Abstract
PC12 cells are derived from a rat pheochromocytoma (Greene and Tischler, 1976 ). They have high affinity receptors for nerve growth factor (NGF) and, in response to NGF, cease division, extend long neuronal processes that can support action potentials and have been used as models of neuronal cells maintained in tissue culture (Danaher et al., 2000 ; Greene and Tischler, 1976 ; Su et al., 1999 ; Thomselli et al., 1990 ). We have previously demonstrated that after 2 weeks of NGF differentiation, infection of PC12 cells with HSV-1 results in a limited productive infection, which is followed by the establishment of a quiescent state. The quiescent HSV genomes inside the nucleus can become active and release infectious progeny following treatment with reactivation stimuli (Su et al., 1999 ). The NGF-differentiated PC12 culture system provides an opportunity to study HSV-1neuronal cell interactions in a homogeneous population of cells in tissue culture.
Here, we report that compared with uninfected cells, and contrary to expectations, HSV-1-infected neuronal-like PC12 cells in culture remained attached to the culture flask substratum longer than uninfected cells. Following HSV-1 infection, NGF-differentiated PC12 cell detachment was prevented or delayed, and the infected culture adhered to the culture flask beyond 10 weeks of seeding. Studies with an LAT null mutant, a DNA polymerase mutant and UV-inactivated viruses suggested that detachment prevention was dependent on de novo gene expression but it appeared to be LAT- and viral DNA replication-independent. Although the precise mechanism whereby HSV-1 prevents PC12 detachment is not yet known, the observation is striking. Several important facts are clear: LAT gene expression and virus replication are dispensable, yet viral gene expression appears to be necessary.
Cells.PC12 cells [American Type Culture Collection (ATCC), Rockville, MD] were grown in RPMI 1640 supplemented with 10% heat-inactivated horse serum and 5% heat-inactivated fetal bovine serum (PC12 medium). CV-1 cells (ATCC), PolB3 cells [an HSV-1 DNA polymerase-expressing cell line, kindly provided by Dr Charles Hwang, State University of New York (Hwang et al., 1997 )] and CHOHveA cells (stably transfected CHO cells with a β-galactosidase (β-gal) reporter under the control of the ICP4 promoter, a gift from Professor Patricia Spear, NorthWestern University, Chicago, IL) were maintained in Eagle's minimal essential medium plus 5% calf serum.
Viruses and virus stock preparation.
HSV-1 strain 17, the McKrae strain and the LAT null mutant (dLAT2903) and its rescuant, dLAT2903R (Perng et al., 1994 ), were grown in CV-1 cells. To prepare virus stock, CV-1 cells were infected with virus at an m.o.i. of 0·1 and harvested when 95% of the infected culture displayed a cytopathic effect. Infected cells were frozen, thawed, sonicated and then aliquoted. Virus titre was determined by a standard plaque assay on CV-1 monolayers under methylcellulose. DNA polymerase mutant HP66 (Marcy et al., 1990 ), kindly provided by Dr Donald Coen (Harvard Medical school, Boston, MA) was grown and titred in the PolB3 cell line.
Differentiation of PC12 cells.
To differentiate PC12 cells, 1x105 cells were seeded on 25 cm2 culture flasks coated with poly-L-orinithine (Sigma). The following day, cells were incubated in PC12 medium containing 100 ng/ml of 2·5S NGF (Collaborative Biomedical Products) for 1 week. The medium was replaced every 3 days. On day 7, fluorodeoxyuridine (5-Flu) (Sigma) was added to a concentration of 20 µM for 3 days to eliminate undifferentiated PC12 cells. This 3 day treatment has been tested previously and shown to eliminate all cells in cultures of undifferentiated PC12 cells (data not shown). Fresh NGF-supplemented medium was replaced thereafter.
Establishment of long-term quiescent HSV-1 infection.
Differentiated PC12 cultures were infected with HSV-1 strain 17 at an m.o.i. of 20 (2x106 p.f.u./flask). Following a 1 h incubation at 37 °C, cultures were treated with 3 ml sodium citrate buffer, pH 3, for 3060 s to inactivate residual virus, as previously described (Su et al., 1999 ). The buffer was removed and flasks were rinsed once with PC12 medium. After low-pH treatment, cultures were incubated at 37 °C with fresh medium containing NGF.
Terminal deoxynucleotidyltransferase-mediated UTP end-labelling (TUNEL) staining.
