Entry pathways of herpes simplex virus type 1 into human keratinocytes are dynamin- and cholesterol-dependent

Herpes simplex virus type 1 (HSV-1) can enter cells via endocytic pathways or direct fusion at the plasma membrane depending on the cell line and receptor(s). Most studies into virus entry have used cultured fibroblasts but since keratinocytes represent the primary entry site for HSV-1 infection in its human host, we initiated studies to characterize the entry pathway of HSV-1 into human keratinocytes. Electron microscopy studies visualized free capsids in the cytoplasm and enveloped virus particles in vesicles suggesting viral uptake both by direct fusion at the plasma membrane and by endocytic vesicles. The ratio of the two entry modes differed in primary human keratinocytes and in the keratinocyte cell line HaCaT. Inhibitor studies further support a role for endocytosis during HSV-1 entry. Infection was inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin, which demonstrates the requirement for host cholesterol during virus entry. Since the dynamin-specific inhibitor dynasore and overexpression of a dominant-negative dynamin mutant blocked infection, we conclude that the entry pathways into keratinocytes are dynamin-mediated. Electron microscopy studies confirmed that virus uptake is completely blocked when the GTPase activity of dynamin is inhibited. Ex vivo infection of murine epidermis that was treated with dynasore further supports the essential role of dynamin during entry into the epithelium. Thus, we conclude that HSV-1 can enter human keratinocytes by alternative entry pathways that require dynamin and host cholesterol.     

The proliferative and multipotent epidermal progenitor cells for human skin reconstruction in vitro and in vivo

ObjectivesThe skin exhibits tremendous regenerative potential, as different types of progenitor and stem cells regulate skin homeostasis and damage. However, in vitro primary keratinocytes present with several drawbacks, such as high donor variability, short lifespan, and limited donor tissue availability. Therefore, more stable primary keratinocytes are needed to generate multiple uniform in vitro and in vivo skin models.ResultsWe identified epidermal progenitor cells from primary keratinocytes using Integrin beta 1 (ITGB1) an epidermal stem cell marker markedly decreased after senescence in vitro. Epidermal progenitor cells exhibited unlimited proliferation and the potential for multipotent differentiation capacity. Moreover, they could completely differentiate to form an organotypic skin model including conversed mesenchymal cells in the dermis and could mimic the morphologic and biochemical processes of human epidermis. We also discovered that proliferation and the multipotent differentiation capacity of these cells relied on ITGB1 expression. Eventually, we examined the in vitro and in vivo wound healing capacity of these epidermal progenitor cells.ConclusionsOverall, the findings suggest that these stable and reproducible cells can differentiate into multiple lineages, including human skin models. They are a potentially powerful tool for studying skin regeneration, skin diseases, and are an alternative for in vivo experiments.

Our stable and reproducible epidermal progenitor cells from human epidermis have proliferation and multipotent differentiation potentials, regeneration capacity and could generate in vivo mimic 3D skin model not only for regeneration therapy but also for alternative animal experiments.     

Herpes Simplex Virus type 1 infects Langerhans cells and the novel epidermal dendritic cell,Epi-cDC2s,via different entry pathways

Skin mononuclear phagocytes (MNPs) provide the first interactions of invading viruses with the immune system. In addition to Langerhans cells (LCs), we recently described a second epidermal MNP population, Epi-cDC2s, in human anogenital epidermis that is closely related to dermal conventional dendritic cells type 2 (cDC2) and can be preferentially infected by HIV. Here we show that in epidermal explants topically infected with herpes simplex virus (HSV-1), both LCs and Epi-cDC2s interact with HSV-1 particles and infected keratinocytes. Isolated Epi-cDC2s support higher levels of infection than LCs in vitro, inhibited by acyclovir, but both MNP subtypes express similar levels of the HSV entry receptors nectin-1 and HVEM, and show similar levels of initial uptake. Using inhibitors of endosomal acidification, actin and cholesterol, we found that HSV-1 utilises different entry pathways in each cell type. HSV-1 predominantly infects LCs, and monocyte-derived MNPs, via a pH-dependent pathway. In contrast, Epi-cDC2s are mainly infected via a pH-independent pathway which may contribute to the enhanced infection of Epi-cDC2s. Both cells underwent apoptosis suggesting that Epi-cDC2s may follow the same dermal migration and uptake by dermal MNPs that we have previously shown for LCs. Thus, we hypothesize that the uptake of HSV and infection of Epi-cDC2s will stimulate immune responses via a different pathway to LCs, which in future may help guide HSV vaccine development and adjuvant targeting.     

