HSV LAT AND NEURONAL SURVIVAL

International Reviews of Immunology, 23: 187 198, 2004 Copyright # Taylor & Francis Inc. ISSN: 0883-0185 print/1563-5244 online DOI: 10.1080=08830180490265592 HSV LAT AND NEURONAL SURVIVAL DAVID C. BLOOM Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, Florida, USA Herpes Simplex Virus (HSV) establishes a latent infection within sensory neurons and periodically reactivates in response to stress. HSV s ability to inhabit neurons for the life of the host involves a number of virally encoded functions that tightly regulate the latency-reactivation cycle, preventing uncontrolled spread of reactivating virus and large-scale death of neurons. The HSV latency-associated transcript (LAT) is a complex transcription unit expressed primarily in neurons containing latent genomes. While mutational analyses indicate LAT is nonessential for viral replication, the 5 0 exon of LAT greatly enhances reactivation. Several studies have also identified LAT mutations that reduce establishment of latency and enhance virulence. Recently, LAT has also been shown to inhibit cell death through by blocking caspase-8 and caspase-9 pathways. While blocking apoptosis is not essential for either establishment of latency or reactivation, it likely augments these processes and may contribute to HSV s long-term persistence and spread. Keywords: HSV, LAT, apoptosis, latency, herpesvirus, latency-associated transcript Herpes Simplex Virus types 1 and 2 (HSV-1 and HSV-2) are large enveloped viruses containing double-stranded DNA genomes of approximately 152 kb in size. HSV-1 is the primary cause of oral=facial lesions (cold sores) in humans, whereas HSV-2 lesions are primarily restricted to the genital tract. While this review focuses on HSV-1 because it has been more extensively studied, it is likely that the general concepts apply to both viruses since they share a great deal of similarity in their biological features, including a high degree of sequence similarity at the nucleic acid level (for reviews see Taylor et al. [1] and Wagner and Bloom [2]). Address correspondence to David C. Bloom, Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Box 100266, Gainesville, FL 32610-0266. E-mail: dbloom@ufl.edu 187

188 D. C. Bloom Even though HSV-1 has a broad tropism and can productively infect a wide variety of cell types and animals, its hallmark is the ability to establish a lifelong latent infection within neurons of the peripheral nervous system. While the acute (lytic) reproductive cycle is readily studied in cell culture, latency can only be established with difficulty in primary neuronal cultures [3,4], and perhaps in differentiated rat pheochromocytoma PC-12 cells [5]. Much of the work on the latent phase of the infection, therefore, relies on experimental infection of animal models (for a review see Wagner and Bloom [2]). Primary models used to study infection in vivo are the mouse and rabbit (HSV-1) and the guinea pig (HSV-2). For HSV-1, animals are typically inoculated by the ocular route, allowing the virus to replicate within epithelial tissues and enter the local nerve termini for fast axonal transport to the neuronal nuclei. Once inside neurons of the sensory ganglion (typically the trigeminal ganglion, but virus can also be recovered from the superior cervical and pterypalantine ganglia) the virus either initiates an acute infection resulting in the production of progeny virions, or the viral DNA circularizes and establishes a latent infection. While the precise mechanism of this acute= latent switch is unknown, it is believed to involve the repression of viral immediate early gene transcription. Data suggest that at least part of this process is under neuronal control, and that latency may be preferentially established in certain phenotypic classes of sensory neurons [6 8]. During HSV-1 latency, lytic gene transcription is tightly repressed and the only transcript that abundantly accumulates is the latencyassociated transcript (LAT). There is no direct evidence that LAT encodes a peptide, and the only consistent phenotype displayed by a number of LAT mutants in both the mouse and rabbit models is a reduction in the frequency of reactivation. Recent data strongly suggests that LAT is capable of inhibiting neuronal cell death, and may do so by inhibiting both the receptor-mediated (caspase-8) and mitochondrialmediated (caspase-9) pathways [9 11]. This chapter will review data supporting a role for LAT in blocking neuronal cell death and apoptosis, and discuss the impact of this inhibition on the pathobiology of HSV infection, particularly its impact on latency and reactivation. EVIDENCE THAT LAT PROMOTES CELL SURVIVAL It is generally agreed that an element encoded within the HSV-1 LAT plays some role in promoting neuronal survival. This consensus has emerged from observations that recombinants containing large deletions encompassing the LAT promoter and extending into the 2.0 kb