Detached NGF-differentiated PC12 cells were sedimented on poly-L-orinithine-coated slides, fixed with 4% formaldehyde, permeabilized with 0·2% Triton X-100 in PBS, and then stained with the Fluorescein-labelled Apoptosis Detection System (Promega), according to manufacturer's specifications. Cells were observed and photographed using fluorescence microscopy.
Preparation and characterization of UV-irradiated viruses.
CV-1 cells were infected with HSV-1 strain 17 at an m.o.i. of 0·1. Forty-eight hours later, infected cells were scraped off into culture medium, centrifuged, frozen, thawed and sonicated, as described above. Cell-free virions were isolated by centrifugation in a 20% sucrose gradient. Virions resuspended in 1x PBS were filtered via a 0·45 µm filter and then aliquoted into four wells. Each well of virions was subjected to UV irradiation (12000 µJ) for 0, 1·5, 2 or 2·5 min, using a UV-Stralinker (Stratagene). After UV-irradiation, each aliquot was tested for: (i) infectivity on a CV-1 cell monolayer by standard plaque assay; (ii) the ability to transactivate the ICP4 promoter activity in CHOHevA cells as below; and (iii) the ability to prevent detachment.
CHOHevA cell cultures were prepared and infected with each aliquot of UV-treated viruses at an m.o.i. of 1 (dilution based on the virus titre obtained for 0 min UV aliquot). Six hours after infection, cultures were fixed and stained for β-gal activity, as previously described (Su et al., 2000 ). The number of positive-stained (blue) cells was counted in an area of approximately 3 mm2 for each infected culture under a microscope. The number of positive-stained cells in the 0 min aliquot was considered to be 100%.
HSV-1 infection prevents detachment of NGF-differentiated PC12 cells from the culture surfaceIn the course of studying NGF-differentiated PC12 cells, we found that HSV-1-infected NGF-differentiated PC12 cells consistently remained attached in culture for longer periods of time than the uninfected controls. Uninfected NGF-differentiated PC12 cells began to detach from the culture flasks 5 weeks after seeding, whereas infected NGF-differentiated cells remained attached to the culture flasks. To quantify this observation, a single end state time point (using four culture flasks per group) was analysed 48 days after seeding. NGF-differentiated PC12 cells were infected at an m.o.i. of 20 with HSV-1 strain 17, or left uninfected. Uninfected and HSV-1-infected cells that remained in the culture flask on day 48 after seeding were trypsinized, collected and counted by trypan blue staining. It was found that an average of 92% of the uninfected, NGF-differentiated PC12 cells were lost from the surface of the culture flask after 7 weeks of seeding. In contrast, only 11% of the HSV-1 strain 17-infected cells were lost from the surface of the culture flask.
Since a single end state time point showed a significant difference in the adherence of uninfected and infected NGF-differentiated PC12 cells, it was of interest to determine the kinetics of the detachment of uninfected PC12 cells detaching from the culture flask and further quantify the results. The kinetics of cells detaching from the flasks was studied by observing the change in number of cells in pre-selected areas over time. NGF-differentiated PC12 cultures were infected with HSV-1 strain 17 at an m.o.i. of 20, or left uninfected. Morphology and cell numbers were followed by serial photomicroscopy, as a function of time, after infection. Briefly, areas of approximately 3 mm2 were marked off on the bottom of flasks prior to seeding, so that photographs of the same area could be taken. Using this method permits morphology and cell counting assessments of the same cells over a period of many weeks (Fig. 1A).
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It was noted that cell migration occurred following NGF treatment and continued for 34 weeks after seeding. Therefore, cell number per field varied by as much as ±20% within the first 3 weeks. However, the cell number was relatively stable 4 weeks after seeding, and remained so until detachment began. To reduce the confounding influence of cell migration on counts of cell number per field, ten separate fields in five culture flasks were counted per experimental group and the average numbers of cells were calculated with a standard deviation. At the end of the experiment, the amount of total DNA in each flask was measured to corroborate the results obtained from the cell counting.
