The Proteome and Secretome of Cortical Brain Cells Infected With Herpes Simplex Virus.
ABSTRACT: Infections of the brain with herpes simplex virus type 1 (HSV-1) cause life-threatening Herpes simplex encephalitis (HSE) characterized by viral replication in neurons and neuro-inflammation including an infiltration of peripheral immune cells. HSV-1 reprograms host cells to foster its own replication and for immune evasion, but eventually the immune responses clear the infection in most patients. However, many survivors suffer from long-term neuronal damage and cannot regenerate all brain functions. HSV-1 influences the physiology of neurons, astrocytes, oligodendrocytes and microglia, and significantly changes their protein expression and secretion pattern. To characterize temporal changes upon HSV-1 infection in detail, we inoculated mixed primary cultures of the murine brain cortex, and performed quantitative mass spectrometry analyses of the cell-associated proteome and the secretome. We identified 28 differentially regulated host proteins influencing inflammasome formation and intracellular vesicle trafficking during endocytosis and secretion. The NIMA-related kinase 7 (NEK7), a critical component of the inflammasome, and ArfGap1, a regulator of endocytosis, were significantly up-regulated upon HSV-1 infection. In the secretome, we identified 71 proteins including guidance cues regulating axonal regeneration, such as semaphorin6D, which were enriched in the conditioned media of HSV-1 infected cells. Modulation of inflammasome activity and intracellular membrane traffic are critical for HSV-1 cell entry, virus assembly, and intracellular spread. Our proteome analysis provides first clues on host factors that might dampen the inflammasome response and modulate intracellular vesicle transport to promote HSV infection of the brain. Furthermore, our secretome analysis revealed a set of proteins involved in neuroregeneration that might foster neuronal repair processes to restore brain functions after clearance of an HSV-1 infection.
Project description:Herpes simplex virus 1 (HSV-1) is a common pathogen infecting the majority of people worldwide at some stage in their lives. The early host response to viral infection is initiated by the cells of the innate immune response, including macrophages. Here, we have characterized the secretome of HSV-1-infected human primary macrophages using high-throughput quantitative proteomics. We identified and quantified 516 distinct human proteins with high confidence from the macrophage secretome upon HSV-1 infection, and the secretion of 411 proteins was >2-fold increased upon beta interferon (IFN-?) priming and/or HSV-1 infection. Bioinformatics analysis of the secretome data revealed that most of the secreted proteins were intracellular, and almost 80% of the proteins whose secretion increased more than 2-fold were known exosomal proteins. This strongly suggests that nonclassical, vesicle-mediated protein secretion is activated in IFN-?-primed and HSV-1-infected macrophages. Proteins related to immune and inflammatory responses, interferon-induced proteins, and endogenous danger signal proteins were efficiently secreted upon IFN-? priming and HSV-1 infection. The secreted IFN-induced proteins include interferon-induced tetratricopeptide protein 2 (IFIT2), IFIT3, signal transducer and activator of transcription 1 (STAT1), and myxovirus resistance protein A (MxA), implicating that these proteins also have important extracellular antiviral functions. Proinflammatory cytokine interleukin-1? was not released by HSV-1-infected macrophages, demonstrating that HSV-1 can antagonize inflammasome function. In conclusion, our results provide a global view of the secretome of HSV-1-infected macrophages, revealing host factors possibly having a role in antiviral defense.
Project description:Herpes simplex virus-1 (HSV-1) infections of the brain cause a severe neuroinflammatory condition – Herpes simplex encephalitis (HSE). During the acute phase, HSV-1 changes the host cells to facilitate its own replication and to escape host immunity. However, the host reacts with a severe neuroinflammation which may be beneficial for viral clearance or detrimental inducing neuronal damage and hindering regeneration. Indeed, surviving HSE-patients often suffer from an incomplete regeneration with memory deficits and other neuropsychiatric symptoms. HSV-1 infects different cell types such as neurons, astrocytes, and oligodendrocytes all of which participate in the host response. This includes a changed paracrine signaling. However, the host response has not been characterized on a global molecular level so far. Here, we infected primary mixed cortical cells with HSV-1 and analyzed the proteome and the secretome of the host. We identified 28 differentially regulated proteins in the host proteome involved in endocytosis, vesicle trafficking at the golgi-apparatus, and inflammasome formation. In the secretome, we identified 71 differentially regulated proteins with a subset involved in axonal regeneration. Endocytosis and vesicle trafficking are critical for viral entry and envelopment. The suppression of inflammasome activity is crucial for viral spread. Thus, our proteomic analysis hints for potential molecules regulating viral production and spread in brain cells. Moreover, the secretome analysis gives rise to a set of proteins involved in neuroregeneration.
Project description:Infection with herpes simplex viruses is one of the most ancient diseases described to affect humans. Infection with these viruses produces vexing effects to the host, which frequently recur. Infection with herpes simplex viruses is lifelong, and currently there is no vaccine or drug to prevent or cure infection. Prevalence of herpes simplex virus 2 (HSV-2) infection varies significantly depending on the geographical region and nears 20% worldwide. Importantly, HSV-2 is the first cause of genital ulcers in the planet. HSV-2 affects approximately 500 million people around the globe and significantly increases the likelihood of acquiring the human immunodeficiency virus (HIV), as well as its shedding. Thus, controlling HSV-2 infection and spread is of public health concern. Here, we review the diseases produced by herpes simplex viruses, the factors that modulate HSV-2 infection, the relationship between HSV-2 and HIV and novel therapeutic and prophylactic microbicides/antivirals under development to prevent infection and pathological outcomes produced by this virus. We also review mutations associated with HSV-2 resistance to common antivirals.
