Latency-associated Transcript Intron Research Articles

Termination of Transcription of LAT Increases the Amounts of ICP0 mRNA but Does Not Alter the Course of HSV-1 Infection in Latently Infected Murine Ganglia.

On entering sensory ganglia, herpes simplex viruses 1 (HSV-1) establishes a latent infection with the synthesis of a latency associated transcript (LAT) or initiates productive infection with expression of a set of immediate early viral proteins. The precise mechanisms how expression of α genes is suppressed during the latency are unknown. One mechanism that has been proposed is illustrated in the case of ICP0, a key immediate early viral regulatory protein. Specifically, the 2kb LAT intron is complementary to the 3' terminal portion of ICP0 mRNA. To test the hypothesis that accumulation of LAT negatively affects the accumulation of ICP0 mRNA, we inserted a DNA fragment encoding two poly(A) sequences into LAT to early terminate LAT transcript without interrupting the complementary sequence of ICP0 transcript (named as SR1603). Comparisons of the parent (SR1601) and mutant (SR1603) HSV-1 viruses showed the following: Neurons harboring latent SR1603 virus accumulated equivalent amounts of viral DNA but higher amounts of ICP0 mRNA and lower amounts of LAT, when compared to neurons harboring the SR1601 virus. One notable difference between the two viruses is that viral RNA accumulation in explanted ganglia harboring SR1603 virus initiated significantly sooner than that in neurons harboring SR1601 virus, suggesting that ICP0 may act as an activator of viral gene expression in permissive cells. Collectively, these data suggest that increased ICP0 mRNA by suppressed LAT did not affect the establishment of latency in latently infected murine ganglia.

Investigating the effect of Herpes Simplex Virus 1 Latency-Associated Non-Coding RNAs on the Human Neuronal Transcriptome

Herpes simplex virus 1 (HSV-1) is a prevalent neurotropic virus that persists for the host’s lifetime due to HSV-1 establishing latency in sensory neurons. During latency, the only abundantly transcribed HSV-1 gene is the latency-associated transcript (LAT), which is processed into the 1.5kb or 2.0kb major LAT intron and several microRNAs. These latency-associated non-coding RNAs (ncRNAs) have been reported to impact the establishment, maintenance and reactivation from latency. However, the molecular mechanisms of these ncRNAs are not fully characterised, especially in the context of human neurons. This study investigated how the latency-associated ncRNAs affect the human neuronal transcriptome. We developed an experimental system to deliver the latency-associated ncRNAs to human neurons, differentiated from SH-SY5Y neuroblastoma cells. The cells were infected with a replication-defective HSV-1 mutant, in1382, that establishes a quiescent infection in which LAT is strongly expressed. Alternatively, we utilised lentiviruses engineered to express the first 3.1kb of LAT, without or with mutations in splice sites that prevents splicing of the major LAT intron, or five HSV-1 microRNAs, shown to be abundant in latently infected human ganglia. Following RNA-Seq of uninfected versus infected or transduced SH-SY5Y cells, we identified 178 host genes that had significant differential expression in response to in1382 quiescent infection and lentivirus delivery of LAT or the latency-associated microRNAs. A subset of these were validated by PCR. This work provides insight into possible roles of the latency-associated ncRNAs in neuronal cell biology and latency that could aid future investigations examining how HSV-1 latency affects human neurons.

Investigating the impact of Herpes simplex virus type 1 latency-associated non-coding RNAs on apoptosis in human neuronal cells

Herpes simplex virus 1 (HSV-1) establishes latency in sensory neurons, allowing it to persist for the lifetime of the host. During latency, the only abundantly transcribed HSV-1 gene is the latency-associated transcript (LAT), which is processed into the 2.0 kb major LAT intron and several microRNAs. These non-coding RNAs (ncRNAs) have been reported to influence latency, possibly through limiting apoptosis of infected cells. As these studies have used animal models or non-neuronal cell culture, we have developed a differentiated human neuroblastoma (SH-SY5Y cells) model to examine their effect in human neuronal cells. We have infected these neuronal cultures with replication-defective HSV-1, which establishes a quiescent infection and strongly expresses the latency ncRNAs. We show that quiescent HSV-1 infection reproducibly protects differentiated SH-SY5Y from etoposide-induced apoptosis. We are also further defining the contribution of different LAT ncRNAs using recombinant lentiviruses to drive expression of the LAT intron or microRNAs. Furthermore, we are also currently exploring the mechanisms of this anti-apoptosis effect, and broader virus-neuron interactions by characterising whether the human neuronal transcriptome is altered by LAT RNA expression. Improving our understanding of the molecular interactions underpinning HSV-1 latency in neurons could help develop novel therapies to target HSV-1 latency.

