Stability and circularization of herpes simplex virus type 1 genomes in quiescently infected PC12 cultures

Ying-Hsiu Su1, Michael J. Moxley1, Alan K. Ng1, Judy Lin1, Robert Jordan1, Nigel W. Fraser2 and Timothy M. Block1

Jefferson Center for Biomedical Research of Thomas Jefferson University, 700 E. Butler Ave, Doylestown, PA 18901-2697, USA1
Department of Microbiology, University of Pennsylvania, Philadelphia, PA, USA2

Author for correspondence: Ying-Hsiu Su. Fax +1 215 489 4920. e-mail yinghsiu.su{at}mail.tju.edu


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Herpes simplex virus type 1 (HSV-1) DNA has been shown to exist as a linear, double-stranded molecule in the virion and as a non-linear (endless), episomal, nucleosomal form in latently infected trigeminal ganglia. The kinetics of the formation and appearance of endless viral genomes and the stability of linear genomes in neuronal cells are not well understood. Nerve growth factor (NGF)-differentiated PC12 cells can sustain long-term, quiescent infections with HSV-1. In this report, the structure and stability of HSV-1 viral DNA in NGF-differentiated PC12 cells was studied as a function of time following infection using both wild-type and replication-defective virus. Unexpectedly, unencapsidated linear genomes were stable in the nucleus of NGF-differentiated PC12 cells for up to 2–3 weeks following infection, beyond the period at which there is no detectable viral gene expression. However, following infection with wild-type HSV, the majority of quiescent viral genomes were in an endless form after 3–4 weeks. These data suggest that the stability and fate of HSV-1 DNA in non-mitotic neuronal-like cells is different from that in productively infected cells and thus there is a significant cellular role in this process. The relevance to the virus life-cycle in neurones in vivo is discussed.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Herpes simplex virus type 1 (HSV-1) genomes are found in at least three different physical states: linear, circular and concatemeric. In the virion, genomes are linear duplexes of approximately 150 kb (Roizman, 1979 ). The linear genomes assume a circular conformation within hours after infection of permissive cells (Deshmane et al., 1995 ; Garber et al., 1993 ; Poffenberger & Roizman, 1985 ). Concatemers of viral genomes have been detected during the replication phases of infection (Bataille & Epstein, 1997 ; Jacob et al., 1979 ; Severini et al., 1994 ).

Viral genomes derived from trigeminal ganglia (TG) of latently infected animals appear to be predominantly maintained in circular or concatenated forms (Efstathiou et al., 1986 ; Rock & Fraser, 1983 ). These circular or concatenated forms have been called ‘endless’ DNA, because terminal restriction fragments characteristic of linear duplex chromosomes cannot be detected, or are greatly reduced from the equimolar amounts expected from a linear genome. In the mouse model of HSV-1 latency, the majority of endless latent HSV-1 DNA is extrachromosomal (Mellerick & Fraser, 1987 ) and is associated with nucleosomes in a chromatin structure (Deshmane & Fraser, 1989 ). It has been hypothesized that this form of latent DNA may contribute towards suppression of productive viral gene expression and maintenance of latency (Deshmane & Fraser, 1989 ). Thus, it seems likely that the structure of the viral DNA relates to its function and there is considerable interest in understanding the processes involved in converting linear into endless HSV genomes.

In tissue culture models of long-term HSV infection, the structure of the quiescent HSV genome has been determined to be predominantly endless (Block et al., 1994 ; Jamieson et al., 1995 ; Wilcox et al., 1997 ). However, in one study by Wigdahl et al. (1984) , the quiescent viral genome was determined to be in a linear state. That system of tissue culture quiescence required the use of antiviral compounds and high temperatures to maintain the non-replication state, and the relationship between the suppression of viral gene expression and genomic structure was unclear.

Nerve growth factor (NGF)-differentiated PC12 cells cease dividing, extend long neuritic processes and acquire many properties associated with neurones of the peripheral nervous system (Greene & Tischler, 1976 ). It has been shown that the overall DNA repair efficiency in PC12 cells declines following NGF differentiation (Hanawalt et al., 1992 ), which resembles the observation of a general deficiency in repair of DNA in neurones. Thus, it seems possible that the metabolism of non-cellular DNA (e.g. virion DNA) in mitotic and non-mitotic cells is different. We have previously reported that HSV-1 can establish a long-term, quiescent infection in NGF-differentiated PC12 cells and that the quiescent virus can be induced by co-cultivation (Su et al., 1999 ). It was of interest to study the structure and stability of the HSV genome in a neurone-like environment in a quiescent state. Here we report that there is a long lag in time between infection and predominance of endless viral DNA during the establishment of latency in NGF-differentiated PC12 cultures. The delayed predominance of endless quiescent HSV DNA appears to be due to the slow degradation of linear DNA.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus and cells.
PC12 cells from ATCC were grown in RPMI 1640 supplemented with 10% heat-inactivated horse serum and 5% heat-inactivated foetal bovine serum (PC12 medium). CV-1 cells (ATCC) were maintained in Eagle's minimal medium with 5% calf serum. HSV-1 strain 17 was prepared in CV-1 cells and HSV-1 mutant HP66, containing a 2·3 kb deletion in the locus of the DNA polymerase (Marcy et al., 1990 ), kindly provided by Don Coen (Harvard Medical School, Boston, MA, USA), was prepared in the complementing cell line polB3 (Hwang et al., 1997 ; kindly provided by Charles Hwang, SUNY Health Science Center, Syracuse, NY, USA).

