A comprehensive understanding of the putative synergy between human papillomavirus (HPV) and Epstein–Barr virus (EBV) in laryngeal squamous cell carcinoma (LSCC) necessitates an integrated perspective on their distinct yet complementary oncogenic paradigms, which converge to form a collaborative “two-hit” model. The basis of this model rests on the independent pathogenic mechanisms of each virus. High-risk HPVs, most notably HPV16, promote carcinogenesis via their early oncoproteins E6 and E7 (Gupta et al., 2018; Chow, 2020).

The E6 protein inactivates the key tumor-suppressor p53 through ubiquitin-mediated degradation, impairing the DNA damage response and apoptotic pathways, while E7 targets the retinoblastoma protein (pRb) for degradation. This liberation of E2F transcription factors thereby drives unchecked cell cycle progression (Yang et al., 2019; Zhang et al., 2025). Beyond overriding cell cycle checkpoints, a pivotal role of E6/E7 lies in the profound disruption of the host epithelial differentiation program—a process central to both the viral life cycle and the cross talk with EBV (Nawandar et al., 2015).

EBV, in turn, utilizes a distinct oncogenic toolkit. It infects epithelial cells via receptors such as Ephrin type-A receptor 2 (EPHA2), a receptor tyrosine kinase that transduces pro-tumorigenic signals upon viral engagement (Locard-Paulet et al., 2016; Bu et al., 2022). Following infection, EBV can establish latency, during which proteins such as latent membrane protein 1 (LMP1) act as constitutively active signaling molecules. These activate pathways, including NF-κB, to enhance cell survival and proliferation (Cao et al., 2021). The switch from latency to lytic replication is tightly linked to terminal epithelial differentiation and is directly modulated by host factors, such as Krüppel-like factor 4 (KLF4) (Nawandar et al., 2015).

The proposed synergistic model posits that HPV infection acts as the initial priming hit. By expressing E6 and E7, HPV establishes a differentiation-defective microenvironment in the basal epithelial layer. This altered cellular state directly interferes with EBV’s differentiation-dependent lytic replication, favoring a shift toward persistent latency—characterized by the sustained expression of latent oncoproteins such as LMP1 (Makielski et al., 2016; Guidry et al., 2019). This HPV-mediated reprogramming creates a permissive niche for EBV persistence, constituting the first hit.

Within this preconditioned microenvironment, the second hit unfolds through functional cross talk between persistent HPV oncoproteins and latent EBV factors, thereby amplifying oncogenic effects. For instance, co-expression of HPV E6 and EBV LMP1 synergistically compromises DNA damage response pathways while hyperactivating pro-survival signals such as NF-κB, thereby exacerbating genomic instability and apoptosis resistance (Shimabuku et al., 2014). Furthermore, EBV entry via EPHA2 in coinfected cells may provide an additional layer of early synergy: engagement of the receptor by viral glycoproteins aberrantly activates its intrinsic pro-invasive and pro-angiogenic signaling, potentially accelerating the early stages of malignant progression (Chatzikalil et al., 2024; Lee et al., 2024).

In summary, this “two-hit” framework outlines a plausible biological sequence: HPV delivers the first hit by profoundly reprogramming the epithelial niche and disrupting host differentiation, which in turn facilitates the establishment of EBV latency. Subsequent collaborative cross talk between viral proteins from both pathogens amplifies impairments across key cellular hallmarks, collectively reducing the threshold for malignant transformation and accelerating tumor progression in HPV–EBV-coinfected laryngeal epithelium (Figure 1A).

