CD8 T cell control of HSV reactivation from latency is abrogated by viral inhibition of MHC class I

In humans, herpes simplex virus (HSV) establishes latency in sensory nerve ganglia from where it periodically reactivates, whereas in murine models, the virus efficiently establishes latency but rarely reactivates. HSV inhibits MHC class I antigen presentation to CD8 T cells efficiently in humans but poorly in mice, and whether this is a crucial determinant of HSV's ability to reactivate in humans remains uncertain. To test this, we generated a panel of recombinant HSVs that inhibit presentation by murine MHC class I mimicking the effect in humans. Antigen-specific CD8 T cells prevent the in vivo reactivation of wild-type HSV. Despite their presence in the ganglia of latently infected mice, CD8 T cells do not prevent the reactivation of recombinant HSVs that inhibit murine MHC class I in mice. These findings suggest that efficient inhibition of MHC class I by HSV is a key factor in its ability to reactivate in humans.     

Localization of sequences in a protein (ORF2) encoded by the latency-related gene of bovine herpesvirus 1 that inhibits apoptosis and interferes with Notch1-mediated trans-activation of the bICP0 promoter

Bovine herpesvirus 1 (BHV-1) infection induces clinical symptoms in the upper respiratory tract, inhibits immune responses, and can result in life-threatening secondary bacterial infections. Following acute infection, BHV-1 establishes latency in sensory neurons within trigeminal ganglia. Periodically, reactivation from latency occurs, resulting in virus transmission. The latency-related (LR) RNA is abundantly expressed in latently infected sensory neurons, suggesting that LR gene products regulate the latency-reactivation cycle. An LR mutant virus with stop codons at the amino terminus of the first open reading frame (ORF) in the LR gene (ORF2) does not reactivate from latency, in part because it induces higher levels of apoptosis in infected neurons. ORF2 inhibits apoptosis in transiently transfected cells, suggesting that it plays an important role in the latency-reactivation cycle. ORF2 also interacts with Notch1 or Notch3 and consequently inhibits their ability to trans-activate the bICP0 early and glycoprotein C promoters. In this study, we identified ORF2 sequences that were necessary for inhibiting cold shock-induced apoptosis or Notch1-mediated trans-activation of the bICP0 early promoter and stimulation of productive infection. Relative to ORF2 sequences necessary for inhibiting apoptosis, distinct domains in ORF2 were important for interfering with Notch1-mediated trans-activation. Five consensus protein kinase A and/or protein kinase C phosphorylation sites within ORF2 regulate the steady-state levels of ORF2 in transfected cells. A nuclear localization signal in ORF2 was necessary for inhibiting Notch1-mediated trans-activation but not apoptosis. In summary, ORF2 has more than one functional domain that regulates its stability and functional properties.     

Failure of thymidine kinase-negative herpes simplex virus to reactivate from latency following efficient establishment

Thymidine kinase-negative mutants of herpes simplex virus did not reactivate from latency in mouse trigeminal ganglia, even when their latent viral loads were comparable to those that permitted reactivation by wild-type virus. Thus, reduced establishment of latency does not suffice to account for the failure to reactivate.     

PML‐NB‐dependent type I interferon memory results in a restricted form of HSV latency

Herpes simplex virus (HSV) establishes latent infection in long‐lived neurons. During initial infection, neurons are exposed to multiple inflammatory cytokines but the effects of immune signaling on the nature of HSV latency are unknown. We show that initial infection of primary murine neurons in the presence of type I interferon (IFN) results in a form of latency that is restricted for reactivation. We also find that the subnuclear condensates, promyelocytic leukemia nuclear bodies (PML‐NBs), are absent from primary sympathetic and sensory neurons but form with type I IFN treatment and persist even when IFN signaling resolves. HSV‐1 genomes colocalize with PML‐NBs throughout a latent infection of neurons only when type I IFN is present during initial infection. Depletion of PML prior to or following infection does not impact the establishment latency; however, it does rescue the ability of HSV to reactivate from IFN‐treated neurons. This study demonstrates that viral genomes possess a memory of the IFN response during de novo infection, which results in differential subnuclear positioning and ultimately restricts the ability of genomes to reactivate.     

