Herpes Simplex Virus Type 1 Pathogenesis Research Articles

A dual fluorescent herpes simplex virus type 1 recombinant reveals divergent outcomes of neuronal infection.

Critical stages of lytic herpes simplex virus type 1 (HSV-1) replication are marked by the sequential expression of immediate early (IE) to early (E), then late (L) viral genes. HSV-1 can also persist in neuronal cells via a non-replicative, transcriptionally repressed infection called latency. The regulation of lytic and latent transcriptional profiles is critical to HSV-1 pathogenesis and persistence. We sought a fluorescence-based approach to observe the outcome of neuronal HSV-1 infection at the single-cell level. To achieve this goal, we constructed and characterized a novel HSV-1 recombinant that enables discrimination between lytic and latent infection. The dual reporter HSV-1 encodes a human cytomegalovirus-immediate early (hCMV-IE) promoter-driven enhanced yellow fluorescent protein (eYFP) to visualize the establishment of infection and an endogenous mCherry-VP26 fusion to report lytic replication. We confirmed that viral gene expression, replication, and spread of infection are not altered by the incorporation of the fluorescent reporters, and fluorescent protein (FP) detection virtuously reports the progression of lytic replication. We demonstrate that the outcome of HSV-1 infection of compartmentalized primary neurons is determined by viral inoculating dose: high-dose axonal inoculation proceeds to lytic replication, whereas low-dose axonal inoculation establishes a latent HSV-1 infection. Interfering with low-dose axonal inoculation via small molecule drugs reports divergent phenotypes of eYFP and mCherry reporter detection, correlating with altered states of viral gene expression. We report that the transcriptional state of neuronal HSV-1 infection is variable in response to changes in the intracellular neuronal environment.IMPORTANCEHerpes simplex virus type 1 (HSV-1) is a prevalent human pathogen that infects approximately 67% of the global human population. HSV-1 invades the peripheral nervous system, where latent HSV-1 infection persists within the host for life. Immunological evasion, viral persistence, and herpetic pathologies are determined by the regulation of HSV-1 gene expression. Studying HSV-1 gene expression during neuronal infection is challenging but essential for the development of antiviral therapeutics and interventions. We used a recombinant HSV-1 to evaluate viral gene expression during infection of primary neurons. Manipulation of cell signaling pathways impacts the establishment and transcriptional state of HSV-1 latency in neurons. The work here provides critical insight into the cellular and viral factors contributing to the establishment of latent HSV-1 infection.

Manipulation of Oxidative Stress Responses by Non-Thermal Plasma to Treat Herpes Simplex Virus Type 1 Infection and Disease.

Herpes simplex virus type 1 (HSV-1) is a contagious pathogen with a large global footprint, due to its ability to cause lifelong infection in patients. Current antiviral therapies are effective in limiting viral replication in the epithelial cells to alleviate clinical symptoms, but ineffective in eliminating latent viral reservoirs in neurons. Much of HSV-1 pathogenesis is dependent on its ability to manipulate oxidative stress responses to craft a cellular environment that favors HSV-1 replication. However, to maintain redox homeostasis and to promote antiviral immune responses, the infected cell can upregulate reactive oxygen and nitrogen species (RONS) while having a tight control on antioxidant concentrations to prevent cellular damage. Non-thermal plasma (NTP), which we propose as a potential therapy alternative directed against HSV-1 infection, is a means to deliver RONS that affect redox homeostasis in the infected cell. This review emphasizes how NTP can be an effective therapy for HSV-1 infections through the direct antiviral activity of RONS and via immunomodulatory changes in the infected cells that will stimulate anti-HSV-1 adaptive immune responses. Overall, NTP application can control HSV-1 replication and address the challenges of latency by decreasing the size of the viral reservoir in the nervous system.

The Potential Role of Herpes Simplex Virus Type 1 and Neuroinflammation in the Pathogenesis of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease affecting ~50 million people worldwide. To date, there is no cure and current therapies have not been effective in delaying disease progression. Therefore, there is an urgent need for better understanding of the pathogenesis of AD and to rethink possible therapies. Herpes simplex virus type 1 (HSV1) has recently received growing attention for its potential role in sporadic AD. The virus is a ubiquitous human pathogen that infects mucosal epithelia and invades the peripheral nervous system (PNS) of its host to establish a reactivable, latent infection. Upon reactivation, HSV1 spreads back to the epithelium and initiates a new infection, causing epithelial lesions. Occasionally, the virus spreads from the PNS to the brain after reactivation. In this review, we discuss current work on the pathogenesis of AD and summarize research results that support a potential role for HSV1 in the infectious hypothesis of AD. We also highlight recent findings on the neuroinflammatory response, which has been proposed to be the main driving force of AD, starting early in the course of the disease. Relevant rodent models to study neuroinflammation in AD and novel therapeutic approaches are also discussed. Throughout this review, we focus on several aspects of HSV1 pathogenesis, including its primary role as an invader of the PNS, that should be considered in the etiology of AD. We also point out some of the contradictory data and remaining knowledge gaps that require further research to finally fully understand the cause of AD in humans.

