Gamma interferon can prevent herpes simplex virus type 1 reactivation from latency in sensory neurons

We recently demonstrated that CD8(+) T cells could block herpes simplex virus type 1 (HSV-1) reactivation from latency in ex vivo trigeminal ganglion (TG) cultures without destroying the infected neurons. Here we establish that CD8(+) T-cell prevention of HSV-1 reactivation from latency is mediated at least in part by gamma interferon (IFN-gamma). We demonstrate that IFN-gamma was produced in ex vivo cultures of dissociated latently infected TG by CD8(+) T cells that were present in the TG at the time of excision. Depletion of CD8(+) T cells or neutralization of IFN-gamma significantly enhanced the rate of HSV-1 reactivation from latency in TG cultures. When TG cultures were treated with acyclovir for 4 days to insure uniform latency, supplementation with recombinant IFN-gamma blocked HSV-1 reactivation in 80% of cultures when endogenous CD8(+) T cells were present and significantly reduced and delayed HSV-1 reactivation when CD8(+) T cells or CD45(+) cells were depleted from the TG cultures. The effectiveness of recombinant IFN-gamma in blocking HSV-1 reactivation was lost when its addition to TG cultures was delayed by more than 24 h after acyclovir removal. We propose that when the intrinsic ability of neurons to inhibit HSV-1 gene expression is compromised, HSV-specific CD8(+) T cells are rapidly mobilized to produce IFN-gamma and perhaps other antiviral cytokines that block the viral replication cycle and maintain the viral genome in a latent state.     

Nerve growth factor deprivation results in the reactivation of latent herpes simplex virus in vitro.

Primary sympathetic neuronal cultures were maintained for up to 5 weeks after inoculation with herpes simplex virus (HSV) without evidence of viral infection. Upon deprivation of nerve growth factor, the cultures produced infectious HSV, indicating that the cultures harbored latent HSV. This study demonstrates a function of nerve growth factor in the maintenance of HSV latency.     

Encephalitis resulting from reactivation of latent herpes simplex virus in mice.

Herpes simplex virus (HSV) encephalitis was produced in mice from reactivation of latent virus. Two experimental models were used: the trigeminal model after corneal inoculation of HSV, and the hypoglossal model after tongue inoculation of HSV. In the trigeminal model, cyclophosphamide treatment induced reactivation of latent virus in ganglia but not in central nervous system tissue. Spread of the reactivated virus from ganglia to brain occurred only in mice deficient in anti-HSV antibody. In the hypoglossal model, sectioning of the hypoglossal nerve provoked chromatolysis in the corresponding central nervous system motor neurons and occasionally reactivated latent HSV in the brains of mice. These results suggest that HSV encephalitis can result from the spread of reactivated virus from ganglia to brain and also from in situ reactivation in brain.     

Herpes simplex virus type 1 immediate-early protein Vmw110 reactivates latent herpes simplex virus type 2 in an in vitro latency system.

Reactivation of latent herpes simplex virus type 2 (HSV-2) by the immediate-early protein Vmw110 was studied by using an in vitro latency system. Adenovirus recombinants that express Vmw110 reactivated latent HSV-2. An HSV-1 mutant possessing a deletion in a carboxy-terminal region of Vmw110 reactivated latent HSV-2, whereas mutant FXE, which has a deletion in the second exon, did not. Therefore, Vmw110 alone is required to reactivate latent HSV-2 in vitro, and the region of Vmw110 defined by the deletion in FXE is important for this process.     

Role for gamma interferon in control of herpes simplex virus type 1 reactivation

Observation of chronic inflammatory cells and associated high-level gamma interferon (IFN-gamma) production in ganglia during herpes simplex type 1 (HSV-1) latent infection in mice (E. M. Cantin, D. R. Hinton, J. Chen, and H. Openshaw, J. Virol. 69:4898-4905, 1995) prompted studies to determine a role of IFN-gamma in maintaining latency. Mice lacking IFN-gamma (GKO mice) or the IFN-gamma receptor (RGKO mice) were inoculated with HSV-1, and the course of the infection was compared with that in IFN-gamma-competent mice with the same genetic background (129/Sv//Ev mice). A time course study showed no significant difference in trigeminal ganglionic viral titers or the timing of establishment of latency. Spontaneous reactivation resulting in infectious virus in the ganglion did not occur during latency in any of the mice. However, 24 h after the application of hyperthermic stress to mice, HSV-1 antigens were detected in multiple neurons in the null mutant mice but in only a single neuron in the 129/Sv//Ev control mice. Mononuclear inflammatory cells clustered tightly around these reactivating neurons, and by 48 h, immunostaining was present in satellite cells as well. The incidence of hyperthermia-induced reactivation as determined by recovery of infectious virus from ganglia was significantly higher in the null mutant than in control mice: 11% in 129/Sv//Ev controls, 50% in GKO mice (P = 0.0002), and 33% in RGKO mice (P = 0.03). We concluded that IFN-gamma is not involved in the induction of reactivation but rather contributes to rapid suppression of HSV once it is reactivated.     