Induction of reactivation of herpes simplex virus in murine sensory ganglia in vivo by cadmium.

Herpes simplex viruses maintained in a latent state in sensory neurons in mice do not reactivate spontaneously, and therefore the factors or procedures which cause the virus to reactivate serve as a clue to the mechanisms by which the virus is maintained in a latent state. We report that cadmium sulfate induces latent virus to reactivate in 75 to 100% of mice tested. The following specific findings are reported. (i) The highest frequency of induction was observed after two to four daily administrations of 100 micrograms of cadmium sulfate. (ii) Zinc, copper, manganese, or nickel sulfate administered in equimolar amounts under the same regimen did not induce viral reactivation; however, zinc sulfate in molar ratios 25-fold greater than those of cadmium induced viral replication in 2 of 16 ganglia tested. (iii) Administration of zinc, nickel, or manganese prior to the cadmium sulfate reduced the incidence of ganglia containing infectious virus. (iv) Administration of cadmium daily during the first week after infection and at 2-day intervals to 13 days after infection resulted in the recovery from ganglia of infectious virus in titers 10- to 100-fold higher than those obtained from animals given saline. Moreover, infectious virus was recovered as late as 11 days after infection compared with 6 days in mice administered saline. (v) Administration of cadmium immediately after infection or repeatedly after establishment of latency did not exhaust the latent virus harbored by sensory neurons, inasmuch as the fraction of ganglia of mice administered cadmium and yielding infectious virus was similar to that observed in mice treated with saline. We conclude that induction of cadmium tolerance precludes reactivation of latent virus. If the induction of metallothionein genes was the sole factor required to cause reactivation of latent virus, it would have been expected that all metals which induce metallothioneins would also induce reactivation, which was not observed. The results therefore raise the possibility that in addition to inducing the metallothionein genes, cadmium inactivates the factors which maintain the virus in latent state.     

Expression of the herpes simplex virus 1 alpha transinducing factor (VP16) does not induce reactivation of latent virus or prevent the establishment of latency in mice.

A feature of the cascade regulation of herpes simplex virus 1 gene expression in productive infection is that the first genes to be expressed, the alpha genes, are transactivated by a structural component of the virion designated as the alpha transinducing factor (alpha TIF). In this study, we have tested the hypothesis that latent infection of sensory neurons results from the failure of alpha TIF, a tegument protein, to be transported from the nerve endings to the nucleus of the sensory neuron. Two viruses were constructed. The first recombinant virus (R6003) contained a second copy of the alpha TIF gene placed under the control of a metallothionein promoter. The second recombinant virus (R6004) is identical to R6003 except for the presence of a stop codon inserted at amino acid 70 of the second alpha TIF gene. The metallothionein promoter inserted into the viral genome was shown to be expressed, and alpha TIF mRNA was detected by in situ hybridization of sections of trigeminal ganglia of mice infected with R6003, both untreated and those given cadmium injections. In all experiments, there were no significant differences in the recovery of latent virus from mice infected with R6003 or R6004, whether injected with cadmium or not. Cadmium administration at the time of infection and at intervals thereafter did not preclude establishment of latency. In another series of experiments, transgenic mice expressing the metallothionein-driven alpha TIF did not differ from nontransgenic siblings with respect to the incidence of latent virus in trigeminal ganglia. We conclude that the absence of alpha TIF cannot alone account for the establishment of latency.     

水痘-带状疱疹病毒的基因分型与分子进化

水痘-带状疱疹病毒(Varicella-zoster virus,VZV)又称人类疱疹病毒3型,属疱疹病毒科,与单纯疱疹病毒HSV-1、HSV-2一起归入α亚科。人类是其唯一的自然宿主,对其普遍易感。VZV引起的原发感染表现为水痘,并在宿主的感觉神经节内潜伏,再激活时可引起带状疱疹。近年来VZV分子流行病学的研究涉及流行病学、病毒学、生物信息学等相关领域,通过监测、研究VZV的基因变异,区分疫苗株或野生株引起的感染,探讨世界范围内各VZV病毒株的系统发育关系和各遗传支之间的分子进化史。现将近年来有关VZV不同的地理分布和遗传支进化的研究状况综述如下。     

Reactivation of latent herpes simplex virus by adenovirus recombinants encoding mutant IE-0 gene products.

