INTRODUCTION

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During primary infection of sensory ganglia with herpes simplex virus type 1 (HSV-1), there is an early divergence of the latent and productive pathways of viral gene transcription (17, 28, 41, 43). Since neither viral gene expression nor viral DNA replication appears to be necessary for HSV to establish a latent infection (2, 3, 14, 28, 37, 38, 43), it is likely that host cell factors play important roles in regulating establishment of the latent state. Experiments carried out in vitro suggest that host expression of Oct-1, Oct-2, gamma interferon, and nerve growth factor (NGF) may affect the outcome of infection with HSV by either direct or indirect interaction with the transcriptional regulatory complex responsible for transcription of the HSV immediate-early (IE) genes (7, 23, 31, 44, 45). However, many of these experiments were carried out with nonneuronal cell lines, transformed cell lines, and hybrid cell lines. Thus, the relevance of these studies to our understanding of the regulation of HSV infection in sensory neurons in vivo remains in question.

Primary sensory neurons are a diverse population of cells that can be classified according to cellular morphology, physiological response properties, neuropeptide content, synthesis of cytoplasmic enzymes, and expression of cell surface receptors and glycoconjugates (36). It has long been our working hypothesis that different populations of host sensory neurons may be capable of differentially regulating the outcome of an infection with HSV; in a previous study, we presented preliminary data indicating that a latent pattern of viral gene expression was more likely to occur in some murine dorsal root ganglion (DRG) neuronal phenotypes (SSEA3 immunoreactive) than in others (LD2 immunoreactive) (28). In the present study, we extend these findings by examining patterns of viral gene expression in eight different populations of primary sensory neurons. Distinguishing features of the neuronal populations that we have chosen to study include expression of (i) Trk_A, the high-affinity NGF receptor, which is primarily expressed in neurons that convey nociceptive information (40); (ii) the neuropeptides, calcitonin gene-related peptide (CGRP) and substance P (SP); (iii) b-NOS (brain nitric oxide synthase); (iv) the developmentally regulated globoseries glycoconjugate SSEA3 (9); (v) the neurofilament protein recognized by monoclonal antibody (MAb) RT97, which specifically labels the "large light" neuron population (20); and (vi) the lactoseries glycoconjugates recognized by MAbs KH10 and A5 (10). MAbs KH10 and A5 identify the same populations of primary sensory neurons as MAbs LD2 and 1B2 (10), antisera used in prior studies of HSV-infected sensory ganglia (19, 27, 28).

The results of our study indicate that although all neuronal populations of the trigeminal ganglion (TG) are capable of supporting a productive infection with HSV, some neuronal populations (e.g., KH10 immunoreactive) are much more permissive for this course of infection than others. Of the TG neuronal populations studied, HSV infection of the A5-immunoreactive population was the most likely to result in a latent pattern of viral infection. Our results were reproducible independent of inoculation site (footpad or eye), survival time (7 or 21 days), HSV latency-associated transcript (LAT) expression, and method of detecting latently infected neurons (in situ hybridization [ISH] for LAT versus immunohistochemistry [IH] for -galactosidase [-Gal]). These findings highlight the key role that the host neuron may play in regulating the repertoire of viral gene expression during establishment of HSV latent infection. They also highlight the importance of considering the complex neuronal composition of primary sensory ganglia in interpreting the results of in vivo studies of HSV infection.

	MATERIALS AND METHODS

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Cells and virus stocks. Viral stocks were propagated in rabbit skin cells maintained in minimal essential medium supplemented with 5% fetal calf serum, 250 mg of penicillin per ml, and 250 mg of streptomycin per ml. KOS, KOS/62, and KOS/1 were used in the course of these studies. KOS/62 and KOS/1 have been described in detail elsewhere (11, 26, 28, 35). KOS/62 has a deletion of the 5' ends of both copies of the LAT coding region with an insertion of the Escherichia coli lacZ gene immediately downstream of both copies of the LAT TATA box. As assayed by Northern blot analysis, KOS/62 does not express LATs (42). KOS/1 has a single LAT promoter-lacZ cassette inserted in the glycoprotein C coding region. Viral titers in the range of 10⁸ to 10⁹ PFU/ml were typically obtained for the three viruses.

