Biology subjects

Longnecker, R.

Publications and source records attributed to Longnecker, R..

10 recordsLinked to original sources

bioRxiv2026

Glycoprotein C Mutations Regulate the Plaque Size of Fusion-Defective Herpes Simplex Virus

Herpes simplex virus 1 (HSV-1) entry and virus-induced cell fusion require the coordinated activity of the essential glycoproteins gD, gH/gL, and gB. Mutations in this core fusion machinery reduce viral entry, fusion, and spread. Previously, we showed that introducing three mutations in gB (gB3A) or replacing HSV-1 gB with saimiriine herpesvirus 1 (SaHV-1) gB resulted in fusion-defective, small-plaque viruses. Serial passage of these viruses selected mutations in gH and gC, and we demonstrated that the gH mutations partially restored fusion function. In this study, we examined whether mutations in gC contribute to plaque formation, fusion, and entry. We generated an additional fusion-defective, small-plaque gD chimeric virus by replacing the profusion domain of HSV-1 gD with the corresponding region from SaHV-1 gD (gDchi). Serial passage of this virus partially restored plaque size and also selected for a mutation in gC. This study demonstrates that the selected gC mutants contributed to the partial restoration of plaque size. Plaque size of all three fusion-defective viruses was enhanced by exogenous expression of the corresponding selected gC mutants, whereas exogenous expression of wild-type gC reduced plaque size of the passaged isolates carrying gC mutations. Although gC mutants did not enhance fusion or entry mediated by the wild-type core fusion machinery, both wild-type and mutant gC enhanced cell-cell fusion when coexpressed with fusion-defective gH mutants. These results indicate that gC functions as a conditional accessory regulator that can partially compensate for defects in the HSV-1 core fusion machinery. IMPORTANCEHerpes simplex virus 1 (HSV-1) entry is mediated by a core fusion machinery comprised of glycoproteins gD, gH/gL, and gB. The contributions of additional viral glycoproteins to this process are not well defined. Glycoprotein C (gC) is known to function in cell attachment to heparan sulfate and immune evasion but is dispensable for viral entry under standard conditions. Here, we show that gC can act as a conditional accessory regulator of HSV-1 entry and fusion. Using serial passage of fusion-defective viruses, we demonstrate that mutations in gC are repeatedly selected and can partially compensate for defects in entry and fusion. These findings indicate that gC contributes to viral fusion under suboptimal conditions and highlight a role for this accessory glycoprotein in the adaptability of herpesvirus entry.

microbiology↗

bioRxiv2025

Species-Specific gB Ectodomain Interactions and Cytoplasmic Domain Stability Regulate Herpes Simplex Virus Fusion

Entry of herpesviruses into cells requires coordinated action of multiple viral glycoproteins, including gH/gL and gB which comprise the core fusion machinery conserved in herpesviruses. The gH/gL heterodimer activates the gB fusion protein, triggering its refolding from a prefusion to a postfusion form to drive membrane merger. The cytoplasmic tail domain (CTD) of gB is proposed to act as an inhibitory clamp that stabilizes the prefusion state, with interactions between gH and gB CTDs destabilizing this clamp. We previously found that herpes simplex virus 1 (HSV-1) and saimiriine herpesvirus 1 (SaHV-1) gB homologs are functionally interchangeable but mediate reduced fusion when coexpressed with heterotypic gH/gL. To map the regions of gB responsible for species-specific interactions, we generated HSV-1/SaHV-1 gB chimeras by swapping ectodomain, membrane-proximal region (MPR), transmembrane domain (TMD), and CTD segments. Our results show that homotypic CTD interactions alone are insufficient to trigger fusion, suggesting that gH/gL contacts the ectodomain of gB. We show that the HSV-1 gB CTD is hyperfusogenic relative to the SaHV-1 CTD, whereas the HSV-1 MPR is hypofusogenic relative to the SaHV-1 MPR. Together, these findings suggest that functional interaction between gH/gL-gB occur on both sides of the membrane and that gB maintains a balance between promotion and restraint of fusion through coordinated contributions of its domains.

