Gamma interferon impedes the establishment of herpes simplex virus type 1 latent infection but has no impact on its maintenance or reactivation in mice

Murine models of gamma interferon (IFN-gamma) deficiency demonstrate the role of this cytokine in attenuating acute herpes simplex virus (HSV) disease; however, the effect of IFN-gamma on the establishment and maintenance of neuronal latency and viral reactivation is not known. Using the IFN-gamma knockout (GKO) model of IFN-gamma deficiency and sensitive quantitative PCR methods, we show that IFN-gamma significantly reduces the ganglion content of latent HSV-1 in BALB/c mice, which in turn delays viral time to reactivation following UV irradiation. Similar effects were not seen in the C57BL/6 strain. These results indicate that IFN-gamma significantly attenuates latent HSV infection in the mouse model of ocular infection but has no impact on the maintenance of latency or virus reactivation.     

Influence of galectin-9/Tim-3 interaction on herpes simplex virus-1 latency

After HSV-1 infection, CD8(+) T cells accumulate in the trigeminal ganglion (TG) and participate in the maintenance of latency. However, the mechanisms underlying intermittent virus reactivation are poorly understood. In this study, we demonstrate the role of an inhibitory interaction between T cell Ig and mucin domain-containing molecule 3 (Tim-3)-expressing CD8(+) T cells and galectin 9 (Gal-9) that could influence HSV-1 latency and reactivation. Accordingly, we show that most K(b)-gB tetramer-specific CD8(+) T cells in the TG of HSV-1-infected mice express Tim-3, a molecule that delivers negative signals to CD8(+) T cells upon engagement of its ligand Gal-9. Gal-9 was also upregulated in the TG when replicating virus was present as well during latency. This could set the stage for Gal-9/Tim-3 interaction, and this inhibitory interaction was responsible for reduced CD8(+) T cell effector function in wild-type mice. Additionally, TG cell cultures exposed to recombinant Gal-9 in the latent phase caused apoptosis of most CD8(+) T cells. Furthermore, Gal-9 knockout TG cultures showed delayed and reduced viral reactivation as compared with wild-type cultures, demonstrating the greater efficiency of CD8(+) T cells to inhibit virus reactivation in the absence of Gal-9. Moreover, the addition of recombinant Gal-9 to ex vivo TG cultures induced enhanced viral reactivation compared with untreated controls. Our results demonstrate that the host homeostatic mechanism mediated by Gal-9/Tim-3 interaction on CD8(+) T cells can influence the outcome of HSV-1 latent infection, and manipulating Gal-9 signals might represent therapeutic means to inhibit HSV-1 reactivation from latency.     

单纯疱疹病毒Ⅱ型潜伏、激活细胞模型建立

【目的】建立单纯疱疹病毒Ⅱ型(HSV-2)潜伏感染人神经母细胞瘤细胞株SH-SY5Y及激活的细胞模型。【方法】分别加入20,40,60,80,100,120,140μmol/L的阿昔洛韦(ACV),观察对SH-SY5Y细胞生物性状的影响;在ACV存在的情况下,分别将含有0.1、1、10、100MOI的病毒液接种SH-SY5Y细胞,运用相差显微镜观察病毒对细胞的影响,确定潜伏的建立;分别用41℃、42℃、43℃、44℃、45℃加热0.5h、1.0h、1.5h、2.0h、2.5h,观察加热时间及温度诱导HSV-2在SH-SY5Y细胞中激发的最适条件;加入25、50、75、100、125μmol/L福斯高林(Forskolin)诱导病毒在细胞中激活,探讨诱导的最佳浓度;对HSV-2在SH-SY5Y细胞中的潜伏及激发进行验证并测序;运用相差显微镜观察病毒激活后细胞形态的变化。【结果】60μmol/LACV的存在最适合HSV-2在SH-SY5Y细胞中建立潜伏状态;1-10MOI的感染量均能取得较好的病毒潜伏及激发效果;通过观察,病毒在SH-SY5Y细胞中最长可潜伏14d;43℃,1.5h及75μmol/LForskolin均为诱导病毒潜伏激发的最佳条件;相差显微镜观察病毒激发后细胞病变,从24h到72h,细胞变性、坏死的程度、数量随感染时间延长而增加;HSV-2LAT、gG基因PCR扩增及电泳结果,证实病毒在细胞中的潜伏及激活。【结论】初步在人神经母细胞瘤细胞株SH-SY5Y上建立了HSV-2潜伏感染及激活的细胞模型,为下一步研究HSV-2的潜伏与激发机理,了解HSV-2的致病机制打下基础。     

