Faculty Bibliography

How type I and II interferons prevent periodic reemergence of latent pathogens in tissues of diverse cell types remains unknown. Using homogeneous neuron cultures latently infected with herpes simplex virus 1, we show that extrinsic type I or II interferon acts 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.

Herpes simplex virus type (HSV) establishes a latent reservoir in neurons of human peripheral nerves. In this quiescent state the viral genome persists as a circular, histone-associated episome and transcription of viral lytic-cycle genes is largely suppressed through epigenetic processes. Periodically latent virus undergoes reactivation whereby lytic genes are activated and viral replication occurs. In this GEM we review recent evidence that mechanisms governing the initial transcription of lytic genes are distinct from those of de novo infection and directly link reactivation to neuronal stress response pathways. We also discuss evidence that lytic cycle gene expression can be uncoupled from the full reactivation program, arguing for a less sharply bimodal definition of latency.

To facilitate studies of herpes simplex virus 1 latency, cell culture models of quiescent or latent infection have been developed. Using deep sequencing, we analyzed the expression of viral microRNAs (miRNAs) in two models employing human fibroblasts and one using rat neurons. In all cases, the expression patterns differed from that in productively infected cells, with the rat neuron pattern most closely resembling that found in latently infected human or mouse ganglia in vivo.

We describe a primary neuronal culture system suitable for molecular characterization of herpes simplex virus type 1 (HSV-1) infection, latency, and reactivation. While several alternative models are available, including infections of live animal and explanted ganglia, these are complicated by the presence of multiple cell types, including immune cells, and difficulties in manipulating the neuronal environment. The highly pure neuron culture system described here can be readily manipulated and is ideal for molecular studies that focus exclusively on the relationship between the virus and host neuron, the fundamental unit of latency. As such it allows for detailed investigations of both viral and neuronal factors involved in the establishment and maintenance of HSV-1 latency and in viral reactivation induced by defined stimuli.

After replicating in surface epithelia, herpes simplex virus type-1 (HSV-1) enters the axonal terminals of peripheral neurons. The viral genome translocates to the nucleus, where it establishes a specialized infection known as latency, re-emerging periodically to seed new infections. Studies using cultured neuron models that faithfully recapitulate the molecular hallmarks of latency and reactivation defined in live animal models have provided fresh insight into the control of latency and connections to neuronal physiology. With this comes a growing appreciation for how the life cycles of HSV-1 and other herpesviruses are governed by key host pathways controlling metabolic homeostasis and cell identity.

Latent herpes simplex virus-1 (HSV1) genomes in peripheral nerve ganglia periodically reactivate, initiating a gene expression program required for productive replication. Whether molecular cues detected by axons can be relayed to cell bodies and harnessed to regulate latent genome expression in neuronal nuclei is unknown. Using a neuron culture model, we found that inhibiting mTOR, depleting its regulatory subunit raptor, or inducing hypoxia all trigger reactivation. While persistent mTORC1 activation suppressed reactivation, a mutant 4E-BP (eIF4E-binding protein) translational repressor unresponsive to mTORC1 stimulated reactivation. Finally, inhibiting mTOR in axons induced reactivation. Thus, local changes in axonal mTOR signaling that control translation regulate latent HSV1 genomes in a spatially segregated compartment.

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility. Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA(+) neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor(1). A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

Herpes simplex virus type-1 (HSV-1) establishes latency in peripheral neurons, creating a permanent source of recurrent infections. The latent genome is assembled into chromatin and lytic cycle genes are silenced. Processes that orchestrate reentry into productive replication (reactivation) remain poorly understood. We have used latently infected cultures of primary superior cervical ganglion (SCG) sympathetic neurons to profile viral gene expression following a defined reactivation stimulus. Lytic genes are transcribed in two distinct phases, differing in their reliance on protein synthesis, viral DNA replication and the essential initiator protein VP16. The first phase does not require viral proteins and has the appearance of a transient, widespread de-repression of the previously silent lytic genes. This allows synthesis of viral regulatory proteins including VP16, which accumulate in the cytoplasm of the host neuron. During the second phase, VP16 and its cellular cofactor HCF-1, which is also predominantly cytoplasmic, concentrate in the nucleus where they assemble an activator complex on viral promoters. The transactivation function supplied by VP16 promotes increased viral lytic gene transcription leading to the onset of genome amplification and the production of infectious viral particles. Thus regulated localization of de novo synthesized VP16 is likely to be a critical determinant of HSV-1 reactivation in sympathetic neurons.

HYPOTHESIS: Reactivation of herpes simplex virus type 1 (HSV-1) in geniculate ganglion neurons (GGNs) is an etiologic mechanism of Bell's palsy (BP) and delayed facial palsy (DFP) after otologic surgery. BACKGROUND: Several clinical studies, including temporal bone studies, antibody, titers, and intraoperative studies, suggest that reactivation of HSV-1 from latently infected GGNs may lead to both BP and DFP. However, it is difficult to study these processes in humans or live animals. METHODS: Primary cultures of GGNs were latently infected with Patton strain HSV-1 expressing a green fluorescent protein-late lytic gene chimera. Four days later, these cultures were treated with trichostatin A (TSA), a known chemical reactivator of HSV-1 in other neurons. Cultures were monitored daily by fluorescent microscopy. Titers of media from lytic, latent, and latent/TSA treated GGN cultures were obtained using plaque assays on Vero cells. RNA was harvested from latently infected GGN cultures and examined for the presence of viral transcripts using reverse transcription-polymerase chain reaction. RESULTS: Latently infected GGN cultures displayed latency-associated transcripts only, whereas lytically infected and reactivated latent cultures produced other viral transcripts, as well. The GGN cultures displayed a reactivation rate of 65% after treatment with TSA. Media from latently infected cultures contained no detectable infectious HSV-1, whereas infectious virus was observed in both lytically and latently infected/TSA-treated culture media. CONCLUSION: We have shown that cultured GGNs can be latently infected with HSV-1, and HSV-1 in these latently infected neurons can be reactivated using TSA, yielding infectious virus. These results have implications for the cause of both BP and DFP

The four Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded interferon (IFN) regulatory factor homologues (vIRF1 to vIRF4) are used to counter innate immune defenses and suppress p53. The vIRF genes are arranged in tandem but differ in function and expression. In KSHV-infected effusion lymphoma lines, K10.5/vIRF3 and K11/vIRF2 mRNAs are readily detected during latency, whereas K9/vIRF1 and K10/vIRF4 mRNAs are upregulated during reactivation. Here we show that the K10/vIRF4 promoter responds to the lytic switch protein RTA in KSHV-infected cells but is essentially unresponsive in uninfected cells. Coexpression of RTA with vIRF4 is sufficient to restore regulation, a property not shared by other vIRFs. The K9/vIRF1 promoter behaves similarly, and production of infectious virus is enhanced by the presence of vIRF4. Synergy requires the DNA-binding domain (DBD) and C-terminal IRF homology regions of vIRF4. Mutations of arginine residues within the putative DNA recognition helix of vIRF4 or the invariant cysteines of the adjacent CxxC motif abolish cooperation with RTA, in the latter case by preventing self-association. The oligomerization and transactivation functions of RTA are also essential for synergy. The K10/vIRF4 promoter contains two transcription start sites (TSSs), and a 105-bp fragment containing the proximal promoter is responsive to vIRF4/RTA. Binding of a cellular factor(s) to this fragment is altered when both viral proteins are present, suggesting a possible mechanism for transcriptional synergy. Reliance on coregulators encoded by either the host or viral genome provides an elegant strategy for expanding the regulatory potential of a master regulator, such as RTA.