Mdv1-miR-M7-5p, located in the newly identified first intron of the latency-associated transcript of Marek’s disease virus, targets the immediate-early genes ICP4 and ICP27

Marek's disease virus serotype 1 (MDV-1) is an oncogenic alphaherpesvirus causing fatal T-cell lymphoma in chickens. MDV latency is characterized by the production of latency-associated transcripts (LATs), a family of non-protein-coding spliced RNAs. A cluster of four microRNAs (cluster mdv1-miR-M8-M10) was identified, but not formally mapped, at the predicted LAT 5' end. We established a LAT cDNA library from latently MDV-infected cell line MSB-1. We identified 22 highly variable LATs, which were due to the extensive alternative splicing of a total of 14 introns. RACE PCR confirmed the predicted 3' end and allowed identification of the 5' end, 400 nt upstream of the previously predicted LAT end. The LATs share their transcription start site with the microRNA-expressing transcript described previously, localizing the microRNAs to the first LAT intron and identifying the LATs as the primary transcripts of the microRNAs. We identified MDV immediate-early (IE) genes ICP4 and ICP27 as putative targets of mdv1-miR-M7-5p, the third microRNA of the cluster mdv1-miR-M8-M10. Endogenously expressed mdv1-miR-M7-5p in MSB-1 cells reduced luciferase activity significantly when microRNA-responsive elements from ICP4 or ICP27 were cloned in the 3' UTR of the firefly luciferase gene. ICP27 protein levels were decreased by 70 % when the mdv1-miR-M7-5p precursor was co-expressed with an ICP27 expression plasmid. Additionally, we showed a negative correlation between the decreased expression of mdv1-miR-M7-5p and an increase in ICP27 expression during virus reactivation. Our results suggest that, by targeting two IE genes, MDV microRNAs produced from LAT transcripts may contribute to establish and/or maintain latency.

The Stable 2-Kilobase Latency-Associated Transcript of Herpes Simplex Virus Type 1 Can Alter the Assembly of the 60S Ribosomal Subunit and Is Exported from Nucleus to Cytoplasm by a CRM1-Dependent Pathway

During latency of herpes simplex virus type 1 in the neurons of the peripheral nervous system, the major transcript detected is the 2-kb latency-associated transcript (LAT) intron. During lytic infection, this intron has been shown to associate with ribosomes, suggesting a role in modifying the translational machinery of infected cells. In this study we show, using LAT-transfected cells, that the interaction of the intron with the 60S ribosomal subunit leads to irreversible changes in the sedimentation profile of this subunit in the nucleus. Furthermore, the 2-kb LAT intron is transported to the cytoplasm as part of the 60S ribosomal subunit, using a CRM1-dependent pathway.

CTCF-Dependent Chromatin Boundary Element between the Latency-Associated Transcript and ICP0 Promoters in the Herpes Simplex Virus Type 1 Genome

Cells latently infected with herpes simplex virus (HSV) contain nucleosomal DNA similar to that of host cell chromatin. Recent studies have demonstrated that histones in the latency-associated transcript (LAT) promoter and intron regions contain histone modifications permissive for transcription. However, those histones associated with the lytic-specific ICP0 gene, which lies only 5 kb away, contain modifications typical of silenced chromatin. How this active chromatin is kept separate from the repressed chromatin in the nearby ICP0 region remains crucial to the understanding of the HSV lytic cycle. In this study, we show that the LAT intron region contains an insulator. Specifically, we show that an 800-bp region from the LAT intron can block enhancers in both tissue culture cells and Drosophila melanogaster embryos. Importantly, the 800-bp HSV insulator protects a LAT transgene from positional effects in Drosophila eye tissue. The 800-bp region contains nine copies of 16-bp repeats. In vitro electrophoretic mobility shift assay revealed that CTCF interacts with the CTCCC sequence within the repeats. In vivo chromatin immunoprecipitation assay demonstrated that CTCF interacts with these repeats in latently infected trigeminal ganglion neurons. The deletion of these repeats impaired insulator activity in human K562 cells and Drosophila embryos. Finally, double-spaced RNA knockdown of CTCF disrupts enhancer-blocking activity of the LAT insulator in transfected Drosophila S3 cells. These results strongly support the hypothesis that the 800-bp DNA in the LAT intron region works as a chromatin boundary during latency to separate active chromatin associated with the LAT promoter region from repressed chromatin in the ICP0 gene.

