A Model of In Vivo HSV-1 DNA Transport Using Murine Retinal Ganglion Cells.

Mammalian nervous tissues are heterogeneous. The retina, brain, spinal cord, and peripheral sensory and autonomic ganglia are each composed of neuronal and glial cell partners embedded in a connective tissue bed and supplied by vascular and immune cells. This complicated structure presents many challenges to neuroscientists and cell biologists (e.g., how to carry out a quantitative study of neurons surrounded by the hormonal and immune stimuli of supporting cells). A reductionist view has led investigators to study tissue slices and cultures of isolated neurons in vitro, subtracting the immune and vascular components to simplify the problem. Recently, investigators have extended the approach and produced organoids which are derived from embryonic neurons from induced pluripotent stem cells (Muffat et al., Proc Natl Acad Sci U S A 115:7117-7122, 2018).Using this approach advances have been made in the study of viral infections of the nervous system. For example, by using a genetically modified carrier virus, they can compare the effect of different viral envelope proteins on viral tropism and viral response pathways. However, the timed delivery of hormonal stimuli and interactions with immune cells remain problematic.We present an alternative method for studying these issues using the axonal transport of Herpes simplex virus in mature retinal neurons in vivo. Using genetically identical mice and carefully controlling the delivery of virus, an investigator can obtain insight into the transport of virus to and from the neuron cell body within the cellular environment of an intact, mature animal. This allows confirmation and extension of results seen in vitro.

The Basic Domain of Herpes Simplex Virus 1 pUS9 Recruits Kinesin-1 To Facilitate Egress from Neurons.

UNLABELLED: The alphaherpesviral envelope protein pUS9 has been shown to play a role in the anterograde axonal transport of herpes simplex virus 1 (HSV-1), yet the molecular mechanism is unknown. To address this, we used an in vitro pulldown assay to define a series of five arginine residues within the conserved pUS9 basic domain that were essential for binding the molecular motor kinesin-1. The mutation of these pUS9 arginine residues to asparagine blocked the binding of both recombinant and native kinesin-1. We next generated HSV-1 with the same pUS9 arginine residues mutated to asparagine (HSV-1pUS9KBDM) and then restored them being to arginine (HSV-1pUS9KBDR). The two mutated viruses were analyzed initially in a zosteriform model of recurrent cutaneous infection. The primary skin lesion scores were identical in severity and kinetics, and there were no differences in viral load at dorsal root ganglionic (DRG) neurons at day 4 postinfection (p.i.) for both viruses. In contrast, HSV-1pUS9KBDM showed a partial reduction in secondary skin lesions at day 8 p.i. compared to the level for HSV-1pUS9KBDR. The use of rat DRG neuronal cultures in a microfluidic chamber system showed both a reduction in anterograde axonal transport and spread from axons to nonneuronal cells for HSV-1pUS9KBDM. Therefore, the basic domain of pUS9 contributes to anterograde axonal transport and spread of HSV-1 from neurons to the skin through recruitment of kinesin-1. IMPORTANCE: Herpes simplex virus 1 and 2 cause genital herpes, blindness, encephalitis, and occasionally neonatal deaths. There is also increasing evidence that sexually transmitted genital herpes increases HIV acquisition, and the reactivation of HSV increases HIV replication and transmission. New antiviral strategies are required to control resistant viruses and to block HSV spread, thereby reducing HIV acquisition and transmission. These aims will be facilitated through understanding how HSV is transported down nerves and into skin. In this study, we have defined how a key viral protein plays a role in both axonal transport and spread of the virus from nerve cells to the skin.

In vivo HSV-1 DNA transport studies using murine retinal ganglion cells.

The mammalian retina, brain, spinal cord, and peripheral ganglia are all heterogeneous tissues. Each is composed of neuronal and glial cell partners embedded in a connective tissue bed and supplied by vascular and immune cells. This complicated structure presents many challenges to neuroscientists and cell biologists, e.g., how to carry out a quantitative study of neurons in a mature animal surrounded by the hormonal and immune stimuli. A reductionist view leads investigators to study single neurons in vitro, subtracting the immune and vascular components and simplifying the problem. While this has advantages, it limits relevance of the study. We present a method for studying the axonal transport of Herpes simplex virus in mature neurons in situ. Using genetically identical mice and carefully controlling the delivery of virus, an investigator can obtain insight into the transport of virus to and from the neuron cell body within the cellular environment of an intact animal.