microbiology↗

bioRxiv2025

Zyxin Restricts Viral Fusion and Entry across Multiple Virus Families

The entry of enveloped viruses into host cells requires fusion of the viral envelope with a host cell membrane. The composition and dynamics of cellular membranes are impacted by the underlying cytoskeleton. Zyxin, a cytosolic protein that bridges the actin cytoskeleton with adhesion receptor proteins, was recently identified as an antiviral factor that antagonizes HSV-1 entry. To determine if zyxin specifically interferes with membrane fusion, we examined its impact in a quantitative cell-cell fusion assay. HSV-1 entry proteins exhibited enhanced fusion activity with retinal pigment epithelial (RPE) cells when the cells were knocked out (KO) for zyxin. Zyxin-KO cells also showed enhanced fusion activity with pseudorabies virus (PRV), paramyxovirus and rhabdovirus fusion proteins. Additionally, the size of plaques formed following infection with members of each viral family were increased in the absence of zyxin. Bulk RNA sequencing of wild-type (WT) and zyxin-KO RPE cells identified 18 genes that enrich into several ontology groups of interest, including regulation of membrane potential, herpes simplex virus 1 infection (CCL2 and ZNF14), NABA core matrisome (CCL2 and ZNF14), extracellular matrix organization (CTSK, SERPINE1 and TGFB2), cell-cell adhesion (RAC2 and TGFB2), anchoring fibril formation, and regulation of the MAPK cascade (ACKR3 and TGFB2). These results highlight zyxin as a broadly active antiviral factor and suggest potential therapeutic implications for viral infections. IMPORTANCEEnveloped viruses enter host cells by fusing their envelope with a cellular membrane. This process is triggered when a viral glycoprotein(s) engages with a cellular membrane receptor. Less well understood are the cytoplasmic factors that indirectly govern viral fusion and entry. We report that zyxin, a cellular protein that bridges cell-adhesion receptors with the underlying actin cytoskeleton, antagonizes the fusion and entry of several enveloped viruses.

microbiology↗

bioRxiv2024

Structures of Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus virions reveal species-specific tegument and envelope features

Epstein-Barr virus (EBV) and Kaposis sarcoma-associated herpesvirus (KSHV) are classified into the gammaherpesvirus subfamily of Herpesviridae, which stands out from its alpha- and betaherpesvirus relatives due to the tumorigenicity of its members. Although structures of human alpha- and betaherpesviruses by cryogenic electron tomography (cryoET) have been reported, reconstructions of intact human gammaherpesvirus virions remain elusive. Here, we structurally characterize extracellular virions of EBV and KSHV by deep learning-enhanced cryoET, resolving both previously known monomorphic capsid structures and previously unknown pleomorphic features beyond the capsid. Through subtomogram averaging and subsequent tomogram-guided sub-particle reconstruction, we determined the orientation of KSHV nucleocapsids from mature virions with respect to the portal to provide spatial context for the tegument within the virion. Both EBV and KSHV have an eccentric capsid position and polarized distribution of tegument. Tegument species span from the capsid to the envelope and may serve as scaffolds for tegumentation and envelopment. The envelopes of EBV and KSHV are less densely populated with glycoproteins than those of herpes simplex virus 1 and human cytomegalovirus, representative members of alpha- and betaherpesviruses, respectively. This population density of glycoproteins correlates with their relative infectivity against HEK293T cells. Also, we observed fusion protein gB trimers exist within triplet arrangements in addition to standalone complexes, which is relevant to understanding dynamic processes such as fusion pore formation. Taken together, this study reveals nuanced yet important differences in the tegument and envelope architectures among human herpesviruses and provides insights into their varied cell tropism and infection. ImportanceDiscovered in 1964, Epstein-Barr virus (EBV) is the first identified human oncogenic virus and the founding member of the gammaherpesvirus subfamily. In 1994, another cancer-causing virus was discovered in lesions of AIDS patients and later named Kaposis sarcoma-associated herpesvirus (KSHV), the second human gammaherpesvirus. Despite the historical importance of EBV and KSHV, technical difficulties with isolating large quantities of these viruses and the pleiomorphic nature of their envelope and tegument layers have limited structural characterization of their virions. In this study, we employed the latest technologies in cryogenic electron microscopy (cryoEM) and tomography (cryoET) supplemented with an artificial intelligence-powered data processing software package to reconstruct 3D structures of the EBV and KSHV virions. We uncovered unique properties of the envelope glycoproteins and tegument layers of both EBV and KSHV. Comparison of these features with their non-tumorigenic counterparts provides insights into their relevance during infection.