{blacksquare} Differentiation of PC12 cells.
To differentiate PC12 cells, 1x105 cells were seeded on 25 cm2 culture flasks coated with poly-L-ornithine (Sigma). The following day, the 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, 20 µM 5-fluoro-2'-deoxyuridine (FUDR) (Sigma) was added for 3 days to eliminate undifferentiated PC12 cells. Fresh NGF-supplemented medium was replaced thereafter.

{blacksquare} Establishment of long-term HSV-1 infection.
Differentiated PC12 cultures were infected with HSV-1 strain 17 or mutant HP66 at an m.o.i. of 20 (2x106 p.f.u./flask). Following a 1 h incubation at 37 °C, the cultures were treated with 3 ml sodium citrate buffer (pH 3) for 30–60 s to inactivate residual virus, modified as described (Su et al., 2000 ). The buffer was removed and the flasks were rinsed with PC12 medium once. After low-pH treatment, cultures were incubated at 37 °C with fresh medium containing NGF.

{blacksquare} Nuclear DNA isolation and Southern blot hybridization.
To isolate nuclear DNA, cells were scraped into medium and collected by centrifugation at 1500 r.p.m. for 10 min at 4 °C. The cell pellet was resuspended in nuclei lysis buffer (1 mM CaCl2, 60 mM KCl, 15 mM NaCl, 3 mM MgCl2, 10 mM Tris, pH 7·5, 5% sucrose) containing NP-40 and the nuclei were collected by centrifugation at 1500 r.p.m. for 10 min at 4 °C. The nuclear pellet was then resuspended in nuclei lysis buffer containing 0·1% of deoxycholate to strip off nuclear membrane-associated proteins. After deoxycholate treatment, nuclear DNA was isolated by SDS–proteinase K digestion, phenol–chloroform extraction and ethanol precipitation.

Southern blot hybridization was performed by digesting the purified DNA with the restriction endonuclease BamHI. The digested DNA was resolved on a 1·5% agarose gel and transferred to a nylon membrane by capillary transfer. The membrane was hybridized with the selected 32P-labelled probe of interest. The autoradiographic image was generated and quantified by Bio-Rad phosphoimager analysis.

{blacksquare} Viral capsid preparation and micrococcal nuclease (MN) digestion of HSV DNA.
HSV-1-infected CV-1 cells were scraped into medium and collected by centrifugation at 1500 r.p.m. for 10 min at 4 °C. The cells were lysed by sonication at 40% power (Heat System Ultrasonicator) for 1 min. Cell debris was separated from virions by centrifugation at 2000 r.p.m. for 15 min at 4 °C. HSV-1 virions were pelleted by centrifugation at 28000 r.p.m. using an SW41 rotor for 1 h at 10 °C. Isolated virions were then lysed in virion lysis buffer (0·25% Triton X-100, 10 mM EDTA, 10 mM Tris, pH 8·0) and viral capsids were isolated by centrifugation through a 20% sucrose cushion at 28000 r.p.m. using an SW55 rotor for 50 min at 4 °C.

To perform MN digestion, samples containing intact capsids, nuclei isolated as described in Greenberg & Bender (1997) and naked purified viral DNA (strain 17) were incubated with 3 units of MN for 0 or 20 min at 37 °C. Following MN treatment, DNA from each digest was isolated and amplified by PCR using primers specific for the HSV-1 icp27 gene (Su et al., 2000 ).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
HSV DNA structure in productive infection in NGF-differentiated PC12 cells
Monomeric linear HSV genomes can be distinguished from those that are either circular or long concatemers by examining restriction fragments following complete digestion with BamHI (Rock & Fraser, 1983 ). As shown graphically in Fig. 1, BamHI restriction of a linear HSV-1 genome will result in terminal fragments s and p of 2·6 and 3·2 kb, respectively, which resolve in equimolar amounts relative to the internal 5·8 kb sp fragment (Rock & Fraser, 1983 ). In contrast, circular molecules will be devoid of terminal s and p fragments.