As shown in Fig. 1(B), limited cell division continued after NGF treatment (compare day 10 to day 0), as a small population of PC12 cells did not respond optimally to NGF treatment. This is expected and replicating cells were eliminated by 5-Flu treatment on day 10, as previously described (Su et al., 1999 ). Elimination of mitotic cells by 5-Flu accounts for the drop in cell number from day 10 to day 13. To obtain a similar baseline for both uninfected and infected cultures, cell counts were taken prior to viral infection on day 13 after seeding. The number of cells counted at day 13 was taken as 100%, as day 13 was when infection took place. As expected, there was no appreciable cell death or gross morphological alteration observed in HSV-1-infected cultures after infection (Fig. 1B, days 1319), despite modest HSV replication during the first week following infection (105 p.f.u./25 cm2 flask) (Su et al., 1999 ).
Compared with uninfected cells, HSV-1 infection enabled PC12 cells to remain adherent in culture flasks for a significantly longer period of time than the uninfected counterparts (Fig. 1B, days 4054). Uninfected NGF-differentiated PC12 cells detached from the observation fields between 37 and 54 days after seeding, while no significant loss was seen in the HSV-1-infected cultures (Fig. 1A). In fact, more than 90% of the HSV-1-infected, NGF-differentiated PC12 cells remained attached to the culture flask 102 days after seeding, at which point the experiment was terminated.
These results were further quantified by isolating total DNA remaining in the flasks. The amount of DNA, and therefore the relative number of cells, was more than tenfold higher in the HSV-1-infected NGF-differentiated PC12 cultures, as compared with the uninfected NGF-differentiated PC12 cultures (data not shown). This was similar to the results obtained by direct cell counts (Fig. 1B).
The above results were observed in six independent experiments conducted in the same manner. Thus, it appears that the HSV-1 infection prevented cell detachment from the culture flask surface that would otherwise occur by day 50 following NGF differentiation of PC12 cells.
Viability of detached PC12 cells
Since the number of uninfected NGF-differentiated PC12 cells remaining attached to the culture flask dropped by week 7 after seeding to less than 10% of the number seeded, it was of interest to determine whether detachment was caused by cell death. Therefore, on day 42 after seeding, uninfected cells that had spontaneously detached from the culture flask and HSV-1-infected cells that had been mechanically detached from the flask were collected and stained with trypan blue. Surprisingly, only 5% of the uninfected cells stained positive with the trypan blue (Table 1), which indicated that the majority of the cells were physically viable. This was similar to the results obtained with mechanically detached HSV-1-infected cells, where 5% of cells also stained positive with trypan blue. As a positive control, undifferentiated PC12 cells, permeabilized with 0·2% Triton X-100 and incubated with trypan blue, were uniformly positively stained.
Table 1. Viability of detached NGF-differentiated PC12 cells
To further explore the physiology of the cell by examining the number of apoptotic cells in the culture, a TUNEL assay was performed on detached, uninfected, NGF-differentiated PC12 cells. Detached, uninfected, NGF-differentiated PC12 cells were tested for apoptosis immediately after detachment by a fluorescein-labelled apoptosis detection system. As with trypan blue staining, less than 5% of the uninfected NGF-differentiated PC12 cells examined scored positive for TUNEL staining (Table 1). This percentage of TUNEL staining was also seen in the infected NGF-differentiated PC12 cells, which had been detached by mechanical force. Since the majority of detached NGF-differentiated PC12 cells were not stained with trypan blue and scored negative in the TUNEL assay, there was no evidence that the uninfected NGF-differentiated PC12 cells were detaching as a consequence of cell death.
HSV-1 LAT null mutant infection of NGF-differentiated PC12 cells
The LATs are the only viral gene family consistently detected during HSV-1 neuronal latency (Rock et al., 1987 ; Stevens et al., 1987 ) and NGF-differentiated PC12 cell long-term quiescent infection (Su et al., 1999 ). LAT gene expression has been suggested to have various effects on host cellular function and viral gene regulation. It was of interest to determine whether LAT expression was necessary for HSV-1 to prevent detachment of infected NGF-differentiated PC12 cells. dLAT2903, an HSV-1 LAT null mutant that does not produce LAT RNAs (Perng et al., 1994 ), was used to determine the role of LAT expression in infected PC12 cells. NGF-differentiated PC12 cells were infected with HSV-1 strain 17, wild-type McKrae strain (parent strain of dLAT2903), dLAT2903, dLAT2903R (rescued dLAT2903), or left uninfected. Morphology and cell number were observed as a function of time by serial photography of ten pre-selected fields in five culture flasks each (as described in Fig. 1A).