Animals and inoculations. Four-week-old female Swiss Webster mice were anesthetized by intraperitoneal injection with 1.5% pentobarbital. Following corneal scarification, eyes and the ocular adnexa were inoculated with 15 µl of viral stock (10⁸ PFU/ml). Footpad inoculation was carried out with 30 µl of viral stock (15). Following inoculation, mice were allowed to recover from anesthesia and monitored daily.

Tissue preparation. Three to 21 days postinoculation (p.i.) mice were euthanized by carbon dioxide inhalation and thoracotomy. Cardiac perfusion with 0.1 M phosphate-buffered saline (PBS; pH 7.2) was performed immediately, followed by perfusion with 4% paraformaldehyde in 0.1 M PBS. Dissected sensory ganglia from groups of five mice were combined and immersion fixed in 4% paraformaldehyde at 4°C for either 30 min (for immunofluorescence) or 4 to 6 h (for combined IH-ISH). All fixed tissue was subsequently equilibrated with 10% sucrose, embedded in OCT (Miles Inc., Ind.), and snap frozen in liquid nitrogen. Serial sections (6 µm) were collected as five alternate sets onto Superfrost* Plus slides (Fisher). Cut tissue was stored at 20°C for immunofluorescence and 70°C for combined IH-ISH.

Primary antisera. The following rabbit polyclonal antisera were used in the course of this study: anti-CGRP (Amersham, Arlington Heights, Ill.), anti-SP (Incstar Corp., Stillwater, Minn.), anti-b-NOS (Signal Transduction Laboratory Lexington, Ky.), fluorescein isothiocyanate (FITC)-conjugated rabbit anti-HSV (DAKO, Carpinteria, Calif.), and anti-Trk_A (a gift from Douglas Clary and Louis Reichardt). The Trk_A-specific antiserum has been characterized previously (1). Mouse MAbs anti-SSEA3, KH10, A5, and RT97 were obtained from the Developmental Studies Hybridoma Bank (Iowa City, Iowa). Antisera KH10 and A5 identify the same populations of neurons as antisera LD2 and 1B2, respectively (10). Mouse monoclonal anti--Gal antiserum (Boehringer Mannheim) was labeled with biotin, using a biotinylation kit from Amersham.

IH. For dual immunofluorescence studies, of -Gal and neuronal cell markers tissue sections were incubated with primary antisera for 16 to 48 h at 4°C, washed with 1% normal goat serum in PBS and then incubated with secondary antisera (FITC-conjugated goat anti-rabbit immunoglobulin G [IgG] [Vector Laboratories, Burlingame, Calif.] or FITC-conjugated goat anti-mouse IgM [Vector]) for 40 min at room temperature (RT). Slides were then sequentially incubated with biotinylated mouse anti--Gal antiserum for 1 h, Rhodamine₆₀₀ Avidin D (Vector) diluted 1:1,200 for 40 min, biotinylated goat anti-avidin D (Vector Laboratories) diluted 1:200 for 40 min, and again Rhodamine₆₀₀ Avidin D for 40 min. The slides were then washed and mounted with glass coverslips by using Vectashield (Vector Laboratories). For dual immunofluorescence studies of HSV antigen and neuronal cell markers, a similar protocol was used except that the secondary antiserum for the neuronal markers was trimethyl rhodamine isocyanate (TRITC)-conjugated goat anti-rabbit IgG or TRITC-conjugated goat anti-mouse IgM (TAGO Inc., Burlingame, Calif.) and FITC-conjugated rabbit anti-HSV-1 (DAKO) was used to detect HSV antigen-positive cells. To minimize bias in the selection and evaluation of experimental tissue, sets of tissue representing every fifth tissue section (see "Tissue preparation" above) were stained and analyzed with a given combination of antisera. Results were then expressed as percentage of ganglionic neurons positive for a given combination of antisera; the total number of neurons analyzed for any combination of antisera is given in Tables 1 to 4. Data were expressed in this fashion for ease of comparison to results obtained by combined IH-ISH.

NADPH diaphorase histochemistry. Tissue sections were washed in 0.1 M PBS (pH 8.0), incubated in a solution containing 1 mM NADPH (Sigma), 2 mM nitroblue tetrazolium (Sigma), and 0.3% Triton X-100 in 0.1 M PBS (pH 8.0) for 25 min at 37°C, and washed in PBS again to stop the reaction. Stained sections were assessed by light microscopy.

