Anti-Glycoprotein H Antibody Impairs the Pathogenicity of Varicella-Zoster Virus in Skin Xenografts in the SCID Mouse Model

Varicella-zoster virus (VZV) infection is usually mild in healthy individuals but can cause severe disease in immunocompromised patients. Prophylaxis with varicella-zoster immunoglobulin can reduce the severity of VZV if given shortly after exposure. Glycoprotein H (gH) is a highly conserved herpesvirus protein with functions in virus entry and cell-cell spread and is a target of neutralizing antibodies. The anti-gH monoclonal antibody (MAb) 206 neutralizes VZV in vitro. To determine the requirement for gH in VZV pathogenesis in vivo, MAb 206 was administered to SCID mice with human skin xenografts inoculated with VZV. Anti-gH antibody given at 6 h postinfection significantly reduced the frequency of skin xenograft infection by 42%. Virus titers, genome copies, and lesion size were decreased in xenografts that became infected. In contrast, administering anti-gH antibody at 4 days postinfection suppressed VZV replication but did not reduce the frequency of infection. The neutralizing anti-gH MAb 206 blocked virus entry, cell fusion, or both in skin in vivo. In vitro, MAb 206 bound to plasma membranes and to surface virus particles. Antibody was internalized into vacuoles within infected cells, associated with intracellular virus particles, and colocalized with markers for early endosomes and multivesicular bodies but not the trans-Golgi network. MAb 206 blocked spread, altered intracellular trafficking of gH, and bound to surface VZV particles, which might facilitate their uptake and targeting for degradation. As a consequence, antibody interference with gH function would likely prevent or significantly reduce VZV replication in skin during primary or recurrent infection.Varicella-zoster virus (VZV) causes chicken pox (varicella) upon primary infection. Lifelong latency is established in neurons of the sensory ganglia, and reactivation leads to shingles (herpes zoster) (1). Disease is usually inconsequential in immunocompetent people but can be severe in immunocompromised patients. The current prophylaxis for these high-risk individuals exposed to VZV is high-titer immunoglobulin to VZV administered within 96 h of exposure. This prophylaxis does not always prevent disease, but the severity of symptoms and mortality rates are usually reduced (32).Glycoprotein H (gH) is a type 1 transmembrane protein that is required for virus-cell and cell-cell spread in all herpesviruses studied (12, 15, 24, 26). gH is an important target of the host immune system. Individuals who have had primary infection with VZV or herpes simplex virus (HSV), the most closely related human alphaherpesvirus, have humoral and cellular immunity against gH (1, 56). Immunization of mice with a recombinant vaccinia virus expressing VZV gH and its chaperone, glycoprotein L (gL), induced specific antibodies capable of neutralizing VZV in vitro (28, 37). Immunization of mice with purified HSV gH/gL protein resulted in the production of neutralizing antibodies and protected mice from HSV challenge (5, 44), and administration of an anti-HSV gH monoclonal antibody (MAb) protected mice from HSV challenge (16). Antibodies to HSV and Epstein-Barr virus gH effectively neutralize during virus penetration but not during adsorption in vitro, indicating an essential role for gH in the fusion of viral and cellular membranes but not in initial attachment of the virus to the cell (18, 33).Anti-gH MAb 206, an immunoglobulin G1 (IgG1) antibody which recognizes a conformation-dependent epitope on the mature glycosylated form of gH, neutralizes VZV infection in vitro in the absence of complement (35). MAb 206 inhibits cell-cell fusion in vitro, based on reductions in the number of infected cells and the number of infected nuclei within syncytia, and appears to inhibit the ability of virus particles to pass from the surface of an infected epithelial cell to a neighboring cell via cell extensions (8, 35, 43). When infected cells were treated with MAb 206 for 48 h postinfection (hpi), virus egress and syncytium formation were not apparent, but they were evident within 48 h after removal of the antibody, suggesting that the effect of the antibody was reversible and that there was a requirement for new gH synthesis and trafficking to produce cell-cell fusion. Conversely, nonneutralizing antibodies to glycoproteins E (gE) and I (gI), as well as an antibody to immediate-early protein 62 (IE62), had no effect on VZV spread (46).Like that of other herpesviruses, VZV entry into cells is presumed to require fusion of the virion envelope with the cell membrane or endocytosis followed by fusion. One of the hallmarks of VZV infection is cell fusion and formation of syncytia (8). Cell fusion can be detected as early as 9 hpi in vitro, although VZV spread from infected to uninfected cells is evident within 60 min (45). In vivo, VZV forms syncytia through its capacity to cause fusion of epidermal cells. Syncytia are evident in biopsies of varicella and herpes zoster skin lesions during natural infection and in SCIDhu skin xenografts (34). VZV gH is produced, processed in the Golgi apparatus, and trafficked to the cell membrane, where it might be involved in cell-cell fusion (11, 29, 35). gH then undergoes endocytosis and is trafficked back to the trans-Golgi network (TGN) for incorporation into the virion envelope (20, 31, 42). Since VZV is highly cell associated in vitro, little is known about the glycoproteins required for entry, but VZV gH is present in abundance in the skin vesicles during human chickenpox and zoster (55).Investigating the functions of gH in the pathogenesis of VZV infection in vivo is challenging because it is an essential protein and VZV is species specific for the human host. The objective of this study was to investigate the role of gH in VZV pathogenesis by establishing whether antibody-mediated interference with gH function could prevent or modulate VZV infection of differentiated human tissue in vivo, using the SCIDhu mouse model. The effects of antibody administration at early and later times after infection were determined by comparing infectious virus titers, VZV genome copies, and lesion formation in anti-gH antibody-treated xenografts. In vitro experiments were performed to determine the potential mechanism(s) of MAb 206 interference with gH during VZV replication, virion assembly, and cell-cell spread. The present study has implications for understanding the contributions of gH to VZV replication in vitro and in vivo, the mechanisms by which production of antibodies to gH by the host might restrict VZV infection, and the use of passive antibody prophylaxis in patients at high risk of serious illness caused by VZV.     