Tissue-Specific Splicing of the Herpes Simplex Virus Type 1 Latency-Associated Transcript (LAT) Intron in LAT Transgenic Mice

To study the regulation of herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) expression and processing in the absence of other cis and trans viral functions, a transgenic mouse containing the region encompassing the LAT promoter (LAP1) and the LAT 5' exon through the 2.0-kb intron was created. LAT expression was detectable by reverse transcriptase PCR (RT-PCR) in a number of tissues, including the dorsal root ganglia (DRG), trigeminal ganglia (TG), brain, skin, liver, and kidney. However, when the accumulation of the 2.0-kb LAT intron was analyzed at the cellular level by in situ hybridization, little or no detectable accumulation was observed in the brain, spinal cord, kidney, or foot, although the 2.0-kb LAT intron was detected at high levels (over 90% of neurons) in the DRG and TG. Northern blot analysis detected the stable 2.0-kb LAT intron only in the sensory ganglia. When relative amounts of the spliced and unspliced LAT within the brain, liver, kidney, spinal cord, TG, and DRG were analyzed by real-time RT-PCR, splicing of the 2.0-kb LAT intron was significantly more efficient in the sensory ganglia than in other tissues. Finally, infection of both transgenic mice and nontransgenic littermates with HSV-1 revealed no differences in lytic replication, establishment of latency, or reactivation, suggesting that expression of the LAT transgene in trans has no significant effect on those functions. Taken together, these data indicate that the regulation of expression and processing of LAT RNA within the mouse is highly cell-type specific and occurs in the absence of other viral cis- and trans-acting factors.

The stable 2-kb LAT intron of herpes simplex stimulates the expression of heat shock proteins and protects cells from stress

During latency of herpes simplex virus type 1 (HSV-1), the major viral transcript that is detected is the latency-associated transcript (LAT) 2-kb intron. This intron is excised from a larger (∼10 kb) primary transcript. Infection of cells with HSV expressing LATs showed that their presence increased accumulation of Hsp70 protein specifically during cold shock. Transfection of cells with a plasmid, expressing the 2-kb LAT intron, showed increased translation compared to cells transfected with plasmids deleted in regions of the intron. Cold shock of cells expressing the intron resulted in an up-regulation of Hsp70 protein, but not Hsp70 mRNA. Furthermore, these cells showed increased cell viability. Using plasmid deletion mutants in the LAT gene sequence, we have shown that the effect requires full-length LAT intron. These findings show that a function of the stable 2-kb LAT intron is to protect cells from cold-induced stress and that this may be mediated via Hsp70.

Stable Levels of Long-Term Transgene Expression Driven by the Latency-Associated Transcript Promoter in a Herpes Simplex Virus Type 1 Vector

Previous gene transfer studies of the herpes simplex virus type 1 (HSV-1) using the latency-associated transcript (LAT) promoter have reported a decrease in transgene expression in the brain over time, but the extent of this decrease has not been measured and it is unknown if expression eventually stabilizes. We examined LAT promoter-mediated transgene expression in the mouse brain for 1 year following intracranial injection with a HSV-1 vector expressing human β-glucuronidase (GUSB). The vector genome copy number remained stable from 2 to 52 weeks. Quantitative reverse transcriptase PCR detected a peak of LAT intron expression at 2 weeks (corresponding to the end of the acute phase of viral infection), followed by stable expression during latency (13–52 weeks). The number of GUSB-positive cells also had a peak in the acute phase and then was stable during latency (13–52 weeks). GUSB enzymatic activity was maintained at 11% of normal at 6 and 12 months, indicating that the LAT promoter is capable of driving stable transgene expression in the brain.