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Fig. 1. Maps of the HSV-1 genome as linear (A) and circular (B) forms. A linear map of the HSV-1 genome (strain 17), divided into unique long (UL) and unique short (US) segments with open boxes (TRL, TRS, terminal long and short repeats, respectively; IRL, IRS, internal long and short repeats, respectively) identifying the regions of the genome that are repeats. Solid boxes under the linear map show the genomic locations of the terminal BamHI s and p fragments and the internal sp fragment, with the molecular masses indicated. Note that the BamHI s and p fragments exist only as linked sp fragments (two copies) in the circular form.

 
The ability to distinguish linear from circular or endless viral genomes is shown in Fig. 2. Virion-derived HSV-1 genomic DNA contains, as expected, equimolar amounts of s+p:sp BamHI fragments (Fig. 2, lane 1). Briefly, linear viral genomes digested with BamHI and probed with radiolabelled HSV DNA BamHI sp fragment produced 2·6 and 3·2 kb terminal restriction fragments designated s and p, respectively, in an amount approximately equimolar (sp:p+s ratio of 1·03; see Fig. 2) to the internal 5·8 kb sp fragment, as determined by phophorimager intensity analysis.



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Fig. 2. Kinetics of HSV-1 DNA circularization in NGF-differentiated PC12 cells. NGF-differentiated PC12 cells (NGF-PC12) were infected with HSV-1 strain 17 at an m.o.i. of 3 in the presence of 400 µg/ml PAA. As controls, PC12 and CV-1 cells were also infected with strain 17 at an m.o.i. of 3 in the presence of PAA. Nuclear DNA from infected cells was harvested at 3 and 6 h p.i. Nuclear DNA or virion DNA was digested with BamHI, resolved by electrophoresis through 2% agarose, transferred to nylon membranes and hybridized to a 32P-labelled BamHI sp fragment, as described in Block et al. (1994) . The autoradiographic image was produced by a Bio-Rad phosphoimager. The mobility of the BamHI s, p and sp fragments are indicated. The intensities of the bands shown in the table are provided in arbitrary units and were determined by the phosphoimager analysis. The ratio of the intensity values of the sp:p+s fragments is also given. BKG, background, below the level of detection.

 
To generate a source of circular or endless viral genome, DNA was examined from CV-1 and non-NGF-treated PC12 cells, infected with HSV-1 strain 17 under conditions that enrich for endless viral DNA, at an m.o.i. of 3 and where viral DNA replication was inhibited with 400 µg/ml of phosphonoacetic acid (PAA). Since both CV-1 and non-NGF-treated cells are permissive for virus replication, and since replication of the viral genome results in an amplification of linear viral genomic forms, PAA was included to enrich for endless DNA. As shown in Fig. 2 (lanes 2 and 3), the terminal fragments of the HSV-1 genome were greatly reduced to almost undetectable levels shortly after infection of permissive cells, consistent with the rapid circularization of the genome. Circularization of viral DNA occurs passively, even in the absence of viral DNA replication, as reported by Garber et al. (1993) .

Nouspikel & Hanawalt (2000) have previously suggested that, compared with mitotic cells, non-mitotic cells have reduced DNA repair capability, implying that DNA metabolism in non-dividing neuronal cells might be different from dividing cells. To determine whether circularization of HSV-1 genomes occurs in the absence of viral DNA replication in NGF-differentiated, non-mitotic, neurone-like PC12 cells during initial infection, PC12 cells were infected with HSV-1 strain 17 at an m.o.i. of 3 on day 12 after NGF treatment. As before, viral DNA replication was inhibited with 400 µg/ml of PAA during infection. As shown in Fig. 2 (lanes 4 and 5), HSV viral DNA assumed a predominantly endless form in NGF-differentiated PC12 cells within 3 h of infection, even in the absence of virus replication.

In addition to providing a source of reference endless DNA for subsequent analysis in this report, these data extend the work of others (Deshmane et al., 1995 ; Garber et al., 1993 ; Poffenberger & Roizman, 1985 ) in showing that, as with productively infected mitotic cells, HSV-1 DNA can assume an endless (presumably circular) state within hours of infection of NGF-differentiated PC12 cells, even in the absence of viral DNA replication.

HSV DNA structure in long-term quiescent infection in NGF-differentiated PC12 cells
To study further the structure of the HSV genome in NGF-differentiated PC12 cells as a function of time after infection, PC12 cells were treated for 10 days with 100 ng/ml NGF and for 3 days with 20 µM of the anti-mitotic agent FUDR prior to HSV-1 strain 17 infection at an m.o.i. of 20, as described previously (Su et al., 1999 ). Note that, unlike the situation described in Fig. 2, these conditions permitted virus replication (i.e. PAA was not included). Also, to characterize and quantify viral genomic forms, we focused on times exceeding day 10 post-infection (p.i.), since infected cells between days 1 and 10 p.i. contain multiple (circular, linear and replicating) forms of the viral genome (data not shown), which complicate interpretation.