We have previously shown that adenovirus recombinants expressing functional ICP0 reactivate latent herpes simplex virus type 2 (HSV-2) in an in vitro latency system. This study demonstrated that ICP0, independent of other HSV gene products, is sufficient to reactivate latent HSV-2 in this in vitro system. To assess the effects of defined mutations in the sequence encoding ICP0 (IE-0) on reactivation, seven in-frame insertion and three in-frame deletion mutants were moved into an adenovirus expression vector. Each recombinant directed the synthesis of stable ICP0 of the correct size. The transactivation activity of the mutated sequences in these recombinants was similar to that when they were tested in plasmids. When these recombinants were examined for their ability to reactivate in the in vitro latency system, mutants with dramatic defects in transactivation (Ad-0/125, Ad-0/89, Ad-0/2/7, and Ad-0/88/93) were unable to reactivate latent HSV-2 independent of the multiplicity of infection. An exception to this correlation was the finding that Ad-0/89, which transactivated poorly, was able to reactivate latent virus after prolonged incubation whereas other transactivation-deficient mutants could not. Moreover, the presence of ICP4 did not compensate for the inability of any of the recombinants tested to reactivate HSV-2. These results show that (i) the transactivation domains of ICP0 are also used in reactivation, (ii) the presence of another essential HSV regulatory protein ICP4 does not alter the pattern of reactivation by ICP0, and (iii) mutations in some regions of IE-0 previously shown to affect viral growth and plaque formation did not alter its ability to reactivate in this in vitro system.     

Gamma interferon blocks gammaherpesvirus reactivation from latency in a cell type-specific manner

Gammaherpesviruses are important pathogens whose lifelong survival in the host depends critically on their capacity to establish and reactivate from latency, processes regulated by both viral genes and the host immune response. Previous work has demonstrated that gamma interferon (IFN-gamma) is a key regulator of chronic infection with murine gammaherpesvirus 68 (gammaHV68), a virus that establishes latent infection in B lymphocytes, macrophages, and dendritic cells. In mice deficient in IFN-gamma or the IFN-gamma receptor, gammaHV68 gene expression is altered during chronic infection, and peritoneal cells explanted from these mice reactivate more efficiently ex vivo than cells derived from wild-type mice. Furthermore, treatment with IFN-gamma inhibits reactivation of gammaHV68 from latently infected wild-type peritoneal cells, and depletion of IFN-gamma from wild-type mice increases the efficiency of reactivation of explanted peritoneal cells. These profound effects of IFN-gamma on chronic gammaHV68 latency and reactivation raise the question of which cells respond to IFN-gamma to control chronic gammaHV68 infection. Here, we show that IFN-gamma inhibited reactivation of peritoneal cells and spleen cells harvested from mice lacking B lymphocytes, but not wild-type spleen cells, suggesting that IFN-gamma may inhibit reactivation in a cell type-specific manner. To directly test this hypothesis, we expressed the diphtheria toxin receptor specifically on either B lymphocytes or macrophages and used diphtheria toxin treatment to deplete these specific cells in vivo and in vitro after establishing latency. We demonstrate that macrophages, but not B cells, are responsive to IFN-gamma-mediated suppression of gammaHV68 reactivation. These data indicate that the regulation of gammaherpesvirus latency by IFN-gamma is cell type specific and raise the possibility that cell type-specific immune deficiency may alter latency in distinct and important ways.     

