Circular RNAs (circRNAs) represent a class of highly stable, covalently closed RNA molecules increasingly recognized as important regulators of brain aging and neurodegenerative disease. Growing evidence also implies circRNAs in viral infection, suggesting a potential intersection between viral neuropathogenesis and neurodegeneration. However, no studies have yet directly integrated circRNAs, neurotropic viral infections, and neurodegenerative disorders within a single mechanistic framework. To date, specific circRNAs have been linked to the progression of Alzheimer's disease and Parkinson's disease, where they regulate central pathological processes including amyloid-β clearance, neuroinflammation, synaptic plasticity, neuronal apoptosis, and oxidative stress. Moreover, it has been established that both host cells and viruses produce circRNAs during infection. Virus-derived circRNAs can enhance viral replication, promote immune evasion, and support latency. In contrast, host circRNAs contribute to antiviral defense by acting as microRNA sponges, interacting with viral proteins, or encoding peptides with antiviral activity, mechanisms particularly explored in viral oncogenesis. In this review, we will evaluate the most updated research evidence on the role of circRNAs in major neurodegenerative diseases and neurotropic viral infections. Considering the growing concern regarding the long-term neurological consequences of viral infections, including chronic neuroinflammation, viral reactivation, and post-viral syndromes, dysregulated circRNAs may represent a mechanistic link between viral infection and associated neurodegenerative processes. Finally, we will discuss future directions for identifying circRNAs-based biomarkers and developing circRNAs-targeted therapeutic strategies for age-related and virus-associated neurological disorders.
N-Myc and STAT interactor (NMI), initially identified as a Myc- and STAT-associated protein, is increasingly recognized as a regulatory node in innate antiviral immunity. Current evidence indicates that NMI does not exert a fixed antiviral or proviral effect. Instead, its functional output is shaped by interacting partners, signaling pathway context, subcellular localization, and stage of infection. In selected acute RNA virus models, NMI suppresses IRF7-dependent type I interferon (IFN-I) signaling. In the IAV model, this involves an NMI-IFP35 complex coupled to the TRIM21-IRF7 axis that promotes IRF7 degradation, suppresses IFN-I responses, and thereby facilitates viral replication. In foamy virus infection, by contrast, NMI directly binds the viral transactivator Tas, retains it in the cytoplasm, and suppresses viral transcription, thus acting as a host restriction factor. In human cytomegalovirus (HCMV) infection, NMI appears to function as a host pathway component targeted by viral antagonism, as the viral protein UL23 interferes with the NMI-STAT1/IFN-γ axis and dampens antiviral gene expression. In addition, extracellular NMI and IFP35 can function as damage-associated molecular patterns that amplify inflammation. Together, these findings support the view that NMI acts as a context-dependent molecular switch rather than a unidirectional effector in innate antiviral immunity.
Virus entry leads to the delivery of the viral genome into the target cell for replication and viral gene expression. This critical step is therefore the target of many host immune mechanisms, as well as antiviral drugs. Various experimental systems have been developed to investigate virus entry and test virus entry inhibitors. Often, these systems are either cell-based or employ recombinant proteins to characterize the interaction between viral glycoproteins and their receptors, as well as determine the structural implications of virus attachment and entry. In this study, we have developed a quantitative virus particle fusion assay that allows for the measurement of viral membrane fusion events triggered by the binding of viral glycoproteins to their receptors. Specifically, we produced HIV-1 lentiviral particles incorporating HIV-1-Env/LgBiT-Vpr or CD4-CCR5/HiBiT-Vpr and recorded high levels of reconstituted NanoLuc luminescence when these virus particles are co-incubated. Background levels of luminescence were detected when these virus particles did not carry Env or CD4-CCR5, indicating that HIV-1 Env and CD4/CCR5 are essential for particle fusion and NanoLuc reconstitution. We further showed that HIV-1 entry inhibitors targeting Env, CD4, and CCR5, all potently inhibit lentiviral particle fusion. Lastly, the antiviral proteins SERINC5 and IFITM3, which are known to inhibit HIV-1 entry, also significantly diminish fusion in the context of this assay. Taken together, these data demonstrate that this quantitative lentiviral particle fusion assay can be used for discovering viral entry inhibitors, generating insights into the molecular mechanisms of virus entry, and elucidating the role of host factors in viral entry.