Impact of Varicella-Zoster Virus on Dendritic Cell Subsets in Human Skin during Natural Infection

Varicella-zoster virus (VZV) causes varicella and herpes zoster, diseases characterized by distinct cutaneous rashes. Dendritic cells (DC) are essential for inducing antiviral immune responses; however, the contribution of DC subsets to immune control during natural cutaneous VZV infection has not been investigated. Immunostaining showed that compared to normal skin, the proportion of cells expressing DC-SIGN (a dermal DC marker) or DC-LAMP and CD83 (mature DC markers) were not significantly altered in infected skin. In contrast, the frequency of Langerhans cells was significantly decreased in VZV-infected skin, whereas there was an influx of plasmacytoid DC, a potent secretor of type I interferon (IFN). Langerhans cells and plasmacytoid DC in infected skin were closely associated with VZV antigen-positive cells, and some Langerhans cells and plasmacytoid DC were VZV antigen positive. To extend these in vivo observations, both plasmacytoid DC (PDC) isolated from human blood and Langerhans cells derived from MUTZ-3 cells were shown to be permissive to VZV infection. In VZV-infected PDC cultures, significant induction of alpha IFN (IFN-α) did not occur, indicating the VZV inhibits the capacity of PDC to induce expression of this host defense cytokine. This study defines changes in the response of DC which occur during cutaneous VZV infection and implicates infection of DC subtypes in VZV pathogenesis.Varicella-zoster virus (VZV) is a highly species-specific human herpesvirus that causes the diseases varicella (chicken pox) and herpes zoster (shingles). Varicella results from the primary phase of infection and is characterized by a diffuse rash of vesiculopustular lesions that appear in crops and usually resolve within 1 to 2 weeks (7, 26). Primary infection is initiated by inoculation of mucosal sites, such as the upper respiratory tract and the conjunctiva, with infectious virus, usually contained within respiratory droplets (3, 23). Following inoculation, there is a 10- to 21-day incubation period during which VZV is transported to the regional lymph nodes; however, it remains unclear which cell types are responsible for transport of VZV during natural infection (3). It has been hypothesized that dendritic cells (DC) of the respiratory mucosa may be among the first cells to encounter VZV during primary infection and are capable of virus transport to the draining lymph nodes (1, 45). It is postulated that within lymph nodes, VZV undergoes a period of replication, resulting in a primary cell-associated viremia, during which time virus is transported to the reticuloendothelial organs, where it undergoes another period of replication that results in a secondary cell-associated viremia and virus transport to the skin (3, 23). However, VZV has recently been shown to have tropism for human tonsillar CD4⁺ T lymphocytes (37), and it has been demonstrated that these T lymphocytes express skin homing markers that may allow them to transport VZV directly from the lymph node to the skin during primary viremia (38). Once the virus reaches the skin, it infects cutaneous epithelial cells, resulting in distinctive vesiculopustular lesions.During the course of primary infection, VZV establishes a lifelong latent infection within the sensory ganglia, from which virus may reactivate years later to cause herpes zoster (22, 42, 53). VZV reactivation results in the production of new infectious virus and a characteristic vesiculopustular rash, which differs from that of varicella insofar as the distribution of the lesions is typically unilateral and covers only 1 to 2 dermatomes (8). In both primary and reactivated VZV infection of human skin, VZV antigens are detectable in the epidermis and dermis (2, 30, 46, 47, 49, 52), and although some studies have examined the immune infiltrate present in these lesions, most have focused on T lymphocytes, macrophages, and NK cells (40, 48, 50, 51, 58). The role of DC subsets in VZV infection in human skin has not been previously explored in vivo.Our laboratory provided the first evidence that VZV could productively infect human immature and mature monocyte-derived dendritic cells (MDDC) in vitro (1, 45), and Hu and Cohen (2005) showed that VZV ORF47 was critical for replication of virus in human immature DC but not mature DC (29). However, whether DC become directly infected during natural VZV skin infection and the impact VZV infection may have on DC subsets has yet to be elucidated. The two subsets of DC that are normally present in the skin and which may be involved in the pathogenesis of VZV infection are the Langerhans cells (LC) of the epidermis and dermal DC (DDC) (60). LC are present in an immature state in uninfected skin and in upper respiratory tract epithelium. Upon capture of foreign antigens, LC have the capacity to migrate from the periphery to the lymph nodes, where they seek interaction with T lymphocytes (60). Although the location of cutaneous DC suggests that they are a DC subset likely to be involved in the pathogenesis of VZV infection, other subsets of DC, such as the blood-derived myeloid DC (MDC) and plasmacytoid DC (PDC), are also potentially important in the pathogenesis of VZV infection. Of particular interest are PDC, since these cells are important in innate antiviral immune responses due to their ability to recruit to sites of inflammation and secrete high levels of alpha interferon (IFN-α) (6, 18, 56). PDC also participate in adaptive immune responses through their secretion of cytokines and chemokines that promote activation of effector cells, including NK cells, NKT cells, B lymphocytes, and T lymphocytes, and also through their capacity to present antigen to T lymphocytes (9, 63). Whether PDC and LC can be infected with VZV and their roles during infection have not been previously studied.In this study, we sought to identify and compare the subsets of DC present in human skin lesions following natural VZV infection and to assess DC permissiveness to VZV infection. We utilized immunohistochemical (IHC) and immunofluorescent (IFA) staining to characterize DC subsets within the skin of multiple patients with either varicella or herpes zoster, and identified profound changes in the frequency of LC and PDC as a consequence of cutaneous VZV infection. In addition, some LC and PDC costained with a range of VZV antigens indicative of productive infection. PDC isolated from human blood and LC derived from the MUTZ-3 cells were shown to be permissive to productive VZV infection in vitro. This study defines changes in the type and distribution of DC during natural cutaneous VZV infection and implicates infection of specific DC subsets in VZV pathogenesis.     