HSV LAT and Neuronal Survival 189 FIGURE 1 Map of the HSV-1 genome and the HSV-1 LAT region. The organization of the HSV-1 genome is depicted at the top. The unique regions of the genome (U L and U S ) are depicted by lines, and the repeat segments (R L and R S ) are depicted by black and open boxes, respectively. The expanded region represents the entire long repeat (R L ) and a portion of the short repeat (R S ). Illustrated are the locations of the latency associated transcript (LAT) and three other viral genes (ICP0, gamma 34.5 and ICP4) that are encoded on the opposite strand and overlap with the LAT. LAT intron ( Figure 1) establish latent infections in 1=2to1=3asmany neurons as wild-type or rescue viruses [12,13], and that infection with these LAT deletion mutants results in greater loss of neurons from the trigeminal ganglia (TG) than observed in wild-type virus infections. Rabbit Trigeminal Ganglia Neurons Exhibit Large Numbers of TUNEL and PARP Cleavage Products Following Infection with HSV-1 Recombinants with Large LAT Deletions The first evidence that a function encoded within the LAT region promotes cell survival was put forth by Perng et al. [9] using a recombinant of HSV-1 McKrae (dlat2903) which contains a deletion spanning 161 to þ1667, relative to the transcriptional start of the primary 8.5 kb LAT (see Figure 2). This study demonstrated that large numbers of TUNEL-positive neurons could be observed in rabbit TG acutely infected (day 7 postinfection (p.i.)) with dlat2903, compared with only a few TUNEL-positive cells in TGs from rabbits infected with the rescue of dlat2903 or the parent strain, HSV-1 McKrae. TUNEL results were further confirmed by the demonstration that p85 cleavage products of poly-adp-ribose polymerase (PARP) were detectable in > 25% of TG neurons infected with the dlat2903, whereas no PARP-positive cells were detected in TGs infected with wild-type McKrae. This suggested that a function encoded within the LAT region ultimately resulted in the inhibition of caspase-3 induced PARP cleavage, initiated during an HSV-1 infection.

190 D. C. Bloom FIGURE 2 Analysis of the HSV-1 LAT region for the region capable of blocking apoptosis. The location of the HSV-1 LAT promoter, 662 bp 5 0 exon region and the 2.0 kb intron are illustrated at the top. Panel A summarizes analysis of HSV-1 recombinants containing deletions in the LAT region and their ability to induce detectable apoptosis in ganglia by TUNEL analysis. The bars and solid lines represent the span of the deletion in each of the recombinants. Recombinants that demonstrate an increase in TUNEL-positive cells over wild-type (or rescue) are indicated by open boxes, whereas the recombinants that demonstrated no increase in TUNEL-positive cells are indicated by the black boxes. Panel B summarizes analysis of plasmids containing regions of the HSV-1 LAT by transient transfection assay for their ability to protect cells from induced apoptosis. The open boxes indicate the regions of LAT that were present in each plasmid. Only plasmids that were able to protect > 50% of the cells from induced apoptosis are indicated. Note that the first three plasmids shown demonstrated the highest level of protection, whereas the pdxcmi construct showed less than 100% protection. Panel C depicts the consensus region where the LAT antiapoptosis phenotype is likely to reside. The black portion of the box represents 100% congruity between the in vitro and in vivo data sets, whereas the grey region on either side represents where partially protecting phenotypes have been observed. A Region of the HSV-1 LAT Protects Cells from Induced Apoptosis in Transient Transfection Assays While questions were initially raised concerning the specificity of the anti-parp p85 antisera, and detection of cytoplasmic staining [14],