Lupeol impairs herpes simplex virus type 1 replication by inhibiting the promoter activity of the viral immediate early gene α0.

Herpes simplex virus type 1 (HSV-1) is an important human pathogenic virus. It is urgent to develop novel antiviral targets because of the limited treatment options and the emergence of drug resistant strains. In this study, we tested the antiviral activity of lupeol, a triterpenoid compound, against HSV-1 and acyclovir (ACV) resistant strains. Lupeol significantly inhibited HSV-1 (F strain) and ACV-resistant strains including HSV-1/106, HSV-1/153, and HSV-1/Blue. Lupeol activity of the HSV-1α0 and α4 promoters, therefore down regulating the expression of the α0, α4, and α27 genes. Collectively, lupeol showed strong antiviral activity against HSV-1 and ACV-resistant strains, and could be a promising therapeutic candidate for HSV-1 pathogenesis. Keywords: herpes simplex virus 1; lupeol; ACV-resistant strains; promoter.

Vaccine-induced antibodies target sequestered viral antigens to prevent ocular HSV-1 pathogenesis, preserve vision, and preempt productive neuronal infection

The cornea is essential for vision yet highly sensitive to immune-mediated damage following infection. Generating vaccines that provide sterile immunity against ocular surface pathogens without evoking vision loss is therefore clinically challenging. Here, we tested a prophylactic live-attenuated vaccine against herpes simplex virus type 1 (HSV-1), a widespread human pathogen that can cause corneal blindness. Parenteral vaccination of mice resulted in sterile immunity to subsequent HSV-1 challenge in the cornea and suppressed productive infection of the nervous system. This protection was unmatched by a relevant glycoprotein subunit vaccine. Efficacy of the live-attenuated vaccine involved a T-dependent humoral immune response and complement C3 but not Fcγ-receptor 3 or interferon-α/β signaling. Proteomic analysis of viral proteins recognized by antiserum revealed an unexpected repertoire dominated by sequestered antigens rather than surface-exposed envelope glycoproteins. Ocular HSV-1 challenge in naive and subunit-vaccinated mice triggered vision loss and severe ocular pathologies including corneal opacification, scar formation, neovascularization, and sensation loss. However, corneal pathology was absent in mice receiving the live-attenuated vaccine concomitant with complete preservation of visual acuity. Collectively, this is the first comprehensive report of a prophylactic vaccine candidate that elicits resistance to ocular HSV-1 infection while fully preserving the cornea and visual acuity.

Herpes Simplex Virus Type 1 Engages Toll Like Receptor 2 to Recruit Macrophages During Infection of Enteric Neurons.

Herpes simplex virus type 1 (HSV-1) is a widespread neurotropic pathogen responsible for a range of clinical manifestations. Inflammatory cell infiltrate is a common feature of HSV-1 infections and has been implicated in neurodegeneration. Therefore, viral recognition by innate immune receptors (i.e., TLR2) and the subsequent inflammatory response are now deemed key players in HSV-1 pathogenesis. In this study we infected with HSV-1 the enteric nervous system (ENS) of wild-type (WT) and TLR2 knock-out (TLR2ko) mice to investigate whether and how TLR2 participates in HSV-1 induced neuromuscular dysfunction. Our findings demonstrated viral specific transcripts suggestive of abortive replication in the ENS of both WT and TLR2ko mice. Moreover, HSV-1 triggered TLR2-MyD88 depend signaling in myenteric neurons and induced structural and functional alterations of the ENS. Gastrointestinal dysmotility was, however, less pronounced in TLR2ko as compared with WT mice. Interesting, HSV-1 caused up-regulation of monocyte chemoattractant protein-1 (CCL2) and recruitment of CD11b+ macrophages in the myenteric ganglia of WT but not TLR2ko mice. At the opposite, the myenteric plexuses of TLR2ko mice were surrounded by a dense infiltration of HSV-1 reactive CD3+CD8+INFγ+ lymphocytes. Indeed, depletion CD3+CD8+ cells by means of administration of anti-CD8 monoclonal antibody reduced neuromuscular dysfunction in TLR2ko mice infected with HSV-1. During HSV-1 infection, the engagement of TLR2 mediates production of CCL2 in infected neurons and coordinates macrophage recruitment. Bearing in mind these observations, blockage of TLR2 signaling could provide novel therapeutic strategies to support protective and specific T-cell responses and to improve neuromuscular dysfunction in pathogen-mediated alterations of the ENS.