microbiology↗

bioRxiv2022

Unique functional responses differentially map onto genetic subtypes of dopamine neurons

Dopamine neurons are characterized by their response to unexpected rewards, but they also fire during movement and aversive stimuli. Dopamine neuron diversity has been observed based on molecular expression profiles; however, whether different functions map onto such genetic subtypes remains unclear. Here, we establish that three genetic dopamine subtypes within the substantia nigra pars compacta each have a unique set of responses to rewards, aversive stimuli, accelerations and decelerations, and these signaling patterns are highly-correlated between somas and axons within subtypes. Remarkably, reward responses were not detected in one subtype, which instead displayed acceleration-correlated signaling. Our findings establish a connection between functional and genetic dopamine subtypes and demonstrate that molecular expression patterns can serve as a common framework to dissect dopaminergic functions.

neuroscience↗

bioRxiv2022

Multiple sites on glycoprotein H (gH) functionally interact with the gB fusion protein to promote fusion during herpes simplex virus (HSV) entry

Enveloped virus entry requires fusion of the viral envelope with a host cell membrane. Herpes simplex virus type 1 (HSV-1) entry is mediated by a set of glycoproteins that interact to trigger the viral fusion protein glycoprotein B (gB). In the current model, receptor-binding by gD signals a gH/gL heterodimer to trigger a refolding event in gB that fuses the membranes. To explore functional interactions between gB and gH/gL, we used a bacterial artificial chromosome (BAC) to generate two HSV-1 mutants that show a small plaque phenotype due to changes in gB. We passaged the viruses to select for restoration of plaque size and analyzed second-site mutations that arose in gH. HSV-1 gB was replaced either by gB from saimiriine herpesvirus type 1 (SaHV-1) or by a mutant form of HSV-1 gB with three alanine substitutions in domain V (gB3A). To shift the selective pressure away from gB, the gB3A virus was passaged in cells expressing gB3A. Sequencing of passaged viruses identified two interesting mutations in gH, including gH-H789 in domain IV and gH-S830N in the cytoplasmic tail (CT). Characterization of these gH mutations indicated they are responsible for the enhanced plaque size. Rather than being globally hyperfusogenic, both gH mutations partially rescued function of the specific gB version present during their selection. These sites may represent functional interaction sites on gH/gL for gB. gH-H789 may alter the positioning of a membrane-proximal flap in the gH ectodomain, whereas gH-S830 may contribute to an interaction between the gB and gH CTs. IMPORTANCEEnveloped viruses enter cells by fusing their envelope with the host cell membrane. Herpes simplex virus type 1 (HSV-1) entry requires the coordinated interaction of several viral glycoproteins, including gH/gL and gB. gH/gL and gB are essential for virus replication and both proteins are targets of neutralizing antibodies. gB fuses the membranes after being activated by gH/gL, but the details of how gH/gL activates gB are not known. This study examined the gH/gL-gB interaction using HSV-1 mutants that displayed reduced virus entry due to changes in gB. The mutant viruses were grown over time to select for additional mutations that could partially restore entry. Two mutations in gH (H789Y and S830N) were identified. The positions of the mutations in gH/gL may represent sites that contribute to gB activation during virus entry.

microbiology↗

bioRxiv2019

Two pathways of p27Kip1 degradation are required for murine lymphoma driven by Myc and EBV latent membrane protein 2A