Construction of a Herpes Simplex Virus Type 1 Mutant with Only a Three-Nucleotide Change in the Branchpoint Region of the Latency-Associated Transcript (LAT) and the Stability of Its Two-Kilobase LAT Intron

Previous studies using a eukaryotic expression system indicated that the unusual stability of the latency-associated transcript (LAT) intron was due to its nonconsensus branchpoint sequence (T.-T Wu, Y.-H. Su, T. M. Block, and J. M. Taylor, Virology, 243:140-149, 1998). The present study investigated the role of the branchpoint sequence in the stability of the intron expressed from the herpes simplex virus type 1 (HSV-1) genome and the role of LAT intron stability in the HSV-1 life cycle. A branchpoint mutant called Sy2 and the corresponding rescued viruses, SyRA and SyRB, were constructed. To preserve the coding sequence of the immediate early gene icp0, which overlaps with the branchpoint region of the 2-kb LAT, a 3-nucleotide mutation into the branchpoint region of the 2-kb LAT was introduced, resulting in a branchpoint that is 85% identical to the consensus intron branchpoint sequence of eukaryotic cells. As anticipated, there was a 90- to 96-fold reduction in 2-kb LAT accumulation following productive infection in tissue culture and latent infection in mice with Sy2, as determined by Northern blot analysis. These results clearly suggest that the accumulation of the 2-kb intron in tissue culture and in vivo is, at least in part, due to the nonconsensus branchpoint sequence of the LAT intron. Interestingly, a failure to accumulate LAT was associated with greater progeny production of Sy2 at a low multiplicity of infection (0.01) in tissue culture, but not in mice. However, the ability of mutant Sy2 to reactivate from trigeminal ganglia (TG) derived from latently infected mice was indistinguishable from that of wild-type virus, as assayed in the mouse TG explant reactivation system.

A non-consensus branch point plays an important role in determining the stability of the 2-kb LAT intron during acute and latent infections of herpes simplex virus type-1

Herpes simplex virus type 1 (HSV-1) establishes lifelong latent infection in sensory neurons of the peripheral nervous system. During HSV latency, the latency-associated transcripts (LATs) are the only viral transcripts abundantly expressed. The most abundant form of LATs is a 2-kb stable intron spliced from a primary transcript (mLAT). It has been previously reported that a non-consensus branch point influences the stability of the intron (in vitro) in cells transfected with plasmid constructs (J. Virol. 71 (1997) 5849; J. Virol. 71 (1997) 4199). However, it is unknown whether this branch point is important in determining LAT stability in vivo (in the context of virus). To study the role of this stable intron in HSV-1 infection, we have constructed a mutant virus KOS-CONS in which the branch point has been mutated to consensus branch point nucleotides. The accumulation of the 2-kb intron in KOS-CONS-infected cells was greatly reduced. The LAT intron was not detectable in KOS-CONS-infected mouse trigeminal ganglia (TG) during acute and latent phase infection by Northern blot analysis. Replication of the KOS-CONS and the wild-type KOS viruses on Vero cells was determined to be similar, as was the level of HSV-1 DNA in mouse trigeminal ganglia during acute and latent phase infection. Using the mouse TG explant model, the reactivation pattern of both viruses was shown to be similar. Our data suggest that the unique branch point plays a significant role in determining the stability of LAT intron in vivo, but that the stability of the intron does not appear to affect HSV-1 replication, the establishment of latency, or viral reactivation.

Establishment and maintenance of HSV latent infection is mediated through correct splicing of the LAT primary transcript

To study the effect of the splicing of HSV-1 latency-associated transcript (LAT) on viral latency, we constructed two mutant viruses (FHλ+ and FHλ−) in which the 168-bp HpaI– HpaI fragment within the 2-kb LAT intron was replaced by a 447-bp bacteriophage lambda sequence. The lambda DNA was inserted in opposite orientations in FHλ+ and FHλ−. The mutation in FHλ+ disrupted the splicing of LAT primary transcript and altered both LAT exon and intron, whereas the mutation in FHλ− virus preserved the wild-type splice sites and the wild-type exon. Quantitative PCR analysis revealed that during latency there was a reduction in the number of viral genomes in mouse trigeminal ganglia infected with FHλ+ but not in those infected with FHλ−. The decrease in the latent genome numbers was not due to a defect in viral replication during the acute stage of infection. Furthermore, trigeminal ganglia from mice latently infected with FHλ+ displayed a slower reactivation kinetics compared to those infected with the parental strain. To elucidate the mechanism, we examined the antiapoptotic properties of these LAT constructs. A plasmid containing the pHλ+ construct was found to be less protective for cells against apoptosis than plasmid containing the wild-type or pHλ− constuct. These results suggest that the splicing of LAT primary transcript, and thus the correctly spliced exon product, play an important role in promoting the establishment and/or maintenance of viral latency.

