The molecular mechanisms directing the reactivation of the latent HSV genome remain unknown. A detailed knowledge of the timing and progression of primary reactivation in the ganglia is required to rationally explore these mechanisms. Only by understanding the similarities and differences among models of reactivation will it be possible to develop a cohesive experimental effort among groups working toward understanding this process. In this report, we characterize the reactivation of latent HSV in ganglia in two widely used model systems, comparing the timing of infectious virus production, the quantity of virus produced, and the number of neurons undergoing reactivation. The extent and similarity of physiological changes in the ganglion resulting from the different induction triggers utilized (hyperthermic stress and axotomy followed by explant) were also evaluated. The results of our study provide essential baseline information to facilitate both the interpretation of past studies of HSV reactivation and the design for future studies.
Three major points emerged from this study. First, the physiologic state of explanted ganglionic neurons differs extensively from that of neurons in ganglia induced to reactivate in vivo. In explanted ganglia, neurons exhibited marked morphological changes soon after explant, atypical expression and/or localization of most cell cycle- and stress-related proteins examined, and DNA fragmentation in neuronal nuclei. The number of neurons exhibiting these changes increased as the time postexplant increased. Importantly, these changes were also observed in uninfected explanted ganglia and were absent from ganglia undergoing quantitatively similar reactivation in vivo. Thus, we conclude that these changes resulted from axotomy and explant and not from the initiation or progression of virus reactivation.
The unusual expression in neurons of G1
/S-phase protein markers (e.g., cdk2 and cdk4), the cytoplasmic location of cytochrome c
, and TUNEL-positive nuclei following explantation indicate that axotomy followed by explant of the ganglia results in the initiation and progression of apoptosis in neurons. Indeed the observed changes are consistent with other reports detailing the neuronal response to axotomy (1
). Standard culture conditions for ganglion explant were used (9
), and several adjustments to the medium did not significantly alter the outcome.
Second, it is reasonable to anticipate that within a given period of time a greater number of neurons would reactivate in response to the extreme stimulus of axotomy and explant; however, this did not prove to be the case. The profound physiological changes in explanted ganglia did not influence either the number of neurons undergoing reactivation or the number of PFU recovered from ganglia latently infected with wild-type strain 17syn+ or KOS during the first 22 h. In both cases, reactivation occurred only in very rare neurons (one to seven per ganglion with most positive ganglia containing one or two positive neurons). Prior work demonstrates that these ganglia each contained on the order of 6,000 latently infected neurons with a total number of viral genomes in the range of 105
). Thus, reactivation is an exceedingly rare event occurring in fewer than 1 out of 1,000 latently infected cells.
The rarity of reactivation events that we and others have observed in vivo (15
) is consistent with the hypothesis that, at any given time, the latent pool contains a small subset of neurons that are competent to undergo reactivation in response to the appropriate signals. We have found that, despite repeated exposure to hyperthermic stress, the reactivation frequency within a group of mice is maintained through time (unpublished observation). That is, similar numbers of neurons reactivate regardless of whether the animal has been exposed to a reactivation stimulus just once or multiple times over a period of weeks or months. Assuming that neurons that produce infectious virus do not survive (15
), this observation indicates either that there is a mechanism for some latently infected cells to become reactivation competent at any given time or that all latent genomes are equally competent with a very low probability of reactivating.
Most latently infected neurons contain between 1 and 100 HSV genomes, but very rare neurons contain significantly more than 1,000, suggesting that the latent genome copy number may influence reactivation (19
). It may be that there is a threshold of genome copies required for reactivation. In this model some mechanism, perhaps occult replication of the viral genome in latently infected neurons, (19
), results in a few reactivation-competent neurons at any given time. However, our findings do not exclude the alternate hypothesis that every viral genome has an equal, but very low (~1:105
), probability of reentering the lytic pathway. It follows from this hypothesis that 1 out of 10 neurons latently infected with 100 HSV genome copies (1,000 total HSV genomes) would have the same probability of reactivating as would a single neuron containing 1,000 latent viral genomes. In this model there would be no need of a clocking mechanism that would permit only a few neurons to be reactivation competent at any given time. Neither the nature of “competency” nor the mechanism by which this occurs is understood. Although a role for viral genome copy number is an attractive hypothesis (19
) and there are data consistent with this hypothesis (23
), the relationship between copy number and reactivation competency has yet to be fully delineated.