Project description:The purpose of this study was to determine which genes are differentially regulated virus infection in RAW264.7 cells. Cells were infected with Vesicular Stomatitis Virus (VSV) or herpes simplex virus 1 (HSV-1) for 6h. Then the differentially regulated genes were analyzed, focusing on F-box proteins and E3 ubiquitin ligases. RAW264.7 cells were infected with Vesicular Stomatitis Virus (VSV, MOI=1) or herpes simplex virus 1 (HSV-1, MOI=5) for 6h. Equal amounts of RNA were assayed for gene expression using Affymetrix mouse 430 2.0 arrays.
Project description:The purpose of this study was to determine what are the effects of Src deficiency on innate antiviral response upon virus infection in RAW264.7 cells. Wild type and Src-/- RAW264.7 cells were infected with vesicular stomatitis virus (VSV) or herpes simplex virus 1 (HSV-1) for 6h. Then the differentially regulated genes were analyzed. Wild type and Src-/- RAW264.7 cells were infected with vesicular stomatitis virus (VSV, MOI=1) or herpes simplex virus 1 (HSV-1, MOI=5) for 6h. Equal amounts of RNA were assayed for gene expression using Affymetrix mouse 430 2.0 arrays.
Project description:The proinflammatory cytokines interleukin (IL)-1? and IL-18 are products of activation of the inflammasome, an innate sensing system, and important in the pathogenesis of herpes simplex virus type 1 (HSV-1). The release of IL-18 and IL-1? from monocytes/macrophages is critical for protection from HSV-1 based on animal models of encephalitis and genital infection, yet if and how HSV-1 activates inflammasomes in human macrophages is unknown. To investigate this, we utilized both primary human monocyte derived macrophages and human monocytic cell lines (THP-1 cells) with various inflammasome components knocked-out. We found that HSV-1 activates inflammasome signaling in proinflammatory primary human macrophages, but not in resting macrophages. Additionally, HSV-1 inflammasome activation in THP-1 cells is dependent on nucleotide-binding domain and leucine-rich repeat-containing receptor 3 (NLRP3), apoptosis-associated speck-like molecule containing a caspase recruitment domain (ASC), and caspase-1, but not on absent in melanoma 2 (AIM2), or gamma interferon-inducible protein 16 (IFI16). In contrast, HSV-1 activates non-canonical inflammasome signaling in proinflammatory macrophages that results in IL-1?, but not IL-18, release that is independent of NLRP3, ASC, and caspase-1. Ultraviolet irradiation of HSV-1 enhanced inflammasome activation, demonstrating that viral replication suppresses inflammasome activation. These results confirm that HSV-1 is capable of activating the inflammasome in human macrophages through an NLRP3 dependent process and that the virus has evolved an NLRP3 specific mechanism to inhibit inflammasome activation in macrophages.
Project description:Herpes simplex virus 1 (HSV-1), a leading cause of genital herpes, infects oral or genital mucosal epithelial cells before infecting the peripheral sensory nervous system. The spread of HSV-1 beyond the sensory nervous system and the resulting broader spectrum of disease are not well understood. Using a mouse model of genital herpes, we found that HSV-1-infection-associated lethality correlated with severe fecal and urinary retention. No inflammation or infection of the brain was evident. Instead, HSV-1 spread via the dorsal root ganglia to the autonomic ganglia of the enteric nervous system (ENS) in the colon. ENS infection led to robust viral gene transcription, pathological inflammatory responses, and neutrophil-mediated destruction of enteric neurons, ultimately resulting in permanent loss of peristalsis and the development of toxic megacolon. Laxative treatment rescued mice from lethality following genital HSV-1 infection. These results reveal an unexpected pathogenesis of HSV associated with ENS infection.
Project description:Herpes simplex virus type 1 (HSV-1), an enveloped DNA virus, plays a key role in varieties of diseases including recurrent cold sores, keratoconjunctivitis, genital herpes and encephalitis in humans. Great efforts have been made in developing more effective and less side-effects anti-herpes simplex virus agents, including traditional Chinese herbal medicines. In the present study, we evaluated the antiviral efficacy of Rheum tanguticum nanoparticles against HSV-1 in vitro and in vivo. R. tanguticum nanoparticles could inactivate the HSV-1 virions and block the viral attachment and entry into cells. Time-of-addition assay indicated that R. tanguticum nanoparticles could interfere with the entire phase of viral replication. Besides, R. tanguticum nanoparticles showed the ability to inhibit the mRNA expression of HSV-1 immediate early gene ICP4 and early gene ICP8 as well as the expression of viral protein ICP4 and ICP8. Moreover, R. tanguticum nanoparticles have been proved to protect mice against HSV-1 induced lethality by decreasing the viral load and alleviated pathological changes in brain tissues. In conclusion, we demonstrated that R. tanguticum nanoparticles could inhibit HSV-1 infection through multiple mechanisms. These results suggest that R. tanguticum nanoparticles may have novel roles in the treatment of HSV-1 infection.
Project description:Genital herpes is a venereal disease caused by herpes simplex virus (HSV). Although HSV symptoms can be reduced with antiviral drugs, there is no cure. Moreover, because HSV infected individuals are often unaware of their infection, it is highly likely that they will transmit HSV to their sexual partner. Once infected, an individual has to live with HSV for their entire life, and HSV infection can lead to meningitis, encephalitis, and neonatal herpes as a result of vertical transmission. In addition, HSV infection increases the rates of human immunodeficiency virus (HIV) infection and transmission. Because of the high burden of genital herpes, HSV vaccines have been developed, but none have been very successful. In this review, we discuss the current status of genital herpes vaccine development.
Project description:Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1-/- mice results in replication of HSV-1 and Asah1-/- mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol.