ICP0, ICP4, or VP16 expressed from adenovirus vectors induces reactivation of latent herpes simplex virus type 1 in primary cultures of latently infected trigeminal ganglion cells.

In a previous study, we demonstrated that infected-cell polypeptide 0 (ICP0) is necessary for the efficient reactivation of herpes simplex virus type 1 (HSV-1) in primary cultures of latently infected trigeminal ganglion (TG) cells (W. P. Halford and P. A. Schaffer, J. Virol. 75:3240-3249, 2001). The present study was undertaken to determine whether ICP0 is sufficient to trigger HSV-1 reactivation in latently infected TG cells. To test this hypothesis, replication-defective adenovirus vectors that express wild-type and mutant forms of ICP0 under the control of a tetracycline response element (TRE) promoter were constructed. Similar adenovirus vectors encoding wild-type ICP4, wild-type and mutant forms of the HSV-1 origin-binding protein (OBP), and wild-type and mutant forms of VP16 were also constructed. The TRE promoter was induced by coinfection of Vero cells with the test vector and an adenovirus vector that expresses the reverse tetracycline-regulated transactivator in the presence of doxycycline. Northern blot analysis demonstrated that transcription of the OBP gene in the adenovirus expression vector increased as a function of doxycycline concentration over a range of 0.1 to 10 microM. Likewise, Western blot analysis demonstrated that addition of 3 microM doxycycline to adenovirus vector-infected Vero cells resulted in a 100-fold increase in OBP expression. Wild-type forms of ICP0, ICP4, OBP, and VP16 expressed from adenovirus vectors were functional based on their ability to complement plaque formation in Vero cells by replication-defective HSV-1 strains with mutations in these genes. Adenovirus vectors that express wild-type forms of ICP0, ICP4, or VP16 induced reactivation of HSV-1 in 86% +/- 5%, 86% +/- 5%, and 97% +/- 5% of TG cell cultures, respectively (means +/- standard deviations). In contrast, vectors that express wild-type OBP or mutant forms of ICP0, OBP, or VP16 induced reactivation in 5% +/- 5%, 8% +/- 0%, 0% +/- 0%, and 13% +/- 6% of TG cell cultures, respectively. In control infections, an adenovirus vector expressed green fluorescent protein efficiently in TG neurons but did not induce HSV-1 reactivation. Therefore, expression of ICP0, ICP4, or VP16 is sufficient to induce HSV-1 reactivation in latently infected TG cell cultures. We conclude that this system provides a powerful tool for determining which cellular and viral proteins are sufficient to induce HSV-1 reactivation from neuronal latency.

ICP0 is required for efficient reactivation of herpes simplex virus type 1 from neuronal latency.

Relative to wild-type herpes simplex virus type 1 (HSV-1), ICP0-null mutant viruses reactivate inefficiently from explanted, latently infected mouse trigeminal ganglia (TG), indicating that ICP0 is not essential for reactivation but plays a central role in enhancing the efficiency of reactivation. The validity of these findings has been questioned, however, because the replication of ICP0-null mutants is impaired in animal models during the establishment of latency, such that fewer mutant genomes than wild-type genomes are present in latently infected mouse TG. Therefore, the reduced number of mutant viral genomes available to reactivate, rather than mutations in the ICP0 gene per se, may be responsible for the reduced reactivation efficiency of ICP0-null mutants. We have recently demonstrated that optimization of the size of the ICP0 mutant virus inoculum and transient immunosuppression of mutant-infected mice with cyclophosphamide can be used to establish wild-type levels of ICP0-null mutant genomes in latently infected TG (W. P. Halford and P. A. Schaffer, J. Virol. 74:5957-5967, 2000). Using this procedure to equalize mutant and wild-type genome numbers, the goal of the present study was to determine if, relative to wild-type virus, the absence of ICP0 function in two ICP0-null mutants, n212 and 7134, affects reactivation efficiency from (i) explants of latently infected TG and (ii) primary cultures of latently infected TG cells. Although equivalent numbers of viral genomes were present in TG of mice latently infected with either wild-type or mutant viruses, reactivation of n212 and 7134 from heat-stressed TG explants was inefficient (31 and 37% reactivation, respectively) relative to reactivation of wild-type virus (KOS) (95%). Similarly, n212 and 7134 reactivated inefficiently from primary cultures of dissociated TG cells plated directly after removal from the mouse (7 and 4% reactivation, respectively), relative to KOS (60% reactivation). The efficiency and kinetics of reactivation of KOS, n212, and 7134 from cultured TG cells (treated with acyclovir to facilitate the establishment of latency) in response to heat stress or superinfection with a nonreplicating HSV-1 ICP4(-) mutant, n12, were compared. Whereas heat stress induced reactivation of KOS from 69% of latently infected TG cell cultures, reactivation of n212 and 7134 was detected in only 1 and 7% of cultures, respectively. In contrast, superinfection with the ICP4(-) virus, which expresses high levels of ICP0, resulted in the production of infectious virus in nearly 100% of cultures latently infected with KOS, n212, or 7134 within 72 h. Thus, although latent mutant viral genome loads were equivalent to that of wild-type virus, in the absence of ICP0, n212 and 7134 reactivated inefficiently from latently infected TG cells during culture establishment and following heat stress. Collectively, these findings demonstrate that ICP0 is required to induce efficient reactivation of HSV-1 from neuronal latency.

Optimized viral dose and transient immunosuppression enable herpes simplex virus ICP0-null mutants To establish wild-type levels of latency in vivo.

The reduced efficiency with which herpes simplex virus type 1 (HSV-1) mutants establish latent infections in vivo has been a fundamental obstacle in efforts to determine the roles of individual viral genes in HSV-1 reactivation. For example, in the absence of the "nonessential" viral immediate-early protein, ICP0, HSV-1 is severely impaired in its ability to (i) replicate at the site of inoculation and (ii) establish latency in neurons of the peripheral nervous system. The mouse ocular model of HSV latency was used in the present study to determine if the conditions of infection can be manipulated such that replication-impaired, ICP0-null mutants establish wild-type levels of latency, as measured by viral genome loads in latently infected trigeminal ganglia (TG). To this end, the effects of inoculum size and transient immunosuppression on the levels of acute replication in mouse eyes and of viral DNA in latently infected TG were examined. Following inoculation of mice with 2 x 10(3), 2 x 10(4), 2 x 10(5), or 2 x 10(6) PFU/eye, wild-type virus replicated in mouse eyes and established latency in TG with similar efficiencies at all four doses. In contrast, increasing the inoculum size of the ICP0-null mutants n212 and 7134 from 2 x 10(5) to 2 x 10(6) PFU/eye significantly decreased the levels of infectious virus detected in the tear films of mice from days 4 to 9 postinfection. In an attempt to establish the biological basis for this finding, the effect of viral dose on the induction of the host proinflammatory response was examined. Quantitative reverse transcription-PCR demonstrated that increasing the inoculum of 7134 from 2 x 10(4) to 2 x 10(6) PFU/eye significantly increased the expression of proinflammatory (interleukin 6), cell adhesion (intercellular adhesion molecule 1), and phagocyte-associated (CD11b) genes in mouse eyes 24 h postinfection. Furthermore, transient immunosuppression of mice with cyclophosphamide, but not cyclosporin A, significantly enhanced both the levels of acute n212 and 7134 replication in the eye and the levels of mutant viral genomes present in latently infected TG in a dose-dependent manner. Thus, the results of this study demonstrate that acute replication in the eye and the number of ICP0-null mutant genomes in latently infected TG can be increased to wild-type levels for both n212 and 7134 by (i) optimization of inoculum size and (ii) transient immunosuppression with cyclophosphamide.

The inherent quantitative capacity of the reverse transcription-polymerase chain reaction.

The quantitative capacity of the reverse transcription-polymerase chain reaction (RT-PCR) is generally underestimated. In this study, PCR and RT-PCR products were amplified from serially diluted DNA and RNA templates, respectively, using a 35-cycle PCR. In the approximate 30- to 100-fold range of template input above the lower limit of detection, herpes simplex virus ICP27 RT-PCR product yield was dependent on the logarithm of template mRNA input (r2 = 0.99). Likewise, regression analysis indicated that yields of interleukin-12 p40, herpes simplex virus DNA polymerase, and interferon-gamma PCR products were dependent on the logarithm of template DNA input over 40- (r2 = 0.98), 60- (r2 = 0.96), and 100-fold (r2 = 0.99) ranges, respectively. This quantitative relationship appears to derive from the competition for reactants between specific PCR products and nonspecific primer-dimers that occurs at limiting concentrations of template. Although primer-dimers are not generally considered a common feature of PCR, 30 of 32 primer pairs tested in this study produced primer-dimer amplification in the absence of template. Because the coefficient of variation in replicate PCRs was typically 10-20% in the linear range, the precision of PCR was sufficient to measure 4-fold differences in template concentration. Thus, with statistically adequate sample numbers, an appropriate standard curve, and the inherent quantitative capacity of the method, differences in the abundance of a mRNA species are measurable by 35-cycle RT-PCR.

The persistent elevated cytokine mRNA levels in trigeminal ganglia of mice latently infected with HSV-1 are not due to the presence of latency associated transcript (LAT) RNAs.

Trigeminal ganglia (TG) from mice latently infected with wild type HSV-1 contain detectable levels of cytokine transcripts that are not present in TG from uninfected mice. This suggests that during HSV-1 neuronal latency, the immune system is stimulated by the production of one or more viral proteins. Since the LAT (latency associated transcript) gene is essential for wild type levels of spontaneous reactivation and is the only highly active viral gene during latency, the stimulation of cytokines may indicate the presence of a LAT encoded latency protein. We therefore compared the cytokine transcript profiles in the TG of mice latently infected with wild type and LAT negative viruses. Mice were latently infected with either: (1) the LAT null mutant dLAT2903; (2) its marker rescued virus dLAT2903R; or (3) the parental wild type HSV-1 strain McKrae. As expected, reactivation following explant cultivation of TG from latently infected mice was significantly decreased with dLAT2903 (P < 0.05)(40 +/- 8%, n = 24) compared with dLAT2903R (85 +/- 7.6%, n = 36) or the parental virus (70 +/- 10.0%, n = 36). The relative levels of various cytokines was determined by RT-PCR analysis of TG extracts. None of the cytokine transcripts detected in mice latently infected with the wild type or marker rescued viruses were missing or decreased in mice latently infected with the LAT null mutant 30 or 60 days post infection. There were also no differences in the HSV-1 antibody titers induced by the LAT negative virus compared to the LAT positive viruses. Thus, although LAT facilitated reactivation of HSV-1 from explanted mouse TG, expression of LAT during latency did not appear to be involved in persistent cytokine expression in TG. This suggests that during latency, HSV-1 does not produce a highly antigenic abundant LAT encoded protein.

Cytokine and chemokine production in HSV-1 latently infected trigeminal ganglion cell cultures: effects of hyperthermic stress.

