Invasion of Herpes Simplex Virus 1 into Murine Dermis: Role of Nectin-1 and Herpesvirus Entry Mediator as Cellular Receptors during Aging

Skin is a major target tissue of herpes simplex virus 1 (HSV-1), and we are only beginning to understand how individual receptors contribute to the initiation of infection in tissue. We recently demonstrated the impact of the receptors nectin-1 and herpesvirus entry mediator (HVEM) for entry of HSV-1 into murine epidermis. Here, we focus on viral invasion into the dermis, a further critical target tissue in vivo. In principle, murine dermal fibroblasts are highly susceptible to HSV-1, and we previously showed that nectin-1 and HVEM can act as alternative receptors. To characterize their contribution as receptors in dermal tissue, we established an ex vivo infection assay of murine dermis. Only after separation of the epidermis from the dermis, we observed single infected cells in the upper dermis from juvenile mice at 5 h postinfection with increasing numbers of infected cells at later times. While nectin-1-expressing cells were less frequently detected, we found HVEM expressed on most cells of juvenile dermis. The comparison of infection efficiency during aging revealed a strong delay in the onset of infection in the dermis from aged mice. This observation correlated with a decrease in nectin-1-expressing fibroblasts during aging while the number of HVEM-expressing cells remained stable. Accordingly, aged nectin-1-deficient dermis was less susceptible to HSV-1 than the dermis from control mice. Thus, we conclude that the reduced availability of nectin-1 in aged dermis is a key contributor to a decrease in infection efficiency during aging. IMPORTANCE HSV-1 is a prevalent human pathogen which invades skin and mucocutaneous linings. So far, the underlying mechanisms of how the virus invades tissue, reaches its receptors, and initiates infection are still unresolved. To unravel the mechanical prerequisites that limit or favor viral invasion into tissue, we need to understand the contribution of the receptors that are involved in viral internalization. Here, we investigated the invasion process into murine dermis with the focus on receptor availability and found that infection efficiency decreases in aging mice. Based on studies of the expression of the receptors nectin-1 and HVEM, we suggest that the decreasing number of nectin-1-expressing fibroblasts leads to a delayed onset of infection in the dermis from aged compared to juvenile mice. Our results imply that the level of infection efficiency in murine dermis is closely linked to the availability of the receptor nectin-1 and can change during aging

Endocytic Internalization of Herpes Simplex Virus 1 in Human Keratinocytes at Low Temperature

Herpes simplex virus 1 (HSV-1) can adopt a variety of pathways to accomplish cellular internalization. In human keratinocytes representing the natural target cell of HSV-1, both direct plasma membrane fusion and endocytic uptake have been found. The impact of either pathway in successful infection, however, remains to be fully understood. To address the role of each internalization mode, we performed infection studies at low temperature as a tool to interfere with endocytic pathways. Interestingly, successful HSV-1 entry in primary human keratinocytes and HaCaT cells was observed even at 7 degrees C, although it was delayed compared to infection at 37 degrees C. Moreover, ex vivo infection of murine epidermis demonstrated that virus entry at 7 degrees C is accomplished not only in cultured cells but also in tissue. Control experiments with cholera toxin B confirmed a block of endocytic uptake at 7 degrees C. In addition, uptake of dextran by macropinosomes and phagocytic uptake of latex beads were also inhibited at 7 degrees C. Infection of nectin-1-deficient murine keratinocytes affirmed that the entry at 7 degrees C was receptor dependent. Strikingly, the lysosomotropic agent ammonium chloride strongly inhibited HSV-1 entry, suggesting a role for endosomal acidification. Ultrastructural analyses in turn revealed free capsids in the cytoplasm as well as virus particles in vesicles after infection at 7 degrees C, supporting both plasma membrane fusion and endocytic internalization as already observed at 37 degrees C. Overall, entry of HSV-1 at 7 degrees C suggests that the virus can efficiently adopt nectin-1-dependent unconventional vesicle uptake mechanisms in keratinocytes, strengthening the role of endocytic internalization for successful infection. IMPORTANCE The human pathogen herpes simplex virus 1 (HSV-1) relies on multiple internalization pathways to initiate infection. Our focus is on the entry in human keratinocytes, the major in vivo target during primary and recurrent infection. While antivirals reduce the severity of clinical cases, there is no cure or vaccine against HSV. To develop strategies that interfere with virus penetration, we need to understand the various parameters and conditions that determine virus entry. Here, we addressed the impact of virus internalization via vesicles by blocking endocytic processes at low temperature. Intriguingly, we detected entry of HSV-1 even at 7 degrees C, which led to infection of primary keratinocytes and epidermal tissue. Moreover, electron microscopy of human keratinocytes at 7 degrees C support that internalization is based on fusion of the viral envelope with the plasma membrane as well as vesicle membranes. These results provide novel insights into conditions that still allow endocytic internalization of HSV-1

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice

Ex vivo Human Skin Infection with Herpes Simplex Virus 1

Although herpes simplex virus 1 (HSV-1) is a well-studied virus, how the virus invades its human host via skin and mucosa to reach its receptors and initiate infection remains an open question. For studies of HSV-1 infection in skin, mice have been used as animal models. Murine skin infection can be induced after injection or scratching of the skin, which provides insights into disease pathogenesis but is clearly distinct from the natural entry route in human tissue. To explore the invasion route of HSV-1 on the tissue level, we established an ex vivo infection assay using skin explants. Here, we detail a protocol allowing the investigation of how the virus overcomes mechanical barriers in human skin to penetrate in keratinocytes and dermal fibroblasts. The protocol includes the preparation of total skin samples, skin shaves, and of separated epidermis and dermis, which is followed by incubation in virus suspension. The ex vivo infection assay allows the visualization, quantification, and characterization of single infected cells in the epidermis and dermis prior to viral replication and the virus-induced tissue damage. Hence, this experimental approach enables the identification of primary viral entry portals

Entry of Herpes Simplex Virus 1 into Epidermis and Dermal Fibroblasts Is Independent of the Scavenger Receptor MARCO

To enter host cells, herpes simplex virus 1 (HSV-1) initially attaches to cell surface glycosammoglycans, followed by the requisite binding to one of several cellular receptors, leading to viral internalization. Although virus-receptor interactions have been studied in various cell lines, the contributions of individual receptors to uptake into target tissues such as mucosa, skin, and cornea are not well understood. We demonstrated that nectin-1 acts as a major receptor for HSV-1 entry into murine epidermis, while herpesvirus entry mediator (HVEM) can serve as an alternative receptor. Recently, the macrophage receptor with collagenous structure (MARCO) has been described to mediate adsorption of HSV-1 to epithelial cells. Here, we investigated the impact of MARCO on the entry process of HSV-1 into the two major cell types of skin, keratinocytes in the epidermis and fibroblasts in the underlying dermis. Using ex vivo infection of murine epidermis, we showed that HSV-1 entered basal keratinocytes of MARCO(-/-)epidermis as efficiently as those of control epidermis. In addition, entry into dermal fibroblasts was not impaired in the absence of MARCO. When we treated epidermis, primary keratinocytes, or fibroblasts with poly(l), a ligand for class A scavenger receptors, HSV-1 entry was strongly reduced. As we also observed reducing effects of poly(l) in the absence of both MARCO and scavenger receptor A1, we concluded that the inhibitory effects of poly(l) on HSV-1 infection are not directly linked to class A scavenger receptors. Overall, our results support that HSV-1 entry into skin cells is independent of MARCO. IMPORTANCE During entry into its host cells, the human pathogen herpes simplex virus (HSV) interacts with various cellular receptors. Initially, receptor interaction can mediate cellular adsorption, followed by receptor binding that triggers viral internalization. The intriguing question is which receptors are responsible for the various steps during entry into the natural target tissues of HSV? Previously, we demonstrated the role of nectin-1 as a major receptor and that of HVEM as an alternative receptor for HSV-1 to invade murine epidermis. As MARCO has been described to promote infection in skin, we explored the predicted role of MARCO as a receptor that mediates adsorption to epithelial cells. Our infection studies of murine skin cells indicate that the absence of MARCO does not interfere with the efficiency of HSV-1 entry and that the inhibitory effect on viral adsorption by poly(l), a ligand of MARCO, is independent of MARCO

Mechanical Barriers Restrict Invasion of Herpes Simplex Virus 1 into Human Oral Mucosa

Oral mucosa is one of the main target tissues of the human pathogen herpes simplex virus 1 (HSV-1). How the virus overcomes the protective epithelial barriers and penetrates the tissue to reach its receptors and initiate infection is still unclear. Here, we established an ex vivo infection assay with human oral mucosa that allows viral entry studies in a natural target tissue. The focus was on the susceptibility of keratinocytes in the epithelium and the characterization of cellular receptors that mediate viral entry. Upon ex vivo infection of gingiva or vestibular mucosa, we observed that intact human mucosa samples were protected from viral invasion. In contrast, the basal layer of the oral epithelium was efficiently invaded once the connective tissue and the basement membrane were removed. Later during infection, HSV-1 spread from basal keratinocytes to upper layers, demonstrating the susceptibility of the stratified squamous epithelium to HSV-1. The analysis of potential receptors revealed nectin-1 on most mucosal keratinocytes, whereas herpesvirus entry mediator (HVEM) was found only on a subpopulation of cells, suggesting that nectin-1 acts as primary receptor for HSV-1 in human oral mucosa. To mimic the supposed entry route of HSV-1 via microlesions in vivo, we mechanically wounded the mucosa prior to infection. While we observed a limited number of infected keratinocytes in some wounded mucosa samples, other samples showed no infected cells. Thus, we conclude that mechanical wounding of mucosa is insufficient for the virus to efficiently overcome epithelial barriers and to make entry-mediating receptors accessible. IMPORTANCE To invade the target tissue of its human host during primary infection, herpes simplex virus (HSV) must overcome the epithelial barriers of mucosa, skin, or cornea. For most viruses, the mechanisms underlying the invasion into the target tissues of their host organism are still open. Here, we established an ex vivo infection model of human oral mucosa to explore how HSV can enter its target tissue. Our results demonstrate that intact mucosa samples and even compromised tissue allow only very limited access of HSV to keratinocytes. Detailed understanding of barrier functions is an essential precondition to unravel how HSV bypasses the barriers and approaches its receptors in tissue and why it is beneficial for the virus to use a cell-cell adhesion molecule, such as nectin-1, as a receptor

