeo B, Esclatine A, Zhang W, Roizman B (2003) The stress-inducible immediate- infection is mediated through correct splicing of the LAT primary transcript. Virology early responsive gene IEX-1 is activated in cells infected with herpes simplex virus 1, 312(1):233–244. but several viral mechanisms, including 3′ degradation of its RNA, preclude expression 3. Zabolotny JM, Krummenacher C, Fraser NW (1997) The herpes simplex virus type 1 of the gene. J Virol 77(11):6178–6187. 2.0-kilobase latency-associated transcript is a stable intron which branches at a gua- 29. Taddeo B, Zhang W, Roizman B (2009) The virion-packaged endoribonuclease of herpes nosine. J Virol 71(6):4199–4208. simplex virus 1 cleaves mRNA in polyribosomes. Proc Natl Acad Sci USA 106(29):12139–12144. 4. Alvira MR, Goins WF, Cohen JB, Glorioso JC (1999) Genetic studies exposing the 30. Rooney JF, et al. (1991) Prevention of ultraviolet light-induced herpes labialis by splicing events involved in herpes simplex virus type 1 latency-associated transcript sunscreen. Lancet 338(8780):1419–1422. production during lytic and latent infection. J Virol 73(5):3866–3876. 31. BenMohamed L, et al. (2015) Decreased reactivation of a herpes simplex virus type 1 5. Drolet BS, et al. (1998) The region of the herpes simplex virus type 1 LAT gene in- (HSV-1) latency-associated transcript (LAT) mutant using the in vivo mouse UV-B volved in spontaneous reactivation does not a functional protein. Virology model of induced reactivation. J Neurovirol, 10.1007/s13365-015-0348-9. 242(1):221–232. 32. Kriesel JD, et al. (1997) Anti–interleukin-6 antibodies inhibit herpes simplex virus re- 6. Farrell MJ, Dobson AT, Feldman LT (1991) Herpes simplex virus latency-associated activation. J Infect Dis 175(4):821–827. transcript is a stable intron. Proc Natl Acad Sci USA 88(3):790–794. 33. Kriesel JD, Ricigliano J, Spruance SL, Garza HH, Jr, Hill JM (1997) Neuronal reactivation 7. Preston CM, Efstathiou S (2009) Molecular basis of HSV latency and reactivation. of herpes simplex virus may involve interleukin-6. J Neurovirol 3(6):441–448. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis, eds Arvin A, et al. 34. Vicetti Miguel RD, et al. (2010) 17-beta estradiol promotion of herpes simplex virus (Cambridge Univ Press, Cambridge), 1st Ed, pp 602–615. type 1 reactivation is estrogen -dependent. J Virol 84(1):565–572. 8. Roizman B, Whitley RJ (2013) An inquiry into the molecular basis of HSV latency and 35. Rivera L, Beuerman RW, Hill JM (1988) Corneal nerves contain intra-axonal HSV-1 reactivation. Annu Rev Microbiol 67:355–374. after virus reactivation by epinephrine iontophoresis. Curr Eye Res 7(10):1001–1008. 9. Du T, Zhou G, Roizman B (2011) HSV-1 gene expression from reactivated ganglia is 36. Kiryu-Seo S, et al. (2008) Neuronal injury-inducible gene is synergistically regulated by disordered and concurrent with suppression of latency-associated transcript and ATF3, c-Jun, and STAT3 through the interaction with Sp1 in damaged neurons. J Biol miRNAs. Proc Natl Acad Sci USA 108(46):18820–18824. Chem 283(11):6988–6996. 10. Du T, Zhou G, Roizman B (2012) Induction of apoptosis accelerates reactivation of 37. Hua B, et al. (2006) A splice variant of stress response gene ATF3 counteracts NF- latent HSV-1 in ganglionic organ cultures and replication in cell cultures. Proc Natl kappaB–dependent anti-apoptosis through inhibiting recruitment of CREB-binding Acad Sci USA 109(36):14616–14621. protein/p300 coactivator. J Biol Chem 281(3):1620–1629. 11. Du T, Zhou G, Roizman B (2013) Modulation of reactivation of latent herpes simplex 38. Jiang X, et al. (2011) The herpes simplex virus type 1 latency-associated transcript can

