In Vitro TH-mediated HSV-1 Latency Cell Culture Model

In vitro TH-mediated HSV-1 latency cell culture model 1

The final type of model involves the infection of standard tissue culture cells, usually human fibroblasts, with HSV-1 mutants that are impaired for immediate early (IE) gene expression and thus do not kill cells (Preston and Nicholl, 1997; Samaniego et al. LAP1 is insufficient to mediate long-term latent phase expression, because insertion of reporter genes downstream of LAP1 results in only transient latent phase gene expression. Inducible cyclic AMP early repressor produces reactivation of latent herpes simplex virus type 1 in neurons in vitro. The impacts of TH on virus-mediated pathophysiology was discussed but not extensively studied. In vitro TH-mediated HSV-1 latency cell culture model. We tested this approach in an in vitro HSV latency model using the engineered homing endonuclease (HE) HSV1m5, which recognizes a sequence in the HSV-1 gene UL19, encoding the virion protein VP5. Presence of progeny virus in the cell culture supernatant is indicated on the right (PFU: Plaque forming unit; +++ indicates 100 PFU/ml; ++ indicates 10 PFU/ml; – indicates no virus detected). HE-mediated mutagenesis, we used the replication-deficient HSV-1 mutant virus d106, which is deleted for four genes encoding the immediate early protein ICP4, 22, 27, and 47 but retains ICP0 and expresses GFP under the CMV promoter from the locus of the ICP27 gene (UL 54).

In vitro TH-mediated HSV-1 latency cell culture model 2In this study we assess the role of the HSV-1 latency-associated transcript in the control of viral genome silencing and reactivation in mouse nervous tissue and individual neurons. Finally, using a fluorescent mouse model of infection to isolate and culture single latently infected neurons, we also show that reactivation occurs at a greater frequency from cultures harbouring LAT-negative HSV-1. A cell-based model of HSV-1 latent infection was developed and characterized. This system utilizes a pure culture of sympathetic neurons and allows for the molecular dissection of latency in a neuron autonomous environment. This in vitro system recapitulates the pivotal features of latency in vivo, including the exhibition of spontaneous reactivation. Using this system, the role of neurotrophin signaling-mediated HSV-1 latency was investigated. HSV-1 LAT expression was observed to influence the number of latently infected neurons in trigeminal but not dorsal root ganglia. We conclude that the HSV-1 LATs facilitate the long-term stability of the latent cell population within the infected host and that interpretation of LAT establishment phenotypes is influenced by infection methodology. We have previously described the ROSA26R reporter mouse model of infection allowing historical marking of neuron infection via the use of HSV-1 strain SC16 recombinants expressing Cre recombinase (29). 2.5 g/ml) and 1 nonessential amino acids (PAA) for long-term culture.

Latent herpes simplex virus-1 (HSV1) genomes in peripheral nerve ganglia periodically reactivate, initiating a gene expression program required for productive replication. HSV productive (lytic) growth in a primary neuron cell culture model system that faithfully exhibits key hallmarks of latency as defined in animal models (Camarena et al. Persistent rheb-mediated mTORC1 activation is sufficient to maintain latency and prevent inducible reactivation. Using a primary neuronal culture model of HSV-1 latency and reactivation, we show that continuous signaling through the phosphatidylinositol 3-kinase (PI3-K) pathway triggered by nerve growth factor (NGF)-binding to the TrkA receptor tyrosine kinase (RTK) is instrumental in maintaining latent HSV-1. Significantly, we find that a continuous neuronal signaling program mediated by NGF through the TrkA receptor, PI3-kinase (PI3-K) p110 isoform, PDK1, and Akt is required to suppress HSV productive (lytic) growth and maintain latency. Nevertheless, human CNS cell-based models of anti-HSV-1 immunity are of particular importance as responses to any given neurotropic virus may differ between humans and animals. HiPSC-mediated study of antiviral immunity in both healthy controls and patients with HSV-1 encephalitis will be a powerful to. The human embryonic carcinoma cell line NT2 has been used as an in vitro model in studies of CNS neurons anti-HSV-1 immunity.

Plos Pathogens: The HSV-1 Latency-associated Transcript Functions To Repress Latent Phase Lytic Gene Expression And Suppress Virus Reactivation From Latently Infected Neurons

In vitro TH-mediated HSV-1 latency cell culture model 3The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Because infection is rarely fatal and HSV establishes latency, over one third of the world’s population has recurrent HSV infections and, therefore, the capability of transmitting HSV during episodes of productive infection. As with primary HSV-1 infection, recurrent infection may occur in the absence of clinical symptoms. Viral shedding as detected by culture lasts 10-12 days, and lesions resolve over 16-20 days. Animal studies suggest that activated macrophages, interferons, and, to a lesser extent, natural killer cells are important in limiting initial HSV infection, whereas humoral immunity and cell-mediated immunity are important in controlling both initial and recurrent infections. Project 1. HSV latency in cultured neurons: who’s in control, the virus or the host? We are now using this in vitro system to understand the role of the virus-encoded transcription factor VP16 and its cellular cofactor HCF-1 in overcoming epigenetic barriers to reactivation. HSV infects epithelial cells in the mucosa or skin, then enters peripheral nerve endings and travels intraaxonally to the sensory ganglia. Using mouse models of HSV infection, it is possible to derive detailed mechanisms of host resistance in different anatomical compartments. Adoptive transfer experiments of primed T cells from local LNs indicate an important role for CD4 T cells in resolving cutaneous infections, probably mediated by recruitment and activation of macrophages (2). This indicates that as the virus moves from one compartment to another, i. Following primary ocular infection, HSV-1 remains latent in the sensory neurons of trigeminal ganglia (TG) for the life of the host, with periodic stress-induced reactivation that produces progeny viruses in the eye causing potentially blinding recurrent corneal herpetic disease. As observed in animal models, herpes virus-specific T-cell responses have been reported to both protect against disease as well as cause disease 8.

Control Of Viral Latency In Neurons By Axonal Mtor Signaling And The 4e-bp Translation Repressor