Expression and nuclear localization of the TATA-box-binding protein during baculovirus infection

The TATA-box-binding protein (TBP) plays a key role in initiating eukaryotic transcription and is used by many viruses for viral transcription. We previously reported increased TBP levels during infection with the baculovirus Autographa californica multicapsid nuclear polyhedrovirus (AcMNPV). The TBP antiserum used in that study, however, cross-reacted with a baculoviral protein. Here, we reported that increased amounts of nuclear TBP were detected upon infection of Spodoptera frugiperda and TN-368 cells with a TBP-specific antiserum. TBP levels increased until 72 h post-infection (p.i.), whilst tbp transcripts decreased by 16 h p.i., which suggested a virus-induced influence on the TBP protein levels. To address a potential modification of the TBP degradation pathway during infection, we investigated the possible role of viral ubiquitin. Infection studies with AcMNPV recombinants carrying a mutated viral ubiquitin gene revealed that the TBP increase during infection was not altered. In addition, pulse-chase experiments indicated a high TBP half-life of similar to 60 h in uninfected cells, suggesting that a virus-induced increase of TBP stability was unlikely. This increase in TBP correlated with a redistribution to nuclear domains resembling sites of viral DNA synthesis. Furthermore, we observed colocalization of TBP with host RNA polymerase (RNAP) II, but only until 8 h p.i., whilst TBP, but not RNAPII, was present in the enlarged replication domains late during infection. Thus, we suggested that AcMNPV adapted a mechanism to accumulate the highly stable cellular TBP at sites of viral DNA replication and transcription

Invasion of Herpes Simplex Virus Type 1 into Murine Epidermis: An Ex Vivo Infection Study

Herpes simplex virus type 1 (HSV-1) invades its human host via the skin or mucosa. We aim to understand how HSV-1 overcomes the barrier function of the host epithelia, and for this reason, we established an ex vivo infection assay initially with murine skin samples. Here, we report how tissue has to be prepared to be susceptible to HSV-1 infection. Most efficient infection of the epidermis was achieved by removing the dermis. HSV-1 initially invaded the basal epidermal layer, and from there, spreading to the suprabasal layers was observed. Strikingly, in resting stage hair follicles, only the hair germ was infected, whereas the quiescent bulge stem cells (SCs) were resistant to infection. However, during the growth phase, infected cells were also detected in the activated bulge SCs. We demonstrated that cell proliferation was not a precondition for HSV-1 invasion, but SC activation was required as shown by infection of aberrantly activated bulge SCs in integrin-linked kinase (ILK)-deficient hair follicles. These results suggest that the status of the bulge SCs determines whether HSV-1 can reach its receptors, whereas the receptors on basal keratinocytes are accessible irrespective of their proliferation status

Epithelial Barriers in Murine Skin during Herpes Simplex Virus 1 Infection: The Role of Tight Junction Formation

Herpes simplex virus 1 has to overcome skin or mucosa barriers to infect its human host. The impact of the various barrier functions on successful viral invasion is not known. On ex vivo infection of murine skin, we observed efficient invasion only via the basal epidermal layer when the dermis was removed. Here, we investigated how wounding and intercellular junction formation control successful viral entry. After wounding of skin samples or removal of the stratum corneum, infected cells were rarely detected. On the basis of infection studies in epidermis from IFN-stimulated mice, we assume that mechanical wounding does not lead to an antiviral state that impedes infection. When we infected human skin equivalents, we observed entry only into unstratified keratinocytes or after wounding of fully stratified cultures. Reduced infection of keratinocytes after calcium-induced stratification confirmed the impact of junction formation. To assess the effect of functional tight junctions, stratified cultures of polarity regulator partitioning-defective-3-or E-cadherin-deficient keratinocytes were infected. As the number of infected cells strongly increased with enhanced paracellular permeability, we conclude that the formation of functional tight junctions interferes with viral entry indicating that next to the stratum corneum tight junctions are a major physical barrier for herpes simplex virus 1 invasion into tissue