The stable 2.0-kilobase intron of the herpes simplex virus type 1 latency-associated transcript does not function as an antisense repressor of ICP0 in nonneuronal cells.

During latency, herpes simplex virus expresses a unique set of latency-associated transcripts (LATs). As the 2.0-kb LAT intron is complementary to, and overlaps, the 3' end of the ICP0 transcript, it has been suggested that the stable LAT intron might function as an antisense repressor of ICP0 expression. We tested this hypothesis in cell culture by dissociating cis- and trans-acting effects of the 2.0-kb LAT, using a series of complementary strategies. Initially, we constructed 293T cell lines that stably express the nuclear 2.0-kb LAT intron to determine whether LAT accumulation in trans affects ICP0 expression. ICP0 mRNA and protein expression profiles were studied (i) following infections with a viral mutant containing wild-type LAT and ICP0 sequences but having deletions of other immediate-early (IE) genes, thus preventing the progression of viral early gene expression, (ii) at early time points after infection with wild-type virus, before viral LAT expression, and (iii) by plasmid transfections. Northern and Western blot analysis showed that trans expression of the 2.0-kb LAT intron does not affect ICP0 mRNA expression, stability, accumulation, splicing, or translation. In addition, suppression of viral replication by overexpression of the 2.0-kb LAT, which has been detected previously in neuronal cell lines, was not found in these nonneuronal cell lines. However, deletion of the latency-active promoter (LAP) region of the virus resulted in overexpression of IE genes, which occurred soon after infection, before viral LAT expression had commenced. This was not complemented by the expression of LAT in trans, suggesting that the LAP deletion affected transcriptional regulation of the IE genes in cis. We conclude that the function of the highly conserved LAT intron is unlikely to involve a direct-acting anti-ICP0 antisense mechanism but that the LAT region could affect ICP0 mRNA expression from the viral genome.

The 2-kilobase intron of the herpes simplex virus type 1 latency-associated transcript has a half-life of approximately 24 hours in SY5Y and COS-1 cells.

The herpes simplex virus type 1 (HSV-1) 2-kb latency-associated transcript (LAT) is a stable intron, which accumulates in cells both lytically and latently infected with HSV-1. We have used a tetracycline-repressible expression system to determine the half-life of the 2-kb LAT RNA intron in the human neuroblastoma cell line SY5Y. Using Northern hybridization analyses of RNA isolated from transiently transfected SY5Y cells over time after repression of LAT expression, we measured the half-life of the 2-kb LAT to be approximately 24 h. Thus, unlike typical introns that are rapidly degraded in a matter of seconds following excision, the 2-kb LAT intron has a half-life similar to those of some of the more stable cellular mRNAs. Furthermore, a similar half-life was measured for the 2-kb LAT in transiently transfected nonneuronal monkey COS-1 cells, suggesting that the stability of the 2-kb LAT is neither cell type nor species specific. Previously, we found that the determinant responsible for the unusual stability of the 2-kb LAT maps to the 3' terminus of the intron. At this site is a nonconsensus intron branch point located adjacent to a predicted stem-loop structure that is hypothesized to prevent debranching by cellular enzymes. Here we show that mutations which alter the predicted stem-loop structure, such that branching is redirected, either reduce or abolish the stability of the 2-kb LAT intron.

Regions of the herpes simplex virus type 1 latency-associated transcript that protect cells from apoptosis in vitro and protect neuronal cells in vivo.