The establishment of a primary trigeminal ganglion (TG) cell culture latently infected with herpes simplex virus type 1 (HSV-1) has been useful in studying stress-induced reactivation of the latent virus. However, the immune profile of this culture system prior to and after stress has never been established. In the present manuscript, cytokine and chemokine production were measured in primary cultures of TG cells obtained from uninfected and HSV-1 latently infected mice. Supernates from TG cell cultures contained detectable interleukin (IL)-6 but not IL-1beta, IL-2, IL-10, interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha as determined by ELISA. The basal level of IL-6 in uninfected TG cell cultures was 20.5 +/- 2.3 ng/ml, whereas latently infected TG cells produced significantly less IL-6 (12.1 +/- 1.9 ng/ml). Supernates from TG cell cultures also contained detectable levels of C-10, MCP-1 and eotaxin but little to no MIP-1alpha, MIP-1beta, or MIP-2. While there were no differences in the basal level of MCP-1 and eotaxin in TG cell cultures from HSV-1-infected and uninfected mice, C10 levels were significantly higher in TG cultures originating from infected mice compared to uninfected ones (5.86 +/- 0.61 ng/ml compared to 1.18 +/- 0.16 ng/ml). Hyperthermic stress (43 degrees C, 180 min), which induces reactivation of latent HSV-1 from TG cell cultures, significantly reduced IL-6 and C-10 levels from both uninfected and latently infected TG cell cultures. However, there was no correlation between cytokine/chemokine levels and HSV-1 reactivation. Immunofluorescent studies showed TG cell cultures contained 10% MAC-3+ staining cells (macrophage specific) but no dendritic cells. By comparison, cells from freshly isolated TG contained 6% positive dendritic cells but < 1% MAC-3 + cells. Both in vivo and in vitro TG consisted of a low percentage of CD3+ and CD8+ cells. Hyperthermic stress (43 degrees C for 3 h) eliminated the lymphocyte population as determined by RT-PCR. Whereas no spontaneous reactivation has been reported in mice, spontaneous reactivation occurred in 4.5% (10/220) of TG cell cultures surveyed over a 20 day period. Collectively, the dichotomy between HSV-1 replication and reactivation comparing the in vitro and in vivo HSV-1 latency models may reside, in part, to the differences in the levels of cytokines, chemokines and immune cell populations within the microenvironment of the in vitro and in vivo TG.

Role of the hypothalamic pituitary adrenal axis and IL-6 in stress-induced reactivation of latent herpes simplex virus type 1.

Hyperthermic stress induces reactivation of herpes simplex virus type 1 (HSV-1) in latently infected mice and also stimulates corticosterone release from the adrenals via activation of the hypothalamic pituitary adrenal axis. In the present study, we tested the hypothesis that stress-induced elevation of corticosterone potentiates HSV-1 reactivation in latently infected mice. Because of the putative role of IL-6 in facilitating HSV-1 reactivation in mice, the effect of hyperthermic stress and cyanoketone treatment on IL-6 expression in the trigeminal ganglion was also measured. Preadministration of cyanoketone, a glucocorticoid synthesis inhibitor, blocked the stress-induced elevation of corticosterone in a dose-dependent manner. Furthermore, inhibition of corticosterone synthesis was correlated with reduced levels of HSV-1 reactivation in latently infected mice. Hyperthermic stress elicited a transient rise in IL-6 mRNA levels in the trigeminal ganglion, but not other cytokine transcripts investigated. In addition, there was a significant reduction in MAC-3+, CD8+, and DX5+ (NK cell marker) cells in the trigeminal ganglion of latent HSV-1-infected mice 24 h after stress. Cyanoketone blocked the stress-induced rise in IL-6 mRNA and protein expression in the trigeminal ganglion latently infected with HSV-1. Collectively, the results indicate that the activation of the hypothalamic pituitary adrenal axis plays an important role in stimulating IL-6 expression and HSV-1 reactivation in the trigeminal ganglion following hyperthermic stress of mice.

Immunization with HSV-1 antigen rapidly protects against HSV-1-induced encephalitis and is IFN-gamma independent.