Efficient reactivation of latent herpes simplex virus from mouse central nervous system tissues

For decades, numerous ex vivo studies have documented that latent herpes simplex virus (HSV) reactivates efficiently from ganglia, but rarely from the central nervous systems (CNS), of mice when assayed by mincing tissues before explant culture, despite the presence of viral genomes in both sites. Here we show that 88% of mouse brain stems reactivated latent virus when they were dissociated into cell suspensions before ex vivo explant culture. The efficient reactivation of HSV from the mouse CNS was demonstrated with more than one viral strain, viral serotype, and mouse strain, further indicating that the CNS can be an authentic latency site for HSV with the potential to cause recurrent disease.     

Involvement of cellular death signals in the reactivation of herpes simplex virus type 1 and lambda bacteriophage from a latent state

Herpes simplex virus (HSV) 1 has adapted to the human host through two modes of infection, the acute-transient infection that may cause diseases (such as encephalitis) and the latent state, which is a source for recurrent infection and disease. While much information has been gathered on the cellular and molecular concomitants of establishment and maintenance of HSV-1 latent state, the biological basis of viral reactivation is still unclear. Despite their obvious differences, HSV-1 and the bacterial temperate virus, the bacteriophage lambda, shares four distinct features that may help understand the viral latency phenomenon: (i) two modes of life cycle and a decision point to choose either latency (HSV-1) and lysogeny (bacteriophage lambda), or active replication, that results in cell destruction, (ii) establishment of lysogeny/latency of the respective virus is associated with protection from cell death, (iii) immunity/resistance to super-infection, (iv) agents that trigger mammalian and bacterial cell death also induce reactivation of both HSV-1 and lambda bacteriophage. Thus, despite their differences, these two viruses might display analogous mechanism(s) of reactivation. Based on clinical and experimental data, we propose in this hypothesis that while HSV-1 latency, like bacteriophage lambda lysogeny, is associated with protection from cell death and restriction to super-infection, viral reactivation from the latent state is triggered by exogenous stress signals that interfere with cellular viability and may eventually lead to cell death.     

Involvement of an early human cytomegalovirus function in reactivation of quiescent herpes simplex virus type 2

We have previously described an in vitro system in which the function lacking for herpes simplex virus type 2 (HSV-2) replication can be induced by human cytomegalovirus (HCMV). The mechanism of this reactivation of quiescent HSV-2 by HCMV has been further defined. The HCMV function(s) responsible for HSV-2 stimulation was examined temporally, and the fraction of cells in quiescent cultures producing HSV-2 after superinfection was determined. Using independent biological, genetic and molecular techniques we have made the following observations. (i) As early as 12 h after HCMV superinfection, HSV-2 RNA was expressed in latently infected cells. (ii) At 24 h after HCMV superinfection, a time when newly synthesized HCMV was not yet apparent, infectious HSV-2 was produced by reactivated cultures. (iii) Four HCMV temperature-sensitive mutants, which are DNA-negative at nonpermissive temperature and represent four different complementation groups, induced reactivation of HSV-2 at 39.5 degrees C. (iv) Early after HCMV superinfection, 1.6% of quiescent cells could be induced to transcribe HSV-2 information. (v) Early after HCMV superinfection, 0.3% of cells in the quiescent cultures could be induced to yield infectious HSV-2. The finding that a significant interaction can occur between HCMV and quiescent HSV-2 in an in vitro model is noteworthy in light of the knowledge that both of these herpesviruses often reside simultaneously in the human host.     

In vitro nuclear egress of herpes simplex virus type 1 capsids

During their life cycles, viruses typically undergo many transport events throughout the cell. These events depend on a variety of both viral and host proteins and are often not fully understood. Such studies are often complicated by asynchronous infections and the concurrent presence of various viral intermediates in the cells, making it difficult to molecularly define each step. In the case of the herpes simplex virus type 1, the etiological agent of cold sores and many other illnesses, the viral particles undergo an intricate series of transport steps during its life cycle. Upon entry by fusion with a cellular membrane, they travel to the host cell nucleus where the virus replicates and assembles new viral particles. These particles then travel across the two nuclear envelopes and transit through the trans-Golgi network before finally being transported to and released at the cell surface. Though viral components and some host proteins modulating these numerous transport events have been identified, the details of these processes remain to be elucidated. To specifically address how the virus escapes the nucleus, we set up an in vitro model that reproduces the unconventional route used by herpes simplex type 1 virus to leave nuclei. This has not only allowed us to clarify the route of capsid egress of the virus but is now useful to define it at the molecular level.     