Recent studies have suggested that the latency-associated transcript (LAT) region of herpes simplex virus type 1 (HSV-1) is effective at blocking virus-induced apoptosis both in vitro and in the trigeminal ganglia of acutely infected rabbits (Inman et al., J. Virol. 75:3636-3646, 2001; Perng et al., Science 287:1500-1503, 2000). By transfecting cells with a construct expressing the Pst-Mlu segment of the LAT, encompassing the LAT exon 1, the stable 2.0-kb intron, and 5' part of exon 2, we confirmed that this region was able to diminish the onset of programmed cell death initiated by anti-Fas and camptothecin treatment. In addition, caspase 8-induced apoptosis was specifically inhibited in cells expressing the Pst-Mlu LAT fragment. To further delineate the minimal region of LAT that is necessary for this antiapoptotic function, LAT mutants were used in our cotransfection assays. In HeLa cells, the plasmids lacking exon sequences were the least effective at blocking apoptosis. However, similar to previous work (Inman et al., op. cit.), our data also indicated that the 5' end of the stable 2.0-kb LAT intron appeared to contribute to the promotion of cell survival. Furthermore, cells productively infected with the 17N/H LAT mutant virus, a virus deleted in the LAT promoter, exon 1, and about half of the intron, exhibited a greater degree of DNA fragmentation than cells infected with wild-type HSV-1. These data support the finding that the exon 1 and 2.0-kb intron region of the LAT transcription unit display an antiapoptotic function both in transfected cells and in the context of the virus infection in vitro. In trigeminal ganglia of mice acutely infected with the wild-type virus, 17, and 17DeltaSty, a virus lacking most of exon 1, apoptosis was not detected in cells that were positive for virus particles. However, dual staining was observed in cells from mice infected with 17N/H virus, indicating that the LAT antiapoptotic function demonstrated in cells transfected by LAT-expressing constructs may also play a role in protecting cells from virus-induced apoptosis during acute viral infection in vivo.

The region of the HSV-1 latency-associated transcript required for epinephrine-induced reactivation in the rabbit does not include the 2.0-kb intron.

Previous studies have localized the region of the latency-associated transcript (LAT) of HSV-1 responsible for epinephrine-induced reactivation in the rabbit eye model to the first 1.5 kb of the primary transcript. This region extends from the 5prime prime or minute exon of the primary LAT transcript through the 5prime prime or minute half of the LAT 2.0-kb intron. To determine whether the 5prime prime or minute end of the LAT intron contributes to the induced reactivation phenotype, three recombinant viruses containing deletions within this portion of the LAT intron were constructed. The three recombinants, containing deletions spanning a combined region of 969 bp at the 5prime prime or minute end of the LAT intron, reactivated with the wild-type frequency of 17syn+. These results indicate that the elements governing induced reactivation reside within the first 699 bp of the primary LAT transcript encoding the 5prime prime or minute LAT exon.

Herpes simplex virus type 1 2-kilobase latency-associated transcript intron associates with ribosomal proteins and splicing factors.

During latency of herpes simplex virus type 1 in sensory neurons, the transcription of viral genes is restricted to the latency-associated transcripts (LATs). The stable 2-kb LAT intron has been characterized previously and has been shown to accumulate to high levels in the nuclei of infected neurons. However, in productively infected tissue culture cells, this unique intron is also found in the cytoplasm. Although deletion mutant analysis has suggested that the region of the gene from which the intron is spliced plays a role in maintenance of latency or in reactivation from latency, no well-defined function has been ascribed specifically to the 2-kb LAT intron. Nevertheless, previous work has shown that it associates with 50S particles in the cytoplasm of acutely infected cells. Our studies tested the ability of the 2-kb LAT to dissociate from cytoplasmic protein complexes under various salt conditions. Results indicated that this association, which had been speculated to be mRNA-like, is actually more similar to the affinity of rRNAs for translational complexes. Furthermore, by immunoprecipitation analysis, we demonstrate that the 2-kb LAT associates with ribosomal as well as with splicing complexes in infected cells. Our results suggest that the 2-kb LAT is processed similarly to mRNAs in the nuclei of infected cells. However, in the cytoplasm, the 2-kb LAT may play a structural role in the ribosomal complex, similar to that of the cellular rRNAs, and therefore affect the functioning of the translational machinery.

Analysis of protein expression from within the region encoding the 2.0-kilobase latency-associated transcript of herpes simplex virus type 1.