Enhanced contact allergen- and UVB-induced keratinocyte apoptosis in the absence of CD95/Fas/Apo-1

FAS/CD95/Apo-1 is a ubiquitously expressed cell-surface receptor involved in the initiation of programmed cell death. Its function in epidermal keratinocytes has been incompletely defined. Available evidence from in vitro studies points to important roles of Fas in the pathogenesis of contact dermatitis and in keratinocyte apoptosis induced by ultraviolet light. To define functions of Fas in the epidermis in vivo, we have generated mice with epidermis-specific deletion of the fas gene and tested its requirement for 2,4-dinitrofluorobenzene-induced contact dermatitis and for ultraviolet light B (UVB)-induced keratinocyte apoptosis. We report here our unexpected finding that keratinocyte apoptosis induced by both a contact allergen and UVB irradiation was significantly enhanced in Fas-negative epidermis. Expression of Fas by epidermal keratinocytes was neither necessary for the normal development of contact hypersensitivity of the skin, nor required for keratinocyte apoptosis following UVB irradiation. Our study results thus show that in the epidermis in vivo Fas exerts antiapoptotic effects that outweigh its proapoptotic role in contact hypersensitivity responses of the skin and in the tissue response of the epidermis to UVB irradiation.     

Repression of gene expression upon infection of cells with herpes simplex virus type 1 mutants impaired for immediate-early protein synthesis.

Herpes simplex virus type 1 (HSV-1) mutants defective in immediate-early (IE) gene expression do not readily enter productive replication after infection of tissue culture cells. Instead, their genomes are retained in a quiescent, nonreplicating state in which the production of viral gene products cannot be detected. To investigate the block to virus replication, we used the HSV-1 triple mutant in1820K, which, under appropriate conditions, is effectively devoid of the transactivators VP16 (a virion protein), ICP0, and ICP4 (both IE proteins). Promoters for the HSV-1 IE ICP0 gene or the human cytomegalovirus (HCMV) major IE gene, cloned upstream of the Escherichia coli lacZ coding sequences, were introduced into the in1820K genome. The regulation of these promoters and of the endogenous HSV-1 IE promoters was investigated upon conversion of the virus to a quiescent state. Within 24 h of infection, the ICP0 promoter became much less sensitive to transactivation by VP16 whereas the same element, when used to transform Vero cells, retained its responsiveness. The HCMV IE promoter, which is not activated by VP16, also became less sensitive to the HCMV functional homolog of VP16. Both elements remained available for transactivation by HSV-1 IE proteins at 24 h postinfection, showing that the in1820K genome was not irreversibly inactivated. The promoters controlling the HSV-1 ICP4, ICP22, and ICP27 genes also became essentially unresponsive to transactivation by VP16. The ICP0 promoter was induced when hexamethylene bisacetamide was added to cultures at the time of infection, but the response to this agent was also lost by 24 h after infection. Therefore, promoter elements within the HSV-1 genome are actively repressed in the absence of IE gene expression, and repression is not restricted specifically to HSV-1 IE promoters.     

Herpes simplex virus type 1 pathogenicity in footpad and ear skin of mice depends on Langerhans cell density, mouse genetics, and virus strain.

Skin Langerhans cells have been shown to be very efficient in presenting antigens to T-helper cells and stimulating the immune response. The present study demonstrates their essential role in the control of primary herpetic infections in the skin. Two unrelated stimuli (abrasion and steroids) were shown to cause depletion of the Langerhans cells in the murine epidermis, and both caused enhancement of the virulence of herpes simplex type 1 (HSV-1) in the skin. The Langerhans cell density was found to be lower in the skin of the ear than in the footpad. HSV-1 was consistently more virulent when injected into the ear epidermis than in the footpad. Thus, HSV-1 pathogenicity in mouse skin depends on the mouse age and strain, the virus strain, and the state of the epidermal Langerhans cells. These findings are discussed in relation to the antigen-presenting cell function of the Langerhans cells.     