CD8(+) T-cell attenuation of cutaneous herpes simplex virus infection reduces the average viral copy number of the ensuing latent infection

During an initial encounter with herpes simplex virus type 1 (HSV-1) it takes several days for an adaptive immune response to develop and for herpes-specific CD8(+) T cells to infiltrate sites of infection. By this time the virus has firmly established itself within the innervating sensory nervous system where it then persists indefinitely. Preventing the establishment of viral latency would require blocking the skin to nervous system transmission of the virus. We wished to examine if CD8(+) T cells present early during acute HSV-1 infection could block this transmission. We show that effector CD8(+) T cells failed to prevent the establishment of HSV latency even when present prior to infection. This lack of blocking likely reflects the delayed infiltration of the CD8(+) T cells into the infected skin. Examination of the kinetics of HSV-1 infection highlighted the rapidity at which the virus infects the sensory ganglia and singled out early viral replication within the skin as an important factor in determining the magnitude of the ensuing latent infection. Though unable to prevent the establishment of latency, CD8(+) T cells could reduce the average viral copy number of the residual latent infection by dampening the skin infection and thus limiting the skin-to-nerve transmission of virus.     

Molecular biology of herpes simplex virus type 1 latency in the nervous system

Herpes simplex virus (HSV) is one of the best studied examples of viral ability to remain latent in the human nervous system and to cause recurrent disease by reactivation. Intensive effort was directed in recent years to unveil the molecular viral mechanisms and the virus-host interactions associated with latent HSV infection. The discovery of the state of the latent viral DNA in nervous tissues and of the presence of latency-associated gene expression during latent infection, both differing from the situation during viral replication, provided important clues relevant to the pathogenesis of latent HSV infection. This review summarizes the current state of knowledge on the site of latent infection, the molecular phenomena of latency, and the mechanisms of the various stages of latency: acute infection, establishment and maintenance of latency, and reactivation. This information paved the way to recent trials aiming to use herpes viruses as vectors to deliver genes into the nervous system, an issue that is also addressed in this review.     

A herpes simplex virus type 1 mutant containing a nontransinducing Vmw65 protein establishes latent infection in vivo in the absence of viral replication and reactivates efficiently from explanted trigeminal ganglia.

Vmw65, a herpes simplex virus type 1 (HSV-1) tegument protein, in association with cellular proteins, transactivates viral immediate early genes. In order to examine the role of Vmw65 during acute and latent infection in vivo, a mutant virus (in1814), containing a 12-base-pair insertion in the Vmw65 gene, which lacks the transactivating function of Vmw65 (C. I. Ace, T. A. McKee, J. M. Ryan, J. M. Cameron, and C. M. Preston, J. Virol. 63:2260-2269, 1989) was examined in mice. Following corneal inoculation, the parental virus (17+) and the revertant (1814R) replicated effectively in eyes and trigeminal ganglia with 30 to 60% mortality. At either equal PFU or equal particle numbers, in1814 did not replicate in trigeminal ganglia and none of the infected mice died. Although in1814 did not replicate following corneal inoculation, it established latent infection in trigeminal ganglia. HSV-1 in1814 reactivated at explant as efficiently and rapidly as did 17+ and 1814R. Even low amounts of inoculated in1814 (10(2) PFU) were sufficient to establish latent infection in some animals. Since infectious in1814 was not detected at any time in mouse trigeminal ganglia, in1814 provided a unique opportunity to determine how soon after primary infection latency begins. Latent in1814 infection was detected shortly after virus reached the sensory ganglia, between 24 to 48 h postinfection. Thus, though Vmw65 may be required for lytic infection in vivo, it is dispensable for the establishment of and reactivation from latent infection. These data support the hypotheses that the latent and lytic pathways of HSV-1 are distinct and that latency is established soon after infection without a requirement for viral replication. However, the levels of Vmw65 reaching neuronal nuclei may be a critical determinant of whether HSV-1 forms a lytic or latent infection.     

The immune response to ocular herpes simplex virus type 1 infection

Herpes simplex virus type 1 (HSV-1) is a prevalent microbial pathogen infecting 60% to 90% of the adult world population. The co-evolution of the virus with humans is due, in part, to adaptations that the virus has evolved to aid it in escaping immune surveillance, including the establishment of a latent infection in its human host. A latent infection allows the virus to remain in the host without inducing tissue pathology or eliciting an immune response. During the acute infection or reactivation of latent virus, the immune response is significant, which can ultimately result in corneal blindness or fatal sporadic encephalitis. In fact, HSV-1 is one of the leading causes of infectious corneal blindness in the world as a result of chronic episodes of viral reactivation leading to stromal keratitis and scarring. Significant inroads have been made in identifying key immune mediators that control ocular HSV-1 infection and potentially viral reactivation. Likewise, viral mechanisms associated with immune evasion have also been identified and will be discussed. Lastly, novel therapeutic strategies that are currently under development show promise and will be included in this review. Most investigators have taken full advantage of the murine host as a viable working in vivo model of HSV-1 due to the sensitivity and susceptibility to viral infection, ease of manipulation, and a multitude of developed probes to study changes at the cellular and molecular levels. Therefore, comments in this review will primarily be restricted to those observations pertaining to the mouse model and the assumption (however great) that similar events occur in the human condition.     

Herpes simplex virus 1 ICP0 phosphorylation site mutants are attenuated for viral replication and impaired for explant-induced reactivation

In cell culture experiments, phosphorylation appears to be a critical regulator of the herpes simplex virus 1 (HSV-1) immediate-early (IE) protein, ICP0, which is an E3 ubiquitin ligase that transactivates viral gene expression. Three major regions of phosphorylation in ICP0 (amino acids 224 to 232, 365 to 371, and 508 to 518) have been identified, and mutant viruses that block phosphorylation sites within each region (termed Phos 1, 2, and 3, respectively) have been constructed. Previous studies indicated that replication of Phos 1 is significantly reduced compared to that of wild-type virus in cell culture (C. Boutell, et al., J. Virol. 82:10647-10656, 2008). To determine the effects these phosphorylation site mutations have on the viral life cycle in vivo, mice were ocularly infected with wild-type HSV-1, the Phos mutants, or their marker rescue counterparts. Subsequently, viral replication, establishment of latency, and viral explant-induced reactivation of these viruses were examined. Relative to wild-type virus, Phos 1 eye titers were reduced as much as 7- and 18-fold on days 1 and 5 postinfection, respectively. Phos 2 eye titers showed a decrease of 6-fold on day 1 postinfection. Titers of Phos 1 and 2 trigeminal ganglia were reduced as much as 16- and 20-fold, respectively, on day 5 postinfection. Additionally, the reactivation efficiencies of Phos 1 and 2 were impaired relative to wild-type HSV-1, although both viruses established wild-type levels of latency in vivo. The acute replication, latency, and reactivation phenotypes of Phos 3 were similar to those of wild-type HSV-1. We conclude from these studies that phosphorylation is likely a key modulator of ICP0's biological activities in a mouse ocular model of HSV-1 infection.     

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.