Herpes simplex virus type 1 (HSV-1) infection of mice frequently culminates in fatal encephalitis. Intraperitoneal administration of heat-inactivated HSV-1 0-5 days before infection (active immunization) protected mice from encephalitis. In addition, active immunization 2-5 days before ocular infection with HSV-1 reduced the frequency of establishment of latent HSV-1 infection in the trigeminal ganglion (TG). However, intraperitoneal administration of heat-inactivated HSV-1 did not induce interferon (IFN) production in the peritoneum or serum, as determined by bioassay and ELISA. Intraperitoneal administration of heat-attenuated HSV-1 elicited IFN-gamma but not type I IFN production in the peritoneum. The production of IFN-gamma correlated with the infiltration of CD4 and CD8 cells in the peritoneum as determined by RT-PCR. In addition, there was a significant increase in interleukin (IL)-12 p40, IL-12p35, IL-6, IL-10, and IFN-gamma mRNA in peritoneal cells, as determined by RT-PCR following immunization with heat-attenuated HSV-1, which was not observed using heat-inactivated HSV-1. The results suggest that resistance to HSV-1 is induced rapidly following immunization with viral antigen but that protection against encephalitis is independent of the cytokines that are generated in the peritoneum.

Pathogenesis and Molecular Biology of HSV Latency and Ocular Reactivation in the Rabbit.

Research on herpesvirus infections has commanded the attention of a diverse group of scientists for over a century. Until the advent of the human immunodeficiency virus, the herpes simplex viruses (HSV) were the most intensively studied of all viruses. During the early part of the nineteenth century, long before the infectious agent responsible for cold sores (fever blisters) was identified, studies suggested that damage to the trigeminal nerve could induce peripheral herpetic vesicles (1). Gruter demonstrated that a particle of material from a herpetic blister inoculated into a rabbit eye could cause herpes and that, in this way, the disease could be transmitted in series from one rabbit to another (2). Following Gruter's basic discovery, research conducted in many parts of the world showed a variety of clinical forms of herpes to be similarly transmissible by inoculation (3). At roughly the same time, Goodpasture proposed that "the virus remains in the ganglia in a latent state after the local lesion has healed" and discussed in detail the pathology of herpetic infection in humans and animals (4,5).

Acyclovir blocks cytokine gene expression in trigeminal ganglia latently infected with herpes simplex virus type 1.

We have previously found that interleukin (IL)-2, IL-10, interferon (IFN)-gamma, RANTES, and tumor necrosis factor (TNF)-alpha mRNA transcription remain elevated in the trigeminal ganglia (TG) of herpes simplex virus type 1 (HSV-1) latently infected mice up to 120 days postinoculation (p.i.). To determine if this phenomenon was dependent on HSV-1 DNA replication after the establishment of latency (i.e., reactivation), cytokine gene expression was compared in TG of acyclovir-treated and untreated latently infected mice. Oral acyclovir treatment (begun 16 days p.i.) had no effect on serum levels of total anti-HSV-1 antibodies. However, there was a significant reduction in the titer of antibody specific for glycoprotein D and glycoprotein B but not glycoprotein H/L 120 days PI in the acyclovir-treated compared to vehicle-treated mice. These differences were not significant at earlier time points (i.e., days 34 and 60 p.i.). Consistent with these findings, acyclovir had no effect on cytokine gene expression in latently infected TG 35 and 60 days p.i. However, 120 days p.i., IFN-gamma and TNF-alpha mRNA were approaching baseline levels in TG of acyclovir-treated mice, but remained significantly elevated in untreated controls (i.e., IFN-gamma mRNA levels were sixfold higher in TG of untreated mice). Therefore, viral DNA replication appears to provide an antigenic stimulus for persistent cytokine gene expression in latently infected TG.

Innate and acquired immunity to herpes simplex virus type 1.