As expected, after 7 weeks in culture, the majority of uninfected NGF-differentiated PC12 cells had detached from the culture flask, whereas HSV-1 strain 17-infected NGF-differentiated PC12 cells remained attached to the culture flask over the time observed (Fig. 3). Interestingly, McKrae-, dLAT2903- and dLAT2903R-infected NGF-differentiated PC12 cells also did not detach from the culture flask (Fig. 2). It was noted that there appeared to be a 25% increase in cell number in McKrae-infected cultures between day 13 and day 15. However, this difference was not statistically significant and was probably due to cell migration following NGF treatment, as mentioned above. This finding indicates that LAT expression is not needed to prevent detachment and that prevention of detachment is not HSV-1 strain-specific.
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Role of de novo virus gene expression in mediating longevity of PC12 cells in culture
Infection with human cytomegalovirus, a member of the Herpesviridae family, has been reported to result in activation of signal transduction pathways, through the receptorligand engagement (Singh et al., 2001 ; Yurochko et al., 1997 ; Zhu et al., 1997 ) in the absence of de novo viral gene expression. Thus, it remained possible that viral gene expression was unnecessary for HSV-1 to prevent NGF-differentiated PC12 cell detachment from culture flasks. It has been shown that UV-inactivated HSV-1 results in virus that retains virion-associated functions, but does not induce viral gene expression and is unable to produce progeny (Schaffer et al., 1973 ). Therefore, UV-inactivated HSV-1 strain 17 was used to determine the role of de novo gene expression.
Cell-free HSV-1 virions were subjected to sufficient UV light to reduce infectivity by more than 6 logs, but retain virion-associated transactivation function (Fig. 4A). NGF-differentiated PC12 cells were infected with HSV-1 strain 17 (0 min of UV inactivation), UV-inactivated virus HSV-1 strain 17 (2 min of UV inactivation), or left uninfected. After infection, morphology and cell numbers were observed by serial photomicroscopy of pre-selected areas (as in Fig. 1A). As expected, after 55 days in culture most of the uninfected PC12 cells had detached from the culture flasks (Fig. 4B), while the majority of the HSV-1-infected cells remained adhered. Notably, most of the PC12 cells in flasks inoculated with UV-inactivated virus were lost by 55 days. Thus, the cells in the UV-inactivated flasks behaved similarly to those in uninfected cultures. Since UV-inactivated virus is defective in viral gene expression, this data suggests that de novo viral gene expression is necessary for HSV-1 to prevent the detachment of NGF-differentiated PC12 cells.
Role of HSV-1 replication in prevention of detachment
Because HSV-1 replication is dependent on de novo viral gene expression, and data from the UV-inactivated HSV-1 implied that some gene expression was necessary to prevent detachment of infected NGF-differentiated PC12 cells, it was of interest to test whether virus replication was, itself, necessary to prevent detachment. An HSV-1 DNA polymerase mutant (HP66), lacking the ability to replicate, was investigated for its ability to prevent cell detachment. NGF-differentiated PC12 cells were infected with either HSV-1 strain 17, HP66, or left uninfected. Morphology and cell number were observed as a function of time by serial photography of ten pre-selected fields in five culture flasks each (as in Fig. 1A). As a control for the infectivity of HP66 in NGF-differentiated culture, the content of HSV-1 DNA inside nuclei was determined by Southern hybridization 6 h after infection. The amount of HP66 DNA uncoated in the nucleus was comparable with that of HSV-1 strain 17 (data not shown). Thus, the ability of HP66 and HSV-1 strain 17 to enter NGF-differentiated PC12 cell culture was comparable.
As shown in Fig. 4, less than 10% of HP66- and HSV-1-infected PC12 cells had detached from the culture flask by day 55 after seeding, while 90% of the uninfected cells had detached by that time. Since HP66-infected NGF-differentiated PC12 cells remained attached to the culture flasks, it appears that HSV-1 replication is not needed to prevent detachment.