Over 39.9 million individuals are living with HIV worldwide. There is a need to develop novel therapeutics to improve treatment or cure HIV. In this study, we evaluated HIV reservoir-targeting chimeric antigen receptor (CAR)/CXCR5 natural killer (NK) cells armored with IL-15 and PD-1 knockout as well as control activated NK cells for their safety and efficacy by treating antiretroviral therapy (ART)-suppressed, HIV-infected humanized DRAGA mice following ART interruption. There were no adverse health outcomes associated with cell infusions. At 56 days post-treatment (DPT), 62.5% of CAR NK-treated and 50% of control NK-treated groups had viral loads below the detection limit compared with 0% of the saline control group. CAR NK-treated animals had, on average, 1.88 times higher peak NK cell levels than control NK-treated animals, and elevated levels of NK cells persisted up to 28 DPT in treated animals. Importantly, animals that had undetectable viral loads at 56 DPT had earlier viral rebound post-ART interruption that coincided with high levels of NK cells, suggesting that timing of treatment with viral recrudescence may play a role in efficacy. This is the first study evaluating NK cell therapies in hDRAGA mice and demonstrates the promise of NK cell therapies for curing HIV.
Endolysosomal compartments act as critical sorting hubs for viral entry, trafficking, and uncoating, yet the contribution of lipid-modifying enzymes to these processes remains incompletely understood. Acid sphingomyelinase (ASM), a key regulator of endolysosomal membrane composition, has been implicated in host-pathogen interactions, and is actively engaged in human adenovirus (HAdV) infection. Here, we confirm ASM activity as an essential host determinant of efficient HAdV infection and demonstrate that its pharmacological inhibition synergizes with direct-acting antiviral therapy. Analysis of publicly available human datasets revealed age-associated changes in expression of host cell determinants for HAdV entry like ASM expression in respiratory epithelial tissues and colonic tissue, suggesting that ASM-dependent endolysosomal functions are modulated across the human lifespan. Using epithelial cell models of HAdV infection, we show that viral entry and replication critically depend on ASM activity. Pharmacological inhibition of ASM by functional inhibitors such as fluoxetine resulted in a dose-dependent suppression of viral replication and significantly reduced infection rates in single-cycle infection assays, indicating disruption of early stages of the viral life cycle. Mechanistically, ASM inhibition impaired HAdV uptake and trafficking by reducing viral internalization and co-localization with early endosomes. Importantly, combined treatment with fluoxetine and the viral DNA polymerase inhibitor brincidofovir acts synergistically in reducing HAdV titer. These findings demonstrate that targeting host-controlled endosomal entry pathways can potentiate the antiviral efficacy of direct-acting agents. Together, our study validates ASM as a central regulator of HAdV entry and identifies combined host- and virus-directed therapy as a promising strategy to suppress HAdV infection.
Intrinsic cellular factors that inhibit herpesvirus infection remain incompletely defined. Here, we identify TRIM5α as a restriction factor for herpes simplex virus type 1 (HSV-1). TRIM5α-mediated restriction requires its ubiquitin ligase activity, PRY-SPRY domain, and the ability to oligomerize. Mechanistically, we show that TRIM5α directly engages capsid protein VP19C and promotes the stability of the VP19C-VP23 complex and its nuclear accumulation. VP19C also activates NF-κB synergistically with TRIM5α and independently. HSV-1 counteracts this host defense by triggering proteasome-dependent TRIM5α degradation. In addition, we show that Cyclophilin A (CypA), which is incorporated into HSV-1 virions, also binds to VP19C, but enhances infection. As with HIV-1 and orthopoxviruses, the proviral activity of CypA is disrupted by cyclosporin A (CsA), but unlike the situation with these other viruses, the proviral activity of CypA is independent of TRIM5α. Notably, CsA and its non-immunosuppressive derivatives also exhibit anti-HSV-1 activity in neuronal cell lines, suggesting a potential therapy for HSV-1 encephalitis. TRIM5α and CypA also interact with orthologs of VP19C in other alpha, beta and gamma human herpesviruses. These findings reveal two distinct host pathways acting on the herpesvirus capsid and provide a foundation for comparing how TRIM5α and CypA modulate infection of unrelated virus families, offering new directions to identify shared principles of host recognition and viral evasion.