Herpes Simplex Virus Type 1 Glycoprotein E Mediates Retrograde Spread from Epithelial Cells to Neurites

In animal models of infection, glycoprotein E (gE) is required for efficient herpes simplex virus type 1 (HSV-1) spread from the inoculation site to the cell bodies of innervating neurons (retrograde direction). Retrograde spread in vivo is a multistep process, in that HSV-1 first spreads between epithelial cells at the inoculation site, then infects neurites, and finally travels by retrograde axonal transport to the neuron cell body. To better understand the role of gE in retrograde spread, we used a compartmentalized neuron culture system, in which neurons were infected in the presence or absence of epithelial cells. We found that gE-deleted HSV-1 (NS-gEnull) retained retrograde axonal transport activity when added directly to neurites, in contrast to the retrograde spread defect of this virus in animals. To better mimic the in vivo milieu, we overlaid neurites with epithelial cells prior to infection. In this modified system, virus infects epithelial cells and then spreads to neurites, revealing a 100-fold retrograde spread defect for NS-gEnull. We measured the retrograde spread defect of NS-gEnull from a variety of epithelial cell lines and found that the magnitude of the spread defect from epithelial cells to neurons correlated with epithelial cell plaque size defect, indicating that gE plays a similar role in both types of spread. Therefore, gE-mediated spread between epithelial cells and neurites likely explains the retrograde spread defect of gE-deleted HSV-1 in vivo.Herpes simplex virus type 1 (HSV-1) is an alphaherpesvirus that characteristically infects skin and mucosal surfaces before spreading to sensory neurons, where it establishes a lifelong persistent infection. The virus periodically returns to the periphery via sensory axons and causes recurrent lesions as well as asymptomatic shedding. This life cycle requires viral transport along axons in two directions: toward the neuron cell body (retrograde direction) and away from the neuron cell body (anterograde direction).Many studies of alphaherpesvirus neuronal spread have focused on pseudorabies virus (PRV), a virus whose natural host is the pig. Three PRV proteins, glycoprotein E (gE), gI, and Us9, have been shown to mediate anterograde neuronal spread both in animal models of infection and in cultured neurons. However, these three proteins are dispensable for retrograde spread (3, 8, 11, 12, 31, 46). In contrast, numerous animal models of infection have shown that HSV-1 gE is required for retrograde spread from the inoculation site to the cell bodies of innervating neurons (4, 9, 44, 56). In the murine flank model, wild-type (WT) virus replicates in the skin and then infects sensory neurons and spreads in a retrograde direction to the dorsal root ganglia (DRG). In this model, gE-deleted HSV-1 replicates in the skin but is not detected in the DRG (9, 44). This phenotype differs from gE-deleted PRV, which is able to reach the DRG at WT levels (8). Thus, unlike PRV, gE-deleted HSV-1 viruses have a retrograde spread defect in vivo.HSV-1 gE is a 552-amino-acid type I membrane protein found in the virion membrane as well as in the trans-Golgi and plasma membranes of infected cells (1). gE forms a heterodimer with another viral glycoprotein, gI. The gE/gI complex is important for HSV-1 immune evasion through its Fc receptor activity. gE/gI binds to the Fc domain of antibodies directed against other viral proteins, sequestering these antibodies and blocking antibody effector functions (27, 32, 40). Additionally, gE/gI promotes spread between epithelial cells. Viruses lacking either gE or gI form characteristically small plaques in cell culture and small inoculation site lesions in mice (4, 9, 18, 40, 58). In animal models, gE and gI also mediate viral spread in both anterograde and retrograde directions (4, 19, 44, 56).In order to better understand the role of gE in HSV-1 retrograde neuronal spread, we employed a compartmentalized neuron culture system that has been used to study directional neuronal spread of PRV and West Nile virus (12, 14, 45). In the Campenot chamber system, neurites are contained in a compartment that is separate from their corresponding cell bodies. Therefore, spread in an exclusively retrograde direction can be measured by infecting neurites and detecting spread to neuron cell bodies.HSV-1 replication requires retrograde transport of incoming viral genomes to the nucleus. In neurites, fusion between viral and cellular membranes occurs at the plasma membrane (43, 48). Upon membrane fusion, the capsid and a subset of tegument proteins (the inner tegument) dissociate from glycoproteins and outer tegument proteins, which remain at the plasma membrane (28, 38). Unenveloped capsids and the associated inner tegument proteins are then transported in the retrograde direction to the nucleus (7, 48, 49).For both neurons and epithelial cells, retrograde transport is dependent upon microtubules, ATP, the retrograde microtubule motor dynein, and the dynein cofactor dynactin (22, 34, 49, 52). Several viral proteins interact with components of the dynein motor complex (23, 39, 60). However, none of these proteins suggest a completely satisfactory mechanism by which viral retrograde transport occurs, either because they are not components of the complex that is transported to the nucleus (UL34, UL9, VP11/12) or because capsids lacking that protein retain retrograde transport activity (VP26) (2, 17, 21, 28, 37). This implies that additional viral proteins are involved in retrograde trafficking.We sought to better characterize the role of gE in retrograde spread and found that gE is dispensable for retrograde axonal transport; however, it promotes HSV-1 spread from epithelial cells to neurites. This epithelial cell-to-neuron spread defect provides a plausible explanation for the retrograde spread defect of gE-deleted HSV-1 in animal models of infection.     

Anterograde Spread of Herpes Simplex Virus Type 1 Requires Glycoprotein E and Glycoprotein I but Not Us9