Secreted Herpes Simplex Virus-2 Glycoprotein G Modifies NGF-TrkA Signaling to Attract Free Nerve Endings to the Site of Infection

Herpes simplex virus type 1 (HSV-1) and HSV-2 are highly prevalent viruses that cause a variety of diseases, from cold sores to encephalitis. Both viruses establish latency in peripheral neurons but the molecular mechanisms facilitating the infection of neurons are not fully understood. Using surface plasmon resonance and crosslinking assays, we show that glycoprotein G (gG) from HSV-2, known to modulate immune mediators (chemokines), also interacts with neurotrophic factors, with high affinity. In our experimental model, HSV-2 secreted gG (SgG2) increases nerve growth factor (NGF)-dependent axonal growth of sympathetic neurons ex vivo, and modifies tropomyosin related kinase (Trk)A-mediated signaling. SgG2 alters TrkA recruitment to lipid rafts and decreases TrkA internalization. We could show, with microfluidic devices, that SgG2 reduced NGF-induced TrkA retrograde transport. In vivo, both HSV-2 infection and SgG2 expression in mouse hindpaw epidermis enhance axonal growth modifying the termination zone of the NGF-dependent peptidergic free nerve endings. This constitutes, to our knowledge, the discovery of the first viral protein that modulates neurotrophins, an activity that may facilitate HSV-2 infection of neurons. This dual function of the chemokine-binding protein SgG2 uncovers a novel strategy developed by HSV-2 to modulate factors from both the immune and nervous systems.     

Efficiency of Cell-Free and Cell-Associated Virus in Mucosal Transmission of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus

Effective strategies are needed to block mucosal transmission of human immunodeficiency virus type 1 (HIV-1). Here, we address a crucial question in HIV-1 pathogenesis: whether infected donor mononuclear cells or cell-free virus plays the more important role in initiating mucosal infection by HIV-1. This distinction is critical, as effective strategies for blocking cell-free and cell-associated virus transmission may be different. We describe a novel ex vivo model system that utilizes sealed human colonic mucosa explants and demonstrate in both the ex vivo model and in vivo using the rectal challenge model in rhesus monkeys that HIV-1-infected lymphocytes can transmit infection across the mucosa more efficiently than cell-free virus. These findings may have significant implications for our understanding of the pathogenesis of mucosal transmission of HIV-1 and for the development of strategies to prevent HIV-1 transmission.     

Tropism of and Innate Immune Responses to the Novel Human Betacoronavirus Lineage C Virus in Human Ex Vivo Respiratory Organ Cultures

Since April 2012, there have been 17 laboratory-confirmed human cases of respiratory disease associated with newly recognized human betacoronavirus lineage C virus EMC (HCoV-EMC), and 7 of them were fatal. The transmissibility and pathogenesis of HCoV-EMC remain poorly understood, and elucidating its cellular tropism in human respiratory tissues will provide mechanistic insights into the key cellular targets for virus propagation and spread. We utilized ex vivo cultures of human bronchial and lung tissue specimens to investigate the tissue tropism and virus replication kinetics following experimental infection with HCoV-EMC compared with those following infection with human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome coronavirus (SARS-CoV). The innate immune responses elicited by HCoV-EMC were also investigated. HCoV-EMC productively replicated in human bronchial and lung ex vivo organ cultures. While SARS-CoV productively replicated in lung tissue, replication in human bronchial tissue was limited. Immunohistochemistry revealed that HCoV-EMC infected nonciliated bronchial epithelium, bronchiolar epithelial cells, alveolar epithelial cells, and endothelial cells. Transmission electron microscopy showed virions within the cytoplasm of bronchial epithelial cells and budding virions from alveolar epithelial cells (type II). In contrast, there was minimal HCoV-229E infection in these tissues. HCoV-EMC failed to elicit strong type I or III interferon (IFN) or proinflammatory innate immune responses in ex vivo respiratory tissue cultures. Treatment of human lung tissue ex vivo organ cultures with type I IFNs (alpha and beta IFNs) at 1 h postinfection reduced the replication of HCoV-EMC, suggesting a potential therapeutic use of IFNs for treatment of human infection.     