Flaviviruses, including Dengue, West Nile, Zika, and Japanese encephalitis viruses, are arthropod-borne RNA viruses that pose an increasing global health threat. This review summarizes the role of nonstructural protein 1 (NS1), a multifunctional glycoprotein found in intracellular and secreted forms, as a key regulator of innate immunity. NS1 modulates several pattern recognition receptor pathways, including TLRs, RLRs, SR-B1-related mechanisms, and inflammasome platforms, thereby altering cytokine and interferon responses. Its effects are virus- and context-dependent. WNV NS1 inhibits TLR3/TRIF signaling, reducing IRF3 activation, type I interferon production, and interferon-stimulated gene expression. In contrast, DENV NS1 is linked to inflammatory signaling, particularly through TLR4. At the cytosolic level, NS1 from DENV, WNV, and ZIKV disrupts RIG-I/MDA5-MAVS signaling and weakens IFN-β induction. NS1 also affects inflammasome pathways: DENV promotes IL-1β release through a CD14-dependent mechanism, ZIKV suppresses cGAS-mediated antiviral signaling, and JEV promotes NLRP3 inflammasome assembly. Overall, NS1 selectively dampens interferon-mediated antiviral defenses while sustaining or enhancing inflammation, contributing to endothelial dysfunction, neuroinflammation, and severe disease.
Japanese encephalitis virus (JEV) is an important neurotropic orthoflavivirus that poses a threat to both human and animal health. However, the mechanism underlying its rapid replication in the central nervous system (CNS) remains poorly understood. In this study, we conducted metabolomic profiling of JEV-infected mouse brains and neurons, revealing a profound reprogramming of central carbon metabolism, particularly an enhancement in nucleotide synthesis. Integrated multi-omics analyses confirmed that JEV infection transcriptionally upregulates key enzymes involved in de novo purine biosynthesis (DNPB), one-carbon (1C) metabolism, and the pentose phosphate pathway (PPP) in neurons. Pharmacological inhibition of the core DNPB enzymes potently suppressed JEV replication in neurons and reduced both viral loads and neuroinflammation in JEV-infected mice, suggesting the essential role of DNPB in JEV replication within CNS. Mechanistically, we delineated the critical functions of both the non-oxidative PPP and MTHFD2-mediated 1C metabolism, which jointly supply essential precursors, such as ribose-5-phosphate and formyl groups, for the de novo biosynthesis of purines required for viral RNA replication. These findings unveil a strategy by which JEV co-opts the host's purine biosynthetic machinery to fulfill the nucleotide demands for its genomic replication, establishing DNPB and its supporting pathways as promising therapeutic targets for infections caused by JEV and other neurotropic viruses.
The genomes of human adenoviruses (HAdVs) are double-stranded DNA bound with viral nucleocapsid protein VII. During HAdV infection, host RNA polymerase can produce viral-associated RNAs (VA RNAs) that are recognized by retinoic acid-inducible gene I (RIG-I). However, it remains unclear whether and how HAdV, as a DNA virus, can evade RNA sensors. Here, we show that nucleocapsid protein VII from HAdV can inhibit type I interferon (IFN) production by antagonizing RIG-I. It is found that protein VII precursor of HAdV/C5 (C5preVII) could impede tripartite motif-containing protein 25 (TRIM25)-mediated K63 ubiquitination of RIG-I by preventing TRIM25 oligomerization. Importantly, we provide in vitro and in vivo evidence that cytoplasm-oriented C5preVII is responsible for TRIM25 inactivation. Moreover, the preVII proteins from diverse HAdVs show evolutionarily conserved immunosuppressive functions. Collectively, our study illustrates the function of HAdV preVII in retinoic acid-inducible gene I-like receptor (RLR) signaling and uncovers an evolutionally conserved mechanism of DNA viruses to evade host RNA sensors.
Severe immune-mediated complications following viral infections and vaccinations, including COVID-19 and anti-SARS-CoV-2 immunization, display remarkable clinical overlap despite occurring in distinct biological contexts. In a previous hypothesis-driven work, we proposed that metabolic incorporation of the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) into human glycoconjugates-defined as xenosialylation-may contribute to post-infectious and post-vaccination immune dysregulation. We further suggest that this phenomenon may represent a form of "immunological chimerism", in which host glycoconjugates incorporate non-self molecular structures that predispose the immune system to varying degrees of immune imbalance. In its most severe manifestation, this process may culminate in a profound state of immune dysregulation characterized by loss of immune tolerance, aberrant antibody responses, cytokine storm, and thrombo-inflammatory pathology, which we define as "immunological marasmus". In the present paper, we extend this conceptual framework by integrating glycobiology, Fc immunoglobulin glycosylation, endothelial biology, and sex-dependent immune regulation into a unified, testable immunopathogenic model. We hypothesize that interindividual differences in the extent, tissue distribution, and persistence of xenosialylation may influence susceptibility to exaggerated innate and adaptive immune responses following antigenic challenge. In this context, immune activation may unmask pre-existing xeno-sialylated self-structures embedded within host glycans, promoting varying degrees of glycan dysregulation, autoantibody production, immunothrombosis and chronic inflammatory sequelae. We further propose circulating anti-Neu5Gc antibodies as functional biomarkers for risk stratification and outline preventive strategies based on dietary modulation of xenosialic acid exposure. Taken together, this expanded model provides a potential mechanistic framework for understanding the shared immunological features of post-viral syndromes and vaccine-related adverse immune reactions, while offering a basis for experimental validation and future approaches to personalized risk mitigation.