Immunization with heat-inactivated herpes simplex virus type 1 (HSV-1) 2-5 days before ocular infection reduced the frequency of establishment of latent HSV-1 infection in the trigeminal ganglion (TG); this induction of resistance coincided with reduced expression of IFN-gamma mRNA in the TG. Immunization with unrelated antigens was not protective. In part, this resistance to nervous system invasion correlated with the appearance of serum antibody to HSV-1. Immunization reduced viral replication in the eye and trigeminal ganglion, and prevented HSV-1 spread to the cerebellum. IFN-gamma was detected in immunized mice 4 days postocular infection as determined by plaque reduction using neutralizing Ab to IFN-alpha/beta and IFN-gamma. Injection of antibody (Ab) to IFN-alpha/beta and IFN-gamma administered at the time of immunization did not affect survival. Anti-IFN-gamma-treated mice had significantly reduced levels of IFN in their serum. Treatment with anti-IFN-alpha/beta Ab resulted in an elevation in viral replication as determined by the expression of latency associated transcripts in the TG of mice. Likewise, there was a significant increase in the CD8, IL-12 (p40), and TNF-alpha mRNA levels in the TG of the anti-IFN-alpha/beta-treated mice TG explant cultures demonstrated that viral load was significantly increased in the TG of anti-IFN-alpha/beta-treated mice relative to TG of control mice 7 days after infection. The results suggest that exposure to viral antigens 2-5 days before infection is an important determinant of the extent of HSV-1 spread to the nervous system. Moreover, the data suggest that both an antibody response and IFN-alpha/beta play a role in limiting the progress of infection from the peripheral tissues to the central nervous system.

Persistent cytokine expression in trigeminal ganglion latently infected with herpes simplex virus type 1.

Following ocular infection, herpes simplex virus type 1 (HSV-1) establishes latency in trigeminal ganglion (TG) neurons. Using reverse transcription-PCR, cytokine gene expression was analyzed in the TGs of mice infected with HSV-1. IL-2, TNF-alpha, IFN-gamma, IL-10, and RANTES mRNAs were readily detected in TGs taken from mice 7 days postinoculation (PI). Likewise, IL-2, IL-6, IL-10, and IFN-gamma protein were detected by ELISA of TG homogenates. Between 5 and 45 days PI, IL-10, IFN-gamma, TNF-alpha, and RANTES mRNAs were detected in nearly 100% of latently infected TGs (latent infection was confirmed by reverse transcription-PCR detection of HSV-1 latency-associated transcripts). T cell-associated cytokine and chemokine mRNAs (IL-2, IL-10, IFN-gamma, and RANTES) were still detected in the majority of latently infected TG samples taken between 60 and 135 days PI. In contrast, these cytokine mRNA species were rarely detected in uninfected TGs. Measurement of serum Abs to HSV-1 at different times revealed that anti-HSV-1 Ab concentrations approached a plateau in mice by 30 days PI but remained at high levels 67 and 125 days PI. Although there was molecular evidence of an ongoing immune response to HSV-1 in latently infected TG, histologic analysis indicated that very few mononuclear cells remained in the ganglion 60 days PI. Collectively, the results suggest that residual lymphocytes encounter viral Ag during HSV-1 latency with sufficient frequency to remain activated. The paradox of a persistent immune response against a latent infection is discussed.

Mechanisms of herpes simplex virus type 1 reactivation.

Primary cultures of trigeminal ganglion (TG) cells from herpes simplex virus type 1 (HSV-1) latently infected mice were used to study reactivation. Expression of HSV-1 latency-associated transcripts was noted in TG cell cultures. Infectious virus appeared in 75% of culture supernatants within 120 h after heat stress. Likewise, HSV-1 lytic-phase mRNA and proteins were detectable 24 h after heat stress. HSV-1 antigen first appeared in neurons after heat stress, indicating the neurons were the source of reactivation. The effect of heat stress duration on reactivation was determined. Reactivation occurred in 0, 40, or 67% of cultures after a 1-, 2-, or 3-h heat stress, respectively. However, 72-kDa heat shock protein expression was induced regardless of heat stress duration. Thus, reactivation was not a direct result of inducing the heat shock response. The capacities of several drugs to induce reactivation were also evaluated. While neither epinephrine, forskolin, nor a membrane-permeable cyclic AMP analog induced reactivation, dexamethasone did so in a dose-dependent manner. Furthermore, dexamethasone pretreatment enhanced the kinetics of heat stress-induced reactivation from TG cells. Collectively, the results indicate that TG cell cultures mimic important aspects of in vivo latency and reactivation. Therefore, this model may be useful for studying signalling pathways that lead to HSV-1 reactivation.