Zebrafish 3-O-Sulfotransferase-4 Generated Heparan Sulfate Mediates HSV-1 Entry and Spread

Rare modification of heparan sulfate (HS) by glucosaminyl 3-O sulfotransferase (3-OST) isforms generates an entry receptor for herpes simplex virus type-1 (HSV-1). In the zebrafish (ZF) model multiple 3-OST isoforms are differentially expressed. One such isoform is 3-OST-4 which is widely expressed in the central nervous system of ZF. In this report we characterize the role of ZF encoded 3-OST-4 isoform for HSV-1 entry. Expression of ZF 3-OST-4 into resistant Chinese hamster ovary (CHO-K1) cells promoted susceptibility to HSV-1 infection. This entry was 3-O sulfated HS (3-OS HS) dependent as pre-treatment of ZF 3-OST-4 cells with enzyme HS lyases (heparinase II/III) significantly reduced HSV-1 entry. Interestingly, co-expression of ZF 3-OST-4 along with ZF 3-OST-2 which is also expressed in brain rendered cells more susceptible to HSV-1 than 3-OST-4 alone. The role of ZF-3-OST-4 in the spread of HSV-1 was also evaluated as CHO-K1 cells that expressed HSV-1 glycoproteins fused with ZF 3-OST-4 expressing effector CHO-K1 cells. Finally, adding further evidence ZF 3-OST-4 mediated HSV-1 entry was inhibited by anti-3O HS G2 peptide. Taken together our results demonstrate a role for ZF 3-OST-4 in HSV-1 pathogenesis and support the use of ZF as a model to study it.

Zebrafish encoded 3-O-sulfotransferase-2 generated heparan sulfate serves as a receptor during HSV-1 entry and spread

Previously we reported the role of zebrafish (ZF) encoded glucosaminyl 3-O-sulfotransferase-3 (3-OST-3) isoform in assisting herpes simplex virus type-1 (HSV-1) entry and spread by generating an entry receptor to HSV-1 envelope glycoprotein D (gD). However, the ability of ZF encoded 3-OST-2 isoform to participate in HSV-1 entry has not been determined although it is predominantly expressed in ZF brain, a prime target for HSV-1 to infect and establish lifelong latency. Here we report the expression cloning of ZF encoded 3-OST-2 isoform and demonstrate HSV-1 entry into resistant Chinese hamster ovary (CHO-K1) cells expressing the clone. Additional significance of ZF encoded 3-OST-2 receptor was demonstrated using medically important isolates of HSV-1. In addition, interference to HSV-1 entry was observed upon co-expression of HSV-1 gD and ZF 3-OST-2. Similarly HSV-1 entry was significantly inhibited by the pre-treatment of cells with enzyme HS lyases (heparinase II/III). Finally, ZF-3-OST-2 expressing CHO-K1 was able to fuse with HSV-1 glycoprotein expressing cells suggesting their role in HSV-1 spread. Taken together our result demonstrates a role for ZF 3-OST-2 in HSV-1 pathogenesis.

An Important Role for Syndecan-1 in Herpes Simplex Virus Type-1 Induced Cell-to-Cell Fusion and Virus Spread

Herpes simplex virus type-1 (HSV-1) is a common human pathogen that relies heavily on cell-to-cell spread for establishing a lifelong latent infection. Molecular aspects of HSV-1 entry into host cells have been well studied; however, the molecular details of the spread of the virus from cell-to-cell remain poorly understood. In the past, the role of heparan sulfate proteoglycans (HSPG) during HSV-1 infection has focused solely on the role of HS chains as an attachment receptor for the virus, while the core protein has been assumed to perform a passive role of only carrying the HS chains. Likewise, very little is known about the involvement of any specific HSPGs in HSV-1 lifecycle. Here we demonstrate that a HSPG, syndecan-1, plays an important role in HSV-1 induced membrane fusion and cell-to-cell spread. Interestingly, the functions of syndecan-1 in fusion and spread are independent of the presence of HS on the core protein. Using a mutant CHO-K1 cell line that lacks all glycosaminoglycans (GAGs) on its surface (CHO-745) we demonstrate that the core protein of syndecan-1 possesses the ability to modulate membrane fusion and viral spread. Altogether, we identify a new role for syndecan-1 in HSV-1 pathogenesis and demonstrate HS-independent functions of its core protein in viral spread.