Thus, at 10, 13 and 24 days after HSV-1 strain 17 infection, total nuclear DNA was isolated and analysed (Fig. 3). At 24 days after infection of NGF-differentiated PC12 cells, the abundance of s and p fragments was very low (Fig. 3, lane 5). This suggested that by 24 days p.i., most of the HSV DNA (73%; Fig. 3) in the long-term NGF-differentiated PC12 cultures was present in an endless form. Surprisingly, even at 10 and 13 days after infection, a significant amount of the viral DNA isolated from NGF-differentiated PC12 cells still had detectable terminal s and p fragments. This suggested that even nearly 2 weeks after infection, a significant fraction of the viral DNA was still in a linear form. Indeed, phosphorimager analysis was used to determine the molar ratio of sp to s fragments and suggested that as much as 80 and 60% of the viral genomes were still in a linear state on days 10 and 13 p.i., respectively. Surprisingly, unlike the situation described in Fig. 2 where viral DNA replication was inhibited with PAA under conditions permissive for virus replication, the major species of viral genome was not a circular (endless) form until 2–3 weeks p.i. This existence of the non-circular form of the wild-type viral genome detected in NGF-differentiated PC12 cells 13 days after infection could have been due to maintenance of the infecting genome in a ‘protected’ (encapsidated) state, continuous low level replication, or the inherent permissiveness of the PC12 cell nucleus to linear DNA. These possibilities are considered in the next sections.



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Fig. 3. Southern blot analysis of DNA from quiescently infected NGF-differentiated PC12 cells. Purified virion DNA (0·002 µg), or 5 µg of PC12 or HSV-1 strain 17-infected PC12 DNA (m.o.i. of 20) at days 10 (D10 PI), 13 (D13 PI) and 24 (D24 PI) p.i. were digested with BamHI and analysed as described in Fig. 2. The ratio (R) of the intensity values of the sp:p+s fragments is also provided. Note that the percentage of circular DNA was calculated based on the assumption that the DNA pool only consisted of circular and linear forms of DNA. The equation used to calculated the percentage of circular DNA was:% circular=0·5(R-1)/[1+0·5(R-1)]x100%. BKG, background, below the level of detection.

 
It was noted that the time after infection until endless DNA became the major viral DNA form varied somewhat with the batches of culture. In one experiment, a significant amount of endless DNA was not dominant until 5 weeks after infection (data not shown).

Susceptibility of long-term quiescent linear HSV-1 DNA to micrococcal nuclease
Since unencapsidated linear genomic DNA might be expected to be degraded, it seemed possible that the linear HSV-1 strain 17 DNA in NGF-differentiated cells at 13 days after infection was still within capsids. The possibility that the presence of linear HSV genomes 13 days after infection was due to protection of the DNA by encapsidation was explored using a nuclease sensitivity assay as follows. Viral DNA within nucleocapsids should be relatively resistant to cleavage by nucleases, as compared with that of naked (uncoated) HSV DNA. Therefore, the ability of MN to digest samples of either naked viral DNA, DNA within nucleocapsids, or viral DNA from the nuclei of long-term, HSV-1 strain 17-infected (m.o.i. of 20), NGF-differentiated PC12 cells was determined. Briefly, samples of DNA were treated with 3 units of MN for 0 or 20 min at 37 °C (Fig. 4). After digestion, DNA was isolated and the presence of intact HSV DNA was determined by PCR using primers specific for the HSV-1 icp27 gene. As shown in Fig. 4 (lane 3), naked viral DNA was eliminated by 20 min digestion with MN, since icp27 DNA could not be detected after 35 cycles of PCR as determined by either ethidium bromide staining (top panel) or Southern blot hybridization of the PCR product (bottom panel). On the other hand, DNA inside nucleocapsids was protected from MN digestion, consistent with the notion that DNA within capsids is relatively nuclease-resistant, with no significant difference in the icp27 signal between 0 and 20 min of MN digestion. Significantly, HSV-1 strain 17 DNA from NGF-differentiated PC12 cells was completely degraded by the MN digestion, i.e. no icp27 signal was observed following 20 min MN treatment (Fig. 4, lane 7). These results suggest that on day 13 p.i., neither the linear nor the endless forms of HSV-l strain 17 DNA in NGF-differentiated PC12 cells were encapsidated.



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Fig. 4. Micrococcal nuclease (MN) digestion of HSV DNA. Viral capsids prepared as in Methods, nuclei isolated as described in Greenberg & Bender (1997) from uninfected PC12 cells (PC12) or from NGF-differentiated PC12 cells 13 days after HSV-1 strain 17 infection (m.o.i. of 20) and naked purified HSV-1 strain 17 viral DNA (17 DNA) were incubated with 3 units of MN for 0 or 20 min at 37 °C. Following MN treatment, DNA was isolated and amplified by PCR with icp27 gene-specific primers. Upper panel: PCR products separated by gel electrophoresis and detected by ethidium bromide staining. Lower panel: Southern blot of ethidium bromide-stained gel hybridized with an oligonucleotide probe specific for internal icp27 gene sequences. PC12+17 DNA and PC12+capsid pairs show reconstruction experiments in which MN digestion was performed on uninfected PC12 cell nuclei mixed with either purified HSV strain 17 DNA or purified HSV-1 capsids, respectively. The result shown is representative of three independent experiments.

 
Linear DNA of a replication-defective HSV-1 mutant is stable for weeks in long-term infection of NGF-differentiated PC12 cells
Linear DNA has previously been shown to be degraded by 72 h in transfected, dividing tissue culture cells (Block et al., 1985 ; Lechardeur et al., 1999 ; Loyter et al., 1982 ; reviewed in Norton, 2000 ). It was therefore surprising to detect a significant amount of linear, unencapsidated HSV-1 DNA on day 13 p.i. (Fig. 3). It seemed possible that linear DNA was either more stable in non-mitotic NGF-differentiated PC12 cells or that linear DNA detected on day 13 p.i. was the result of an ongoing low level of wild-type virus replication. Thus, although there was no detectable infectious virus produced by these cultures by 13 days p.i., it was possible that even abortive replication could be contributing to the population of viral DNA, confounding interpretation and quantification. Therefore, HP66, an HSV-1 replication-defective mutant containing a deletion within the DNA polymerase (Marcy et al., 1990 ), was used. Since the infecting genomes could not replicate (due to the defect in the viral polymerase gene), it was possible to quantify and evaluate the fate of the infecting genomic DNA as a function of time following infection.

NGF-differentiated PC12 cells were prepared as described in Methods and infected with HP66 at an m.o.i. of 20 on day 13 after NGF treatment. Nuclear DNA was harvested after 2 h and on days 1, 10, 20 and 30 p.i. and analysed. As shown in Fig. 5(A), surprisingly, a significant percentage of all uncoated HP66 mutant DNA did not appear to circularize at any of the times examined. The reason for a low efficiency of circularization of HP66 genomic DNA is unclear and is discussed below. To study the stability of linear HP66 DNA inside nuclei, the sum of the terminal fragments (s+p) at day 1 p.i. was set as 100%. The relative amount of terminal HSV-1 DNA, s+p, as a function of the time p.i. was calculated and plotted, as shown in Fig. 5(B). Clearly, input DNA remained mostly linear and was stable, with a half-life of nearly 20 days. Indeed, 34% of input linear DNA was still detectable and linear for up to 30 days after infection.



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Fig. 5. The stability of input linear HP66 genomes in NGF-differentiated PC12 cells. Southern blot analysis of DNA from purified DNA polymerase mutant HP66 virions (V), uninfected PC12 cells (PC12) and nuclei from NGF-differentiated PC12 cells at 2 h and on days 1 (D1), 10 (D10), 20 (D20) and 30 (D30) after infection with HSV-1 DNA polymerase mutant HP66 at an m.o.i. of 20, as described in the text. Purified virion DNA (0·002 µg), or 5 µg of PC12 or infected PC12 DNA were digested with BamHI, resolved on a 2% agarose gel and transferred to a nylon membrane. The membrane was hybridized with the 32P-labelled BamHI sp fragment (A) or a cellular gene GAPD amplicon (not shown) prepared by PCR (Su et al., 2000 ). The autoradiographic image was produced and quantified in a Bio-Rad phosphoimager, as described in Fig. 2. The amount of terminal fragments (s+p) of each sample was normalized to the intensity generated by the GAPD probe to obtain the relative amount of s+p per unit of GAPD intensity. The amount of terminal (s+p) HP66 DNA at D1 p.i. was set as 100%. The relative amount of terminal HP66 DNA, s+p, per PC12 DNA as a function of the time p.i. was plotted, as shown in (B). These data are representative of two independent experiments.

 
Thus, linear viral DNA is remarkably stable within NGF-differentiated PC12 cells.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Although virion DNA exists as a linear molecule, it has been known for some time that in cells latently infected with HSV-1, the DNA exists in an endless form (Rock & Fraser, 1983 ). Here, our data suggest that even when virus replication is inhibited with PAA, endless DNA can be formed shortly after HSV-1 infection of NGF-differentiated PC12 cells (Fig. 2), in a manner similar to that seen in mitotic cells (Garber et al., 1993 ). However, in NGF-differentiated PC12 cells infected with wild-type HSV-1, the ratio of linear to endless viral genomes changes with time after infection, with endless forms becoming predominant after several weeks (Fig. 3). Considering all of the data, it is suggested that, in NGF-differentiated PC12 cells, two forms of viral DNA are generated shortly after infection with wild-type virus: linear and endless (circular). Both forms of viral DNA are stable in these cells, with half-lives exceeding 2 weeks. However, the half-life of circular DNA exceeded 4 weeks and was much longer than that of the linear form. Thus, it is reasoned that over a period of time and in the absence of de novo virus replication, the linear forms are degraded and the circular forms become the only detectable form.

Although the endless viral DNA in NGF-differentiated PC12 cells is more stable than the linear form, we were surprised at the length of the half-life of the linear DNA. This was most apparent in studies with the replication-defective mutant HP66. The half-life of linear, input viral DNA in NGF-differentiated cells resulting from infection with HP66 approached 20 days (Fig. 5). This raises the possibility that linear DNA in general might be able to survive for unusually long periods of time in neurone-like cells.

It has been shown that human neuroblastoma cells are deficient in the removal of bulky adducts and exhibit a low level of repair activity (Jensen & Linn, 1988 ). Terminally differentiated human neurones repair transcribed genes, known as transcription-coupled repair, but display attenuated global DNA repair (Nouspikel & Hanawalt, 2000 ). Furthermore, PC12 cells differentiating into neurone-like cells have indicated a marked decrease in global genome repair but not in the repair of expressed genes (Hanawalt et al. 1992 ). The observation that HSV-1 strain 17 (Fig. 3) and HP66 mutant virus (Fig. 4) show long-term survival of linear genomes would be consistent with the hypothesis that linear DNA in the nucleus of non-dividing neuronal cells has a longer half-life than in mitotically active cells. Perhaps non-dividing neuronal cells do not efficiently eliminate foreign DNA, thus providing an ideal environment to host latent viral DNA in vivo. It is known that latently infected HSV-1 DNA exists in an endless form when DNA was analysed after 4 weeks or longer of infection (Efstathiou et al., 1986 ; Mellerick & Fraser, 1987 ; Rock & Fraser, 1983 ). However, it is not known whether linear viral DNA also lasted weeks after infection in latently infected neurons in vivo. Our result suggests that linear HSV-1 DNA might survive for weeks inside the nuclei of latently infected neurons in vivo.

Wigdahl et al. (1984) has also reported that linear forms of the HSV genome are stable in a tissue culture of rat fetal neurons or human embryo lung fibroblasts for several days, in the absence of replication. However, it was not known if the surviving linear DNA was in the cytoplasm or the nucleus and whether it was encapsidated or naked. Here, we report that the unencapsidated HSV-1 linear DNA survived in the nuclei of NGF-differentiated neurone-like PC12 cells.

It was surprising to find a significant amount of DNA polymerase mutant HP66 DNA remaining in a linear (as opposed to endless) state, weeks after entry into the nucleus of NGF-differentiated cells (Fig. 5), since it circularized following infection of polB3 cells (not shown). It is possible that replication competence influences the ability of the HSV genome to form endless DNA and it was noted that failure to circularize was also seen with an alpha 4 mutant virus, which was unable to replicate (dl120; unpublished observation). On the other hand, to facilitate detection of viral genomes, the replication-minus mutants were both used at a high m.o.i. of 20. The high m.o.i. may have exhausted limiting amounts of host factors needed to assist circularization. It has previously been shown that at a high m.o.i. of infection only a fraction of incoming viral DNA circularizes and enters the replicative pool following uncoating (Jacob & Roizman, 1977 ). Thus, if the amount of a host factor needed for circularization of linear genomes was present in limiting amounts in NGF-differentiated PC12 cells, the high m.o.i. would contribute to the smaller portion of circularization observed. It is also possible that uncoated linear DNA is located in a different compartment of the nucleus from the circularized viral DNA. Therefore, it should be noted that equivalent p.f.u. of wild-type virus and replication-deficient mutant might not mean equivalent DNA template for viral gene expression or DNA replication in future studies.

Other studies suggest that endless DNA formation is not entirely passive and requires both host functions (Umene & Nishimoto, 1996 ) and viral components (Batterson et al., 1983 ). We have also found that when non-permissive long-term NGF-differentiated PC12 cells were infected with HSV-1 strain 17 at an m.o.i. of 20, 22 days after NGF-differentiation (Su et al., 2000 ), the majority of viral DNA was in a linear form for up to 10 days p.i. Under these conditions, there was no viral DNA replication observed (Su et al., 2000 ).