Anterograde neuronal spread (i.e., spread from the neuron cell body toward the axon terminus) is a critical component of the alphaherpesvirus life cycle. Three viral proteins, gE, gI, and Us9, have been implicated in alphaherpesvirus anterograde spread in several animal models and neuron culture systems. We sought to better define the roles of gE, gI, and Us9 in herpes simplex virus type 1 (HSV-1) anterograde spread using a compartmentalized primary neuron culture system. We found that no anterograde spread occurred in the absence of gE or gI, indicating that these proteins are essential for HSV-1 anterograde spread. However, we did detect anterograde spread in the absence of Us9 using two independent Us9-deleted viruses. We confirmed the Us9 finding in different murine models of neuronal spread. We examined viral transport into the optic nerve and spread to the brain after retinal infection; the production of zosteriform disease after flank inoculation; and viral spread to the spinal cord after flank inoculation. In all models, anterograde spread occurred in the absence of Us9, although in some cases at reduced levels. This finding contrasts with gE- and gI-deleted viruses, which displayed no anterograde spread in any animal model. Thus, gE and gI are essential for HSV-1 anterograde spread, while Us9 is dispensable.Alphaherpesviruses are parasites of the peripheral nervous system. In their natural hosts, alphaherpesviruses establish lifelong persistent infections in sensory ganglia and periodically return by axonal transport to the periphery, where they cause recurrent disease. This life cycle requires viral transport along axons in two directions. Axonal transport in the retrograde direction (toward the neuron cell body) occurs during neuroinvasion and is required for the establishment of latency, while transport in the anterograde direction (away from the neuron cell body) occurs after reactivation and is required for viral spread to the periphery to cause recurrent disease. In addition to anterograde and retrograde axonal transport within neurons, alphaherpesviruses spread between synaptically connected neurons and between neurons and epithelial cells at the periphery (19, 22).The alphaherpesvirus subfamily includes the human pathogens herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV), as well as numerous veterinary pathogens such as pseudorabies virus (PRV) and bovine herpesviruses 1 and 5 (BHV-1 and BHV-5). The molecular mechanisms that mediate alphaherpesvirus anterograde axonal transport, anterograde spread, and cell-to-cell spread remain unclear. However, many studies of several alphaherpesviruses have indicated that anterograde transport or anterograde spread involves the viral proteins glycoprotein E (gE), glycoprotein I (gI), and Us9 (2, 5, 7, 9, 11, 13, 16, 26, 30, 31, 41, 46, 51, 52).Glycoproteins E and I are type I membrane proteins that form a heterodimer in the virion membrane and on the surface of infected cells. Although dispensable for the entry of extracellular virus, gE and gI mediate the epithelial cell-to-cell spread of numerous alphaherpesviruses (1, 3, 15, 20, 34, 38, 49, 53, 54). Us9 is a type II nonglycosylated membrane protein with no described biological activity apart from its role in neuronal transport (4, 18, 32). Here, we used several model systems to better characterize the roles of gE, gI, and Us9 in HSV-1 neuronal spread.Animal models to assess alphaherpesvirus neuronal transport (viral movement within a neuron) and spread (viral movement between cells) include the mouse flank and mouse retina models of infection. In the mouse flank model (Fig. ​(Fig.1A),1A), virus is scratch inoculated onto the depilated flank, where it infects the skin and spreads to innervating sensory neurons. The virus travels to the dorsal root ganglia (DRG) in the spinal cord (retrograde direction) and then returns to an entire dermatome of skin (anterograde spread). The virus also is transported in an anterograde direction from the DRG to the dorsal horn of the spinal cord and subsequently spreads to synaptically connected neurons. The production of zosteriform lesions and the presence of viral antigens in the dorsal horn of the spinal cord both are indicators of anterograde spread in this system. PRV gE and Us9 are required for the production of zosteriform disease, while gI is dispensable (7). In the absence of gE, HSV-1 also fails to cause zosteriform disease. However, unlike PRV, HSV-1 gE is required for retrograde spread to the DRG, so the role of gE in HSV-1 anterograde spread could not be evaluated in the mouse flank model (8, 36, 42).Open in a separate windowFIG. 1.Model systems to study HSV-1 neuronal spread. (A) Mouse flank model. Virus was scratch inoculated onto the skin, where it replicates, spreads to innervating neurons, and travels in a retrograde direction to the neuron cell body in the DRG. After replicating in the DRG, the virus travels in an anterograde direction back to the skin and into the dorsal horn of the spinal cord. Motor neurons also innervate the skin, allowing virus to reach the ventral horn of the spinal cord by retrograde transport. (B) Mouse retina model. Virus is injected into the vitreous body, from which it infects the retina as well as other structures of the eye, including the ciliary body, iris, and skeletal muscles of the orbit. From the retina, the virus is transported into the optic nerve and optic tract (OT) (anterograde direction) and then to the brain along visual pathways. Anterograde spread is detected in the lateral geniculate nucleus (LGN) and superior colliculus (SC). From the infected ciliary body, iris, and skeletal muscle, the virus spreads in a retrograde direction along motor and parasympathetic neurons and is detected in the oculomotor and Edinger-Westphal nuclei (OMN/EWN). Only first-order sites of spread to the brain are indicated. (Brain images were modified and reproduced from reference 47 with permission from of the publisher. Copyright Elsevier 1992.) (C) Campenot chamber system. Campenot chambers consist of a Teflon ring that divides the culture into three separate compartments. Neurons are seeded into the S chamber and extend their axons into the M and N chambers. Vero cells are seeded into the N chamber 1 day before infection. Virus is added to the S chamber and detected in the N chamber, a measure of anterograde spread.The mouse retina infection model (Fig. ​(Fig.1B)1B) has the advantage of allowing anterograde and retrograde spread to be studied independently of one another. Virus is delivered to the vitreous body, from which it infects the retina and other structures of the eye. The cell bodies of retinal neurons form the innermost layer of the retina; therefore, the virus infects these neurons directly, and spread from the retina along visual pathways to the brain occurs in an exclusively anterograde direction. In addition, the virus infects the anterior uveal layer of the eye (ciliary body and iris) and skeletal muscles in the orbit. From these tissues, the virus infects innervating parasympathetic and motor neurons and spreads to the brain in a retrograde direction. The localization of viral antigens in specific brain sites indicates whether the virus traveled to the brain along an anterograde or retrograde pathway (21, 25, 26, 39, 44, 51). PRV gE, gI, and Us9 each are essential for anterograde spread to the brain yet are dispensable for retrograde spread (5, 11, 25, 52). Even a strain of PRV lacking all three of these proteins retains retrograde neuronal spread activity (12, 40, 44). In contrast, in the absence of gE, HSV-1 fails to spread to the brain by either the anterograde or retrograde pathway (51).The Campenot chamber system (Fig. ​(Fig.1C)1C) has the advantage of allowing quantitative measurement of anterograde spread. Sympathetic neurons are cultured in a tripartite ring in which neuron cell bodies are contained in a separate compartment from their neurites. Virus is added to neuron cell bodies in one chamber, and anterograde spread to a separate chamber is measured by viral titers (13, 29, 30, 39, 43). Using this system, gEnull, gInull, and Us9null PRV each were shown to have only a partial defect in anterograde spread, while a virus lacking all three proteins was totally defective (13).We sought to quantify the anterograde spread activity of gEnull, gInull, and Us9null HSV-1 using the Campenot chamber system. While gEnull and gInull viruses were completely defective at anterograde spread, we found that a Us9null virus retained wild-type (WT) anterograde spread activity in this system. This observation was unexpected, since others previously had reported that Us9 is required for efficient HSV-1 anterograde transport or spread (26, 41, 46). Therefore, we further characterized the neuronal spread properties of two independent Us9-deleted viruses in the mouse retina and mouse flank models of infection. Our results indicate that gE and gI are essential for HSV-1 anterograde spread, whereas Us9 is dispensable.     