The penile epithelium, encompassing multiple anatomical sites, is the primary location of human immunodeficiency virus (HIV) acquisition in heterosexual men. Although the per-contact risk of penile HIV acquisition is generally low, substantial global discrepancies in HIV prevalence still exist, particularly in low-income regions. In uncircumcised men, the immune milieu of the subpreputial space is a key determinant of HIV risk, with inflammation-mediated epithelial disruption and target cell recruitment facilitating viral infection. Specific bacterial components of the penile microbiome cause local inflammation and enhance susceptibility, while penile circumcision reduces HIV risk by both removing susceptible foreskin tissues and reducing the abundance of these bacteria. The penile urethra is also an important site of HIV acquisition, particularly among circumcised men, but determinants of urethral susceptibility remain poorly understood. Penile-vaginal sex induces transient inflammation and epithelial damage at both the subpreputial space and urethra, likely mediated by mechanical effects and/or the sexual exchange of pro-inflammatory bacteria. This review summarizes knowledge regarding the immunological and microbial determinants of penile HIV acquisition risk, highlights biological factors and sexual practices that shape the penile immune milieu, and discusses current advances in microbiome-targeting interventions as potential HIV prevention strategies.
Extracellular vesicles (EVs) are important mediators of intercellular communication in hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. EVs released from infected hepatocytes can carry viral nucleic acids, proteins, and regulatory non-coding RNAs to immune and nonimmune cells, thereby influencing viral dissemination, immune regulation, and disease progression. In particular, EV-associated cargos modulate antiviral immunity by affecting interferon signaling, natural killer cell function, cytokine production, immune checkpoint pathways, and T-cell exhaustion. These effects may promote viral persistence and immune evasion, although some EV populations can also enhance innate antiviral responses, indicating context-dependent dual roles. EVs also contribute to fibrosis and hepatocarcinogenesis by regulating hepatic stellate cell activation, inflammatory signaling, and tumor microenvironment remodeling. In addition, EV-derived RNAs and proteins show potential as noninvasive biomarkers and therapeutic targets. This review summarizes current evidence on EVs in HBV and HCV infection, with emphasis on immune regulation, viral persistence, disease progression, and translational prospects, while also discussing key challenges such as EV heterogeneity and co-isolation with viral particles.
Immune modulation in the male genital tract during HIV infection is not well understood, despite its role in viral persistence and transmission. In a study using an SIV rhesus macaque model, we assessed viral RNA and 61 cytokines, chemokines, and growth factors in peripheral plasma (PP) and seminal plasma (SP). SIV RNA was found in both plasma samples, but the SP viral loads were more variable and decreased over time, independent of the systemic viremia. Baseline immune mediator profiles exhibited compartment-specific patterns, and principal component analysis demonstrated distinct clustering between PP and SP samples. Unlike the extensive systemic immune activation in PP, SIV infection in SP results in selective modulation involving CXCL1, IL-7, CCL4, and YKL-40, with these changes not correlating with the local viral loads. Our findings suggest that the seminal compartment is immunologically distinct, with selective inflammatory responses, highlighting the mechanisms that may influence viral persistence and transmission.
Viral infection triggers a robust DNA damage response (DDR), reshaping the host chromatin landscape to facilitate viral replication. Here, we uncover a novel mechanism by which alphaherpesviruses exploit the DDR pathway. We demonstrated that herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) induced selective degradation of class I histone deacetylases (HDAC1/2), leading to histone hyperacetylation and subsequent DDR activation. Strikingly, viral infection promoted nuclear export of HDAC1/2, followed by MDM2-mediated K63-linked polyubiquitination and proteasomal degradation in the cytoplasm. Pharmacological inhibition of either DDR signaling or HDAC1/2 nuclear export significantly affected viral replication in vitro and in vivo. Our findings reveal a unique viral strategy to hijack host epigenetic regulation for efficient replication, and identify potential therapeutic targets for alphaherpesvirus infections.