Inhibition of H3K27me3-Specific Histone Demethylases JMJD3 and UTX Blocks Reactivation of Herpes Simplex Virus 1 in Trigeminal Ganglion Neurons

Herpes simplex virus 1 (HSV-1) genomes are associated with the repressive heterochromatic marks H3K9me2/me3 and H3K27me3 during latency. Previous studies have demonstrated that inhibitors of H3K9me2/me3 histone demethylases reduce the ability of HSV-1 to reactivate from latency. Here we demonstrate that GSK-J4, a specific inhibitor of the H3K27me3 histone demethylases UTX and JMJD3, inhibits HSV-1 reactivation from sensory neurons in vitro. These results indicate that removal of the H3K27me3 mark plays a key role in HSV-1 reactivation.     

Immediate-early regulatory gene mutants define different stages in the establishment and reactivation of herpes simplex virus latency.

Using nonsense and deletion mutants of herpes simplex virus type 1, we investigated the roles of three immediate-early proteins (ICP4, ICP27 and ICP0) in the establishment and reactivation of ganglionic latency in a mouse ocular model. DNA hybridization, superinfection-rescue, and cocultivation techniques provided quantitative data that distinguished between the failure of a virus to establish latency in the ganglion and its failure to reactivate. Null mutants with lesions in the genes for ICP4 and ICP27 did not replicate in the eye or in ganglia and failed to establish reactivatable latent infections. Three ICP0 deletion mutants which could replicate in the eye and ganglia varied in their ability to establish and reactivate from the latent state, demonstrating that ICP0 plays a role both in the establishment and the reactivation of latency. The use of viral mutants and a variety of stage-specific assays allowed us to better define the stages in the establishment and reactivation of herpes simplex virus type 1 latency.     

Tracing of sensory neurones and spinal motoneurones of the pigeon by injection of fluorescent dyes into peripheral nerves

The projection of peripheral sensory and motor nerves was investigated in the pigeon (Columba livia) by means of retrogradely transported fluorescent dyes. Two combinations of fluorescent tracers were used that could be identified within the same cell when excited by light of 405 nm: 1) Propidium iodide and Bisbenzimide, which label the cytoplasm orange and the nucleus blue, respectively; 2) Fast Blue, which labels the cytoplasm blue, and Nuclear Yellow, which labels the nucleus (especially the nucleolar ring) yellow. The presence of the tracers in a given cell was confirmed microspectrophotometrically. Following injection of the tracers into peripheral nerves, labelled sensory neurones were seen in the dorsal root ganglia and motoneurones of the spinal cord. The peroneal and tibial nerves projected to L2-L5 and L2-L7, respectively, whereas the median and ulnar nerves projected to C12-Th2 and C13-Th1. Double-labelled sensory neurones were observed when both peroneal and tibial, or median and ulnar nerves were injected with different tracers. This indicates that some sensory neurones possess peripheral processes that dichotomize to pass down two different peripheral nerves. Double labelling was never seen in motoneurones, or in sensory neurones after tracer injection into the sciatic and femoral nerves.     

The perineuronal net and the control of CNS plasticity

Perineuronal nets (PNNs) are reticular structures that surround the cell body of many neurones, and extend along their dendrites. They are considered to be a specialized extracellular matrix in the central nervous system (CNS). PNN formation is first detected relatively late in development, as the mature synaptic circuitry of the CNS is established and stabilized. Its unique distribution in different CNS regions, the timing of its establishment, and the changes it undergoes after injury all point toward diverse and important functions that it may be performing. The involvement of PNNs in neuronal plasticity has been extensively studied over recent years, with developmental, behavioural, and functional correlations. In this review, we will first briefly detail the structure and organization of PNNs, before focusing our discussion on their unique roles in neuronal development and plasticity. The PNN is an important regulator of CNS plasticity, both during development and into adulthood. Production of critical PNN components is often triggered by appropriate sensory experiences during early postnatal development. PNN deposition around neurones helps to stabilize the established neuronal connections, and to restrict the plastic changes due to future experiences within the CNS. Disruption of PNNs can reactivate plasticity in many CNSs, allowing activity-dependent changes to once again modify neuronal connections. The mechanisms through which PNNs restrict CNS plasticity remain unclear, although recent advances promise to shed additional light on this important subject.