Characterization of the Host Immune Response in Human Ganglia after Herpes Zoster

Varicella-zoster virus (VZV) causes varicella (chicken pox) and establishes latency in ganglia, from where it reactivates to cause herpes zoster (shingles), which is often followed by postherpetic neuralgia (PHN), causing severe neuropathic pain that can last for years after the rash. Despite the major impact of herpes zoster and PHN on quality of life, the nature and kinetics of the virus-immune cell interactions that result in ganglion damage have not been defined. We obtained rare material consisting of seven sensory ganglia from three donors who had suffered from herpes zoster between 1 and 4.5 months before death but who had not died from herpes zoster. We performed immunostaining to investigate the site of VZV infection and to phenotype immune cells in these ganglia. VZV antigen was localized almost exclusively to neurons, and in at least one case it persisted long after resolution of the rash. The large immune infiltrate consisted of noncytolytic CD8⁺ T cells, with lesser numbers of CD4⁺ T cells, B cells, NK cells, and macrophages and no dendritic cells. VZV antigen-positive neurons did not express detectable major histocompatibility complex (MHC) class I, nor did CD8⁺ T cells surround infected neurons, suggesting that mechanisms of immune control may not be dependent on direct contact. This is the first report defining the nature of the immune response in ganglia following herpes zoster and provides evidence for persistence of non-latency-associated viral antigen and inflammation beyond rash resolution.Varicella-zoster virus (VZV) is a highly species-specific human alphaherpesvirus that infects a majority of the world''s population. VZV causes two clinically significant diseases; varicella (chicken pox) and herpes zoster (shingles) (5, 8, 19). Varicella is characterized by widespread cutaneous vesicular lesions and is a consequence of primary VZV infection in VZV-naïve individuals. While varicella is a relatively mild disease in immunocompetent children, it can cause significant morbidity in healthy adults and is frequently life threatening in immunocompromised individuals (3, 4, 22). The innate and adaptive immune responses act to eliminate replicating virus during varicella, but not all virus is cleared during this time, with some presumed to access nerve axons in the skin, enabling transport to neurons in sensory ganglia, where the virus is able to establish a lifelong latent infection (5, 8, 12, 13, 20, 32). An alternative possibility is that virus is transported to ganglia via hematogenous spread (36). The ability of VZV to establish latency in the host is critical to the success of this virus as a human pathogen.VZV reactivation from latency (herpes zoster) causes serious disease in older and immunocompromised individuals and is characterized by vesicular skin rash in a dermatomal distribution with preceding and concomitant pain (7, 10, 21, 68). During reactivation, sensory ganglia are sites of viral replication, where an intense inflammatory response is induced and widespread necrosis of glial cells and neurons ensues (14, 19, 27, 71, 72). Before the appearance of the zoster rash, VZV travels along the affected sensory nerves to the skin, where it produces the characteristic rash (10, 53) and neural and dermoepidermal inflammation. Clinically, herpes zoster is associated with severe, acute pain, as well as often prolonged severe pain, or postherpetic neuralgia (PHN), that often requires follow-up medical care for months or even years after the initial attack (29, 62, 73). PHN is estimated to occur in 40% of herpes zoster cases in individuals older than 50 years and 75% of adults older than 75 years (15, 43, 56). It is estimated that 1 million or more individuals are afflicted by herpes zoster each year in the United States (54). Herpes zoster pain, and especially PHN, can be disabling and can have a major negative impact on patients'' quality of life (15). In the coming years, the number of individuals suffering from herpes zoster is predicted to rise, concomitant with the increasing number of patients who are elderly or who are receiving immunosuppressive therapies for cancer or transplantation.New antiviral drugs and a vaccine for herpes zoster have been only partially successful, indicating the need for continuing studies of VZV immunopathogenesis to understand the reasons for this partial success and to provide the foundation for developing new immunotherapeutics and vaccines (38, 39, 65). Antiviral therapy, while effective against the rash and pain of acute herpes zoster, appears at best to prevent only 50% of PHN (16, 23, 24, 45, 75, 76). The zoster vaccine was demonstrated to prevent 51% of herpes zoster and 60% of postherpetic neuralgia in patients over the age of 60, although it appeared to be less effective against zoster in the older age group (54). Remarkably, despite the importance of ganglionic infection to the pathogenesis of herpes zoster and PHN, there have been no reports defining the immune response in human ganglia following natural VZV reactivation. Until now, the lack of available ganglia from patients following an episode of herpes zoster has limited these studies. We have overcome this hurdle by obtaining rare naturally infected human ganglia at autopsy from three donors who, near the time of death, had evidence of herpes zoster but who did not die from herpes zoster. The aim of this study was to undertake a comprehensive immunohistological examination of human ganglia following herpes zoster. Specifically, we utilized immunohistochemical (IHC) and immunofluorescent (IF) staining to characterize the infiltrating immune cell subsets and to assess the presence of VZV antigen within ganglia following herpes zoster. This study provides the first detailed examination of the types and distribution of immune cells present following natural VZV reactivation in human ganglia and provides new insights into the immunological mechanisms that may be important in controlling virus infection following the reactivation of a human herpesvirus infection in human ganglia in vivo.     

Comparison of the Pseudorabies Virus Us9 Protein with Homologs from Other Veterinary and Human Alphaherpesviruses