Highly pathogenic avian influenza A(H5N1) viruses of clade 2.3.4.4b, genotype D1.1, are responsible for widespread outbreaks in poultry and continue to cause sporadic, sometimes severe, human infections. Herein, we characterized a wild-type (WT) influenza A(H5N1) D1.1 isolate (BC-H5N1-WT) and its H275Y neuraminidase (NA) variant (BC-H5N1-H275Y), both of which emerged on farms in British Columbia, Canada, during the fall 2024 outbreak. In vitro analysis assessed replication kinetics in MDCK cells, with supernatants collected at different days post-infection (p.i.) and titrated by TCID50 and qRT-PCR. Neuraminidase inhibitor (NAI) susceptibility was determined by NA inhibition assays, whereas susceptibility to baloxavir acid (BXA) was evaluated by plaque reduction assay. In vivo virulence was evaluated in BALB/c mice infected with serial 10-fold dilutions of each virus to monitor weight loss and mortality. Viral titers in lungs, brain, nose, kidney, spleen, and heart were quantified at day 4 p.i. The BC-H5N1-WT virus was susceptible to the four antivirals tested, whereas BC-H5N1-H275Y displayed resistance to oseltamivir and peramivir but remained susceptible to zanamivir and BXA. The BC-H5N1-WT exhibited significantly higher viral replication titers than BC-H5N1-H275Y at all tested time points and showed larger plaque sizes. In mice, BC-H5N1-WT was more virulent with LD50 values of 1.78 × 103 PFUs compared to 8.71 × 104 PFUs for BC-H5N1-H275Y, and produced higher viral titers in lungs and other organs. Despite the reduced fitness of the resistant H5N1 D1.1 variant, its emergence in the absence of viral selection pressure underscores the need for continued surveillance.
Seasonal influenza in humans is predominantly caused by influenza A virus (IAV) and influenza B virus (IBV), but they differ markedly in host range, evolutionary dynamics, and pandemic potential. Such phenotypic divergence reflects the distinct molecular strategies employed by the two viruses at key stages of their life cycles. Hemagglutinin (HA) of IAV possesses prominent structural plasticity, which endows it with the capacity to recognize both avian-type α2,3-linked and human-type α2,6-linked sialic acid receptors, and the function of IAV polymerase is highly dependent on host acidic nuclear phosphoprotein 32 (ANP32) family proteins. Moreover, IAV utilizes multiple pleiotropic virulence factors, such as the nonstructural protein 1 (NS1), to modulate host immune responses and inflammatory processes, thereby contributing to viral fitness, host adaptation, and pandemic potential. In contrast, the HA of IBV preferentially binds to human-type (α2,6-linked) sialic acid receptors and exhibits a more restricted receptor-binding profile.While IBV polymerase is well adapted to human ANP32A and ANP32B, yet shows poor compatibility with avian ANP32 proteins. Additionally, the immunomodulatory machinery is relatively streamlined, engaging host cell death pathways in a more limited manner that may contribute to generally less extensive inflammatory responses in many experimental and clinical settings. As a result, IBV transmission is largely confined to humans, with a narrow host range and a predominantly seasonal epidemic pattern. In this review, we systematically compare IAV and IBV with respect to four core pathogenic processes, namely viral entry, genome replication efficiency and host factor dependence, immune evasion, and the regulation of host cell death pathways, to explain how these mechanisms collectively shape differences in host range, evolutionary dynamics, and pandemic potential. We particularly emphasize the capacity of IAV to achieve efficient replication in a wide range of host species, a trait that facilitates its multi-host circulation and viral gene reassortment. These insights establish a theoretical framework for enhancing influenza surveillance and guiding the development of next-generation influenza vaccines and antiviral therapeutics.
Achieving a functional cure for HIV-1 requires dismantling the molecular circuits that enable viral latency and immune evasion. Beyond transcriptional silencing and epigenetic repression, post-translational modifications (PTMs) of host and viral proteins operate as dynamic molecular switches that regulate all phases of the viral life cycle, from integration and transcription to immune recognition and reservoir maintenance. This review synthesizes recent discoveries across major PTM types, including phosphorylation, ubiquitination, SUMOylation, acetylation, methylation, glycosylation, and ISGylation, and highlights how these modifications modulate intrinsic and adaptive immunity, chromatin state, protein turnover, and viral RNA metabolism. We further explore how targeting PTM networks offers a promising therapeutic avenue to reverse latency, destabilize reservoirs, or enhance immune clearance with minimal off-target activation. By surveying the HIV-PTM regulatory landscape, we highlight actionable nodes that can inform next-generation strategies for durable remission and immune reprogramming.