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A), expressed in EBV latency, contributes to Burkitt Lymphoma (BL) development in a murine model by acting as a constitutively active B cell receptor (BCR) mimic. Mice expressing both LMP2A and MYC transgenes (LMP2A/{lambda}-MYC) develop tumors significantly faster than mice only expressing MYC ({lambda}-MYC). Previously, we demonstrated the cell cycle inhibitor p27Kip1 is present at significantly lower levels in LMP2A/{lambda}-MYC mice due to increased post-translational degradation. P27Kip1 degradation can occur in the cytoplasm following phosphorylation on serine 10 (S10), or in the nucleus via the SCFSkp2 complex, which depends on Cks1. We previously demonstrated a S10A knock-in of p27Kip1 (p27S10A/S10A), which prevented S10 phosphorylation, failed to significantly delay tumor onset in LMP2A/{lambda}-MYC mice. We also previously demonstrated that a Cks1 knockout partially delayed tumor onset in LMP2A/{lambda}-MYC mice, but onset was still significantly faster than in {lambda}-MYC mice. Here, we have combined both genetic manipulations in what we call p27Super mice. LMP2A/{lambda}-MYC/p27Super mice and {lambda}-MYC/p27Super mice both displayed dramatic delays in tumor onset. Strikingly, tumor development in LMP2A/{lambda}-MYC/p27Super mice was later than in {lambda}-MYC mice and not significantly different from {lambda}-MYC/p27Super mice. The p27Super genotype also normalized G1-S phase cell cycle progression, spleen size, and splenic architecture in LMP2A/{lambda}-MYC mice. Our results reveal both major pathways of p27Kip1 degradation are required for the accelerated BL development driven by LMP2A in our BL model and that blocking both degradation pathways is sufficient to delay Myc-driven tumor development with or without LMP2A.\n\nImportanceBurkitt lymphoma (BL) is a cancer that primarily affects children. The side effects of chemotherapy highlight the need for better BL treatments. Many BL tumors contain Epstein-Barr virus (EBV) and our goal is to determine what makes EBV-positive BL different from EBV-negative BL. This may lead to more specific treatments for both types. All cases of BL require overexpression of MYC. Mice engineered to express an EBV LMP2A along with MYC (LMP2A/{lambda}-MYC mice) develop tumors much more quickly than mice only expressing MYC ({lambda}-MYC mice). Blocking degradation of the cell cycle inhibitor protein p27Kip1 in LMP2A/{lambda}-MYC mice causes tumors to develop later than in {lambda}-MYC mice, showing that p27Kip1 degradation may play a larger role in EBV-positive BL than EBV-negative. Furthermore, our studies suggest the cell cycle may be an attractive target as a treatment option for LMP2A positive cancers in humans.

microbiology↗

bioRxiv2019

Characterization of Sex Differences in Ocular HSV-1 Infection and Herpes Stromal Keratitis Pathogenesis of Wild Type and Herpesvirus Entry Mediator Knockout Mice

Sex differences related to immune response and inflammation play a role in the susceptibility and pathogenesis of a variety of viral infections and disease (S. L. Klein, Bioessays 34:1050-9, 2012). Herpes simplex virus type 1 (HSV-1) causes chronic inflammatory disease in the cornea, an immune privileged tissue, resulting in irreversible damage and blindness in affected individuals (A. Rowe, A. St Leger, S. Jeon, D. K. Dhaliwal, J. E. Knickelbein, and E. Vilain, Prog Retin Eye Res 32:88-101, 2013). Our research focuses on the role of Herpes Viral Entry Mediator (HVEM) as an immune regulator during ocular HSV-1 infection. Mice lacking HVEM (HVEM KO) exhibit lower immune cell infiltrates and less severe ocular disease in the cornea compared to wild type (WT) mice. As sex differences contribute to pathogenesis in many inflammatory diseases, we tested sex as a biological variable in the immune response to HSV-1 infection and Herpes Stromal Keratitis pathogenesis (HSK). Adult male and female WT and HVEM KO mice were inoculated with HSV-1 via corneal scarification and monitored daily for disease course. Viral titers and immune cell infiltrates were collected and analyzed. Our results indicate no significant difference in viral titers in tear film or affected tissues; immune cell infiltration; or clinical symptoms between males and females of either genotype. These results suggest that sex is not a significant biological variable in this experimental model, and that male and female mice can similarly be used in studies of ocular HSV-1 pathogenesis. IMPORTANCESex hormones have only recently been considered as an important factor for the development of certain diseases and as such should continue to be considered as a biological variable. Ocular Herpesvirus Type 1 (HSV-1), and the resulting Herpes Stromal Keratitis, is the leading cause of infectious blindness worldwide. We compared ocular HSV-1 infection and pathogenesis between sexes and found no significance difference between male and female wild type mice or herpesvirus entry mediator knockout mice. Therefore, male and female mice can be used interchangeably in studying ocular HSV-1 pathogenesis.

microbiology↗

bioRxiv2019

Ephrin receptor A4 (EphA4) is a new Kaposi’s sarcoma-associated herpesvirus virus entry receptor

