Enhancer and long-term expression functions of herpes simplex virus type 1 latency-associated promoter are both located in the same region.

During herpes simplex virus type 1 (HSV-1) latent infection in vivo, the latency-associated promoter (LAP) is the only promoter to remain highly active long term. In a previous attempt to characterize LAP activity in vitro and in a mouse model, we showed that a 1.5-kb fragment called the long-term expression element (LTE), located immediately downstream from the transcriptional start site of LAP, was able to (i) increase gene expression in an orientation-independent manner, regardless of the cell type or the promoter used in vitro (enhancer activity) and (ii) keep LAP active during latency in vivo (long-term expression activity) (H. Berthomme, J. Lokensgard, L. Yang, T. Margolis, and L. T. Feldman, J. Virol. 74:3613-3622, 2000). To determine if these two functions could be separated genetically, we conducted a mutational analysis on the LTE and analyzed the effect on the LAP-LTE properties in both transient expression in cell culture and mouse dorsal root ganglia lytic and latent infection. In this report, we show that the first half of the LTE sequence, corresponding to the region previously described as LAP2 or exon1, encodes the enhancer function. This same region is also required to keep the LAP active during latency. These results exclude the intron region as containing any significant enhancer activity or any ability to keep the LAP active during latency. The results also show that these two functions have not been separated, leaving open the possibility that there is no long-term expression function per se but that the enhancer itself may function to keep the LAP active during latency by raising the level of expression to a detectable one. Further mutational analysis will be required to determine if these two potential functions continue to cosegregate.

Evidence for a bidirectional element located downstream from the herpes simplex virus type 1 latency-associated promoter that increases its activity during latency.

Herpes simplex virus type 1 (HSV-1) latent infection in vivo is characterized by the constitutive expression of the latency-associated transcripts (LAT), which originate from the LAT promoter (LAP). In an attempt to determine the functional parts of LAP, we previously demonstrated that viruses harboring a DNA fragment 3' of the LAT promoter itself were able to maintain detectable promoter expression throughout latency whereas viruses not containing this element could not (J. R. Lokensgard, H. Berthomme, and L. T. Feldman, J. Virol. 71:6714-6719, 1997). This element was therefore called a long-term expression element (LTE). To further study the role of the LTE, we constructed plasmids containing a DNA fragment encompassing the LTE inserted into a synthetic intron between the reporter lacZ gene and either the LAT or the HSV-1 thymidine kinase promoter. Transient-expression experiments with both neuronal and nonneuronal cell lines showed that the LTE locus has an enhancer activity that does not activate the cytomegalovirus enhancer but does activate the promoters such as the LAT promoter and the thymidine kinase promoter. The enhancement of these two promoters occurs in both neuronal and nonneuronal cell lines. Recombinant viruses containing enhancer constructs were constructed, and these demonstrated that the enhancer functioned when present in the context of the viral DNA, both for in vitro infections of cells in culture and for in vivo infections of neurons in mouse dorsal root ganglia. In the infections of mouse dorsal root ganglia, there was a very high level of promoter activity in neurons infected with viruses bearing the LAT promoter-enhancer, but this decreased after the first 2 or 3 weeks. By 18 days postinfection, neurons harboring latent virus without the enhancer showed no beta-galactosidase (beta-gal) staining whereas those harboring latent virus containing the enhancer continued to show beta-gal staining for long periods, extending to at least 6 months postinfection, the longest time examined.

Herpes simplex virus latency and the immune response.

Following infection, herpes simplex virus establishes latency in the nervous system and recurrences of lytic replication occur periodically. Molecular events which may determine how virus enters latency, how it is maintained and what occurs during reactivation have been investigated. The role of the immune response in limiting infection of the nervous system, influencing the latent state and removing virus from peripheral sites following reactivation has also been studied.

The latency-associated promoter of herpes simplex virus type 1 requires a region downstream of the transcription start site for long-term expression during latency.