Pseudorabies virus (PRV) Us9 is a small, tail-anchored (TA) membrane protein that is essential for axonal sorting of viral structural proteins and is highly conserved among other members of the alphaherpesvirus subfamily. We cloned the Us9 homologs from two human pathogens, varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1), as well as two veterinary pathogens, equine herpesvirus type 1 (EHV-1) and bovine herpesvirus type 1 (BHV-1), and fused them to enhanced green fluorescent protein to examine their subcellular localization and membrane topology. Akin to PRV Us9, all of the Us9 homologs localized to the trans-Golgi network and had a type II membrane topology (typical of TA proteins). Furthermore, we examined whether any of the Us9 homologs could compensate for the loss of PRV Us9 in anterograde, neuron-to-cell spread of infection in a compartmented chamber system. EHV-1 and BHV-1 Us9 were able to fully compensate for the loss of PRV Us9, whereas VZV and HSV-1 Us9 proteins were unable to functionally replace PRV Us9 when they were expressed in a PRV background.Alphaherpesviruses are classified by their variable host range, short reproductive cycle, and ability to establish latency in the peripheral nervous system (PNS) (36, 37). Commonly studied pathogens of this subfamily include herpes simplex virus (HSV) and varicella-zoster virus (VZV), as well as the veterinary pathogens pseudorabies virus (PRV), equine herpesvirus (EHV), and bovine herpesvirus (BHV). Initial infection begins with the virus entering the host mucosal surfaces and spreading between cells of the mucosal epithelium. Invariably, virus enters the PNS through the infection of peripheral nerves that innervate this region. The virus establishes a latent infection in PNS neurons that can be reactivated and that persists for the life of the host (36). In most natural infections, virus replication in the PNS never spreads to the central nervous system (CNS). However, on rare occasions, invasion of the CNS does occur, resulting in devastating encephalitis (46). Trafficking of virus particles from infected epithelial cells into the axon and subsequent transport to neuronal cell bodies is known as retrograde spread of infection. Trafficking of virus particles that are assembled in the neuronal cell body and subsequently sorted into axons for transport to epithelial cells at the initial site of infection (upon reactivation from latency) is known as anterograde spread of infection.Though the natural host of PRV is swine, the virus infects a wide variety of animals, including rodents, cats, dogs, rabbits, cattle, and chicken embryos, but not higher primates (1, 30, 47). In contrast to the well-contained spread of PRV within its natural host, infection of other mammals is usually lethal. Instead of stopping in the PNS, infection continues on to second-order and third-order neurons in the CNS (reviewed in reference 35). This facet of PRV infection makes it a useful tracer of neuronal connections (18). Work in our lab has identified three PRV proteins, Us9 and the gE/gI heterodimer, which are critical for efficient anterograde spread of infection in vivo (i.e., spread from presynaptic to postsynaptic neurons) (6, 45). The molecular mechanism by which these proteins function has been further elucidated in vitro using primary neuronal cultures of superior cervical ganglion (SCG) harvested from embryonic rat pups. PRV Us9 and, to a lesser extent, gE/gI are required for efficient axonal targeting of viral structural proteins, a necessary step for subsequent anterograde, transneuronal spread (10, 11, 27, 28, 42).PRV Us9 is a type II, tail-anchored (TA) membrane protein that is highly enriched in lipid raft microdomains and resides predominantly in or near the trans-Golgi network (TGN) inside infected cells (5-7, 27). It has homologs in most of the alphaherpesviruses, including VZV (16), HSV-1 (22), HSV-2 (17), EHV-1 (21, 40), EHV-4 (41), BHV-1 (25), and BHV-5 (14). Though several studies have examined individually the Us9 proteins encoded by VZV (16), HSV-1 (4, 22, 34, 39), BHV-1 (13), and BHV-5 (14), several gaps in our understanding of Us9 biology remain, namely, whether all of the PRV Us9 homologs are type II membrane proteins, if the proteins localize to similar subcellular compartments within different cell types, and if they can functionally substitute for the loss of PRV Us9 in axonal sorting and anterograde spread of infection. The aim of this study is to examine PRV Us9 in parallel assays with its homologs from VZV, HSV-1, EHV-1, and BHV-1 to identify potential similarities and differences between these highly conserved alphaherpesvirus proteins.     

Virological Synapse-Mediated Spread of Human Immunodeficiency Virus Type 1 between T Cells Is Sensitive to Entry Inhibition

Human immunodeficiency virus type 1 (HIV-1) can disseminate between CD4⁺ T cells via diffusion-limited cell-free viral spread or by directed cell-cell transfer using virally induced structures termed virological synapses. Although T-cell virological synapses have been well characterized, it is unclear whether this mode of viral spread is susceptible to inhibition by neutralizing antibodies and entry inhibitors. We show here that both cell-cell and cell-free viral spread are equivalently sensitive to entry inhibition. Fluorescence imaging analysis measuring virological synapse lifetimes and inhibitor time-of-addition studies implied that inhibitors can access preformed virological synapses and interfere with HIV-1 cell-cell infection. This concept was supported by electron tomography that revealed the T-cell virological synapse to be a relatively permeable structure. Virological synapse-mediated HIV-1 spread is thus efficient but is not an immune or entry inhibitor evasion mechanism, a result that is encouraging for vaccine and drug design.As with enveloped viruses from several viral families, the human immunodeficiency virus type 1 (HIV-1) can disseminate both by fluid-phase diffusion of viral particles and by directed cell-cell transfer (39). The primary target cell for HIV-1 replication in vivo is the CD4⁺ T-cell (13), which is infectible by CCR5-tropic (R5) and CXCR4-tropic (X4) viral variants (29). R5 HIV-1 is the major transmitted viral phenotype and dominates the global pandemic, whereas X4 virus is found later in infection in ca. 50% of infected individuals, and its presence indicates a poor disease progression prognosis (23). Cell-cell HIV-1 transfer between T cells is more efficient than diffusion-limited spread (8, 16, 32, 38), although recent estimates for the differential range from approximately 1 (42) to 4 (6) orders of magnitude. Two structures have been proposed to support contact-mediated intercellular movement of HIV-1 between T cells: membrane nanotubes (33, 43) and macromolecular adhesive contacts termed virological synapses (VS) (15, 17, 33). VS appear to be the dominant structure involved in T-cell-T-cell spread (33), and both X4 (17) and R5 HIV-1 (6, 15, 42) can spread between T cells via this mechanism.VS assembly and function are dependent on HIV-1 envelope glycoprotein (Env) engaging its primary cellular receptor CD4 (2, 6, 17). This interaction recruits more CD4 and coreceptor to the site of cell-cell contact in an actin-dependent manner (17). Adhesion molecules cluster at the intercellular junction and are thought to stabilize the VS (18). In parallel, viral Env and Gag are recruited to the interface by a microtubule-dependent mechanism (19), where polarized viral budding may release virions into the synaptic space across which the target cell is infected (17). The precise mechanism by which HIV-1 subsequently enters the target T-cell cytoplasm remains unclear: by fusion directly at the plasma membrane, fusion from within an endosomal compartment, or both (4, 6, 15, 25, 34).Viruses from diverse families including herpesviruses (9), poxviruses (22) and hepatitis C virus (44) evade neutralizing antibody attack by direct cell-cell spread, since the tight junctions across which the these viruses move are antibody impermeable. It has been speculated that transfer of HIV-1 across VS may promote evasion from immune or therapeutic intervention with the inference that the junctions formed in retroviral VS may be nonpermissive to antibody entry (39). However, available evidence regarding whether neutralizing antibodies (NAb) and other entry inhibitors can inhibit HIV-1 cell-cell spread is inconsistent (25). An early analysis suggested that HIV-1 T-cell-T-cell spread is relatively resistant to neutralizing monoclonal antibodies (NMAb) (12). A later study agreed with this conclusion by demonstrating a lack of permissivity of HIV-1 T-cell-T-cell spread, measured by transfer of viral Gag, to interference with viral fusion using a gp41-specific NMAb and a peptidic fusion inhibitor (6). In contrast, another analysis reported that anti-gp41-specific NMAb interfered effectively with HIV-1 spread between T cells (26). Inhibitors of the HIV-1 surface glycoprotein (gp120)-CD4 or gp120-CXCR4 interaction reduced X4 HIV-1 VS assembly and viral transfer if applied prior to mixing of infected and receptor-expressing target cells (17, 19), but the effect of these inhibitors has not been tested on preformed VS. Thus, the field is currently unclear on whether direct T-cell-T-cell infectious HIV-1 spread is susceptible or not to antibody and entry inhibitor-mediated disruption of VS assembly, and the related question, whether the VS is permeable to viral entry inhibitors, including NAb. Addressing these questions is of central importance to understanding HIV-1 pathogenesis and informing future drug and vaccine design.Since estimates reported in the literature of the relative efficiency of direct HIV-1 T-cell-T-cell spread compared to cell-free spread vary by approximately 3 orders of magnitude (6, 38, 42), and the evidence for the activity of viral entry inhibitors on cell-cell spread is conflicting, we set out to quantify the efficiency of infection across the T-cell VS and analyze the susceptibility of this structure to NAb and viral entry inhibitors. Assays reporting on events proximal to productive infection show that the R5 HIV-1 T-cell VS is approximately 1 order of magnitude more efficient than cell-free virus infection, and imaging analyses reveal that the VS assembled by HIV-1 is most likely permeable to inhibitors both during, and subsequent to, VS assembly. Thus, we conclude that the T-cell VS does not provide a privileged environment allowing HIV-1 escape from entry inhibition.     