Kaposis sarcoma-associated herpesvirus (KSHV) is a human {gamma}-herpesvirus associated with the development of Kaposis sarcoma (KS). KSHV target cells include endothelial cells, B cells, monocytes, epithelial cells, dendritic cells, macrophages, and fibroblasts. KSHV entry into target cells is a complex multistep process and is initiated by the binding and interaction of viral envelope glycoproteins with the cellular receptors. In our current studies, we have found that EphA4 promotes KSHV gH/gL-mediated fusion and infection better than EphA2 in HEK293T cells indicating that EphA4 is a new KSHV entry receptor. To confirm that epithelial cells express EphA2 and EphA4, we analyzed the expression of EphA2 and EphA4 in epithelial cells, endothelial cells, B cells, monocytes, fibroblasts using RNA-seq data analysis of existing data sets. We found these cell types broadly express both EphA2 and EphA4 with the exception of monocytes and B cells. To confirm EphA4 is important for KSHV fusion and infection, we generated EphA2 and EphA4 single and double knockout cells. We found that both EphA2 and EphA4 play a role in KSHV fusion and infection, since EphA2/EphA4 double knockout cells had the greatest decrease in fusion activity and infection compared to single knockout cells. Fusion and infection of KSHV was rescued in the EphA2/EphA4 double knock cells upon overexpression of EphA2 and/or EphA4. EphA2 binds to both EBV and KSHV gH/gL; however, EphA4 binds only to KSHV gH/gL. Taken together, our results identify EphA4 as a new entry receptor for KSHV.\n\nImportanceThe overall entry mechanism for herpesviruses is not completely known including that for the human {gamma}-herpesviruses Kaposis sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). To fully understand the herpesvirus entry process, functional receptors need to be identified. In our current study, we found that EphA4 can also function for a KSHV entry receptor along with EphA2. Interestingly, we found that EphA4 does not function as an entry receptor for EBV whereas EphA2 does. The discovery of EphA4 as KSHV entry receptor has important implications for KSHV pathogenesis in humans and may prove useful in understanding the unique pathogenesis of KSHV infection in humans and may uncover new potential targets that can be used for the development of novel interventional strategies.

microbiology↗

bioRxiv2018

Natural selection of glycoprotein B (gB) mutations that rescue the small plaque phenotype of a fusion-impaired herpes simplex virus (HSV) mutant

Glycoprotein B (gB) is a conserved viral fusion protein that is required for herpesvirus entry. To mediate fusion with the cellular membrane, gB refolds from a prefusion to a postfusion conformation. We hypothesize that an interaction between the C-terminal arm and the central coiled-coil of the herpes simplex virus 1 (HSV-1) gB ectodomain is critical for fusion. We previously reported that three mutations in the C-terminal arm (I671A/H681A/F683A, called gB3A) greatly reduced cell-cell fusion and that virus carrying these mutations had a small plaque phenotype and delayed entry into cells. By serially passaging gB3A virus, we selected three revertant viruses with larger plaques. These revertant viruses acquired mutations in gB that restore the fusion function of gB3A, including gB-A683V, gB-S383F/G645R/V705I/A855V, and gB-T509M/N709H. V705I and N709H are novel mutations that map to the portion of domain V that enters domain I in the postfusion structure. S383F, G645R, and T509M are novel mutations that map to an intersection of three domains in a prefusion model of gB. We introduced these second-site mutations individually and in combination into wild-type gB and gB3A to examine the impact of the mutations on fusion and expression. V705I and A855V (a known hyperfusogenic mutation) restored the fusion function of gB3A, whereas S383F and G645R dampened fusion and T509M and N709H worked in concert to restore gB3A fusion. The results identify two regions in the gB ectodomain that modulate the fusion activity of gB, potentially by impacting intramolecular interactions and stability of the prefusion and/or postfusion gB trimer.\n\nIMPORTANCEGlycoprotein B (gB) is an essential viral protein that is conserved in all herpesviruses and is required for virus entry. gB is thought to undergo a conformational change that provides the energy to fuse the viral and cellular membranes, however the details of this conformational change and the structure of the prefusion and intermediate conformations of gB are not known. Previously, we demonstrated that mutations in the gB \"arm\" region inhibit fusion and impart a small plaque phenotype. Using serial passage of a virus carrying these mutations, we identified revertants with restored plaque size. The revertant viruses acquired novel mutations in gB that restored fusion function and mapped to two sites in the gB ectodomain. This work supports our hypothesis that an interaction between the gB arm and the core of gB is critical for gB refolding and provides details about the function of gB in herpesvirus mediated fusion and subsequent virus entry.

microbiology↗