Combined IH-ISH. (i) IH. IH was performed by immunoperoxidase staining. To maintain the integrity of the RNA signal for further ISH, gloves were used for all procedures, glassware was rinsed with diethylpyrocarbonate (DEPC)-treated water, and all reagents were prepared with DEPC-treated water. Prior to incubation with primary antisera, all tissue sections were treated with 3% H₂O₂ in KPBS (0.02 M potassium phosphate [pH 7.2 to 7.4], 0.15 M NaCl) to quench endogenous peroxidase activity. Tissue sections were incubated with primary antisera (anti-Trk_A, A5, KH10, anti-SSEA3, anti-SP, and anti-CGRP), diluted with KPBS containing bovine serum albumin (20 µg/ml), heparin (5 mg/ml), Triton X-100 (3 µl/ml), and human placental RNase inhibitor (180 U/ml) for 16 to 36 h at 4°C. Sections were subsequently rinsed with KPBS, incubated with the appropriate secondary antisera (biotin-conjugated goat anti-rabbit IgG or biotin-conjugated goat anti-mouse IgM) for 1 h at RT, rinsed again with KPBS, and incubated with ABC reagent (Vector) for 30 min at RT. The sections were again washed with KPBS and reacted with metal-enhanced diaminobenzidine substrate working solution (Pierce, Rockford, Ill.).

FISH prehybridization. Following IH, tissue sections were prepared for fluorescent ISH (FISH) by sequential incubation with 10% neutral buffered formalin (30 min), 0.001% proteinase K (30 min), 0.1 M triethanolamine (3 min), 0.25% acetic anhydride in 0.1 M triethanolamine (3 min), and prehybridization solution for 2 h at 45°C. Prehybridization solution consisted of 50% formamide, 0.3 M NaCl, 20 mM Tris (pH 8.0), 1 mM EDTA, 1× Denhardt's solution, 500 µg of yeast tRNA per ml, 100 µg of salmon sperm DNA per ml, 0.1% sodium dodecyl sulfate, and 100 mM dithiothreitol. Tissue sections that had not been previously reacted for IH served as controls.

FISH. The digoxigenin-labeled riboprobe was dissolved in hybridization solution, which contained 25 ml of formamide, 3 ml of 5 M NaCl, 1 ml of 1 M Tris (pH 8.0), 100 µl of 0.5 M EDTA, 2.5 ml of 20× Denhardt's solution, 25 mg of yeast tRNA, 5 g of dextran sulfate, 0.5 ml of single-stranded DNA (10 mg/ml), 0.5 ml of 10% sodium dodecyl sulfate, and 0.75 g of dithiothreitol, brought to 37.5 ml with DEPC-treated water. The probe concentration in the hybridization solution was approximately 0.5 µg/ml. The hybridization cocktails were heated to 65°C for 10 min, and aliquots of this cocktail were applied to each slide of prehybridized tissue sections, followed by careful placement of a glass coverslip. Hybridization was then carried out in a humidified chamber at 65°C for 16 to 18 h. Following hybridization, the coverslips were removed by immersion in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and the uncovered sections were rinsed in 2× SSC for 10 min followed by RNase A solution at 37°C for 30 min. Sections were then taken through 2× SSC for 10 min, 1× SSC for 10 min, 0.5× SSC for 10 min, 0.1× SSC for 30 min at 60°C, 0.1× SSC for 3 min, 2× SSC for 2 min, rinse buffer (100 mM Tris, 150 mM NaCl) for 2 min, and 1% blocking reagent for nucleic acid hybridization and detection (Boehringer Mannheim) for 30 min. The sections were then incubated with FITC-conjugated antidigoxigenin antiserum (1:10; Boehringer Mannheim) for 30 min and washed in rinse buffer.