The latency-associated transcript (LAT) promoter of herpes simplex virus type 1 (HSV-1) is unique among the many promoters on the viral genome in that it remains active during the latent state. We have previously shown that a DNA fragment comprising the LAT promoter element through the cap site, when moved from the LAT locus to the glycoprotein C gene, is capable of only short-term expression. These and other data suggested that an HSV DNA element from the repeat region, not included in the LAT promoter itself, might be needed to preserve long-term expression. Based on a number of recombinant viruses, we narrowed our search for this putative element to a region 3' of the LAT transcription start site. In the present study, we have shown that a 1.1-kb DNA fragment containing the putative long-term expression element (LTE) is able to restore latent-phase gene expression to the LAT promoter. The element appeared to function best when it was placed in its natural location, which is 3' of the LAT promoter; however, partial function was obtained when the LTE was inserted upstream of the LAT promoter in the reverse direction. These data indicate that the LAT promoter region is more complex than originally anticipated and that in addition to requiring both core promoter and neuronal transcription factor binding sites, the promoter requires a specific region of DNA to prevent its shutoff during a latent infection.

The POU-domain factor Brn-3.0 recognizes characteristic sites in the herpes simplex virus genome.

The restriction of herpes virus latency to mammalian sensory ganglia has led to a search for tissue-specific regulatory molecules in these neurons which alter viral gene expression. We have recently shown that the POU-domain transcriptional regulator Brn-3.0 is abundantly expressed in the adult trigeminal ganglion. To begin to examine the hypothesis that Brn-3.0 might participate in the regulation of the HSV life-cycle, we used Brn-3.0 POU-domain protein as an affinity matrix, and biochemically screened the entire HSV genome for sites of Brn-3.0 binding. This screen identified several sites of the form TA/TA A T N A N TA/T, which significantly do not include the previously identified HSV octamer sequences. All of the selected sites occur in the <25% of the HSV genome which has not been assigned to open reading frames, suggesting that these sites may be transcriptional regulatory elements recognized by Brn-3.0 or another homeobox factor with similar DNA binding properties. However, these sites do not interact with Brn-3.0 with sufficiently high affinity to directly mediate transcriptional activation by Brn-3.0 alone in transfection assays. The experiments described also provide an effective general method for exhaustive screening of large viral genomes or sub-genomic fragments of eukaryotic DNA for sites of interaction with specific transcription factors.

Expression of the lacZ reporter gene in the rat basal forebrain, hippocampus, and nigrostriatal pathway using a nonreplicating herpes simplex vector.

We recently demonstrated the efficacy of a nonreplicating herpes simplex type 1 virus construct, employing the Moloney murine leukemia virus long terminal repeat promoter, in providing long-term expression of the lacZ gene in rat hippocampal neurons. We now report the utility of this construct in expressing the reporter gene in neurons of the basal forebrain and substantia nigra and examine the spread of the virus to other brain regions. Dorsal and ventrolateral hippocampal formation injection of the virus resulted in numerous beta-gal-expressing cells in the stratum pyramidale, stratum oriens, stratum lacunosum-moleculare, and stratum granulosum. Scattered cells of the medial septum/diagonal band were positively stained following direct injection into this region. More intense staining of the basal forebrain was observed following hippocampal injection as a result of retrograde transport of the virus as shown by PCR analysis of viral DNA. Hippocampal injection also resulted in positive cell staining in several other afferent projection nuclei, namely, the supramammillary bodies, dorsal and caudal linear raphe, and perirhinal/entorhinal cortex. Very few cells were labeled around injection sites in the striatum or substantia nigra. However, substantia nigra zona compacta cells were blue following striatal injection, as were pallidal neurons following nigral injection. These data demonstrate the feasibility of using this virus construct to express foreign genes such as neurotrophic factors in basal forebrain and substantia nigra neurons, taking advantage of retrograde transport of the virus to preserve local anatomy.

A 348-base-pair region in the latency-associated transcript facilitates herpes simplex virus type 1 reactivation.