HPV and EBV coinfection synergistically disrupts genomic stability, and both viruses induce basal DNA damage through viral replication. The coexistence of low-risk HPV (LR-HPV) and EBV triggers limited malignant transformations. Although it increases secondary DNA damage and impairs DNA damage response (DDR) efficiency, leading to somatic mutation accumulation and precancerous lesions, it remains insufficient to drive complete malignant transformation (Uehara et al., 2021). Conversely, co-expression of HR-HPV and EBV is hypothesized to form a potent carcinogenic axis (Figure 1C). When the E6 oncoprotein of HR-HPV is co-expressed with EBV latent membrane protein LMP-1, it significantly suppresses core DDR pathway factors, such as the Ataxia Telangiectasia Mutated and ATM- and Rad3-Related Signaling Axis (ATM/ATR) signaling axis, while activating the NF-κB pathway (Shimabuku et al., 2014). Sustained activation of the NF-κB signaling pathway, primarily mediated by LMP1, promotes cell proliferation, increases resistance to apoptosis, and disrupts the reactive oxygen species (ROS) homeostasis, conferring survival advantages to cancer cells (Xin et al., 2022; Khenchouche et al., 2023). However, LMP1 activation alone is necessary but insufficient for coinfection-driven carcinogenesis. The synergistic action of the HPV E6 protein via mechanisms, including p53 degradation and telomerase activation, can bypass cell cycle checkpoint regulation, establishing sufficient and necessary conditions for malignant transformation. Once established, this transformed state may no longer depend on the initial peak NF-κB activity level (experiments confirmed its subsequent decline), suggesting that E6 and LMP1 may synergistically reprogram cells to achieve transformation autonomy. In animal models (e.g., mouse embryonic fibroblasts), co-expression of E6 and LMP1 significantly enhances tumorigenesis, validating their cascading carcinogenic efficacy in suppressing DDR and hijacking signaling pathways (Shimabuku et al., 2014).

EBV sustains infection through a dual latent lytic life cycle. During latency, the virus persists long-term in B cells or epithelial cells as inclusions with restricted viral gene expression. During the lytic cycle, it undergoes three phases: immediate-early (transactivation of BZLF1/Zta and BRLF1/Rta), early (expression of viral replication proteins), and late (assembly of structural proteins, such as capsid antigens and membrane proteins), culminating in infectious virions. EBV lytic activation is highly dependent on epithelial differentiation progression, directly driven by the host transcription factors KLF4 and BLIMP1, triggering BZLF1/BRLF1 promoters (Nawandar et al., 2015). However, HPV significantly disrupts this process via its oncoproteins, E6/E7–HPV16. E6/E7 blocks epithelial differentiation by downregulating KLF4 target genes, including FLG, LOR, and WNT5A, with E7 specifically delaying early differentiation marker expression, thereby undermining the differentiation signals required for EBV lytic replication (Guidry et al., 2019). Thus, the precise regulation of KLF4 by HPV oncoproteins constitutes a fundamental aspect of the microenvironmental reprogramming model. High-risk HPVs fine-tune KLF4 through multilayered mechanisms: E7 upregulates KLF4 protein levels by suppressing miR-145, while E6 may alter KLF4 activity and stability by interfering with its posttranslational modifications (Gunasekharan et al., 2016). Furthermore, within the context of early coinfection in the basal layer, as described in Section 2.1 and occurring prior to terminal differentiation during active E6/E7-mediated reprogramming, this regulatory process contributes to establishing a cellular environment permissive for EBV lytic gene expression. Collectively, within the differentiation-deficient microenvironment established by HPV, the paucity of host differentiation cues favors a shift of EBV from a lytic replication mode to a latent state. We hypothesize that this shift is not merely a default outcome but a critical adaptive strategy. It ensures viral persistence in an otherwise non-permissive cellular context and, more importantly, establishes the essential foundation for the subsequent immune evasion and sustained oncogenic synergy between latent EBV proteins (e.g., LMP1) and persistent HPV oncoproteins. Furthermore, HPV–EBV coinfection directly increases genomic instability. Integration of HR-HPV genes into the host genome is a key event in HPV-driven carcinogenesis, a process that is potentially amplified by the coexistence of EBV. Studies indicate that the presence of EBV may increase the rate of HPV16 integration sevenfold (odds ratio [OR], 7.11; 95% confidence interval [CI]: 1.70–29.67) (Szostek et al., 2009). Another study found that EBV infection increased the likelihood of HPV16 genomic integration into the host genome fivefold (OR, 5.00; 95% CI: 1.15–21.80) (Kahla et al., 2012).