Post-acute sequelae of SARS-CoV-2 (PASC) occurs in subsets of individuals, including those with pre-existing lung disease. To investigate PASC pathogenesis and therapeutics in a chronic bronchitis mouse model (Scnn1b-Tg), Scnn1b-Tg and WT mice were inoculated with a mouse adapted SARS-CoV-2 virus (SARS-CoV-2MA10) and followed for 60 days. Viral titer, histology, immunohistochemistry (IHC), single-cell RNA sequencing, RNA in situ hybridization, and spatial transcriptomic profiling characterized disease pathologies. Scnn1b-Tg mice inoculated with SARS-CoV-2MA10 exhibited lower viral titers and less weight loss than WT mice. Airway epithelia of Scnn1b-Tg mice were less infected than epithelia of WT mice, reflecting increased airway mucus and enhanced epithelial antiviral activities in Scnn1b-Tg mice. However, Scnn1b-Tg mice subsequently exhibited heterogeneous airway and parenchymal disease with elevated Il33 expression characteristic of human eosinophilic pneumonia. Cohorts of infected mice were administered a monoclonal antibody targeting the IL-33 receptor (ST2) or enteral prednisone. Administration of an anti-ST2 monoclonal antibody mitigated development of eosinophilic pneumonia while enteral prednisone suppressed IL33 expression and disease. The eosinophilic pneumonia in Scnn1b-Tg mice after SARS-CoV-2MA10 infection mimics reports of eosinophilic pneumonia in humans post-SARS-CoV-2, suggesting targeting of IL-33 may be beneficial in treating post-viral eosinophilic pneumonia in humans.
More than four decades of HIV/AIDS studies reveal that generation of effective vaccines against HIV infection requires understanding of a host immunity that protects our DNA against the HIV infection. An effective vaccine can elicit the host immunity to stop HIV RNA expression despite the viral DNA integrated into the host DNA. Vaccination is one of the most successful public health interventions of all time. Recent studies show that humans have an intrinsic immunity against foreign nucleotide gene expression, and we have named this the epigenetic immunity. We propose that chromatin vaccine (cVacc), a foreign DNA in chromatin format, is capable of triggering the immunity of host DNA, the epigenetic immunity, to silence the viral gene expression at its RNA transcription level. We discuss these advances in the field of host - virus interaction, specifically host immunity against the retroviral infections by silencing its gene expression, ranging from nucleotide sensors that distinguish self-nonself to the chromatin features that constrict the HIV reservoir. Bolstered by the progress in the fields of virology, immunology and epigenetics, we propose to maneuver cutting edge technologies to develop next-generation vaccines like cVacc, not only silencing the foreign gene expression, but assimilating it to improve our DNA function as well.
The role of glycoprotein G (gG-2) of herpes simplex virus type 2 (HSV-2) in viral pathogenesis remains poorly understood. gG-2 is cleaved into a secreted form (sgG-2) and a membrane-associated form (mgG-2), but the in vivo function of mgG-2 and the contribution of its glycosylation to immune responses have not been defined. Here, we provide a comprehensive characterization of the N- and O-linked glycosylation profile of mgG-2 and investigate its functional relevance for viral spread and vaccine-induced immunity. Using a mouse genital infection model, we show that an mgG-2-deficient HSV-2 mutant replicates in vaginal epithelial cells but is severely impaired in dissemination to dorsal root ganglia and the central nervous system, identifying mgG-2 as a key determinant of neuronal spread in vivo. In parallel, immunization with recombinant mgG-2 elicited strong humoral and Th1-polarized CD4 + T-cell responses and conferred protection against genital HSV-2 challenge. Importantly, glycosylation of mgG-2 was required for optimal immunogenicity and protection, as deglycosylated variants induced reduced CD4 + T-cell responses and allowed increased viral spread to neuronal tissues. Mechanistically, our findings suggest that glycosylation of mgG-2 modulates antigen recognition and shapes adaptive immune responses that limit viral dissemination after vaccination. Together, these results demonstrate that mgG-2 plays a critical role in HSV-2 pathogenesis and provide a strong rationale for targeting glycosylated mgG-2 in the development of both prophylactic and therapeutic vaccines against HSV-2.