Simian Varicella Virus Induces Apoptosis in Monkey Kidney Cells by the Intrinsic Pathway and Involves Downregulation of Bcl-2 Expression

Simian varicella virus (SVV) causes varicella in primates, becomes latent in ganglionic neurons, and reactivates to produce zoster. SVV produces a cytopathic effect in monkey kidney cells in tissue culture. To study the mechanism by which SVV-infected cells die, we examined markers of apoptosis 24 to 64 h postinfection (hpi). Western blot analysis of virus-infected cell lysates revealed a significant increase in the levels of the cleaved active form of caspase-3, accompanied by a parallel increase in caspase-3 activity at 40 to 64 hpi. Caspase-9, a marker for the intrinsic pathway, was activated significantly in SVV-infected cells at all time points, whereas trace levels of the active form of caspase-8, an extrinsic pathway marker, was detected only at 64 hpi. Bcl-2 expression at the mRNA and protein levels was decreased by 50 to 70% throughout the course of virus infection. Release of cytochrome c, an activator of caspase-9, from mitochondria into the cytoplasm was increased by 200% at 64 hpi. Analysis of Vero cells infected with SVV expressing green fluorescent protein (SVV-GFP) at 64 hpi revealed colocalization of the active forms of caspase-3 and caspase-9 and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining with GFP. A significant decrease in the bcl-2 mRNA levels along with an abundance of mRNA specific for SVV genes 63, 40, and 21 was seen in the fraction of Vero cells that were infected with SVV-GFP. Together, these findings indicate that SVV induces apoptosis in cultured Vero cells through the intrinsic pathway in which Bcl-2 is downregulated.Apoptosis, a regulated form of cell death, plays a critical role in the homeostasis of multicellular organisms. Key features include membrane blebbing, chromatin condensation, and cell shrinkage. UV irradiation, deprivation of growth factors, and viral infection all cause apoptosis in cultured cells. Apoptosis is triggered by sequential activation of a group of cysteine proteases known as caspases. Apoptosis proceeds primarily through two pathways. The extrinsic pathway involves activation of caspase-8 and is initiated by ligand interaction with Fas or death receptors, while the intrinsic pathway is activated by an imbalance between proapoptotic (e.g., Bad and Bax) and antiapoptotic (e.g., Bcl-2 and Bcl-xL) proteins in mitochondria (21), resulting in release of cytochrome c from mitochondria, which in turn activates caspase-9. Bcl-2 plays an important role in cell survival (22, 32). Both caspase-8 and caspase-9 activate caspase-3, which along with other effector caspases, cleave critical cellular proteins, resulting in apoptosis.Simian varicella virus (SVV), the primate counterpart of human varicella zoster virus (VZV), produces a naturally occurring exanthematous disease that mimics human varicella (9, 18). Clinical and pathological changes produced by SVV infection of primates are similar to those produced by human varicella, and both VZV and SVV reactivate from latently infected ganglionic neurons (4, 13, 23, 33). The SVV and VZV genomes share a high degree of nucleotide homology (3, 10), and SVV-specific antibodies cross-react with human VZV in serum neutralization and complement fixation tests (5, 6, 30). Both viruses produce a cytopathic effect in monkey kidney cells in tissue culture (2, 29, 31). VZV has been shown to cause apoptosis in cultured Vero cells, human foreskin fibroblasts, and peripheral blood mononuclear cells isolated from healthy donors but not in primary human dorsal root ganglionic neurons (12, 13, 16, 28). Apoptosis is also seen in peripheral blood mononuclear cells of children infected with VZV in vivo (25). Thus, VZV-induced apoptosis may be cell type specific. The main objectives of this study were to determine if SVV induces apoptosis in cultured Vero cells, a monkey kidney cell line, and to identify the specific pathways.     