One striking observation described here is that quiescent HSV-1 infection prevented 67-week-old neurone-like NGF-differentiated PC12 cells from detaching from the culture flask. Although NGF-differentiated PC12 cells have been widely used as a neuronal-like culture in various studies, the length of time that the cells can be kept adherent in this post-mitotic state in culture flasks has not been extensively studied. This length of time probably varies with culture conditions as well as the homogeneity of cultures. In our experiments, cultures are routinely transiently treated with 5-Flu for 3 days after the first week of NGF incubation to eliminate any residual PC12 cells that are still dividing. This treatment is important in studies involving viral infection. Under these conditions, cultures incubated with 100 ng/ml of 2·5S NGF in serum-containing medium maintained a constant cell number for 67 weeks (Fig. 1). A previous report (Danaher et al., 2000 ) showed that when PC12 cells were cultured with NGF and serum-containing medium, but with no anti-mitotic agent, the NGF-differentiated PC12 cells remained in culture for longer than 8 weeks. Under conditions lacking anti-mitotic agent treatment, cell division continued with a 3-log increase in cell number by day 40 of culture. It is possible that a higher cell density and more heterogeneously differentiated cells provided various growth factors that maintained cells in an indefinitive adherent status. However, under the conditions we described here, the life of attached, relatively homogeneous, well-differentiated PC12 cells was limited in culture. Strikingly, HSV-1 quiescent infection was able to prevent the detachment of well-differentiated PC12 cells in culture.The neurone provides an unusual post-mitotic environment. During normal development, as many as 50% of the neurones are eliminated by apoptosis (reviewed in Milligan et al., 2000 ; Morrison & Hof, 1997 ). Thus, apoptosis and gene regulation appear to be managed and used to achieve specific cell destinies. Neuronal cell death may be tactical, as a part of development or to hinder pathogen infection, or pathological, due to injury or aging. Survival and sustenance of developing neurones is dependent on binding neurotrophic factors, such as NGF, and associating with proper extracellular matrix (ECM). Interestingly, results from both trypan blue and TUNEL staining suggested that the majority of detached cells were viable at the time of their detachment. However, following detachment, the surviving neurone-like PC12 cells were noted to be de-differentiating morphologically and resuming the rounded phenotype characteristic of the loosely adherent parental PC12 cells, even in the presence of NGF-containing medium. Since PC12 cell are transformed, pluripotent cells, detaching from an extracellular matrix might not result in apoptosis, but in morphological de-differentiation into a cell type that can survive in a matrix-free setting.
Since the virus stocks used here were prepared as total cell lysates, it is possible that cell factors contained in the virus stocks might be responsible for the prevention of detachment. However, for the UV experiment, cell-free gradient-purified HSV-1 strain 17 virions were used. The 0 min UV treatment gradient-purified virus prevented detachment (Fig. 3) as well as the total cell extract virus stocks used in the other experiments. Thus, it is unlikely that contaminating cell factors were responsible for HSV-1 infection preventing detachment of PC12 cells.
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Experiments to determine the role of HSV-1 in the prevention of detachment using UV-inactivated virus suggested that de novo gene synthesis was important in the prevention of detachment. The use of the UV-inactivated virus also suggested that a ligandreceptor mediated event, host cell response to foreign DNA and virion-associated function were not sufficient to prevent detachment. Experiments with the HSV-1 DNA polymerase mutant (Fig. 4) and LAT null mutants (Fig. 2) have demonstrated that neither HSV-1 replication nor LAT gene expression were necessary for the prevention of detachment.
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Collectively, the data reported here suggest that HSV-1 de novo gene expression exerts a prolonged, sustained effect in infected NGF-differentiated PC12 cells. The detachment phenomena occurred more than 20 days after infection, when the only detectable viral gene transcript was LAT (Su et al., 1999 ). However, LAT was not needed to prevent detachment (Fig. 2). The effecter of detachment prevention must therefore be the result of an event occurring early after infection.
Infection with HSV-1 has been shown to influence a variety of host cell factors and metabolic pathways (Bruni & Roizman, 1998 ; Everett & Maul, 1994 ; Hammarsten et al., 1996 ; Hill et al., 1995 ; Hobbs & DeLuca, 2001 ; McLauchlan et al., 1989 ; Mossman et al., 2001 ; Phelan et al., 1993 ; Sandri-Goldin et al., 1995 ; Stingley et al., 2000 ). To investigate the effect of an initial viral infection on cell attachment, a rat RNA gene array analysis (Mergen Ltd) was performed to compare the cellular gene profiles of uninfected and HSV-1-infected NGF-differentiated PC12 cultures, 24 days after infection (day 37 after seeding). Out of 1100 genes studied by gene array analysis, there were clearly some significant differences at the level of cellular gene profiles (data not shown). The majority of genes modulated by HSV-1 infection 37 days after seeding were cell-cycle related. It is known that HSV-1 infection interacts with cell-cycle related proteins during the productive infection (Bruni & Roizman, 1998 ; Hobbs & Deluca, 2001 ; Lomonte & Everett, 1999 ; Song et al., 2000 ; Davido et al., 2002 ; Jordan et al., 1999 ). Interestingly, our preliminary microarray data suggested that HSV-1 can interfere with the cell cycle of quiescent long-term-infected cells and, furthermore, that this interaction enhances cell attachment. Further studies are in progress to evaluate the microarray data and the possible correlation between the cell-cycle modulation and the neurone-like cell attachment.
Although the molecular and biochemical mechanisms of HSV-1 de novo gene expression in the prevention of detachment are not known, our results strongly suggest that quiescent HSV-1 infection prolongs neuronal-like cell life in culture. The implication of this interesting observation for in vivo systems will require investigations using animal models. None the less, this novel finding suggests another possible mechanism by which HSV-1 might prolong the life of latently infected neurones in vivo other than LAT-mediated neuronal survival.
We thank Robert Jordan and Pamela Norton for careful reading of the manuscript and helpful comments. This research was supported by NIH grants NS33768-11 to Timothy M. Block and Ying-Hsiu Su, NIH RO1 EY13191, NIH RO1EY12823, NIH RO1EY07566 and NIH RO1EY11629 to Steven L. Wechsler.References
Bruni, R. & Roizman, B. (1998). Herpes simplex virus 1 regulatory protein ICP22 interacts with a new cell cycle-regulated factor and accumulates in a cell cycle-dependent fashion in infected cells. Journal of Virology 72, 8525-8531.
Chen, S. H., Kramer, M., Schaffer, P. A. & Coen, D. M. (1997). A viral function represses accumulation of transcripts from productive cycle genes in mouse ganglia latently infected with herpes simplex virus. Journal of Virology 71, 5878-5884.
Danaher, R. J., Jacob, R. J. & Miller, C. S. (2000). Establishment of a quiescent herpes simplex virus type 1 infection in neurally-differentiated PC12 cells. Journal of NeuroVirology 5, 258-267.
Davido, D. J., Lieb, D. & Schaffer, P. A. (2002). The cyclin-dependent? kinase inhibitor Roscovitine inhibits the transactivating activity and alters the posttranslational modification of herpes simplex virus type 1 ICP0. Journal of Virology 76, 1077-1088.
Deatly, A. M., Spivack, J. G., Lavi, E. & Fraser, N. W. (1987). RNA from an immediate early region of the HSV-1 genome is present in the trigeminal ganglia of latently infected mice. Proceedings of the National Academy of Sciences, USA 84, 3204-3208.
Everett, R. D. & Maul, G. G. (1994). HSV-1 IE protein Vmw110 causes redistribution of PML. EMBO Journal 13, 5062-5069.[Medline]
Garber, D. A., Schaffer, P. A. & Knipe, D. M. (1997). A LAT associated function reduces productive cycle gene expression during acute infection of murine sensory neurons with herpes simplex virus type 1. Journal of Virology 71, 5885-5893.
Greene, L. A. & Tischler, A. S. (1976). Establishment of a nonadrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proceedings of the National Academy of Sciences, USA 73, 2424-2428.
Hammarsten, O., Yao, X. & Elias, P. (1996). Inhibition of topoisomerase II by ICRF-193 prevents efficient replication of herpes simplex virus type 1. Journal of Virology 70, 4523-4529.
Hill, A., Jugovic, P., York, I., Russ, G., Bennink, J., Yewdell, J., Ploegh, H. & Johnson, D. (1995). Herpes simplex virus turns off the TAP to evade host immunity. Nature 375, 411-415.[Medline]
Hill, J. M., Sedarati, F., Javier, R. T., Wagner, E. K. & Stevens, J. G. (1990). Herpes simplex virus latent phase transcription facilitates in vivo reactivation. Virology 174, 117-125.[Medline]
Hobbs, W. E. & DeLuca, N. A. (2001). Perturbation of cell cycle progression and cellular gene expression as a function of herpes simplex virus ICP0. Journal of Virology 73, 8245-8255.
Hobbs, W. E., Brough, D. E., Kovesdi, I. & DeLuca, N. A. (2001). Efficient activation of viral genomes by levels of herpes simplex virus ICP0 insufficient to affect cellular gene expression or cell survival. Journal of Virology 75, 3391-3403.