Quantitation of herpes simplex virus type 1 DNA and latency-associated transcripts in rabbit trigeminal ganglia demonstrates a stable reservoir of viral nucleic acids during latency.

In this investigation we determined the dynamics of herpes simplex virus type 1 (HSV-1) DNA and latency-associated transcripts (LAT) in the latently infected rabbit trigeminal ganglion. Rabbit eyes were infected with either the McKrae strain or the l7Syn+ strain of HSV-1. Rabbits were sacrificed between 5 and 360 days after infection and their trigeminal ganglia were analyzed for the number of HSV DNA genomes and the number of neuronal cells expressing LAT. There was no statistically significant change in the number of HSV genomes or the number of neuronal cells expressing LAT in these ganglia between 20 and 360 days after infection. For both strains, the amount of HSV DNA averaged 16.8 genomes per 100 cells, and 9.2% of the neurons expressed LAT. There were 17 to 34 HSV genomes per LAT-expressing neuronal cell. The number of LAT-expressing neurons did not change over the 360 days. Spontaneous reactivation (HSV-1 recovery in tear film) and recurrence (HSV-1-specific epithelial lesions) occurred during the period of this study; however, these events did not alter the quantity of HSV-1 DNA or the number of LAT-expressing cells. These results suggest that after the latent infection is established, the viral DNA in the ganglia does not replicate to any measurable extent over long periods of latency, since no significant change in the number of HSV genomes occurs. The results also suggest that only a very small number of latently infected neuronal cells are needed to produce infectious HSV-1 during reactivation.

Quantitative analysis of polymerase chain reaction products by dot blot.

Quantitative analysis of polymerase chain reaction (PCR) products is usually accomplished by gel electrophoresis and Southern blotting. We have developed an alternative technique that allows PCR products to be directly quantitated from unfractionated samples. The PCR was used to amplify genomic (endogenous) DNA sequences (actin) and exogenous DNA (herpes simplex virus-1 (HSV-1) ribonucleotide reductase) isolated from the trigeminal ganglia of rabbits to demonstrate the dot blot method of PCR product analysis. Two primer pairs (actin and ribonucleotide reductase) were coamplified, resulting in two different PCR products. Duplicate aliquots of the PCR products were applied to separate nylon membranes and hybridized with 32P-labeled oligonucleotide probes. Each radioactive probe was specific for target (HSV-1 DNA) or control (actin DNA) products. Quantitation using a laser scanning PhosphorImager and ImageQuant software demonstrated that the dot blot method can be used to rapidly analyze a large number of PCR samples.

Functional role and sequence analysis of a lymphocyte orphan opioid receptor.

Pharmacological evidence indicates that lymphocytes express opioid receptors, but this finding has been questioned. By DNA sequencing of reverse transcription-polymerase chain reaction products, we have found that mouse lymphocytes express mRNA encoding an orphan opioid receptor. These mRNA transcripts were detected in the CD4+, CD8+, and CD4- CD8- lymphocyte subpopulations. Northern blot analysis confirmed that splenic lymphocytes express a 1.5-kb orphan opioid receptor mRNA. Fifteen bases encoding Tyr71-Arg75 in the first intracellular loop are alternatively spliced, suggesting that orphan opioid receptor mRNA encodes two receptor subtypes. Treatment of lipopolysaccharide-stimulated lymphocytes with orphan opioid receptor antisense oligonucleotides suppressed polyclonal IgG and IgM production by 50%. Our results provide direct evidence that lymphocytes express an opioid-like receptor gene, and suggest that this receptor plays a functional role in immunocompetence.