A live-attenuated HSV-2 ICP0 virus elicits 10 to 100 times greater protection against genital herpes than a glycoprotein D subunit vaccine

Glycoprotein D (gD-2) is the entry receptor of herpes simplex virus 2 (HSV-2), and is the immunogen in the pharmaceutical industry''s lead HSV-2 vaccine candidate. Efforts to prevent genital herpes using gD-2 subunit vaccines have been ongoing for 20 years at a cost in excess of $100 million. To date, gD-2 vaccines have yielded equivocal protection in clinical trials. Therefore, using a small animal model, we sought to determine if a live-attenuated HSV-2 ICP0 ⁻ virus would elicit better protection against genital herpes than a gD-2 subunit vaccine. Mice immunized with gD-2 and a potent adjuvant (alum+monophosphoryl lipid A) produced high titers of gD-2 antibody. While gD-2-immunized mice possessed significant resistance to HSV-2, only 3 of 45 gD-2-immunized mice survived an overwhelming challenge of the vagina or eyes with wild-type HSV-2 (MS strain). In contrast, 114 of 115 mice immunized with a live HSV-2 ICP0 ⁻ virus, 0ΔNLS, survived the same HSV-2 MS challenges. Likewise, 0ΔNLS-immunized mice shed an average 125-fold less HSV-2 MS challenge virus per vagina relative to gD-2-immunized mice. In vivo imaging demonstrated that a luciferase-expressing HSV-2 challenge virus failed to establish a detectable infection in 0ΔNLS-immunized mice, whereas the same virus readily infected naïve and gD-2-immunized mice. Collectively, these results suggest that a HSV-2 vaccine might be more likely to prevent genital herpes if it contained a live-attenuated HSV-2 virus rather than a single HSV-2 protein.     

Analysis of the role of autophagy in replication of herpes simplex virus in cell culture

The herpes simplex virus type 1 (HSV-1) neurovirulence gene encoding ICP34.5 controls the autophagy pathway. HSV-1 strains lacking ICP34.5 are attenuated in growth and pathogenesis in animal models and in primary cultured cells. While this growth defect has been attributed to the inability of an ICP34.5-null virus to counteract the induction of translational arrest through the PKR antiviral pathway, the role of autophagy in the regulation of HSV-1 replication is unknown. Here we show that HSV-1 infection induces autophagy in primary murine embryonic fibroblasts and that autophagosome formation is increased to a greater extent following infection with an ICP34.5-deficient virus. Elimination of the autophagic pathway did not significantly alter the replication of wild-type HSV-1 or ICP34.5 mutants. The phosphorylation state of eIF2alpha and viral protein accumulation were unchanged in HSV-1-infected cells unable to undergo autophagy. These data show that while ICP34.5 regulates autophagy, it is the prevention of translational arrest by ICP34.5 rather than its control of autophagy that is the pivotal determinant of efficient HSV-1 replication in primary cell culture.     

Characterization of a Proteolytically Stable D-Peptide That Suppresses Herpes Simplex Virus 1 Infection: Implications for the Development of Entry-Based Antiviral Therapy

Uncontrolled herpes simplex virus 1 (HSV-1) infection can advance to serious conditions, including corneal blindness or fatal encephalitis. Here, we describe a highly potent anti-HSV-1 peptide (DG2) that inhibits HSV-1 entry into host cells and blocks all aspects of infection. Importantly, DG2 is highly resistant to proteases and shows minimal toxicity, paving the way for prophylactic or therapeutic application of the peptide in vivo.     

