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.

Methylenetetrahydrofolate reductase C677T and A1298C variants do not affect ongoing pregnancy rates following IVF.

BACKGROUND: There is concern that IVF could compromise normal imprinting and methylation of DNA. Methylenetetrahydrofolate reductase (MTHFR) regulates the flow of folic acid-derived, one-carbon moieties for methylation and is critical to early embryonic development. Therefore, we hypothesized that common polymorphisms in MTHFR could associate with IVF outcome. METHODS: MTHFR C677T and A1298C polymorphism genotyping was performed on 374 subjects for this study, representing 197 couples undergoing IVF in a university setting from July 2005 to January 2006. Analysis of variance (ANOVA), chi-square and/or multivariate analyses were used to assess whether these polymorphisms are associated with embryo quality or with ongoing pregnancy or spontaneous abortion rates. RESULTS: Allele frequencies for C677T ( p=0.67, q=0.33) and A1298C ( p=0.71, q=0.29) were in Hardy-Weinberg equilibrium. The C677T and A1298C variants, either alone or in combination, did not associate with embryo quality or short-term pregnancy outcome. CONCLUSIONS: The common polymorphisms in MTHFR are not associated with embryo quality, as defined by cell number or fragmentation score, or with short-term pregnancy outcomes. Therefore, in our population in which women receive adequate folic acid, MTHFR genotypes are not informative in explaining IVF failure. Further studies, however, examining birth outcomes and the other enzymes in the folic acid pathway are warranted.

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.

Prevention of herpes simplex virus infection and latency by prophylactic treatment with acyclovir in a weanling mouse model.

OBJECTIVE: Acyclovir is an antiviral agent that inhibits acute herpes simplex virus replication and decreases the frequency of reactivation, but it is not currently used to prevent primary disease or the establishment of latency. The purpose of this study was to reexamine the efficacy of acyclovir in preventing acute and latent herpes simplex virus infection. STUDY DESIGN: Mice were infected by footpad inoculation with 2 viral recombinants that express beta-galactosidase. Half of each group was treated prophylactically with intraperitoneal acyclovir and then given acyclovir in the drinking water. Four days after infection, the dorsal root ganglia were removed, fixed, and stained, and the number of cells expressing beta-galactosidase were counted. RESULTS: Compared with placebo, prophylactic acyclovir completely inhibited acute viral replication as evidenced by the absence of beta-galactosidase activity (P < .001) and significantly decreased the number of neurons harboring latent infection (P = .01). CONCLUSION: Acyclovir prophylaxis prevented acute and reduced latent ganglionic infection with herpes simplex virus in a weanling mouse model.

Herpes simplex virus type 1 that exhibits herpes simplex virus type 2 sensitivity to (E)-5-(2-bromovinyl)-2'-deoxyuridine.

A clinical isolate, designated 145, of herpes simplex virus (HSV) had type 1 characteristics as determined by monoclonal antibody immunofluorescence, heat stability of viral thymidine kinase (TK), BamHI restriction endonuclease pattern, and absence of the HSV-2-specific 38-kD protein. However, instead of being sensitive to E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) like HSV-1, isolate 145 displayed a resistance pattern like HSV-2 to the drug as determined by viral replication and viral DNA synthesis. Because BVDU is activated by viral TK phosphorylation, we cloned the TK-containing DNA region from isolate 145 and compared it by restriction mapping using several endonucleases to similar regions of HSV-1 and HSV-2. In each instance, the patterns for HSV-1 and isolate 145 were identical to each other, but distinct from the patterns for the corresponding region of HSV-2, suggesting that the genome TK region of isolate 145 was HSV-1-like.

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.

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.

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.

Physical characterization of the herpes simplex virus latency-associated transcript in neurons.

RNA transfer (Northern) blot analysis was used to perform the physical characterization of the transcript expressed in murine sensory nerve ganglia latently infected with herpes simplex virus type 1. Most of this latency-associated transcript (LAT) was isolated in the poly(A)- fraction from ganglia. A smaller RNA species was also detected at less than 10% the abundance of the major one. LAT was not detected with probes from DNA outside the limits of the larger species. In situ hybridization data correlated well with Northern blot analysis; however, low levels of hybridization were seen with probes immediately outside the region of viral DNA giving positive Northern blot signals. S1 nuclease and primer extension mapping were used to locate the 5' end of the LAT 510 bases to the left of a KpnI site at 0.783 map units. The 3' end of the major latency-associated species was mapped to just within a 310-base-pair SmaI fragment located 660 to 970 base pairs to the right of the SalI site at 0.790 map units. These data were correlated with an analysis of the sequence of the DNA encoding this transcript and its possible function in the latent phase of infection.

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.