Virostatic potential of micro–nano filopodia-like ZnO structures against herpes simplex virus-1

Herpes simplex virus type-1 (HSV-1) entry into target cell is initiated by the ionic interactions between positively charged viral envelop glycoproteins and a negatively charged cell surface heparan sulfate (HS). This first step involves the induction of HS-rich filopodia-like structures on the cell surface that facilitate viral transport during cell entry. Targeting this initial first step in HSV-1 pathogenesis, we generated different zinc oxide (ZnO) micro–nano structures (MNSs) that were capped with multiple nanoscopic spikes mimicking cell induced filopodia. These MNSs were predicted to target the virus to compete for its binding to cellular HS through their partially negatively charged oxygen vacancies on their nanoscopic spikes, to affect viral entry and subsequent spread. Our results demonstrate that the partially negatively charged ZnO-MNSs efficiently trap the virions via a novel virostatic mechanism rendering them unable to enter into human corneal fibroblasts – a natural target cell for HSV-1 infection. The anti-HSV-1 activity of ZnO MNSs was drastically enhanced after creating additional oxygen vacancies under UV-light illumination. Our results provide a novel insight into the significance of ZnO MNSs as the potent HSV-1 inhibitor and rationalize their development as a novel topical agent for the prevention of HSV-1 infection.

Herpes simplex virus type 1 infection induces oxidative stress and the release of bioactive lipid peroxidation by-products in mouse P19N neural cell cultures

To determine whether herpes simplex virus type 1 (HSV-1) infection causes oxidative stress and lipid peroxidation in cultured neural cells, mouse P19 embryonal carcinoma cells were differentiated into cells with neural phenotypes (P19N cells) by retinoic acid and were then infected with HSV-1. Cellular levels of reactive oxygen species (ROS) and the release of lipid peroxidation by-products into the tissue culture medium were then measured by the generation of fluorescent markers hydroxyphenyl fluorescein and a stable chromophore produced by lipid peroxidation products, malondialdehyde (MDA) and hydroxyalkenals (4-HAEs; predominantly 4-hydroxy-2-nonenal [HNE]), respectively. HSV-1 infection increased ROS levels in neural cells as early as 1 h post infection (p.i.) and ROS levels remained elevated at 24 h p.i. This viral effect required viral entry and replication as heat- and ultraviolet light-inactivated HSV-1 were ineffective. HSV-1 infection also was associated with increased levels of MDA/HAE in the culture medium at 2 and 4 h p.i., but MDA/HAE levels were not different from those detected in mock infected control cultures at 1, 6, and 24 h p.i. HSV-1 replication in P19N cells was inhibited by the antioxidant compound ebselen and high concentrations of HNE added to the cultures, but was increased by low concentrations of HNE. These findings indicate that HSV-1 infection of neural cells causes oxidative stress that is required for efficient viral replication. Furthermore, these observations raise the possibility that soluble, bioactive lipid peroxidation by-products generated in infected neural cells may be important regulators of HSV-1 pathogenesis in the nervous system.

Transgenic reporter mouse for bioluminescence imaging of herpes simplex virus 1 infection in living mice

Bioluminescence imaging allows spatial and temporal progression of viral infection to be detected and quantified in living mice, thereby providing a new approach for studies of viral–host pathogenesis. It has been necessary to construct and validate recombinant reporter viruses that express firefly luciferase to investigate viral replication and spread with this imaging technology. This strategy greatly limits the ability to analyze multiple strains of virus and/or existing viral mutants, and reporter viruses also may be attenuated relative to the respective parental viruses. To facilitate bioluminescence imaging of herpes simplex virus type 1 (HSV-1), we developed a transgenic reporter mouse that uses the promoter from HSV-1 thymidine kinase to control expression of firefly luciferase. Infection with HSV-1 activated expression of firefly luciferase in corneal and flank models of infection, and amounts of bioluminescence increased in proportion to increasing input titers of virus. Imaging could detect infection with three different strains of HSV-1 with the following relative rank order of bioluminescence produced at the site of infection: McKrae > 17 > KOS. Corneal infection with as few as 1 × 10 3 pfu strain McKrae was detectable above background levels. By comparison, infection with vaccinia virus did not affect bioluminescence in the reporter mouse. Collectively, these data establish a new transgenic reporter mouse for infection with HSV-1, thereby enabling in vivo bioluminescence imaging studies of HSV-1 pathogenesis without constructing new reporter viruses.