Regardless of mechanism, the finding that this low number of reactivating neurons is initially unaltered by the physiological changes in explanted neurons indicates that the profound changes occurring within the first 22 h after axotomy and explant were not sufficient to overcome the preexisting barriers to reactivation. Indeed, were these changes precipitating events in reactivation one would predict that more neurons would have initiated reactivation in the explanted ganglia during the first 22 h, and this was not the case. It is therefore clear that, from the aspect of host cell signals, explant is an unnecessarily complex environment in which to dissect the molecular pathway(s) leading to reactivation. Importantly, the complexity of the physiologic changes occurring in explanted ganglionic neurons could obviate the need for certain viral functions required for reactivation in vivo and lead to spurious conclusions about the mechanics of reactivation. We have already identified one example of this type of environmental compensation. A 365-bp region of the viral genome that is required for reactivation in vivo is not required for reactivation in the explant setting (unpublished data).
The third observation of importance was that there was cell-to-cell spread of virus within the ganglia by 36 h postexplant, an event not observed in vivo. Viral proteins were expressed in explanted ganglia in clusters of neurons, satellite cells, and cells along axonal tracts. Consistent with an increased number of infected cells, the infectious virus titer in explanted ganglia increased ~1,000-fold from 22 to 48 h postexplant. It was not possible to directly determine the relative contributions of primary reactivation and subsequent secondary infection to the infectious virus output. Acyclovir was added to the explant cultures to determine if primary reactivation events continue to occur after 22 h postexplant and to distinguish between those neurons undergoing primary reactivation and those infected as a result of intraganglionic spread of virus. There was a steady increase in the number of neurons expressing viral proteins postexplant (Fig. ). In the presence of acyclovir, the number of neurons initiating reactivation increased about 10-fold from 22 to 48 h postexplant. However, in the absence of acyclovir, virus titer increased 1,000-fold, indicating that the majority of virus in the explanted ganglion at 48 h is generated through spread within the ganglion. The absence of full immune function is a possible explanation for this increased viral spread (3
), but it is also possible that explant-induced changes in neurons and/or supporting cells increase the permissivity for HSV replication. Regardless, it is quite clear that, distinct from reactivation in vivo, neurons continue to reactivate in explanted ganglia. However, at times beyond 22 h postexplant, most transcriptional activity and viral DNA replication detected in explanted ganglia are the result of productive infection of additional cells and not the result of primary reactivation events.
Several groups have studied HSV gene transcription and translation in explanted ganglia as a model of reactivation (4
). In some cases it was concluded that the transcriptional program during reactivation differed from that seen during lytic infection or that viral DNA replication was required for immediate-early gene expression. When these data are considered in the context of results presented here, caution should be employed in interpreting these earlier studies. It is likely that the transcriptional events that they were attempting to quantify (occurring after 24 to 48 h in culture) were largely the result of virus spread in the explanted ganglia and not primary reactivation events. It should be emphasized that most investigators monitor reactivation in explanted ganglia by methods requiring release of virus from the explanted tissue, an approach not designed to detect the low levels of virus produced in a very few reactivating neurons within the tissue. The fact that reactivation occurs within 22 h postexplant is not generally appreciated but has been reported previously. Using the more sensitive approach that we have utilized for detecting reactivation in ganglia in vivo, namely, directly grinding the ganglia and plating the homogenate onto indicator cells, Klein detected from 1 to 100 PFU in seven of eight TG latently infected with 17syn+ at 24 h postexplant (8
). In addition, we have shown here that strain KOS also reactivates in this time frame, although with reduced frequency.
We conclude that explant reactivation may provide an ancillary system for selected studies of the early events in reactivation. However, clear signs of neuronal degeneration as early as 2 to 3 h postexplant indicate that there are dramatic additional changes in these ganglia that are not associated with the reactivation process. For example, we found that many neurons in ganglia explanted in vitro expressed cdk2 and cdk4, but this did not occur in ganglia induced to reactivate in vitro. On the basis of similar findings in cultivated ganglia in vitro, Schang et al. concluded that neuronal levels of cdk2 were among the factors that determine the outcome of HSV infections of neurons (26
). Our results suggest that this conclusion may not be relevant to the in vivo setting, since cdk2 was not upregulated following hyperthermic stress. We did not detect cdk4 expression in TG neurons in vivo before or after hyperthermic stress, although it was upregulated following explant. In contrast, Schang et al. reported expression of cdk4 in the cytoplasm of normal neurons in vivo (26
). The reason for this discrepancy is not known, but it might be attributed to the antibodies, fixation, and/or unmasking procedures employed. Regardless, we show here that the nuclear cdk4 expression that we detected in explanted ganglia is not a prerequisite or a precipitating event for reactivation in vivo.
It is also not yet clear in what ways the changes induced in neurons by axotomy and explantation, including neuronal apoptosis demonstrated by cytoplasmic cytochrome c and TUNEL staining, would influence the pathway(s) leading to viral reactivation. Our results suggest that these changes relax the barriers to reactivation and may obviate the need for certain virally encoded functions. While such a system could ultimately provide a fertile experimental field, understanding the basic details of the virus-host interactions resulting in reactivation in the least complex and most relevant setting, i.e., in vivo, appears to be the most direct and best approach to understanding this important question.