It has been shown that, in the mouse model of HSV-1 latency, the endless latent DNA is associated with nucleosomes in a chromatin structure (Deshmane & Fraser, 1989 ). This form of DNA is thought to be repressed with regard to viral gene expression. We have shown that linear DNA in the quiescent state is not encapsidated. However, it is not clear whether the linear DNA was also associated with nucleosomes. With highly negatively charged molecules such as DNA, one would predict that it should be wrapped in nucleosomes on entering the nucleus. Histone modification has been shown to play a crucial role in gene regulation at the level of transcription (Boggs et al., 2002 ; Jenuwein & Allis, 2001 ; Peters et al., 2002 ). Experiments are ongoing to determine the role of histone modification in HSV gene silencing during the quiescent state.

Collectively, these results suggest that there are two populations of viral DNA created shortly after infection in neurone-like PC12 cells: linear and endless. Unlike the situation in productively infected cells, linear HSV DNA can survive in NGF-differentiated PC12 cells for a surprisingly long period of time – at least several weeks. Thus, at the very least, the data reported here show that: (a) there can be a long lag period between infection and predominance of the endless form of the HSV genome during the establishment of latency; and (b) linear, nuclease-sensitive forms of the viral genome can survive in neurone-like cells for weeks. Although the significance of these findings to viral pathogenesis has not been determined, the PC12 cell system offers a means of studying the dynamics of viral DNA metabolism during productive and quiescent phases of the virus life-cycle.


   Acknowledgments
 
This work was supported by the NIH award NS33768-11 and, in part, by an appropriation from the Commonwealth of Pennsylvania. We thank Drs Ru-Chi Huang and David Mould (Johns Hopkins University) for providing technical advice in nuclei isolation and MN digestion.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Bataille, D. & Epstein, A. L. (1997). Equimolar generation of the four possible arrangements of adjacent L components in herpes simplex virus type 1 replicative intermediates. Journal of Virology 71, 7736-7743.[Abstract]

Batterson, W., Furlong, D. & Roizman, B. (1983). Molecular genetics of herpes simplex virus. VIII. Further characterization of a temperature-sensitive mutant defective in release of viral DNA and in other stages of the viral reproductive cycle. Journal of Virology 45, 397-407.[Medline]

Block, T., Brzykcy, J., Hastie, N. & Hughes, R. G.Jr (1985). Genetic linkage but independent expression of functional HSV-1 tk and mammalian aprt genes after co-transfer to L cells. Canadian Journal of Microbiology 31, 311-316.[Medline]

Block, T., Barney, S., Masonis, J., Maggioncalda, J., Valyi-Nagy, T. & Fraser, N. W. (1994). Long-term herpes simplex virus infection of nerve growth factor-treated PC12 cells. Journal of General Virology 75, 2481-2487.[Abstract]

Boggs, B. A., Cheung, P., Heard, E., Spector, D. L., Chinault, A. C. & Allis, D. (2002). Differentially methylated forms of histone H3 show unique association patterns with inactive human X chromosomes. Nature Genetics 30, 73-76.[Medline]

Deshmane, S. L. & Fraser, N. W. (1989). During latency, herpes simplex virus type 1 DNA is associated with nucleosomes in a chromatin structure. Journal of Virology 63, 943-947.[Medline]

Deshmane, S. L., Raengsakulrach, B., Berson, J. F. & Fraser, N. W. (1995). The replicating intermediates of herpes simplex virus type 1 DNA are relatively short. Journal of NeuroVirology 1, 165-176.[Medline]

Efstathiou, S., Minson, A. C., Field, H. J., Anderson, J. R. & Wildy, P. (1986). Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans. Journal of Virology 57, 446-455.[Medline]

Garber, D. A., Beverly, S. M. & Coen, D. M. (1993). Demonstration of circularization of herpes simplex virus DNA following infection using pulse field gel electrophoresis. Virology 197, 459-462.[Medline]

Greenberg, M. E. & Bender, T. P. (1997). Nuclei isolation by Dounce homogenizer. In Current Protocols in Molecular Biology, pp. 4.10.4–4.10.8. Edited by F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith & K. Struhl. New York: John Wiley & Sons.

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.[Abstract]

Hanawalt, P. C., Gee, P., Ho, L., Hsu, R. K. & Kane, C. J. M. (1992). Genomic heterogeneity of DNA repair. Role in aging? Annals of the New York Academy of Sciences 663, 17-25.[Abstract]

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.[Abstract]

Jacob, R. J. & Roizman, B. (1977). Anatomy of herpes simplex virus DNA. VIII. Properties of the replicating DNA. Journal of Virology 23, 394-411.[Medline]

Jacob, R. J., Morse, L. S. & Roizman, B. (1979). Anatomy of herpes simplex virus DNA. XII. Accumulation of head-to-tail concatemers in nuclei of infected cells and their role in the generation of the four isomeric arrangements of viral DNA. Journal of Virology 29, 448-457.[Medline]

Jamieson, D. R. S., Robinson, L. H., Daksis, J. I., Nicholl, M. J. & Preston, C. M. (1995). Quiescent viral genomes in human fibroblasts after infection with herpes simplex virus type 1 Vmw65 mutants. Journal of General Virology 76, 1417-1431.[Abstract]

Jensen, I. & Linn, S. (1988). A reduced rate of bulky DNA adduct removal is coincident with differentiation of human neuroblastoma cells induced by nerve growth factor. Molecular and Cellular Biology 8, 3964-3968.[Medline]

Jenuwein, T. & Allis, D. (2001). Translating the histone code. Science 293, 1074-1080.[Abstract/Free Full Text]

Lechardeur, D., Sohn, K. J., Haardt, M., Joshi, P. B., Monck, M., Graham, R. W., Beatty, B., Squire, H., O’Brodovich, H. & Lukacs, G. L. (1999). Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Therapy 6, 482-497.[Medline]

Loyter, A., Scangos, G. A. & Ruddle, F. H. (1982). Mechanisms of DNA uptake by mammalian cells monitored by the use of fluorescent dyes. Proceedings of the National Academy of Sciences, USA 79, 422-426.[Abstract]

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.[Medline]

Mellerick, D. M. & Fraser, N. W. (1987). Physical state of the latent herpes simplex virus genome in a mouse model: evidence for an episomal state. Virology 158, 265-275.[Medline]

Norton, P. A. (2000). Introduction of DNA into cultured mammalian cells. In Gene Transfer Methods: Introducing DNA into Living Cells and Organisms , pp. 65-91. Edited by P. A. Norton & L. F. Steel. Natick, MA:Eaton Publishing.

Nouspikel, T. & Hanawalt, P. C. (2000). Terminally differentiated human neurons repair transcribed genes but display attenuated global DNA repair and modulation of repair gene expression. Molecular and Cellular Biology 20, 1562-1570.[Abstract/Free Full Text]

Peters, A. H. F. M., Mermoud, J. E., O’Carroll, D., Pagani, M., Schweizer, D., Brockdorff, N. & Jenuwein, T. (2002). Histone H3 lysine 9 methylation is an epigenetic imprint of facultative heterochromatin. Nature Genetics 30, 7780.

Poffenberger, K. L. & Roizman, B. (1985). A noninverting genome of a viable herpes simplex virus 1: presence of head-to-tail linkages in packaged genomes and requirements for circularization after infection. Journal of Virology 53, 587-595.[Medline]

Rock, D. & Fraser, N. W. (1983). Detection of HSV-1 DNA in central nervous systems of latently infected mice. Nature 302, 523-525.[Medline]

Roizman, B. (1979). The structure and isomerization of herpes simplex virus genomes. Cell 16, 481-494.[Medline]

Severini, A., Morgan, A. R., Tovell, D. R. & Tyrrel, D. L. J. (1994). Study of the structure of replicative intermediates of HSV-1 DNA by pulsed-field gel electrophoresis. Virology 200, 428-435.[Medline]

Su, Y.-H., Meegalla, R., Chowhan, R., Lausch, R. N., Cubitt, C., Oakes, J. E., Fraser, N. W. & Block, T. M. (1999). Human corneal cells and other fibroblasts can induce reactivation of herpes simplex virus from latently infected PC12 cells. Journal of Virology 73, 4171-4180.[Abstract/Free Full Text]

Su, Y.-H., Moxley, M., Kejariwal, R., Mehta, A., Fraser, N. W. & Block, T. M. (2000). The HSV 1 genome in quiescently infected NGGF differentiated PC12 cells can not be stimulated by HSV superinfection. Journal of NeuroVirology 6, 341-349.[Medline]

Umene, K. & Nishimoto, T. (1996). Replication of herpes simplex virus type 1 DNA is inhibited in a temperature-sensitive mutant of BHK-21 cells lacking RCC-1 (regulator of chromosome condensation) and virus DNA remains linear. Journal of General Virology 77, 2261-2270.[Abstract]

Wigdahl, B. L., Scheck, A. C., Ziegler, R. J., Declercq, E. & Rapp, F. (1984). Analysis of the herpes simplex virus genome during latency in human diploid fibroblasts and rat sensory neurons. Journal of Virology 49, 205-213.[Medline]

Wilcox, C. L., Smith, R. L., Everett, R. D. & Mysofski, D. (1997). The herpes simplex virus type 1 immediate-early protein ICP0 is necessary for the establishment of latent infection. Journal of Virology 71, 6777-6785.[Abstract]

Received 22 May 2002; accepted 12 August 2002.