Point Mutations in Herpes Simplex Virus Type 1 oriL, but Not in oriS, Reduce Pathogenesis during Acute Infection of Mice and Impair Reactivation from Latency

In vitro studies of herpes simplex virus type 1 (HSV-1) viruses containing mutations in core sequences of the viral origins of DNA replication, oriL and oriS, that eliminate the ability of these origins to initiate viral-DNA synthesis have demonstrated little or no effect on viral replication in cultured cells, leading to the conclusion that the two types of origins are functionally redundant. It remains unclear, therefore, why origins that appear to be redundant are maintained evolutionarily in HSV-1 and other neurotropic alphaherpesviruses. To test the hypothesis that oriL and oriS have distinct functions in the HSV-1 life cycle in vivo, we determined the in vivo phenotypes of two mutant viruses, DoriL-I(LR) and DoriS-I, containing point mutations in oriL and oriS site I, respectively, that eliminate origin DNA initiation function. Following corneal inoculation of mice, tear film titers of DoriS-I were reduced relative to wild-type virus. In all other tests, however, DoriS-I behaved like wild-type virus. In contrast, titers of DoriL-I(LR) in tear film, trigeminal ganglia (TG), and hindbrain were reduced and mice infected with DoriL-I(LR) exhibited greatly reduced mortality relative to wild-type virus. In the TG explant and TG cell culture models of reactivation, DoriL-I(LR) reactivated with delayed kinetics and, in the latter model, with reduced efficiency relative to wild-type virus. Rescuant viruses DoriL-I(LR)-R and DoriS-I-R behaved like wild-type virus in all tests. These findings demonstrate that functional differences exist between oriL and oriS and reveal a prominent role for oriL in HSV-1 pathogenesis.

A role for 3- O-sulfotransferase isoform-4 in assisting HSV-1 entry and spread

Many heparan sulfate (HS) 3- O-sulfotransferase (3- OST) isoforms generate cellular receptors for herpes simplex virus type-1 (HSV-1) glycoprotein D (gD). Interestingly, the ability of 3- OST-4 to mediate HSV-1 entry and cell-to-cell fusion has not been determined, although it is predominantly expressed in the brain, a primary target of HSV-1 infections. We report that expression of 3- OST-4 can render Chinese hamster ovary K1 (CHO-K1) cells susceptible to entry of wild-type and a mutant (Rid1) strain of HSV-1. Evidence for generation of gD receptors by 3- OST-4 was suggested by gD-mediated interference assay and the ability of 3- OST-4 expressing CHO-K1 cells to preferentially bind HSV-1 gD, which could be reversed by prior treatment of cells with HS lyases (heparinases-II/III). In addition, 3- OST-4 expressing CHO-K1 cells acquired the ability to fuse with cells-expressing HSV-1 glycoproteins. Demonstrating specificity, the cell fusion was inhibited by soluble 3-O-sulfated forms of HS, but not unmodified HS. Taken together our results suggest a role of 3- OST-4 in HSV-1 pathogenesis.

HSV-1-Based Vectors for Gene Therapy of Neurological Diseases and Brain Tumors: Part I. HSV-1 Structure, Replication and Pathogenesis

The design of effective gene therapy strategies for brain tumors and other neurological disorders relies on the understanding of genetic and pathophysiological alterations associated with the disease, on the biological characteristics of the target tissue, and on the development of safe vectors and expression systems to achieve efficient, targeted and regulated, therapeutic gene expression. The herpes simplex virus type 1 (HSV-1) virion is one of the most efficient of all current gene transfer vehicles with regard to nuclear gene delivery in central nervous system-derived cells including brain tumors. HSV-1-related research over the past decades has provided excellent insight into the structure and function of this virus, which, in turn, facilitated the design of innovative vector systems. Here, we review aspects of HSV-1 structure, replication and pathogenesis, which are relevant for the engineering of HSV-1-based vectors.