Induction and prevention of apoptosis in human HEp-2 cells by herpes simplex virus type 1

Cultured human epithelial cells infected with an ICP27 deletion strain of herpes simplex virus type 1 (HSV-1) show characteristic features of apoptotic cells including cell shrinkage, nuclear condensation, and DNA fragmentation. These cells do not show such apoptotic features when infected with a wild-type virus unless the infections are performed in the presence of a protein synthesis inhibitor. Thus, both types of virus induce apoptosis, but the ICP27-null virus is unable to prevent this process from killing the cells. In this report, we show that this ICP27-deficient virus induced apoptosis in human HEp-2 cells through a pathway which involved the activation of caspase-3 and the processing of the death substrates DNA fragmentation factor and poly(ADP-ribose) polymerase. The induction of apoptosis by wild-type HSV-1 occurred prior to 6 h postinfection (hpi), and de novo viral protein synthesis was not required to induce the process. The ability of the virus to inhibit apoptosis was shown to be effective between 3 to 6 hpi. Wild-type HSV-1 infection was also able to block the apoptosis induced in cells by the addition of cycloheximide, staurosporine, and sorbitol. While U(S)3- and ICP22-deficient viruses showed a partial prevention of apoptosis, deletion of either the U(L)13 or vhs gene products did not affect the ability of HSV-1 to prevent apoptosis in infected cells. Finally, we demonstrate that in UV-inactivated viruses, viral binding and entry were not sufficient to induce apoptosis. Taken together, these results suggest that either gene expression or another RNA metabolic event likely plays a role in the induction of apoptosis in HSV-1-infected human cells.     

The multipotency of adult vibrissa follicle stem cells

Several studies focused on the characterization of bulge keratinocytes have proved that they are multipotent stem cells, being recruited not only to regenerate the hair follicle itself, but also the sebaceous gland and the epidermis. However, due to the difficulty in preparing transplantable cell sheets harvested with conventional enzymatic digestion, there is still no direct evidence of the bulge stem cells’ multipotency. Whether they can respond to adult dermal papilla (DP) signals in recombination experiments also remains unclear. In this study, we addressed this problem by culturing and detaching intact bulge keratinocyte sheets from thermo-responsive culture dishes, only by reducing its temperature. When sheets of mass cultured bulge keratinocytes isolated from rat vibrissa follicles were recombined with fresh adult DPs and sole skin dermis in vivo, regeneration of epidermis and sebaceous gland-like structures, and formation of hair bulb with differentiating inner root sheath and hair cuticle were observed within 3 weeks. However, regardless the expression of stem cells markers like CD34, SA1004 and SA1006, no structures were observed when cloned bulge keratinocytes were used to prepare cell sheets and recombinants, revealing the possible existence of monoclonal stem cells within the bulge region. This report is the first to succeed in harvesting adult bulge keratinocyte sheets. Using these sheets it is demonstrated that bulge stem cells directly respond to adult DP signals to induce hair bulb formation in vivo.     

The PK Domain of the Large Subunit of Herpes Simplex Virus Type 2 Ribonucleotide Reductase (ICP10) Is Required for Immediate-Early Gene Expression and Virus Growth

The large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR), RR1, contains a unique amino-terminal domain which has serine/threonine protein kinase (PK) activity. To examine the role of the PK activity in virus replication, we studied an HSV type 2 (HSV-2) mutant with a deletion in the RR1 PK domain (ICP10ΔPK). ICP10ΔPK expressed a 95-kDa RR1 protein (p95) which was PK negative but retained the ability to complex with the small RR subunit, RR2. Its RR activity was similar to that of HSV-2. In dividing cells, onset of virus growth was delayed, with replication initiating at 10 to 15 h postinfection, depending on the multiplicity of infection. In addition to the delayed growth onset, virus replication was significantly impaired (1,000-fold lower titers) in nondividing cells, and plaque-forming ability was severely compromised. The RR1 protein expressed by a revertant virus [HSV-2(R)] was structurally and functionally similar to the wild-type protein, and the virus had wild-type growth and plaque-forming properties. The growth of the ICP10ΔPK virus and its plaque-forming potential were restored to wild-type levels in cells that constitutively express ICP10. Immediate-early (IE) genes for ICP4, ICP27, and ICP22 were not expressed in Vero cells infected with ICP10ΔPK early in infection or in the presence of cycloheximide, and the levels of ICP0 and p95 were significantly (three- to sevenfold) lower than those in HSV-2- or HSV-2(R)-infected cells. IE gene expression was similar to that of the wild-type virus in cells that constitutively express ICP10. The data indicate that ICP10 PK is required for early expression of the viral regulatory IE genes and, consequently, for timely initiation of the protein cascade and HSV-2 growth in cultured cells.     