Delivery of herpes simplex virus to retinal ganglion cell axon is dependent on viral protein Us9.

PURPOSE: How herpes simplex virus (HSV) is transported from the infected neuron cell body to the axon terminal is poorly understood. Several viral proteins are candidates for regulating the process, but the evidence is controversial. We compared the results of Us9 deletions in two HSV strains (F and NS) using a novel quantitative assay to test the hypothesis that the viral protein Us9 regulates the delivery of viral DNA to the distal axon of retinal ganglion cells in vivo. We also deleted a nine-amino acid motif in the Us9 protein of F strain (Us9-30) to define the role of this domain in DNA delivery. METHODS: The vitreous chambers of murine eyes were infected with equivalent amounts of F or NS strains of HSV. At 3, 4, or 5 days post infection (dpi), both optic tracts (OT) were dissected and viral genome was quantified by qPCR. RESULTS: At 3 dpi, the F strain Us9- and Us9-30 mutants delivered less than 10% and 1%, respectively, of the viral DNA delivered after infection with the Us9R (control) strain. By 4 and 5 dpi, delivery of viral DNA had only partially recovered. Deletion of Us9 in NS-infected mice has a less obvious effect on delivery of new viral DNA to the distal OT. By 3 dpi the NS Us9-strain delivered 22% of the DNA that was delivered by the NS wt, and by 4 and 5 dpi the amount of Us9-viral DNA was 96% and 81%, respectively. CONCLUSIONS: A highly conserved acidic cluster within the Us9 protein plays a critical role for genome transport to the distal axon. The transport is less dependent on Us9 expression in the NS than in the F strain virus. This assay can be used to compare transport efficiency in other neurotropic viral strains.

Characterization of a UL49-null mutant: VP22 of herpes simplex virus type 1 facilitates viral spread in cultured cells and the mouse cornea.

Herpes simplex virus type 1 (HSV-1) virions, like those of all herpesviruses, contain a proteinaceous layer termed the tegument that lies between the nucleocapsid and viral envelope. The HSV-1 tegument is composed of at least 20 different viral proteins of various stoichiometries. VP22, the product of the U(L)49 gene, is one of the most abundant tegument proteins and is conserved among the alphaherpesviruses. Although a number of interesting biological properties have been attributed to VP22, its role in HSV-1 infection is not well understood. In the present study we have generated both a U(L)49-null virus and its genetic repair and characterized their growth in both cultured cells and the mouse cornea. While single-step growth analyses indicated that VP22 is dispensable for virus replication at high multiplicities of infection (MOIs), analyses of plaque morphology and intra- and extracellular multistep growth identified a role for VP22 in viral spread during HSV-1 infection at low MOIs. Specifically, VP22 was not required for either virion infectivity or cell-cell spread but was required for accumulation of extracellular virus to wild-type levels. We found that the absence of VP22 also affected virion composition. Intracellular virions generated by the U(L)49-null virus contained reduced amounts of ICP0 and glycoproteins E and D compared to those generated by the wild-type and U(L)49-repaired viruses. In addition, viral spread in the mouse cornea was significantly reduced upon infection with the U(L)49-null virus compared to infection with the wild-type and U(L)49-repaired viruses, identifying a role for VP22 in viral spread in vivo as well as in vitro.

Genetic and molecular in vivo analysis of herpes simplex virus assembly in murine visual system neurons.

Herpes simplex virus (HSV) infects both epithelial cells and neuronal cells of the human host. Although HSV assembly has been studied extensively for cultured epithelial and neuronal cells, cultured neurons are biochemically, physiologically, and anatomically significantly different than mature neurons in vivo. Therefore, it is imperative that viral maturation and assembly be studied in vivo. To study viral assembly in vivo, we inoculated wild-type and replication-defective viruses into the posterior chamber of mouse eyes and followed infection in retinal ganglion cell bodies and axons. We used PCR techniques to detect viral DNA and RNA and electron microscopy immunohistochemistry and Western blotting to detect viral proteins in specific portions of the optic tract. This approach has shown that viral DNA replication is necessary for viral DNA movement into axons. Movement of viral DNA along ganglion cell axons occurs within capsid-like structures at the speed of fast axonal transport. These studies show that the combined use of intravitreal injections of replication-defective viruses and molecular probes allows the genetic analysis of essential viral replication and maturation processes in neurons in vivo. The studies also provide novel direct evidence for the axonal transport of viral DNA and support for the subassembly hypothesis of viral maturation in situ.

