Herpes simplex virus-1 in the brain. The dark side of a sneaky infection

Herpes simplex virus-1 (HSV-1) establishes latency preferentially in sensory neurons of peripheral ganglia. A variety of stresses can induce recurrent reactivations of the virus, which spreads and then actively replicates to the site of primary infection (usually the lips or eyes). Viral particles produced following reactivation can also reach the brain, causing a rare but severe form of diffuse acute infection, namely herpes simplex encephalitis. Most of the time, this infection is clinically asymptomatic. However, it was recently correlated with the production and accumulation of neuropathological biomarkers of Alzheimer's disease. In this review we discuss the different cellular and molecular mechanisms underlying the acute and long-term damage caused by HSV-1 infection in the brain

The role of HSV1 infection in brain epigenetic aging

Introduction. Aging is naturally characterized by time-dependent deterioration of multiple biological and cellular functions, and complex molecular changes, including epigenetic post translational modifications (PTMs), are hallmarks of aging. Deregulation in the acetylation levels may be involved in various pathologies, including cancer, viral infections and neurodegenerative diseases. Herpes Simplex Virus type-1 (HSV-1), a neurotropic virus capable to establish a latent infection in the host trigeminal ganglia and able to reach the brain following reactivations, is known to interact with host epigenetic complexes, but several aspects have yet to be clarified. This study was aimed to investigate the influence of HSV-1 on the host epigenetic aging in the brain, by evaluating specific PTM levels in in vitro and in vivo experimental models of acute and recurrent HSV-1 infection. Materials and Methods. Primary cultures of neuronal cells were obtained by E17 rat embryo brains and, after 7 days in vitro, infected with HSV-1(0.1 multiplicity of infection) or MOCK solution, then analysed by Western Blot (WB) at 24h (acute infection) or 8 days post-infection (p.i.) to evaluate H3K56 and H4K16 acetylation levels. Long-term infection was obtained treating neurons with Acyclovir (before, during and after infection) to induce virus latency. Entorhinal cortex homogenates from BALB/c mice were analysed in WB for H3K56 and H4K16 acetylation levels. These mice were HSV1 or MOCK inoculated at 2 months of age, and then subjected to several hyperthermia cycles to induce virus reactivations over their life and sacrificed at 13 months of age. The virus presence in the brain was tested by PCR and RT-PCR analysis of viral TK and ICP4 genes, and IF analysis of gB protein expression. Results. We found that HSV1 modulates the levels of H3K56 and H4K16 acetylation during acute infection in neuronal cell cultures. Furthermore, a decrease in acetylation levels of H3K56 was found 8 days p.i. and similar effects were found following recurrent infections in mice. On the contrary, H4K16 acetylation was found only slightly altered in both models. Discussion and Conclusions. These results indicate that HSV-1 infection induce a modulation in H3K56 acetylation during long-term infections, and suggest that the virus may promote epigenetic aging. Further studies will be focused on characterizing how the virus could act on age-related epigenetic hallmarks

Recurrent Herpes simplex virus type 1 (HSV-1) infection modulates neuronal aging marks in in vitro and In vivo models

Herpes simplex virus 1 (HSV-1) is a widespread neurotropic virus establishing a life-long latent infection in neurons with periodic reactivations. Recent studies linked HSV-1 to neurodegenerative processes related to age-related disorders such as Alzheimer’s disease. Here, we explored whether recurrent HSV-1 infection might accelerate aging in neurons, focusing on peculiar marks of aged cells, such as the increase in histone H4 lysine (K) 16 acetylation (ac) (H4K16ac); the decrease of H3K56ac, and the modified expression of Sin3/HDAC1 and HIRA proteins. By exploiting both in vitro and in vivo models of recurrent HSV-1 infection, we found a significant increase in H4K16ac, Sin3, and HDAC1 levels, suggesting that the neuronal response to virus latency and reactivation includes the upregulation of these aging markers. On the contrary, we found a significant decrease in H3K56ac that was specifically linked to viral reactivation and apparently not related to aging-related markers. A complex modulation of HIRA expression and localization was found in the brain from HSV-1 infected mice suggesting a specific role of this protein in viral latency and reactivation. Overall, our results pointed out novel molecular mechanisms through which recurrent HSV-1 infection may affect neuronal aging, likely contributing to neurodegeneration