virus 1 in ganglionic organ cultures by p300/CBP and STAT3. Proc Natl Acad Sci USA protect neuron-derived C1300 and Neuro2A cells from granzyme B-induced apoptosis MICROBIOLOGY 110(28):E2621–E2628. and CD8 T-cell killing. J Virol 85(5):2325–2332. 12. Zhou G, Du T, Roizman B (2013) HSV carrying WT REST establishes latency but re- 39. Ahmed M, Lock M, Miller CG, Fraser NW (2002) Regions of the herpes simplex virus activates only if the synthesis of REST is suppressed. Proc Natl Acad Sci USA 110(6): type 1 latency-associated transcript that protect cells from apoptosis in vitro and E498–E506. protect neuronal cells in vivo. J Virol 76(2):717–729. 13. Hunt D, Raivich G, Anderson PN (2012) Activating transcription factor 3 and the 40. Wilcox BJ, Applegate MD, Portera-Cailliau C, Koliatsos VE (1995) Nerve growth factor nervous system. Front Mol Neurosci 5:7. prevents apoptotic cell death in injured central cholinergic neurons. J Comp Neurol 14. Tsujino H, et al. (2000) Activating transcription factor 3 (ATF3) induction by axotomy 359(4):573–585. in sensory and motoneurons: A novel neuronal marker of . Mol Cell 41. Wilcox CL, Johnson EM, Jr (1987) Nerve growth factor deprivation results in the re- Neurosci 15(2):170–182. activation of latent herpes simplex virus in vitro. J Virol 61(7):2311–2315. 15. Abe T, Oue N, Yasui W, Ryoji M (2003) Rapid and preferential induction of ATF3 42. Dziennis S, Alkayed NJ (2008) Role of signal transducer and activator of transcription transcription in response to low doses of UVA light. Biochem Biophys Res Commun 3 in neuronal survival and regeneration. Rev Neurosci 19(4-5):341–361. 310(4):1168–1174. 43. Li GH, et al. (2009) STAT3 silencing with lentivirus inhibits growth and induces apo- 16. Ackland-Berglund CE, Davido DJ, Leib DA (1995) The roles of the cAMP-response ptosis and differentiation of U251 cells. J Neurooncol 91(2):165–174. element and TATA box in expression of the herpes simplex virus type 1 latency-associated 44. Lee N, Neitzel KL, Devlin BK, MacLennan AJ (2004) STAT3 phosphorylation in injured transcripts. Virology 210(1):141–151. axons before sensory and motor neuron nuclei: Potential role for STAT3 as a retro- 17. Bloom DC, Stevens JG, Hill JM, Tran RK (1997) Mutagenesis of a cAMP response ele- grade signaling transcription factor. J Comp Neurol 474(4):535–545. ment within the latency-associated transcript promoter of HSV-1 reduces adrenergic 45. Ejercito PM, Kieff ED, Roizman B (1968) Characterization of herpes simplex virus reactivation. Virology 236(1):202–207. strains differing in their effects on social behaviour of infected cells. J Gen Virol 2(3): 18. Marquart ME, et al. (2001) A cAMP response element within the latency-associated 357–364. transcript promoter of HSV-1 facilitates induced ocular reactivation in a mouse hy- 46. Thompson RL, Sawtell NM (2010) Therapeutic implications of new insights into the perthermia model. Virology 284(1):62–69. critical role of VP16 in initiating the earliest stages of HSV reactivation from latency. 19. Perng GC, et al. (2000) Virus-induced neuronal apoptosis blocked by the herpes Future Med Chem 2(7):1099–1105. simplex virus latency-associated transcript. Science 287(5457):1500–1503. 47. Shu M, Taddeo B, Roizman B (2013) The nuclear-cytoplasmic shuttling of virion host 20. Peng W, et al. (2003) The gene that encodes the herpes simplex virus type 1 latency- shutoff RNase is enabled by pUL47 and an embedded nuclear export signal and de-

associated transcript influences the accumulation of transcripts (Bcl-xL and Bcl-xS) that fines the sites of degradation of AU-rich and stable cellular mRNAs. J Virol 87(24): encode apoptotic regulatory proteins. J Virol 77(19):10714–10718. 13569–13578. 21. Hai TW, Liu F, Coukos WJ, Green MR (1989) Transcription factor ATF cDNA clones: An 48. Advani SJ, Weichselbaum RR, Roizman B (2000) proteins are posttranslationally extensive family of proteins able to selectively form DNA-binding modified concomitantly with a reduction in nuclear binding activity in cells infected heterodimers. Genes Dev 3(12B):2083–2090. with herpes simplex virus 1. J Virol 74(17):7842–7850. 22. Chen BP, Liang G, Whelan J, Hai T (1994) ATF3 and ATF3 delta Zip: Transcriptional 49. Kristie TM, Roizman B (1986) Alpha 4, the major regulatory protein of herpes simplex repression versus activation by alternatively spliced isoforms. J Biol Chem 269(22): virus type 1, is stably and specifically associated with promoter-regulatory domains 15819–15826. of alpha genes and of selected other viral genes. Proc Natl Acad Sci USA 83(10): 23. Pan YX, Chen H, Kilberg MS (2005) Interaction of RNA-binding proteins HuR and 3218–3222. AUF1 with the human ATF3 mRNA 3′-untranslated region regulates its amino acid 50. Liang Y, et al. (2013) A novel selective LSD1/KDM1A inhibitor epigenetically blocks limitation-induced stabilization. J Biol Chem 280(41):34609–34616. herpes simplex virus lytic replication and reactivation from latency. MBio 4(1): 24. Shu M, Taddeo B, Zhang W, Roizman B (2013) Selective degradation of mRNAs by the e00558-12. HSV host shutoff RNase is regulated by the UL47 tegument protein. Proc Natl 51. Liang Y, Vogel JL, Narayanan A, Peng H, Kristie TM (2009) Inhibition of the histone Acad Sci USA 110(18):E1669–E1675. demethylase LSD1 blocks alpha-herpesvirus lytic replication and reactivation from 25. Shu M, Taddeo B, Roizman B (2015) Tristetraprolin recruits the herpes simplex virion latency. Nat Med 15(11):1312–1317. host shutoff RNase to AU-rich elements in stress response mRNAs to enable their 52. Knipe DM, Cliffe A (2008) Chromatin control of herpes simplex virus lytic and latent cleavage. J Virol 89(10):5643–5650. infection. Nat Rev Microbiol 6(3):211–221. 26. Millhouse S, Kenny JJ, Quinn PG, Lee V, Wigdahl B (1998) ATF/CREB elements in the 53. Messer HG, Jacobs D, Dhummakupt A, Bloom DC (2015) Inhibition of H3K27me3- herpes simplex virus type 1 latency-associated transcript promoter interact with members specific histone demethylases JMJD3 and UTX blocks reactivation of herpes simplex of the ATF/CREB and AP-1 transcription factor families. JBiomedSci5(6):451–464. virus 1 in trigeminal ganglion neurons. J Virol 89(6):3417–3420.

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