Using Homogeneous Primary Neuron Cultures to Study Fundamental Aspects of HSV-1 Latency and Reactivation

Kim, Ju Youn; Shiflett, Lora A.; Linderman, Jessica A.; Mohr, Ian; Wilson, Angus C.

doi:10.1007/978-1-4939-0428-0_11

Cited by 8 publications

(10 citation statements)

References 11 publications

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“…We therefore evaluated stimuli for their ability to drive the re-initiation of a productive infection in hESC-derived neurons quiescently infected (GFP-negative) with VZV, as measured by ORF66GFP expression. NGF signaling has been shown to be required to maintain the quiescent state of HSV in the dissociated rodent sympathetic neuron model [ 24 ] [ 25 ], and can facilitate the maintenance of HSV latency in the murine ganglionic explant model [ 26 ] [ 27 ]. We therefore incubated persistently infected cultures in media lacking the three neurotrophin growth factors added to our neuronal cultures: NGF, BDNF and NT3 (18).…”

Section: Resultsmentioning

confidence: 99%

An In Vitro Model of Latency and Reactivation of Varicella Zoster Virus in Human Stem Cell-Derived Neurons

et al. 2015

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Varicella zoster virus (VZV) latency in sensory and autonomic neurons has remained enigmatic and difficult to study, and experimental reactivation has not yet been achieved. We have previously shown that human embryonic stem cell (hESC)-derived neurons are permissive to a productive and spreading VZV infection. We now demonstrate that hESC-derived neurons can also host a persistent non-productive infection lasting for weeks which can subsequently be reactivated by multiple experimental stimuli. Quiescent infections were established by exposing neurons to low titer cell-free VZV either by using acyclovir or by infection of axons in compartmented microfluidic chambers without acyclovir. VZV DNA and low levels of viral transcription were detectable by qPCR for up to seven weeks. Quiescently-infected human neuronal cultures were induced to undergo renewed viral gene and protein expression by growth factor removal or by inhibition of PI3-Kinase activity. Strikingly, incubation of cultures induced to reactivate at a lower temperature (34°C) resulted in enhanced VZV reactivation, resulting in spreading, productive infections. Comparison of VZV genome transcription in quiescently-infected to productively-infected neurons using RNASeq revealed preferential transcription from specific genome regions, especially the duplicated regions. These experiments establish a powerful new system for modeling the VZV latent state, and reveal a potential role for temperature in VZV reactivation and disease.

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Section: Resultsmentioning

confidence: 99%

An In Vitro Model of Latency and Reactivation of Varicella Zoster Virus in Human Stem Cell-Derived Neurons

et al. 2015

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“…Mouse and rabbit models have been used to characterize the HSV-1 infection, and guinea pigs have been favored for HSV-2 infection, at a pathological level, while in vitro tissue culture and primary cell culture models have been used to study molecular pathways of latency in more detail (2,41,42). Although we have learned a great deal from the mouse and rabbit models of human HSV-1 infection, there are many questions that could not be answered with these models.…”

Section: Discussionmentioning

confidence: 99%

Herpes Simplex Virus 1 Infection of Tree Shrews Differs from That of Mice in the Severity of Acute Infection and Viral Transcription in the Peripheral Nervous System