	RESULTS

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Patterns of viral gene expression during acute infection of the mouse TG. As assayed by immunofluorescent staining using polyclonal antisera to HSV, productive infection of TG neurons with KOS/62 was present as early as 2 days after ocular inoculation and peaked 3 days p.i. By 7 days p.i., productively infected neurons were no longer found in infected ganglia, but a few nonneuronal cells, mostly leukocytes and glia, continued to stain for HSV antigen. Immunofluorescent staining for -Gal, a marker of LAT promoter activity, was also present in ganglionic neurons as early as 2 days after inoculation with KOS/62. Staining for -Gal persisted in ganglionic neurons until at least 21 days p.i., the latest time point studied; however, the intensity of -Gal staining decreased markedly between 7 and 21 days p.i. This progressive drop in lacZ reporter gene expression is similar to that previously described for DRG neurons latently infected with KOS/62 (26).

KOS/62-infected neuronal populations in the mouse TG. Following ocular inoculation with KOS/62, the TG of infected mice were assayed by dual immunofluorescence to characterize differences in TG neuronal populations that hosted productive and latent patterns of viral gene expression. Based on the results of experiments described above, we chose to study productively infected, HSV antigen-positive neurons at 3 days p.i. and latently infected, -Gal-positive neurons at 7 and 21 days p.i. The results of these experiments are summarized in Table 1, and representative examples of dual-stained tissue are presented in Fig. 1.

View larger version (66K): [in this window] [in a new window]   FIG. 1.   Representative examples of KOS/62-infected TG tissue sections stained by dual immunofluorescence to identify neuronal subpopulations expressing -Gal or HSV antigen. At 3 days p.i., HSV antigen expression (A and B) can be seen colocalizing to KH10- and CGRP-immunoreactive neurons (A' and B', respectively). At 7 days p.i., -Gal expression (C and D) can be seen colocalizing to TrkA- and A5-immunoreactive neurons (C' and D' respectively). Magnification bar represents 100 µm.

KOS/1-infected neuronal populations in the mouse TG. Since it has been suggested that LAT may play a role in the establishment of HSV latent infection in the mouse TG (35), we wondered whether the LAT deletion in KOS/62 was playing a role in the preferential establishment of latent infection in neurons with the SSEA3, Trk_A, and A5 phenotypes. To examine this issue, TG of mice were assayed by dual immunofluorescence 7 days following ocular inoculation with KOS/1, and the results were compared to those observed with KOS/62. TG neurons from mice 21 days after inoculation with KOS/1 were not evaluated due to the consistently poor immunofluorescent staining for -Gal at this time point.

KOS/62-infected neuronal populations in the mouse DRG. To determine whether the observed patterns of viral gene expression in different neuronal populations of the TG were unique to the sensory neurons innervating ocular tissues, we studied productive and latent patterns of infection in different neuronal populations of DRG following footpad inoculation with KOS/62. HSV antigen-positive neurons were assayed 4 days p.i.; the peak of productive neuronal infection in the DRG (28) and -Gal-positive neurons were assayed 8 days p.i., at which time we could no longer detect HSV antigen-positive neurons.

KOS-infected neuronal populations in the mouse TG. In the course of these studies, it became clear that the number of -Gal-positive neurons in ganglia latently infected with KOS/62, whether detected by indirect immunofluorescence or by enzymatic reaction, was far less than the number of LAT-positive neurons in ganglia latently infected by KOS as assayed by ISH. This was not surprising since we have previously reported that -Gal expression is not stable in ganglionic neurons latently infected with KOS/62 (26). We were thus concerned that the observed difference in viral gene expression in phenotypically distinct neuronal populations was a consequence of the use of -Gal-expressing viral vectors instead of wild-type HSV.

View larger version (104K): [in this window] [in a new window]   FIG. 2.   Representative examples of KOS-infected TG tissue sections, 21 days p.i., assayed by FISH for LAT (A to G) and immunoperoxidase for specific neuronal subpopulations (A' to G'). Panels A' to G' are examples of neurons labeled with TrkA, CGRP, b-NOS, SP, KH10, A5, and SSEA3 antisera, respectively. Magnification bar represents 100 µm.

Risk of a latent infection in different neuronal populations of the mouse TG. Although the data presented thus far provide valuable insights into which subpopulations of TG neurons are more, or less, likely to harbor latent infection with HSV, a more complete picture could be established if the relative number of TG neurons that host productive and latent patterns of HSV infection following ocular inoculation were known. To obtain an estimate of this ratio, KOS-infected TG were batched in groups of five, sectioned in a serial fashion, and assayed by either immunoperoxidase staining for HSV antigen or ISH for LAT. Light microscopic analysis of every fifth section revealed 3,484 neuronal profiles with HSV antigen expression at the peak of productive infection (3 days p.i.) and 1,201 neuronal profiles with autoradiographic signal for LAT at 21 days p.i.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we characterized the populations of TG neurons that become infected with HSV following peripheral inoculation. Although all neuronal subpopulations were capable of supporting a productive viral infection, some neuronal phenotypes were more permissive for establishment of a latent infection with LAT expression than others. We found this observation to be independent of (i) survival time, (ii) site of inoculation, (iii) viral LAT expression, and (iv) the use of -Gal constructs in lieu of wild-type virus. These findings confirm and extend our earlier observations (28) on the differential regulation viral gene expression in different populations of primary sensory neurons and further support the hypothesis that the host neuron plays a key role in regulating the repertoire of HSV gene expression.

Evidence in support of host cell influence on HSV gene expression comes from a number of different laboratories and investigative approaches. Perhaps the earliest indication of the role that neurons might play a key role in the regulation of viral gene expression comes from the simple observation that HSV readily establishes a latent infection in neurons but not in other cell types. More recently it has become clear that a productive pattern of viral gene expression is critically dependent on transactivation of the viral IE genes by a complex formed between the HSV virion protein Vmw65, the cellular transcription factor Oct-1, and one or more additional host cell factors (18, 31, 32). Host cell factors may similarly act to inhibit productive infection with HSV. For example, Lillycrop et al. (22, 23) have provided evidence that two neuronal isoforms of Oct-2 (Oct 2.4 and Oct 2.5) may act as repressors of HSV IE transcription by binding to octamer-related TAATGARAT motifs in the viral IE promoters. In this way viral IE gene expression, and thus the outcome of neuronal infection with HSV, might be dictated by the balance of specific host transcription factors in a given cell. Strong evidence in support of this possibility comes from studies of transgenic mice containing the promoter regulatory region of the HSV-1 ICP4 gene coupled to the E. coli lacZ reporter gene (30). These mice exhibited neuronal -Gal expression, indicative of ICP4 promoter activity, in the absence of HSV infection and markedly different levels of -Gal expression in different neuronal populations.

One way in which HSV gene expression in neurons might be regulated is through the action of NGF. In this study, we found that a disproportionately large percentage of latently infected primary sensory neurons expressed Trk_A and were thus capable of responding to NGF. In contrast, latent infection was rarely found in KH10-immunoreactive neurons, a population that express little or no Trk_A (L. Yang et al., unpublished data). Thus, our data support the hypothesis that in vivo, the ability to respond to NGF may play some role in determining which HSV-infected neurons progress to a latent infection during primary infection. Since many of the A5- and SSEA3-immunoreactive neurons of the mouse TG coexpress Trk_A (Yang et al., unpublished data), this may explain the tendency of these neurons to progress to a latent infection. The exact mechanism by which NGF exerts its influence on HSV gene expression is not clear, but the same second messengers and signal transduction pathways that are responsible for mediating the effects of NGF on neuronal gene expression appear to play some role in mediating the effects of NGF on HSV gene expression in infected neurons (12, 21, 39). However, neuronal responsiveness to NGF does not completely explain our results since (i) almost half of the HSV-infected Trk_A-immunoreactive neurons exhibited a productive pattern of viral gene expression and (ii) about 30% of latently infected neurons did not express Trk_A.

Another way in which neurons might be capable of regulating HSV gene expression is through the production of NO. The antimicrobial properties of NO, including its ability to inhibit replication of HSV-1 (4, 16), have been well documented (34). A subset of neurons in the TG and DRG produce NO, identifiable by immunological and histochemical staining for the enzyme NOS-1 (also known as b-NOS or n-NOS), but we found no evidence that acute HSV infection of these neurons preferentially resulted in a latent pattern of infection. This might be explained by the relatively small amount of NO produced by neurons compared to that produced by macrophages. Alternatively, since neuronal NO is largely packaged for secretion as a neurotransmitter, the antiviral action of this molecule is likely to be greatest in tissues immediately adjacent to, or innervated by, NOS-positive neurons. In this regard it was interesting that in a number of tissue sections we observed multiple NOS-positive neurons immediately adjacent to latently infected LAT-positive neurons (data not shown). Determination of any role that neuronal NO might play in the outcome of infection with HSV requires further study.