Temporal expression of herpes simplex virus type 1 mRNA in murine retina.

PURPOSE: During maturation of herpes simplex virus type 1 (HSV) in infected murine retinal F strain ganglion cells, new viral components are axonally transported in two phases. The viral envelope protein (gD) appears 48 hr before the capsid protein (VP5). Our hypothesis was that delayed appearance of VP5 mRNA in the infected eye causes the delayed expression of the VP5 protein in the axon. METHODS: HSV was injected into the ocular posterior chamber. Three to 24 hr later, the mice were euthanized, and the posterior eye was isolated. RNA was extracted, DNAase-treated, and used for amplification by reverse transcription-polymerase chain reaction (RT-PCR) using primers specific to gD, VP5 and a tegument protein VP22. RESULTS: VP22 and gD mRNAs are expressed 6 hr and VP5 mRNA is first detected 9 hr after infection. CONCLUSIONS: The results establish that delayed transcription does not play a significant role in the 48-hr delay in VP5 appearance in the retinal axons.

The microvesicle as a vehicle for EMMPRIN in tumor-stromal interactions.

EMMPRIN is a transmembrane glycoprotein expressed at high levels by tumor cells. It has been identified as a tumor-derived factor that can stimulate matrix metalloproteinase expression in fibroblasts and hence facilitate tumor invasion and metastasis. Recent studies have shown that full-length EMMPRIN is released by tumor cells, but the mechanism of release remains unclear. Here, we show that EMMPRIN is released from the surface of NCI-H460 cells via microvesicle shedding. However, these vesicles are unstable and rapidly break down to release bioactive EMMPRIN. Although microvesicle shedding has been considered a constitutive process in tumor cells, our data show that it can be amplified upon cell exposure to PMA, elucidating at least one signalling cascade responsible for EMMPRIN release. This pathway is dependent on protein kinase C, calcium mobilization and mitogen-activated protein kinase kinase (MEK 1/2). Thus, the results outline a novel form of tumor-stromal interaction in which extracellular matrix degradation by fibroblasts is controlled through the microvesicular release of EMMPRIN from tumor cells.

A procedure to deliver herpes simplex virus to the murine trigeminal ganglion.

Although initial herpes simplex virus (HSV) infections of the cornea are relatively easily treated, recurrent infections following reactivation of latent virus in the sensory ganglion cells are more difficult to treat. Untreated infections may result in severe consequences, including corneal scarring, glaucoma, and encephalitis. To develop such treatments, an experimental in vivo model was needed in which HSV can be applied directly to trigeminal ganglion cells. We have previously developed such a model to examine the mechanisms of HSV spread from trigeminal neurons to corneal epithelial cells. The current paper describes in detail the technical steps required for implementation of that model. Immunocytochemistry and electron microscopy have been used to validate the efficacy of the described procedures. This technique will be useful for future in vivo studies of neurotrophic viral infections of trigeminal ganglion cells.

Axonal transport and sorting of herpes simplex virus components in a mature mouse visual system.

The time course for delivery and transport of two major proteins of herpes simplex virus (HSV) has been determined for mature mouse retinal ganglion cell axons in vivo. Twenty-four hours after intravitreal injection of HSV, valacyclovir was introduced into the drinking water of the mice to inhibit subsequent viral replication. Without treatment, viral spread and replication in periaxonal glial cells confound study of axonal transport. At 2 to 5 days after infection, the animals were sacrificed and contiguous segments of the optic pathway were removed. Immunofluorescence microscopy indicated that the number of infected astrocytes was reduced in the proximal optic nerve and eliminated in the optic tract. Western blots of the retina with antibodies for envelope and capsid components, glycoprotein D (gD) and VP5, respectively, revealed that both components were expressed in retinal homogenates by 2 days. Results of reverse transcription-PCR indicated that there was no gD mRNA present in the treated optic tract 5 days after infection. Therefore, we conclude that gD is transcribed from viral mRNA in the retinal ganglion cell bodies. The gD accumulated in the proximal ganglion cell axon by 2 days and reached the most distal segment after 3 days. The VP5 first appeared in the proximal axons at 4 days, about 48 h after the appearance of gD. Thus, gD entered the axon earlier and independent of VP5. These finding confirm the subassembly model of viral transport in neurons and suggest that there is a 4- to 5-day window for initiation of effective antiviral treatment with valacyclovir.