Reconstitution of herpes simplex virus type 1 nuclear capsid egress in vitro

Newly assembled herpesvirus capsids travel from the nucleus to the plasma membrane by a mechanism that is poorly understood. Furthermore, the contribution of cellular proteins to this egress has yet to be clarified. To address these issues, an in vitro nuclear egress assay that reproduces the exit of herpes simplex virus type 1 (HSV-1) capsids from nuclei isolated from infected cells was established. As expected, the assay has all the hallmarks of intracellular transport assays, namely, a dependence on time, energy, and temperature. Surprisingly, it is also dependent on cytosol and was slightly enhanced by infected cytosol, suggesting an implication of both host and viral proteins in the process. The capsids escaped these nuclei by budding through the inner nuclear membrane, accumulated as enveloped capsids between the two nuclear membranes, and were released in cytosol exclusively as naked capsids, exactly as in intact cells. This is most consistent with the view that the virus escapes by crossing the two nuclear membranes rather than through nuclear pores. Unexpectedly, nuclei isolated at the nonpermissive temperature from cells infected with a U(L)26 thermosensitive protease mutant (V701) supported capsid egress. Although electron microscopy, biochemical, and PCR analyses hinted at a likely reconstitution of capsid maturation, DNA encapsidation could not be confirmed by a traditional SQ test. This assay should prove very useful for identification of the molecular players involved in HSV-1 nuclear egress.     

Relaxed repression of herpes simplex virus type 1 genomes in Murine trigeminal neurons

The expression of herpes simplex virus (HSV) genomes in the absence of viral regulatory proteins in sensory neurons is poorly understood. Previously, our group reported an HSV immediate early (IE) mutant (d109) unable to express any of the five IE genes and encoding a model human cytomegalovirus immediate early promoter-green fluorescent protein (GFP) transgene. In cultured cells, GFP expressed from this mutant was observed in only a subset of infected cells. The subset exhibited cell type dependence, as the fractions of GFP-expressing cells varied widely among the cell types examined. Herein, we characterize this mutant in murine embryonic trigeminal ganglion (TG) cultures. We found that d109 was nontoxic to neural cultures and persisted in the cultures throughout their life spans. Unlike with some of the cultured cell lines and strains, expression of the GFP transgene was observed in a surprisingly large subset of neurons. However, very few nonneuronal cells expressed GFP. The abilities of ICP0 and an inhibitor of histone deacetylase, trichostatin A (TSA), to activate GFP expression from nonexpressing cells were also compared. The provision of ICP0 by infection with d105 reactivated quiescent genomes in nearly every cell, whereas reactivation by TSA was much more limited and restricted to the previously nonexpressing neurons. Moreover, we found that d109, which does not express ICP0, consistently reactivated HSV type 1 (KOS) in latently infected adult TG cultures. These results suggest that the state of persisting HSV genomes in some TG neurons may be more dynamic and more easily activated than has been observed with nonneuronal cells.     

Ribonucleotide reductase of herpes simplex virus type 2 resembles that of herpes simplex virus type 1.

The ribonucleotide reductase (ribonucleoside-diphosphate reductase; EC 1.17.4.1) induced by herpes simplex virus type 2 infection of serum-starved BHK-21 cells was purified to provide a preparation practically free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could significantly deplete the substrates. Certain key properties of the herpes simplex virus type 2 ribonucleotide reductase were examined to define the extent to which it resembled the herpes simplex virus type 1 ribonucleotide reductase. The herpes simplex virus type 2 ribonucleotide reductase was inhibited by ATP and MgCl2 but only weakly inhibited by the ATP X Mg complex. Deoxynucleoside triphosphates were at best only weak inhibitors of this enzyme. ADP was a competitive inhibitor (K'i, 11 microM) of CDP reduction (K'm, 0.5 microM), and CDP was a competitive inhibitor (K'i, 0.4 microM) of ADP reduction (K'm, 8 microM). These key properties closely resemble those observed for similarly purified herpes simplex virus type 1 ribonucleotide reductase and serve to distinguish these virally induced enzymes from other ribonucleotide reductases.     

Entry of herpes simplex virus type 1 into primary sensory neurons in vitro is mediated by Nectin-1/HveC

Primary cultures of rat and mouse sensory neurons were used to study the entry of herpes simplex virus type 1 (HSV-1). Soluble, truncated nectin-1 but not HveA prevented viral entry. Antibodies against nectin-1 also blocked infection of rat neurons. These results indicate that nectin-1 is the primary receptor for HSV-1 infection of sensory neurons.     

Detection of multiple strains of latent herpes simplex virus type 1 within individual human hosts

One hundred and fifteen isolates of herpes simplex virus were recovered from parallel explant cultures of trigeminal and vagus ganglia and trigeminal nerve roots derived from 20 unselected human cadavers. Restriction enzyme patterns of strains recovered from 18 of 20 individuals could be differentiated from individual to individual, although all isolates from a single host were identical. Isolates from two individuals differed among themselves in the number and location of certain restriction enzyme sites.