Hwang, Y. T., Liu, B.-Y., Coen, D. M. & Hwang, C. B. C. (1997). Effects of mutations in the Exo III motif of the herpes simplex virus DNA polymerase gene on enzyme activities, viral replication, and replication fidelity. Journal of Virology 71, 7791-7798.
Inman, M., Perng, G.-C., Henderson, G., Ghiasi, H., Nesburn, A. B., Wechsler, S. L. & Jones, C. (2001). Region of herpes simplex virus type 1 latency-associated transcript sufficient for wild-type spontaneous reactivation promotes cell survival in tissue culture. Journal of Virology 75, 3636-3646.
Jordan, R., Schang, L. & Schaffer, P. A. (1999). Transactivation of herpes simplex virus type 1 immediate-early gene expression by virion-associated factors is blocked by an inhibitor of cyclin-dependent protein kinases. Journal of Virology 73, 8843-8847.
Lieb, D. A., Nadeau, K. C., Rundle, S. A. & Schaffer, P. A. (1991). The promoter of the latency-associated transcripts of herpes simplex virus type 1 contains a functional cAMP-response element: role of the latency-associated transcripts and cAMP in reactivation of viral latency. Proceedings of the National Academy of Sciences, USA 88, 48-52.
Lomonte, P. & Everett, R. D. (1999). Herpes simplex virus type 1 immediate-early protein Vmw110 inhibits progression of cell through mitosis and from G1 into S phase of the cell cycle. Journal of Virology 73, 9456-9467.
McLauchlan, J., Simpson, S. & Clements, J. B. (1989). Herpes simplex virus induces a processing factor that stimulates poly(A) site usage. Cell 59, 1093-1105.[Medline]
Marcy, A. I., Yager, D. R. & Coen, D. M. (1990). Isolation and characterization of herpes simplex virus mutants containing engineered mutations at the DNA polymerase locus. Journal of Virology 64, 2208-2216.
Milligan, C. E., Barnes, N. Y. & Urioste, A. S. (2000). Mechanisms of neuronal death during development insights from chick motorneurons. In Vivo 14, 61-82.[Medline]
Morrison, J. H. & Hof, P. (1997). Life and death of neurons in the aging brain. Science 278, 412-419.
Mossman, K. L., MacGregor, J., Rozmus, J. J., Goryachev, A. B., Edwards, A. M. & Smiley, J. R. (2001). Herpes simplex virus triggers and then disarms a host antiviral response. Journal of Virology 75, 750-758.
Perng, G.-C., Dunkel, E. C., Geary, P. A., Slanina, S. M., Ghiasi, H., Kaiwar, R., Nesburn, A. B. & Wechsler, S. L. (1994). The latency-associated transcript gene of herpes simplex virus type 1 (HSV-1) is required for efficient in vivo spontaneous reactivation of HSV-1 from latency. Journal of Virology 68, 8045-8055.
Perng, G.-C., Jones, C., Ciacci-Zanella, J., Stone, M., Henderson, G., Yukht, A., Slanina, S. M., Hofman, F. M., Ghiasi, H., Nesburn, A. B. & Wechsler, S. L. (2000a). Virus-induced neuronal apoptosis blocked by the herpes simplex virus latency-associated transcript. Science 287, 1500-1502.
Perng, G.-C., Slanina, S. M., Yukht, A., Ghiasi, H., Nesburn, A. B. & Wechsler, S. L. (2000b). The latency-associated transcript gene enhances establishment of herpes simplex virus type 1 latency in rabbits. Journal of Virology 74, 1885-1891.
Phelan, A., Carmo-Fonseca, M., McLauchlan, J., Lamond, A. I. & Clements, J. B. (1993). A herpes simplex virus type 1 immediate-early gene product, IE63, regulates small nuclear ribonucleoprotein distribution. Proceedings of the National Academy of Sciences, USA 90, 9056-9060.
Rock, D. L., Nesburn, A. B., Ghiasi, H., Ong, J., Lewis, T. L., Lokensgard, J. R. & Wechsler, S. L. (1987). Detection of herpes simplex virus type 1 latency-associated transcript expression in trigeminal ganglia of rabbits latently infected with herpes simplex virus type 1. Journal of Virology 61, 3820-3826.