HSV LAT and Neuronal Survival 191 the observation that IMR-90 and CV-1 cells transiently transfected with a plasmid containing nt 301-2659 of the LAT primary transcript was able to protect > 75% of the cells from ceramide or fumonisin B 1 - induced apoptosis (relative to transfection with a control plasmid) strongly suggests that this region of the LAT protects against induction of apoptosis [9,14]. The level of antiapoptotic activity afforded by the LAT plasmid was comparable to a plasmid containing the baculovirus inhibitor of apoptosis (CpIAP). Finally, it was demonstrated the LAT region also protected Neuro-2a cells (mouse neuroblastoma) from etoposide-induced apoptosis. Since ceramide and fumonisin B 1 (FB 1 ) induce apoptosis through different pathways (protein kinase C and TNF=FAS, respectively), transient transfection studies suggested that LAT s antiapoptosis function acts through a common downstream effector. Evidence for LAT-Mediated Apoptosis Protection in the Murine Model Initial studies indicated that ocular infection of mice with the large LAT-deletion virus 17DAH ( 1136 to þ 828), resulted in depletion of over half the TG neurons relative to uninfected mice, mice infected with wild-type 17syn þ, or the 17AH rescue [15]. Surprisingly, the authors were unable to detect any increase in TUNEL-positive cells or evidence of the cleaved form of caspase-3, leading them to conclude that the loss of neurons was not due to an apoptotic mechanism. This raised the possibility that the apoptosis observed with the LAT mutants might represent a species-specific effect. Two subsequent studies argue convincingly against this possibility. The first study employed a LAT deletion mutant (17N=H) whose deletion ( 359 to þ1491) overlaps with that of 17DAH but extends further into the intron. 17N=H showed a significant increase in TUNEL-positive TG neurons compared to mice infected with 17synþ or a smaller LAT deletion (17DSty) [10]. A second study using a transgenic mouse containing the LAT promoter and first 3 kb of the primary HSV-2 LAT transcript demonstrated diminished apoptosis, both in cell culture and in vivo [16]. In this study, hepatocytes and fibroblasts from transgenic or nontransgenic mice were treated with TNF or anti-fas antibody. Transgenic mice showed an approximate 50% reduction in numbers of cells exhibiting annexin V staining versus nontransgenic controls. In addition, when transgenic mice were treated with anti- Fas antibody, they demonstrated 10-fold lower serum ALT levels than the nontransgenic group. Only 37% of the transgenic mice died, compared to 100% of the nontransgenic mice. Collectively, these data

192 D. C. Bloom suggest that the HSV LAT contains elements that are capable of blocking apoptosis both in rabbits and mice in vivo. One possible explanation for the authors inability to detect evidence of apoptosis induced by 17AH is the smaller size of this recombinant s deletion. Mapping of the LAT s minimal genetic element required to block apoptosis is discussed in the next section. CELL-SPECIFICITY AND MAPPING OF THE ANTIAPOPTOTIC MEDIATOR WITHIN THE LAT REGION Two issues regarding the LAT s role in blocking apoptosis remain unresolved. The first is to map the minimal elements required for apoptosis protection; the second is the inability to identify a protein or open-reading frame that corresponds to the general region in which the antiapoptotic function resides. While the latter point may imply that the antiapoptotic element acts at the RNA level (or perhaps through enhancement of another element in the region), mapping of this region has been confounded by its cell-type specificity. Early studies demonstrated that a plasmid-encoding LAT could protect CV-1 or IMR-90 cells from both ceramide and FB 1 -induced cell death, and could protect Neuro-2A (mouse neuroblastoma) cells from etoposideinduced death [9]. The first real attempt to map this phenotype was performed by Inman et al., and it showed that a plasmid expressing nts 1 1499 (5 0 exon and the 5 0 half of the intron; Figure 2B) promoted cell survival in neuro-2a and CV-1 cells [17]. The first 811 bp (5 0 exon and 5 0 portion of the intron) was somewhat less efficient in protection, while the first 611 bp showed no protection against apoptosis. A followup study by Ahmed et al. showed that transient transfection of HeLa (human cervical carcinoma) and SY5Y (human neuroblastoma) cells with exon 1, the stable 2.0 kb intron, and the 5 0 portion of exon 2 diminished the onset of anti-fas antibody (HeLa) or camptothecin (HeLa and SY5Y)-induced apoptosis. These results indicated that this region of LAT was capable of blocking the caspase-8 induced apoptotic pathway [10]. The study further revealed that plasmids lacking 5 0 exon sequences were the least effective in blocking apoptosis, confirming that the 5 0 end of the intron contains the element(s) critical for apoptotic protection. Mapping the region of the LAT that blocks apoptosis during viral infection is even more complicated. Infection of cells with 17N=H (which lacks the LAT promoter, exon 1 and the first half of the intron; Figure 2A) resulted in DNA fragmentation, indicative of apoptosis [10]. This indicated that exon 1 and the 2.0 kb intron confer protection from apoptosis in the context of whole virus infections. Additional