Reports from two different groups suggest that LAT deletion viruses, including KOS/62, establish a latent infection in the mouse TG less efficiently than viruses with a wild-type LAT coding region but are as efficient as the wild-type virus at establishing latent infection in the murine DRG (8, 35). This has led to the conclusion that the effect of LAT on the establishment of latency is anatomical site specific. In our studies, we found no evidence for a site-specific effect of LAT on the distribution of latently infected neuronal phenotypes in primary sensory ganglia. We obtained similar results following ocular inoculation with either KOS/62, a LAT deletion virus, or viruses with the wild-type LAT coding region, KOS and KOS/1. We also found that results for the DRG following footpad inoculation with KOS/62 were similar to those obtained following ocular inoculation. Based on these observations, we conclude that LAT plays little or no role in differential regulation of viral gene expression in different populations of primary sensory neurons during establishment of latency. Our findings are of particular interest in light of recent data indicating that a LAT-associated function reduces expression of IE viral gene expression during acute infection of neurons in vitro (24) and in vivo (13).

In the course of this study, we have relied on markers of LAT promoter activity to identify neurons latently infected with HSV. This approach seemed reasonable since for years it has been the standard used in studies of HSV latency. However, it could be argued that the higher rate of colocalization of LAT with some neuronal phenotypes reflects differential accumulation of LAT by these cells rather than their relative permissiveness to harbor a latent infection. In latently infected TG, Maggioncalda et al. (25) found two to three times as many neurons positive for HSV DNA by in situ PCR than were positive for LAT by ISH. Thompson and Sawtell (42) have similarly claimed to have found many more HSV DNA-positive neurons in latently infected TG by contextual analysis than by ISH but failed to demonstrate control ISH data for assays using the same viruses and inoculation conditions as used in this study. Although it has been assumed that differential accumulation of LAT among TG neurons is a consequence of differences in latent viral load (33), differential expression and/or degradation of LAT among different neuronal populations of the TG may also contribute to this process. There are two reasons why we do not believe that differential LAT accumulation among latently infected TG neuronal populations accounts for the higher rate of localization of LAT in TrkA-, SSEA3-, and A5-positive neurons than with other TG neuronal populations. First, despite marked cell-to-cell differences in LAT ISH signal strength in tissue sections of TG latently infected with KOS, we did not observe differences in LAT signal strength among the different neuronal phenotypes studied. Second, we obtained virtually identical colocalization results whether we identified latently infected neurons by ISH for LAT or by immunofluorescent labeling for LAT promoter-driven -Gal expression, despite the markedly lower sensitivity of the immunofluorescence assay for identifying latently infected cells. Assuming that differential LAT accumulation among latently infected TG neuronal populations accounts for the higher rate of LAT colocalization with the Trk_A-, SSEA3-, and A5-positive neurons, then we would have expected a higher rate of LAT colocalization with these neuronal phenotypes in the less sensitive assay for identifying latently infected cells.

In summary, in this study we have demonstrated that some neuronal populations of the TG are more permissive for establishment of a latent infection with LAT expression than others. Of the neuronal populations that were studied, infection of A5-immunoreactive neurons was mostly likely to result in a latent pattern of viral gene expression, whereas infection of KH10-immunoreactive neurons was most likely to result in a productive pattern of infection. The differences in the susceptibility of these two neuronal populations to productive infection with HSV were dramatic. Whereas 23 of 24 KH10-immunoreactive neurons that became infected proceeded to a productive infection, only 1 of 4 HSV-infected A5-immunoreactive neurons followed this course of viral gene expression. These findings lend further support for the hypothesis that the host neuron may play a key role in regulating the repertoire of HSV gene expression and that closer attention to differences in the transcriptional regulation among primary sensory neurons may help us understand how transcription of the HSV genome is differentially regulated in productive and latent infection. Our findings also highlight the importance of attention to the complex neuronal makeup of sensory ganglia in order to rationally plan and interpret in vivo experimental studies with HSV.