Latency-associated transcript (LAT) promoter deletion mutants of herpes simplex virus type 1 have a reduced capacity to reactivate following adrenergic induction in the rabbit eye model. We have mapped a reactivation phenotype within LAT and describe the construction of recombinants in which poly(A) addition sites have been placed at intervals within the LAT region to form truncated LAT transcripts. These mutants localize the induced reactivation phenotype to the 5' end of LAT. To further define this region, we constructed a recombinant containing a 348-bp deletion located 217 bp downstream of the transcription start site of the 8.5-kb LAT. This virus, 17delta348, expresses LAT but exhibits a significantly reduced ability to reactivate following epinephrine iontophoresis into the cornea. Quantitative DNA PCR analysis reveals that 17delta 348 establishes a latent infection within rabbit trigeminal ganglia with the same efficiency as does either the rescuant or wild-type virus. The region deleted in 17delta348 encodes three potential translational initiators (ATGs) which we have mutated and demonstrated to be dispensable for epinephrine-induced reactivation. In addition, three smaller deletions within this region have been constructed and were shown to reactivate at wild-type (parent) frequencies. These studies indicate that an undefined portion of the 348-bp region is required to facilitate induced reactivation. Sequence analysis of this 348-bp region revealed a CpG island which extends into the LAT promoter and which possesses homology to conserved elements within the mouse and human XIST transcript encoded on the X chromosome. Possible implications of these elements in the regulation of LAT expression are discussed.

Targeting and gene expression in spinal cord motor neurons following intramuscular inoculation of an HSV-1 vector.

One problem in devising strategies of gene transfer to the nervous system is targeting specific neuronal populations. To evaluate the potential for using herpes simplex virus (HSV) as a vector for gene transfer to spinal cord motor neurons, the HSV-1 mutant LAT-LTR in which the E. coli beta-galactosidase gene is expressed under control of the HSV LAT core promoter (LAT) and the Moloney murine leukemia virus long terminal repeat (LTR) was inoculated unilaterally into the gastrocnemius muscle. Infectious virus was isolated from the spinal cord on days 3-7 post inoculation (PI). Immunocytochemical labeling of HSV antigen was detected in ipsilateral ventral horn neurons in the spinal cord at day 3 PI and had spread to contiguous spinal cord regions by day 6 PI. No viral antigen was detected at 14 or 28 DPI. beta-galactosidase expression (driven by the LAT-LTR promoter) was detected in neurons of the ventral horn on days 3, 6, 14, and 28 PI. Histological analysis showed mild lesions in the ventral horn on day 3 PI which progressed through days 6, 14 and 28 PI. This study demonstrates the feasibility of gene delivery into spinal motor neurons after injection of an HSV vector at a peripheral muscular site. This approach should prove useful in neurobiological investigations and it suggests a possible application to development of gene therapy for heritable diseases affecting motor neurons.

Long-term expression of a reporter gene from latent herpes simplex virus in the rat hippocampus.

A problem in utilizing herpes simplex virus (HSV) as a vector for expression of foreign genes in CNS neurons has been the inability to facilitate long-term expression of the engineered genes. Previously, we showed that the murine moloney leukemia virus LTR would drive beta-galactosidase (beta-gal) transcription for extended periods from the latent viral genome in sensory, but not motor neurons. In this communication we further evaluate the utility of the LTR promoter for use in long-term expression vectors. Following stereotactic injection of 8117/43 (an ICP4 minus, non-replicating virus with the LTR driving the beta-gal gene, or KD6 (an ICP4 minus non-replicating virus not expressing beta-gal) into the hippocampus of rats, polymerase chain reaction (PCR) analysis of viral DNA after 2 months indicated that latent infections were established. Assaying by both x-gal staining and reverse transcriptase PCR we demonstrate that (1) beta-gal can be detected for at least 6 months in hippocampal neurons, and (2) although the number of beta-gal transcripts in these cells drops considerably by 2 weeks, they can be detected during the period studied. These studies indicate that the LTR promoter is active and affords long-term expression in the CNS, albeit at comparatively low levels compared to those observed at acute times.

In vivo deletion analysis of the herpes simplex virus type 1 latency-associated transcript promoter.