HSV LAT and Neuronal Survival 193 analyses revealed that infection with a 371 bp 5 0 exon deletion recombinant (17DSty) showed no evidence of apoptosis (similar to wild-type virus). This strongly suggested that the region responsible for antiapoptotic effects in the context of whole virus is the same as in the transient transfection assays: the LAT intron. A final point revealed by cell culture studies was that the LAT s ability to block apoptosis is cell specific: the LAT had no effect on survival of either 293 (human epithelial cells immortalized by adenovirus) or COS-7 (CV-1 cells transformed with SV-40) cells [17]. BIOLOGICAL IMPACT OF LAT-MEDIATED APOPTOTIC INHIBITION Elucidation of how the LAT s ability to block apoptosis influences the HSV lifecycle is difficult due to HSV s genetic complexity and the fact that it contains at least five other apoptosis-modulating genes. Immediate early (IE; ICP27), early (E; U S 2 and U S 5), and late (gd and gj) gene products and have been shown to block apoptosis primarily during the acute infection [18 21]. In addition to potential interplay between these antiapoptotic functions, alterations in the normal transcriptional program, such as the accumulation of additional amounts of early or leaky-late transcripts, can alter the induction of apoptosis [22]. Therefore, the implication of the LAT in regulating IE and E gene transcription has to be considered when evaluating the apoptotic capacity of specific mutants [23]. Nonetheless, the following sections discuss evidence for the LAT s involvement during HSV-1 infections and the potential of blocking apoptosis to modulate the infection cycle. Impact on the Acute Infection and Establishment of Latency In vivo, the LAT promoter demonstrates a much higher activity in neurons than in other cell types and is most active in cells that harbor latent genomes but do not express acute antigens [24]. Nonetheless, some LAT is expressed in some productively infected cells with leaky late (, c) or strict late (c) kinetics [25]. HeLa cells treated with anti- Fas antibody 16 h prior to infection with the LAT deletion mutant 17N=H showed an increase in fragmented DNA within 6 h (compared to wild-type virus) [10]. It is difficult, however, to assess the overall contribution of events this late in the infection. Since HeLa cells that are not induced to enter apoptosis do not exhibit protective effects when infected with LAT mutants or wild-type virus [10], it seems likely that the LAT s contribution to protection during the acute infection is minimal and dwarfed by other viral antiapoptotic functions.

194 D. C. Bloom The LAT s contribution during the acute infection is further questionable since it is made late in acutely infected cells, only after a number of the other viral antiapoptosis mediators have been made. It has been proposed that the LAT is involved in establishment of latency, and the ability to block apoptosis enhances neuronal survival for increased efficiency. The LAT is abundantly expressed in murine peripheral ganglia neurons that contain HSV-1 DNA but that do not express acute antigens at 4 days p.i. In addition, approximately 5% of the antigen-expressing cells are positive for LAT expression [24]. LAT could play a role in protecting either of these cell populations: both cells that do not express acute antigens as well as cells that abortively express low levels of acute antigens and therefore have the potential to enter latency. In support of this, HSV-1 recombinants with deletions extending from the LAT promoter through the 2.0 kb intron exhibit a 2- to 4-fold reduction in the total number of latently infected cells per ganglia in the mouse and rabbit models [12,15,26]. Reported increases in TUNEL-positive neurons in the rabbit and mouse during the acute infection, while difficult to quantitate, support the concept that the LAT could directly block apoptosis in certain cell populations during the transition from acute infection to latency [9,10]. Due to the complexity of analyzing these processes in the context of whole virus infections in vivo, these studies have failed to provide evidence of direct inhibition of apoptosis by the LAT. The fact that LAT mutants exhibit leakier transcriptional repression during latency could increase the likelihood of a neuron undergoing apoptosis during establishment. Another point that has not yet been resolved is the fact that the LAT promoter mutants do not seem to exhibit the restricted establishment observed in the LAT intron mutants [27 30]. This suggests that the anti-apoptotic function may map to a different promoter, implying that the anti-apoptosis function is not due to a LAT at all, but rather a co-linear transcript. Finally, even though some evidence suggests that some that certain LAT mutants may establish latency in half as many cells as the wild-type virus, the fact that the LAT mutants do establish a latent infection, in at least some population of neurons, suggests that its role in establishment is not essential. Clearly, more work needs to be done, but it seems likely that the LAT s contribution to enhancing establishment by blocking apoptosis may be modest at best. Impact on Reactivation from Latency From the existing data, it seems more likely that the LAT (or the antiapoptosis element contained within this region) exerts its primary