Role of exosomes in HSV-1-driven neuronal damage

Introduction: Several evidence, including those from our group, support the role of Herpes Simplex Virus-1 (HSV-1) infection in Alzheimer disease (AD) pathogenesis, a neurodegenerative disorder characterized by the accumulation of misfolded proteins in the brain such as beta-amyloid peptides (Abeta) and neurofibrillary tangles. These are mainly composed by hyperphosphorylated forms of tau (ptau), an intracellular protein able to bind and stabilize microtubules only when in unphosphorylated-native form. Recent studies have demonstrated transfer of Abeta and spread of tau/ptau between neurons in the brain of transgenic mouse models of AD, indicating that these pathological proteins can be propagated in the brain. Specifically, these proteins have been found in neuron-derived exosomes, small extracellular vesicles that recently emerged as key players in cellular communication in both health and diseases, including viral infections. Herein, we investigated whether HSV-1 infection in the brain could promote tau spreading among neurons via exosomes

Multiple Herpes Simplex Virus-1 (HSV-1) reactivations induce neurodegenerative and oxidative damages in mouse brains

Introduction Several evidence support the role of oxidative stress in Alzheimer disease (AD) physiopathology, a neurodegenerative disorder characterized by the accumulation in the brain of β−amyloid peptides (Aβs) and neurofibrillary tangles (mainly composed by hyperphosphorylated tau), high level of oxidative stress markers and neuroinflammation (De Chiara et al, 2012, Piacentini et al, 2014). In particular, many redox proteomics studies on AD cerebral tissues led to the identification of oxidatively modified proteins that were consistent with biochemical or pathological alterations of the disease (Nunomura et al, 2001; Zhu et al, 2004; Smith et al, 2007; Droge et al, 2007). Interestingly, HSV-1, a neurotropic virus able to establish a lifelong latent infection in trigeminal ganglion followed by periodic reactivations, has been reported linked both to AD (Piacentini et al, 2015) and to oxidative stress conditions (Nucci et al, 2000; Palamara et al, 1995). Herein we design in vivo studies to investigate whether multiple HSV-1 reactivations induced in the brain the accumulation of oxidative stress hallmarks, particularly those correlated to AD Methods BALB/c mice were inoculated via snout abrasion with HSV-1, virus reactivation was periodically induced by thermal stress, and virus replication in the brain was verified through PCR and RT-PCR analysis of viral TK gene and ICP4 mRNA. These mice showed several signs of neurodegeneration (De Chiara et al 2017). Oxidative stress marker levels, i.e. 4-hydroxynonenal (HNE, marker of lipid peroxidation), 3-nitrotyrosine (3NT, marker of protein nytrosylation) and carbonylated proteins, were measured in brains of mice undergone multiple HSV-1 reactivations by dot-blot In addition, redox proteomic was used to identify those HNE-modified proteins mostly modulated by recurrent HSV-1 reactivations into the brain. Results Following several cycles of viral reactivation, we found in mouse brains: 1) increased levels of HNE, 3-NT, and protein carbonylation, indicating generalized conditions of oxidative stress; 2) thirteen HNE-modified proteins whose levels were significantly modulated in the cortex of HSV-1 infected mice compared to control mice. Interestingly, all these proteins are involved in important cellular processes, such as energy metabolism, protein folding, cell structure, and signal transduction, suggesting that their oxidative modification may affect brain physiology. Some of these proteins are reported to be significantly HNE-modified in AD brains compared to matched controls. Conclusion Overall, these data support the hypothesis that repeated HSV-1 reactivation into the brain may concur to neurodegeneration also inducing oxidative damages

Poly(ADP-ribosylated) proteins in mononuclear cells from patients with type 2 diabetes identified by proteomic studies

Abstract. Aims In diabetes, hyperglycemia increases reactive oxygen species that induce DNA damage and poly(ADP-ribose) polymerase activation. The aim of this study is to characterize the proteomic profile and the role of poly(-ADP-ribosylation) in patients with type 2 diabetes. Methods A proteomic platform based on 2DE and MALDI-ToF spectrometry was applied to peripheral blood mononuclear cells obtained from two different cohorts in which diabetic (n = 14) and normoglycemic patients (n = 11) were enrolled. Results Proteomic maps identified WD repeat protein, 78-kDa glucose-regulated protein precursor and myosin regulatory light chain 2, as unique proteins in diabetic patients; vimentin, elongation factor 2, annexin A1, glutathione S-transferase P, moesin and cofilin-1 as unique in the normoglycemic; and calreticulin, rho GDP-dissociation inhibitor 2, protein disulfide isomerase and tropomyosin alpha-4-chain as differentially expressed between the two cohorts. An enrichment in PARylation in diabetic patients was observed in particular, affecting GAPDH and a-Enolase leading to a decrease in their enzymatic activity. Conclusions As the GAPDH and a-Enolase are involved in energy metabolism, protein synthesis and DNA repair, loss of their function or change in their activity can significantly contribute to the molecular mechanisms responsible for the development of type 2 diabetes. These data along with the proteomic profile associated with the disease may provide new insight into the pathophysiology of type 2 diabetes

Decitabine disrupts EBV genomic epiallele DNA methylation patterns around CTCF binding sites to increase chromatin accessibility and lytic transcription in gastric cancer

ABSTRACT Epstein-Barr virus (EBV) is associated with 10% of human gastric carcinomas, which are distinguished by a CpG island methylator phenotype. In gastric carcinoma tumors and cell lines, the EBV genome also exhibits a high degree of 5-methyl cytosine (5mC) marks, which are propagated by host DNA methyltransferases (DNMT) with each cell cycle. Therefore, we sought to determine the effect of DNMT inhibition by the small molecule Decitabine (DCB) on EBV genomic 5mC and chromatin structure in two tumor-derived gastric cancer cell lines, YCCEL1 and SNU719. Decitabine effects on EBV genomic 5mC, chromatin structure, and viral gene expression were profiled by reduced representation bisulfite sequencing, ATAC-seq, and RNA-seq, respectively. Decitabine treatment resulted in global viral genome hypomethylation and a global increase in open chromatin. The most striking finding resulted from analyzing the methylation pattern from single RRBS sequencing reads, showing that the EBV genome contains a heterogeneous pool of epigenetic states, each of which is eroded upon Decitabine treatment. We observed heterogeneous 5mC epiallele patterns around EBV genomic CTCF binding sites and lytic gene transcriptional start sites. These results highlight the importance of 5mC in maintaining EBV genomic chromatin structure and latency. Furthermore, the presence of 5mC epialleles suggests EBV+ gastric cancers harbor transcriptionally distinct EBV episomes, which may exert distinct functional roles in maintaining latency and driving tumorigenesis. IMPORTANCE Epstein-Barr virus (EBV) latency is controlled by epigenetic silencing by DNA methylation [5-methyl cytosine (5mC)], histone modifications, and chromatin looping. However, how they dictate the transcriptional program in EBV-associated gastric cancers remains incompletely understood. EBV-associated gastric cancer displays a 5mC hypermethylated phenotype. A potential treatment for this cancer subtype is the DNA hypomethylating agent, which induces EBV lytic reactivation and targets hypermethylation of the cellular DNA. In this study, we identified a heterogeneous pool of EBV epialleles within two tumor-derived gastric cancer cell lines that are disrupted with a hypomethylating agent. Stochastic DNA methylation patterning at critical regulatory regions may be an underlying mechanism for spontaneous reactivation. Our results highlight the critical role of epigenetic modulation on EBV latency and life cycle, which is maintained through the interaction between 5mC and the host protein CCCTC-binding factor

Multiple Herpes simplex virus-1 (HSV-1) reactivations induce protein oxidative damage in mouse brain. Novel mechanisms for Alzheimer’s disease progression

Compelling evidence supports the role of oxidative stress in Alzheimer’s disease (AD) pathophysiology. Interestingly, Herpes simplex virus-1 (HSV-1), a neurotropic virus that establishes a lifelong latent infection in the trigeminal ganglion followed by periodic reactivations, has been reportedly linked both to AD and to oxidative stress conditions. Herein, we analyzed, through biochemical and redox proteomic approaches, the mouse model of recurrent HSV-1 infection we previously set up, to investigate whether multiple virus reactivations induced oxidative stress in the mouse brain and affected protein function and related intracellular pathways. Following multiple HSV-1 reactivations, we found in mouse brains increased levels of oxidative stress hallmarks, including 4-hydroxynonenal (HNE), and 13 HNE-modified proteins whose levels were found significantly altered in the cortex of HSV-1-infected mice compared to controls. We focused on two proteins previously linked to AD pathogenesis, i.e., glucose-regulated protein 78 (GRP78) and collapsin response-mediated protein 2 (CRMP2), which are involved in the unfolded protein response (UPR) and in microtubule stabilization, respectively. We found that recurrent HSV-1 infection disables GRP78 function and activates the UPR, whereas it prevents CRMP2 function in mouse brains. Overall, these data suggest that repeated HSV-1 reactivation into the brain may contribute to neurodegeneration also through oxidative damage