Roizman, B. & Sears, A. E. (1996). Herpes simplex viruses and their replication. In Fundamental Virology , pp. 2231-2296. Edited by B. N. Fields & D. M. Knipe. Philadelphia:LippincottRaven.
Sandri-Goldin, R. M., Hibbard, M. K. & Hardwicke, M. A. (1995). The C-terminal repressor region of herpes simplex virus type 1 ICP27 is required for the redistribution of small nuclear ribonucleoprotein particles and splicing factor SC35; however, these alterations are not sufficient to inhibit host cell splicing. Journal of Virology 69, 6063-6076.
Sawtell, N. M. & Thompson, R. L. (1992). Herpes simplex virus type 1 latency-associated transcription unit promotes anatomical site-dependent establishment and reactivation from latency. Journal of Virology 66, 2157-2169.
Schaffer, P. A., Aron, G. M. & Bemyesh-Melnick, M. (1973). Temperature sensitive mutants of HSV 1 isolation, complementation and partial characterization. Journal of Virology 52, 57-71.
Singh, J., Simmen, K., Lopper, M., Frueh, K. & Compton, T. (2001). Modulation of cellular transcriptional activity by HCMV glycoprotein B. In 25th International Herpesvirus Workshop, Portland, Oregon, 2000, abstract 202.
Song, B., Liu, J. J., Yeh, K.-C. & Knipe, D. M. (2000). Herpes simplex virus infection block events in the G1 phase of the cell cycle. Virology 367, 326-334.
Steiner, I., Spivack, J. G., Lirette, R. P., Brown, S. M., Maclean, A. R., Subak-Sharpe, J. H. & Fraser, N. W. (1989). Herpes simplex virus type 1 latency-associated transcripts are evidently not essential for latent infection. EMBO Journal 8, 505-511.[Medline]
Stevens, J. G., Wagner, E. K., Devi-Rao, G. B., Cook, M. L. & Feldman, L. T. (1987). RNA complementary to a herpes virus gene mRNA is prominent in latently infected neurons. Science 235, 1056-1059.
Stingley, S. W., Ramirez, J. G., Simmen, K., Sandri-Goldin, R. M., Ghazal, P. & Wagner, E. K. (2000). Global analysis of herpes simplex virus type 1 transcription using an oligonucleotide-based DNA microarray. Journal of Virology 74, 9916-9927.
Su, Y.-H., Meegalla, R. L., Chowan, R., Cubitt, C., Oakes, J. E., Lausch, R. L., Fraser, N. W. & Block, T. M. (1999). Human corneal cells and other fibroblasts can stimulate the appearance of herpes simplex virus from quiescently infected PC12 cells. Journal of Virology 73, 4171-4180.
Su, Y.-H., Moxley, M., Kejariwal, R., Mehta, A., Fraser, N. W. & Block, T. M. (2000). The HSV 1 genome in quiescently infected NGF differentiated PC12 cells cannot be stimulated by HSV superinfection. Journal of NeuroVirology 6, 341-349.[Medline]
Thomselli, K., Hall, D. E., Flier, L. A., Gehlsen, K. R., Turner, D. C., Carbonetto, S. & Reichardt, L. F. (1990). A neuronal cell line PC12 expresses two β1-class integrins α1β1 and α3b1 that recognize different outgrowth-promoting domains in laminin. Neuron 5, 651-662.[Medline]
Trousdale, M. D., Steiner, I., Spivack, J. G., Deshmane, S. L., Brown, S. M., Maclean, A. R., Subak-Sharpe, J. H. & Fraser, N. W. (1991). In vivo and in vitro reactivation impairment of a herpes simplex type 1 latency-associated transcript variant in a rabbit eye model. Journal of Virology 65, 6989-6993.
Yurochko, A. D., Hwang, E., Rasmussen, L., Keay, S., Pereira, L. & Huang, E. (1997). The human cytomegalovirus UL55 (gB) and UL75 (gH) glycoprotein ligands initiate the rapid activation of Sp1 and NF-κB during infection. Journal of Virology 71, 5051-5059.
Zhu, H., Cong, J. & Shenk, T. (1997). Use of differential display analysis to assess the effect of human cytomegalovirus infection on the accumulation of cellular RNAs: induction of interferon-responsive RNAs. Proceedings of the National Academy of Sciences, USA 94, 13985-13990.
Received 8 January 2002; accepted 5 March 2002.