Wang

et al. 2016

J Virol

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Studies of herpes simplex virus (HSV) infections of humans are limited by the use of rodent models such as mice, rabbits, and guinea pigs. Tree shrews (Tupaia belangeri chinensis) are small mammals indigenous to southwest Asia. At behavioral, anatomical, genomic, and evolutionary levels, tree shrews are much closer to primates than rodents are, and tree shrews are susceptible to HSV infection. Thus, we have studied herpes simplex virus 1 (HSV-1) infection in the tree shrew trigeminal ganglion (TG) following ocular inoculation. In situ hybridization, PCR, and quantitative reverse transcription-PCR (qRT-PCR) analyses confirm that HSV-1 latently infects neurons of the TG. When explant cocultivation of trigeminal ganglia was performed, the virus was recovered after 5 days of cocultivation with high efficiency. Swabbing the corneas of latently infected tree shrews revealed that tree shrews shed virus spontaneously at low frequencies. However, tree shrews differ significantly from mice in the expression of key HSV-1 genes, including ICP0, ICP4, and latency-associated transcript (LAT). In acutely infected tree shrew TGs, no level of ICP4 was observed, suggesting the absence of infection or a very weak, acute infection compared to that of the mouse. Immunofluorescence staining with ICP4 monoclonal antibody, and immunohistochemistry detection by HSV-1 polyclonal antibodies, showed a lack of viral proteins in tree shrew TGs during both acute and latent phases of infection. Cultivation of supernatant from homogenized, acutely infected TGs with RS1 cells also exhibited an absence of infectious HSV-1 from tree shrew TGs. We conclude that the tree shrew has an undetectable, or a much weaker, acute infection in the TGs. Interestingly, compared to mice, tree shrew TGs express high levels of ICP0 transcript in addition to LAT during latency. However, the ICP0 transcript remained nuclear, and no ICP0 protein could be seen during the course of mouse and tree shrew TG infections. Taken together, these observations suggest that the tree shrew TG infection differs significantly from the existing rodent models. IMPORTANCEHerpes simplex viruses (HSVs) establish lifelong infection in more than 80% of the human population, and their reactivation leads to oral and genital herpes. Currently, rodent models are the preferred models for latency studies. Rodents are distant from primates and may not fully represent human latency. The tree shrew is a small mammal, a prosimian primate, indigenous to southwest Asia. In an attempt to further develop the tree shrew as a useful model to study herpesvirus infection, we studied the establishment of latency and reactivation of HSV-1 in tree shrews following ocular inoculation. We found that the latent virus, which resides in the sensory neurons of the trigeminal ganglion, could be stress reactivated to produce infectious virus, following explant cocultivation and that spontaneous reactivation could be detected by cell culture of tears. Interestingly, the tree shrew model is quite different from...

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“…Superior cervical ganglia (SCG) neurons were isolated, cultured and latently-infected as described (Kim et al, 2014; Kobayashi et al, 2012a). Briefly, SCGs isolated from E21 Sprague-Dawley ® rat pups were incubated at 37°C in trypsin (0.1%, Invitrogen) and collagenase (10 μg/ml, Sigma) for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Immune Escape via a Transient Gene Expression Program Enables Productive Replication of a Latent Pathogen

et al. 2017

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SUMMARY How type I/II interferons prevent periodic reemergence of latent pathogens in tissues of diverse cell-types remains unknown. Using homogenous neuron cultures latently-infected with herpes simplex virus-1, we show that extrinsic type I or II interferon act directly on neurons to induce unique gene expression signatures and inhibit the reactivation-specific burst of viral genome-wide transcription called Phase I. Surprisingly, interferons suppressed reactivation only during a limited period early in Phase I preceding productive virus growth. Sensitivity to type II interferon was selectively lost if viral ICP0, which normally accumulates later in Phase I, was expressed before reactivation. Thus, interferons suppress reactivation by preventing initial expression of latent genomes but are ineffective once Phase I viral proteins accumulate, limiting interferon action. This demonstrates that inducible reactivation from latency is only transiently sensitive to interferon. Moreover, it illustrates how latent pathogens escape host immune control to periodically replicate by rapidly deploying an interferon-resistant state.

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Using Homogeneous Primary Neuron Cultures to Study Fundamental Aspects of HSV-1 Latency and Reactivation

Cited by 8 publications

References 11 publications

An In Vitro Model of Latency and Reactivation of Varicella Zoster Virus in Human Stem Cell-Derived Neurons

An In Vitro Model of Latency and Reactivation of Varicella Zoster Virus in Human Stem Cell-Derived Neurons

Herpes Simplex Virus 1 Infection of Tree Shrews Differs from That of Mice in the Severity of Acute Infection and Viral Transcription in the Peripheral Nervous System

Immune Escape via a Transient Gene Expression Program Enables Productive Replication of a Latent Pathogen

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