During herpes simplex virus latency, transcripts accumulate from a single transcription unit of the viral genome. The promoter for these latency-associated transcripts (LAT) has been located, and a number of studies have documented the specific regions of this promoter which are important in transient assays of neuronal cells in culture. To examine the regulation of this promoter from the viral genome, both in vitro and in vivo, a series of seven promoter deletion viruses which drive the expression of the reporter gene beta-galactosidase was constructed. Rabbit skin cells were infected in cell culture with viruses bearing each promoter mutation, and the LAT promoter activity was compared with that obtained by infecting two neuronal cell lines, ND7 cells and C1300 neuroblastoma cells. Mouse dorsal root ganglia were also infected with these recombinant viruses by footpad inoculations, and beta-galactosidase activity was measured. Infected neuronal cells lines and dorsal root ganglia exhibit much more LAT promoter activity than infected rabbit skin cells, suggesting that the region upstream of -250 may contain one or several neuronal specific DNA-binding sites. However, a comparison of LAT promoter activities within the deletion series revealed many differences between neurons of the dorsal root ganglia infected in vivo and the two neuronal cell lines infected in vitro. These results suggest that neurons may vary extensively in the quantity or kind of transcription factors they contain.

Long-term promoter activity during herpes simplex virus latency.

The ability to direct foreign gene expression from the herpes simplex virus type 1 (HSV-1) genome during an acute or latent infection is a subject of increasing importance in the utilization of HSV vectors for gene therapy. Little is known about the types of transcription factors present in neurons or about whether different neuronal populations within a ganglion vary in their complement of these factors. With respect to HSV-1 latency, it is not known how or why the latency-associated transcript (LAT) promoter is able to function continually during latency while all other viral promoters are inactive. To further studies of these two phenomena, we constructed seven recombinant viruses with various promoter constructs driving expression of the lacZ reporter gene. Each construct was inserted into HSV-1 at the glycoprotein C locus, and recombinant viruses were evaluated for the ability to express beta-galactosidase during acute and latent viral infections in murine dorsal root ganglia. During acute infection of murine dorsal root ganglia, the activities of the promoters varied over a wide range. Constructs containing the murine metallothionein promoter (MT1), the phosphoglycerate kinase promoter, the Moloney murine leukemia virus long terminal repeat (LTR), or the region upstream of and including the HSV LAT core promoter (LAT) were active during the acute but not the latent phase of infection. The addition of transcription factor binding sites present in the upstream LAT region to the MT1 and LTR promoters (LAT-MT1 and LAT-LTR, respectively) significantly increased acute-phase expression. Despite these high initial rates of transcription, of all the promoter constructs only LAT-LTR was able to remain transcriptionally active after the establishment of a latent state. Thus, the Moloney murine leukemia virus LTR provides a DNA element which functions to prevent promoter inactivation during latency. An analogous HSV long-term-expression element is evidently not present in the upstream LAT promoter, indicating that the HSV long-term-expression function is provided by a region outside of that which gives high-level neuronal expression during the acute phase of infection.

Effect of the transcription start region of the herpes simplex virus type 1 latency-associated transcript promoter on expression of productively infected neurons in vivo.

It has been previously reported that the latency-associated transcript (LAT) promoter contains a DNA sequence at the LAT transcription start site which resembles the ICP4 consensus DNA binding site and that this site allows ICP4-mediated downregulation of the LAT promoter in transient assays (A. H. Batchelor and P. O'Hare, J. Virol. 64:3269-3279, 1990). We have confirmed these data by showing that an ICP4-expressing plasmid will downregulate lacZ expression from a plasmid containing the LAT promoter and transcription start site (pJA1) and does not downregulate lacZ expression from a plasmid in which the start site has been mutagenized (pWAG15). To determine the role of the LAT transcription start site in regulating LAT promoter activity in the context of the virus, two recombinant viruses, KOS-1 and KOS-15, were studied. KOS-1 contains an 863-bp portion of the LAT promoter, including the LAT cap site, fused to the lacZ gene and inserted into the gC locus (T.P. Margolis, F. Sedarati, A.T. Dobson, L.T. Feldman, and J.G. Stevens, Virology 189:150-160, 1992). The second virus (KOS-15) was constructed in identical fashion, using plasmid pWAG-15, which is not downregulated by ICP4. Vero cells productively infected with KOS-15 produce 10-fold more beta-galactosidase than do those infected with KOS-1. In murine dorsal root ganglia acutely infected with KOS-1, only 1.2% of dorsal root ganglion neurons that expressed viral antigen also expressed beta-galactosidase. In contrast, in KOS-15-infected mice, beta-galactosidase was detected in 18% of viral antigen-positive neurons. Similar findings were observed in trigeminal ganglia acutely infected with KOS-1 and KOS-15. Thus, the region encompassing the LAT transcription start site appears to play an important role in repression of the LAT promoter activity not only in vitro but also in acutely infected neurons in vivo. These results suggest that during productive infection with HSV-1, LAT expression is tightly regulated.

Decreased reporter gene expression during latent infection with HSV LAT promoter constructs.

The latency-associated transcripts (LAT), which code from an 8.5 kb segment of the internal repeat region of the HSV genome, are the only viral transcripts that are present during HSV latent infection. However, little is known about the relative contribution of promoter activity, degradative processes, and elements or regions affecting long term expression of these transcripts in latently infected neurons. To begin to address this question we investigated LAT promoter activity during acute and latent infection. Mouse footpads were infected with KOS/62-3, an engineered herpes simplex virus in which both copies of the LAT promoter are used to drive expression of the Escherichia coli lac Z gene. Four days post-inoculation (p.i.) abundant beta-galactosidase (beta-gal) protein and transcripts were present within ganglionic neurons as assayed by enzyme histochemistry and in situ hybridization. In contrast, by Day 21 (at which time a latent infection had been established) no beta-gal transcripts were present in infected ganglia, even when assayed by the polymerase chain reaction (PCR). These findings indicate a significant drop in LAT promoter activity between Day 4 and Day 21 p.i. To provide confirmatory evidence for this conclusion we infected mice with a second viral construct, KOS/67-7, in which the LAT promoter was used to drive expression of the nerve growth factor (NGF) gene. Four days p.i., abundant NGF antigen and transcripts were present in infected ganglionic neurons, but no evidence of transcription of the cloned NGF gene could be found in latently infected ganglia. Our findings suggest that LAT promoter activity is severely restricted during the latent phase of ganglionic infection.

An HSV-1 mutant lacking the LAT TATA element reactivates normally in explant cocultivation.

In order to examine if mutations within the LAT promoter region of HSV-1 are sufficient to change the reactivation phenotype, a mutant (KOS/29) containing a deletion of the LAT TATA element was used to establish latent infections in mouse ganglia by corneal inoculation. During the acute phase of infection, KOS/29 replicated as efficiently as its wild-type parent. As previously noted, latent KOS/29 infections were totally devoid of LAT gene transcripts (Dobson A. T., Sederati F., Devi-Rao G., Flanagan J., Farrell M. J., Stevens J. G., Wagner E. K., and Feldman L. T., J. Virol. 63, 3844-3851 (1989))). However, unlike other null mutants, KOS/29 reactivated from explanted ganglia with a kinetics similar to that of the LAT competent parent. These data show that the deletion created in KOS/29, removing the LAT TATA promoter element and small upstream and downstream flanking sequences, is not enough to alter the reactivation phenotype and that efficient reactivation can occur in the absence of any detectable LAT expression during latency.

The herpes simplex virus type 1 reactivation function lies outside the latency-associated transcript open reading frame ORF-2.

The latency-associated transcription unit has been shown to be important for in vivo reactivation of herpes simplex virus from the latent state. A recombinant virus was constructed to alter the largest open reading frame in this region. This virus had a wild-type reactivation phenotype, suggesting that herpes simplex virus does not require a protein function from this reading frame for efficient reactivation from latency.

Pathways of viral gene expression during acute neuronal infection with HSV-1.

Pathways of viral gene expression were investigated during the acute phase of sensory ganglionic infection with HSV-1. To facilitate these studies we constructed KOS/62-3, an HSV-1 vector in which the Escherichia coli lac-Z gene was inserted behind both copies of the promoter for the viral latency-associated transcripts. Following footpad inoculation of mice with the virus, acutely infected dorsal root ganglion (DRG) neurons were assayed by dual immunofluorescence for the presence of beta-galactosidase and HSV viral antigens. Most infected neurons stained for either beta-galactosidase or viral antigens. Less than 0.2% of neurons staining for viral antigens also expressed beta-galactosidase, and less than 10% of neurons expressing beta-galactosidase also stained for viral antigen. As a consequence of these findings, we propose that there are essentially two populations of HSV-infected neurons during the acute phase of ganglionic infection. In one population of neurons there is abundant viral protein synthesis but minimal transcription of latency-associated transcripts, whereas in a second population of neurons viral gene expression is severely restricted except for the synthesis of latency-associated transcripts. Since DRG neurons are a heterogeneous population of cells, we further sought to determine whether either pathway of gene expression was more likely to occur in a particular neuronal phenotype. To accomplish this, antibodies were used to characterize the DRG neuronal phenotypes acutely infected with the virus. The results indicated that the pathway of neuronal infection characterized by transcription of abundant latency-associated transcripts and minimal viral protein synthesis was much more likely to occur in DRG neurons expressing the cellular antigen SSEA-3. These data indicate that the neuron plays a major role in regulating the outcome of infection with HSV. Finally, we sought to determine whether DNA replication occurs in the course of establishment of a latent infection. We found that the DNA content of neurons latently infected with KOS(M) strain HSV was not affected by treatment with nucleotide analogues during the acute phase of ganglionic infection, suggesting that viral DNA replication does not occur during the establishment of latent infection.

Herpes simplex virus latency-associated transcript is a stable intron.

The latency-associated transcript (LAT) is the major viral transcript detected by in situ hybridization of mouse and human sensory ganglia latently infected with herpes simplex virus type 1. The last 750 bases of LAT are complementary to infected-cell polypeptide 0, a herpes simplex virus type 1 immediate-early gene that encodes a transactivating protein that may facilitate re-activation of the virus from the latent state. Several laboratories have shown that LAT accumulates in the nucleus and is not polyadenylylated. Recently, we showed that the promoter for LAT lies 688 bases upstream from its 5' end. We report here that LAT is actually a uniquely stable intron. Furthermore, LAT effectively inhibits transactivation of gene expression by infected-cell polypeptide 0 in transient transfection assays.

A latent, nonpathogenic HSV-1-derived vector stably expresses beta-galactosidase in mouse neurons.

A genetically engineered herpes simplex virus variant was constructed for use as a stable gene vector for neurons. To inhibit replication, the agent possessed a deletion in the immediate early gene ICP4, and to minimize reactivation from the latent state, the gene encoding the latency-associated transcript was deleted. The E. coli beta-galactosidase gene under the control of the Maloney murine leukemia virus long terminal repeat promoter was inserted into the ICP4 region. When introduced into the peripheral nervous system, this virus established latent infections and stably expressed beta-galactosidase in primary sensory neurons. Expression of beta-galactosidase over a more limited time period was observed when the latent infection was established in motor neurons of the hypoglossal nucleus. Agents of this general design have considerable potential for use as gene vectors for studies of neuronal function and correction of genetic defects affecting neurons.

Identification of the latency-associated transcript promoter by expression of rabbit beta-globin mRNA in mouse sensory nerve ganglia latently infected with a recombinant herpes simplex virus.

The herpes simplex virus type 1 latency-associated transcript (LAT) is expressed as a major species in latently infected mouse neurons. Previous sequence analysis revealed no obvious promoter elements near the 5' end of the LAT, but a TATA box and other potential promoter elements were found 700 base pairs upstream. A recombinant virus in which the rabbit beta-globin gene was inserted immediately downstream of the TATA box expressed globin mRNA and did not express the LAT. A second recombinant virus, in which this TATA box was removed, was negative for LAT expression in a latent infection. The location of the LAT promoter suggested that RNA upstream of the LAT was synthesized and degraded during latent-phase transcription. Low levels of this RNA were observed by in situ hybridization. In other experiments, RNA from a productive infection was used to detect a transcript extending from the LAT promoter to a polyadenylation signal approximately 8.5 kilobase downstream. These data suggest that the LAT may be processed from a larger transcription unit which begins distal to the TATA box 700 base pairs upstream of the LAT and extends to a polyadenylation signal almost 5 kilobases downstream of the 3' end of the LAT.