HSV LAT and Neuronal Survival 195 influence during the reactivation phase of HSV s life cycle. LAT is present in many (if not all) latently infected neurons, and would be available at the time reactivation is initiated. It would make biological sense that an anti-apoptotic function would enhance exit from latency, especially since induction of reactivation shows high correlation with various physiologic stressors, many of which have been implicated as at least co-factors in apoptosis induction. Unfortunately, only circumstantial data exists supporting the anti-apoptotic activity of the LAT on facilitating reactivation. The primary evidence is from rather large LAT deletion mutants that are unable to block apoptosis, but are also restricted in reactivation. While it has been demonstrated that replacing the region deleted in the recombinant dlat2903 with the latency-related (LR) gene of BHV-1 (which has also been shown to have anti-apoptotic functions) restored spontaneous reactivation to wild-type levels [31], this only proves that the BHV-1 homologue of LAT is capable of restoring reactivation, not necessarily that the LR (and hence LAT) plays a solely apoptotic role in facilitating reactivation. Until the element that mediates the block of apoptosis is cleanly mapped (and identified), it will be difficult to definitively link its effects on blocking apoptosis with reactivation. The main difficulty in making this assignment is that while the region of the genome encoding the LAT that blocks apoptosis overlaps with the region shown to facilitate reactivation, reactivation maps primarily to the 5 0 exon, whereas the apoptosis phenotype resides primarily within the 2.0 kb intron. In addition, the LAT promoter and 5 0 exon mutants do not seem to possess in vivo antiapoptosis activity. So, while data clearly links the LAT function to both reactivation and apoptosis, direct links between these functions suggest they are genetically distinct. The hypothesis that the LAT region is involved in protecting reactivating neurons from apoptosis to increase the efficiency of replication and=or maintain latency remains attractive. Identification of element(s) within the LAT that mediates apoptotic protection would allow that hypothesis to be directly tested. CONCLUSIONS AND UNRESOLVED QUESTIONS A number of studies have clearly demonstrated that the region of the genome encoding the HSV LAT contains a function capable of blocking induced apoptosis. The most compelling data for this function has been generated in cells transiently transfected with plasmids containing various regions of the HSV-1 LAT, and that have mapped this activity to the region corresponding to the 5 0 end of the HSV-1 LAT 2.0 kb intron. In addition, a transgenic mouse containing this region of the

196 D. C. Bloom LAT is also capable of protecting cells from apoptosis inducers. While data indicates that HSV-1 recombinants containing deletions of the region encoding the LAT show a modest increase in the number of apoptotic cells during acute infection of TG, these studies are complicated by the fact that the LAT, in addition to other functions, alters the transcriptional profile of the HSV genome in neurons. In addition, it is not clear that the LAT promoter mutants show increased levels of apoptosis, raising the possibility that the apoptosis phenotype is genetically distinct from the LAT (or LAP1) promoter. Future studies must resolve the following questions: 1. Precisely where does the apoptosis function map within the LAT, and is it transcribed from LAP1? 2. What is the nature of this antiapoptosis element? Since there is little evidence for a protein encoded in this region, is it a functional RNA? 3. What are the cellular targets of this antiapoptotic function? 4. Does the antiapoptosis element function to protect reactivating neurons from apoptosis? 5. Does the antiapoptosis function augment viral reactivation by making the production of new virions more efficient? These questions will be answered in the following years, and clearly this area of investigation has great potential to teach us not only about the pathobiology of HSV infections, but also to provide insight into mechanisms by which viruses block cell death. REFERENCES [1] T.J. Taylor, M.A. Brockman, E.E. McNamee, and D.M. Knipe, Herpes simplex virus, Front. Biosci., 7: d752 764, 2002. [2] E.K. Wagner and D.C. Bloom, Experimental investigation of herpes simplex virus latency, Clin. Micro. Reviews, 10: 419 443, 1997. [3] C.L. Wilcox and E.M. Johnson, Characterization of nerve growth factor-dependent Herpes Simplex Latency in neurons in vitro, J. Virol., 62: 393 399, 1988. [4] J.L. Arthur, C.G. Scarpini, V. Connor, R.H. Lachmann, A.M. Tolkovsky, and S. Efstathiou, Herpes simplex virus type 1 promoter activity during latency establishment, maintenance, and reactivation in primary dorsal root neurons in vitro, J. Virol., 75: 3885 3895, 2001. [5] Y.H. Su, R.L. Meegalla, R. Chowhan, C. Cubitt, J.E. Oakes, R.N. Lausch, N.W. Fraser, and T.M. Block, Human corneal cells and other fibroblasts can stimulate the appearance of herpes simplex virus from quiescently infected PC12 cells, J. Virol., 73: 4171 4180, 1999. [6] T.P. Margolis, C.R. Dawson, and J.H. LaVail, Herpes simplex viral infection of the mouse trigeminal ganglion. Immunohistochemical analysis of cell populations, Invest. Ophthalmol. Vis. Sci., 33: 259 267, 1992.

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