Mutagenesis of Varicella-Zoster Virus Glycoprotein B: Putative Fusion Loop Residues Are Essential for Viral Replication,and the Furin Cleavage Motif Contributes to Pathogenesis in Skin Tissue In Vivo

Glycoprotein B (gB), the most conserved protein in the family Herpesviridae, is essential for the fusion of viral and cellular membranes. Information about varicella-zoster virus (VZV) gB is limited, but homology modeling showed that the structure of VZV gB was similar to that of herpes simplex virus (HSV) gB, including the putative fusion loops. In contrast to HSV gB, VZV gB had a furin recognition motif ([R]-X-[KR]-R-|-X, where | indicates the position at which the polypeptide is cleaved) at residues 491 to 494, thought to be required for gB cleavage into two polypeptides. To investigate their contribution, the putative primary fusion loop or the furin recognition motif was mutated in expression constructs and in the context of the VZV genome. Substitutions in the primary loop, W180G and Y185G, plus the deletion mutation Δ⁴⁹¹RSRR⁴⁹⁴ and point mutation ⁴⁹¹GSGG⁴⁹⁴ in the furin recognition motif did not affect gB expression or cellular localization in transfected cells. Infectious VZV was recovered from parental Oka (pOka)-bacterial artificial chromosomes that had either the Δ⁴⁹¹RSRR⁴⁹⁴ or ⁴⁹¹GSGG⁴⁹⁴ mutation but not the point mutations W180G and Y185G, demonstrating that residues in the primary loop of gB were essential but gB cleavage was not required for VZV replication in vitro. Virion morphology, protein localization, plaque size, and replication were unaffected for the pOka-gBΔ⁴⁹¹RSRR⁴⁹⁴ or pOka-gB⁴⁹¹GSGG⁴⁹⁴ virus compared to pOka in vitro. However, deletion of the furin recognition motif caused attenuation of VZV replication in human skin xenografts in vivo. This is the first evidence that cleavage of a herpesvirus fusion protein contributes to viral pathogenesis in vivo, as seen for fusion proteins in other virus families.Varicella-zoster virus (VZV), an alphaherpesvirus, causes chicken pox (varicella) as a primary infection and shingles (zoster) upon reactivation from infected ganglia in humans (reviewed in reference 16). Although not yet investigated in VZV, herpesvirus entry requires fusion of the virus envelope with cell membranes governed by viral glycoprotein B (gB) and gH/gL, which are conserved across the family Herpesviridae (12, 27, 57). gB is the most conserved glycoprotein, with its function as a fusion protein well documented for several of the herpesviruses (10, 19, 38, 48, 51, 52).Open reading frame 31 (ORF31) codes for the 931 amino acids of VZV gB (18, 37). The successive N- and O-linked glycosylation plus the sialation and sulfation of VZV gB yields a mature protein with a molecular mass of approximately 140 kDa (45). Upon maturation, gB is cleaved, presumably by cellular proteases, into two polypeptides of 66 and 68 kDa. Intracellular trafficking of gB was shown to be dependent upon amino acid motifs in the cytoplasmic domain (24-26). In transfection studies, gB was transported to the cellular surface where it was endocytosed and localized to the trans-Golgi, where envelopment of viral particles is thought to occur.The structures of gB in the two human alphaherpesviruses, VZV and herpes simplex virus type 1 (HSV-1), are likely to be very similar as they have 49% amino acid identity (reviewed in reference 16). The ectodomain of HSV-1 gB was shown to form a spike that consisted of trimers with the structural homology to gG of vesicular stomatitis virus (28). Heldwein et al. (28) proposed that HSV-1 gB is a class II fusion protein based on homology to VSV G. The herpesvirus gB monomer was divided into five domains, I to V. Domain I consisted of a continuous amino acid sequence that folded into a pleckstrin homology-like domain, while domain II was comprised of two discontinuous segments, which also had a pleckstrin homology-like domain. A loop region exposed to the exterior of gB connected domain II with domain III. Domain III was comprised of three discontinuous segments and connected to the external loop by a long α helix that ended in a central coiled coil. Domain IV crowned gB and was connected to domain V, which stretched from the top to the bottom of the gB monomer, forming the core of the trimer making contacts with the two other subunits. The structural homology and lack of furin cleavage suggest that the herpesvirus gB and VSV G proteins have undergone convergent evolution.Although not proven experimentally, VZV gB is likely to be cleaved by the subtilisin-like proprotein convertase furin as the glycoprotein has a furin recognition motif [R]-X-[KR]-R-|-X (where | indicates the position at which the polypeptide is cleaved) (29). The [R]-X-[KR]-R-|-X motif is conserved in gBs for all of the herpesvirus families (5, 9, 21, 36, 40, 53, 63, 64). This site has been shown to be dispensable for the replication of human cytomegalovirus (HCMV), bovine herpesvirus type 1 (BHV-1), and pseudorabies virus (PRV) in vitro (32, 49, 58). Furin site mutants for BHV-1 and PRV show an altered phenotype in vitro, but effects were not examined in vivo. HSV-1 gB is not cleaved and lacks the [R]-X-[KR]-R-|-X motif at the canonical site, which is of interest because HSV-1 is genetically the most closely related human herpesvirus to VZV.Domain I of HSV gB showed structural conservation of putative fusion loops similar to those found in domain IV of the VSV G protein (28). Despite the lack of conserved amino acids within these loops, the hydrophobicity of the residues appears to be conserved for the Herpesviridae (4). Substitution of hydrophobic residues in Epstein-Barr virus gB and linker insertion mutagenesis close to the putative fusion loops of HSV-1 gB abrogated fusion based on in vitro transfection studies (4, 22, 34). However, the effect of substitutions in these putative fusion loops on viral replication has not been characterized. Since the development of fusion assays for VZV has proven elusive, the effect of substitutions in the putative fusion loop using viral mutagenesis to make recombinant viruses provides an alternative approach for identifying functional residues in VZV gB.In contrast to HSV-1, VZV is a human-restricted pathogen (reviewed in reference 16). To study the pathogenesis of VZV in vivo, well-established human xenograft models have been developed using SCID mice (6, 7, 13, 14, 41, 44, 54, 65). Lesions formed by VZV in the skin are similar to those seen in human subjects following primary infection (15, 43). The relevance of the model was demonstrated by studies with the varicella vaccine virus (vOka) that exhibited decreased growth in skin xenografts in vivo but does not cause disease in the healthy human host. In contrast, the vaccine virus and its parent strain, parental Oka (pOka), have indistinguishable replication kinetics in vitro (15, 43).The present study was designed to investigate the effects of structure-based targeted mutations in VZV gB on viral replication in cultured cells and in human skin xenografts in the SCIDhu mouse model. This was performed in the context of infectious virus recovered using the self-excisable bacterial artificial chromosome (BAC) containing the genome of a clinical isolate, Oka (62). The roles of the conserved residues W180 (gB-W180G) and Y185 (gB-Y185G) in the putative fusion loop were evaluated using glycine substitution, and the role of the furin recognition motif (⁴⁹¹RSRR⁴⁹⁴) was assessed by a complete deletion of the furin motif (gBΔ⁴⁹¹RSRR⁴⁹⁴) or a substitution of the arginine residues with glycine (gB⁴⁹¹GSGG⁴⁹⁴) to conserve the carbon backbone.