Overexpression of CIN85 suppresses the growth of herpes simplex virus in HeLa cells

The adaptor protein CIN85 is widely distributed in different tissues and has three Src homology 3 (SH3) domains, a proline-rich region (PRR), and a coiled-coil domain. During studies on the function of CIN85, it was reported to form a complex with herpes simplex virus 1 (HSV-1) infected cell protein 0 (ICP0), which plays a key role in enabling viral replication. Here, we demonstrate that plaque formation by HSV-1 is reduced on HeLa cells expressing CIN85 ectopically. The PRR of CIN85 was found to be essential for the inhibition of virus growth, whereas the three SH3 domains were not required. CIN85 also suppressed HSV-1 growth in Chinese hamster ovary (CHO) cells expressing the receptor for herpes simplex virus entry (herpes virus entry mediator A; HVEM). However, immunoprecipitation experiments showed that CIN85 did not interact with HVEM directly, indicating that CIN85 is not involved in the HSV-1 cell-entry pathway, but rather in another downstream pathway. Collectively, our data indicate that CIN85 might play an important role in HSV-1 infection.     

Herpes simplex virus type 1 ICP27-dependent activation of NF-kappaB

The ability of herpes simplex virus type 1 (HSV-1) to activate NF-kappaB has been well documented. Beginning at 3 to 5 h postinfection, HSV-1 induces a robust and persistent nuclear translocation of an NF-kappaB-dependent (p50/p65 heterodimer) DNA binding activity, as measured by electrophoretic mobility shift assay. Activation requires virus binding and entry, as well as de novo infected-cell protein synthesis, and is accompanied by loss of both IkappaBalpha and IkappaBbeta. In this study, we identified loss of IkappaBalpha as a marker of NF-kappaB activation, and infection with mutants with individual immediate-early (IE) regulatory proteins deleted indicated that ICP27 was necessary for IkappaBalpha loss. Analysis of both N-terminal and C-terminal mutants of ICP27 identified the region from amino acids 21 to 63 as being necessary for IkappaBalpha loss. Additional experiments with mutant viruses with combinations of IE genes deleted revealed that the ICP27-dependent mechanism of NF-kappaB activation may be augmented by functional ICP4. We also analyzed two additional markers for NF-kappaB activation, phosphorylation of the p65 subunit on Ser276 and Ser536. Phosphorylation of both serines was induced upon HSV infection and required functional ICP4 and ICP27. Pharmacological inhibitor studies revealed that both IkappaBalpha and Ser276 phosphorylation were dependent on Jun N-terminal protein kinase activity, while Ser536 phosphorylation was not affected during inhibitor treatment. These results demonstrate that there are several layers of regulation of NF-kappaB activation during HSV infection, highlighting the important role that NF-kappaB may play in infection.     

Herpes simplex virus-induced epithelial damage and susceptibility to human immunodeficiency virus type 1 infection in human cervical organ culture

Normal human premenopausal cervical tissue has been used to derive primary cell populations and to establish ex vivo organ culture systems to study infections with herpes simplex virus (HSV-1 or HSV-2) and human immunodeficiency virus type 1 (HIV-1). Infection with either HSV-1 or HSV-2 rapidly induced multinuclear giant cell formation and widespread damage in mucosal epithelial cells. Subsequent exposure of the damaged mucosal surfaces to HIV-1 revealed frequent co-localization of HSV and HIV-1 antigens. The short-term organ culture system provides direct experimental support for the epidemiological findings that pre-existing sexually transmitted infections, including primary and recurrent herpes virus infections at mucosal surfaces, represent major risk factors for acquisition of primary HIV-1 infection. Epithelial damage in combination with pre-existing inflammation, as described here for overtly normal human premenopausal cervix, creates a highly susceptible environment for the initiation and establishment of primary HIV-1 infection in the sub-mucosa of the cervical transformation zone.