During latent infections of sensory neurons, herpes simplex virus type 1 gene expression is restricted to the latency-associated transcripts (LATs). The association of the stable 2.0-kb LAT intron with polysomes has suggested that it might represent a novel mRNA. In this work, we investigated expression of 2.0-kb LAT open reading frames (ORFs) by inserting the gene for green fluorescent protein (GFP) within the 2.0-kb LAT sequence, both within a LAT expression plasmid and in the context of the virus. Upon transient transfection of cells of both neuronal and nonneuronal origin with LAT-GFP expression vectors, low-level GFP fluorescence was distributed over the cell cytoplasm and likely resulted from infrequent initiation at a GFP AUG codon, on either unspliced or alternately spliced LAT RNAs. A second nucleolar GFP expression pattern which resulted from fusion of GFP to a conserved ORF in exon 1 of the LAT gene was also observed. However, the abundant expression of this fusion protein was dependent upon an artificially added translation initiation codon. Expression was much reduced and restricted to a small subset of transfected cells when this initiator codon was removed. Neither the 2.0-kb LAT-GFP intron itself nor transcripts originating from the latency-associated promoter 2 (LAP2) were responsible for GFP expression. Abundant alternate splicing involving the 1.5-kb LAT splice acceptor and including splicing between the 1.5-kb LAT splice donor and acceptor, was observed in the nonneuronal Cos-1 cell line. Contrary to the results of our transfection studies, GFP expression could not be detected from a LAT-GFP virus at any stage of the infection cycle. Our results suggest that the inhibition of LAT ORF expression during viral infection occurred primarily at the level of translation.

Significantly increased expression of beta-glucuronidase in the central nervous system of mucopolysaccharidosis type VII mice from the latency-associated transcript promoter in a nonpathogenic herpes simplex virus type 1 vector.

Herpes simplex virus (HSV) has the ability to establish life-long latent infections in postmitotic neurons and to remain transcriptionally active, continuously expressing latency-associated transcripts (LAT) while producing minimal disease. These properties have made HSV an excellent candidate for neuronal gene transfer. Previously, we have shown that in mucopolysaccharidosis type VII mice (MPS VII, beta-glucuronidase deficiency) the LAT promoter is capable of expressing beta-glucuronidase (GUSB) in the trigeminal ganglion and the brainstem after latency is established. However, the number of neurons expressing GUSB is much lower than the number expressing 2-kb LAT following a wild-type virus infection. In this study, we have evaluated the effect of the position of the coding sequence relative to the LAT promoter on beta-glucuronidase gene expression in the central nervous system (CNS). Non-neurovirulent (ICP-34.5-deleted HSV-1) vectors were used, allowing direct intracranial injection. Significantly more GUSB activity was detected in brains of MPS VII mice inoculated with a recombinant virus (HSV-LAT-GUSB-JS) in which the GUSB cDNA was inserted near the LAT promoter, compared to viruses where it was inserted farther downstream in either the LAT exon 1 or overlapping exon 1 and the 2-kb LAT intron. This vector produced more than 100 times the number of positive cells than the other constructs. During acute infection, the distribution of viral replication differed from the distribution of GUSB enzyme expression. Viral antigen was predominately present in cells around the site of injection in the caudate putamen and in ependymal cells lining the ventricles. In contrast, GUSB expression was present mainly in cells of the thalamus and hypothalamus, which did not exhibit viral antigen, suggesting that GUSB enzyme activity was expressed from latently but not acutely infected neuronal cells. This vector design should be useful for high-level expression of various genes in the CNS.

Selection of a nonconsensus branch point is influenced by an RNA stem-loop structure and is important to confer stability to the herpes simplex virus 2-kilobase latency-associated transcript.

Herpes simplex virus type 1 latent infection in sensory neurons is characterized by the highly restricted transcription of viral genes. The latency-associated transcripts (LAT) family members are the only transcripts that can be identified in large amounts in latently infected cells. The most abundant LAT species is a 2-kb RNA that results from splicing of a rare primary transcript. Analysis of a LAT mutant virus (TB1) in cell culture revealed an aberrant splicing pattern and production of a stable small (0.95-kb) LAT intron. A panel of deletion constructs expressing truncated LAT in transiently transfected cells mapped the region influencing stability to the 3' end of the LAT intron. This region encompasses the branch point and a putative stable stem-loop hairpin structure immediately upstream of the splice acceptor consensus polypyrimidine tract. Mutagenic analysis of the sequence in this region confirmed our hypothesis that the stem-loop structure is important for efficient splicing by influencing the selection of a nonconsensus branch point. Changes in this structure correlate with changes in branch point selection and production of an unstable 2-kb LAT.