The transforming growth factor beta (TGF-β) signaling pathway may serve as a core hub for EBV and HPV cross-regulation of viral replication and transformation potential. The TGF-β1/Smad pathway is a critical switch in the EBV lytic cycle. It induces latent EBV reactivation by activating BZLF1/Zta expression, while Zta itself can promote TGF-β secretion via Smad-dependent or Smad-independent feedback mechanisms, forming a positive feedback loop that amplifies EBV lytic reactivation (Liang et al., 2002; Iempridee et al., 2011). However, this pathway has a dual effect on cancer progression. TGF-β, a core inducer of epithelial–mesenchymal transition (EMT), drives cancer cell migration and invasion by downregulating E-cadherin and upregulating vimentin (Velapasamy et al., 2018), directly promoting metastasis. Conversely, HPV disrupts TGF-β signaling via its early oncogenes, E5 and E7, synergistically downregulating TGF-βRII expression and blocking TGF-β/Smad signaling. This interference is thought to promote early carcinogenesis and could synergize with EBV to potentially accelerate EMT progression (Figure 1B) (French et al., 2013).

Metabolic reprogramming accompanying cancer progression centers on the Warburg effect (aerobic glycolysis). Aerobic glycolysis is the process by which tumor cells preferentially convert glucose into pyruvate via glycolysis, and subsequently into lactate, even under oxygen-sufficient conditions, bypassing the efficient energy production of mitochondrial oxidative phosphorylation (Paul et al., 2022). This process confers proliferative advantages to cancer cells by enhancing glycolytic flux and lactate accumulation while shaping an immunosuppressive tumor microenvironment (Heawchaiyaphum et al., 2023). HPV and EBV, as key oncogenic viruses, synergistically disrupt host metabolic programs through their encoded oncoproteins (Figure 2). At the glucose uptake level, E6 and E7 proteins of HPV directly activate glucose transporter 1 (GLUT1) expression, degrading the tumor suppressor p53 to release the transcriptional repression of GLUT1 and GLUT4 (Ancey et al., 2018), thereby upregulating GLUT1 via these dual mechanisms. However, LMP1 of EBV activates the mammalian target of rapamycin signaling complex 1-nuclear factor kappa-light-chain enhancer of activated B cells (mTORC1-NF-κB) signaling axis, inducing GLUT1 gene transcription and promoting its membrane localization, significantly enhancing glycolytic flux (Zhang et al., 2017). At the level of glycolytic activation, HPV relies on E6/E7 to elevate c-Myc levels, directly upregulating the glycolytic rate-limiting enzyme hexokinase II (HK-II) (Flier et al., 1987). LMP1 of EBV stabilizes c-Myc via the Phosphoinositide 3-Kinase (PI3-K)/Akt-Glycogen Synthase Kinase 3 Beta (GSK3β)-F-box and WD repeat domain-containing 7 (FBW7) signaling axis pathway, driving HK-II overexpression (the c-Myc-HK-II axis is essential for EBV metabolic reprogramming) while upregulating all glycolytic enzymes (Xiao et al., 2014). Finally, at the metabolic center regulatory level, HPV 16 oncoproteins E6/E7 block the interaction between hypoxia-inducible factor-1α (HIF-1α) and the E3 ubiquitin ligase von Hippel–Lindau (VHL) tumor-suppressor protein, inhibiting ubiquitin-mediated degradation (Knuth et al., 2017). However, EBV disrupts the HIF-1α hydroxylation-dependent degradation pathway via EBNA5 inactivation of prolyl hydroxylase-domain protein 1 (PHD1) and EBNA3 inhibition of prolyl hydroxylase-domain protein 1 (PHD2) (Darekar et al., 2012), with both viruses collectively elevating HIF-1α levels. Additionally, LMP1 of EBV stabilizes Siah 1 protein in human epithelial cells, inducing PHD degradation and preventing VHL-HIF-1α interaction, elevating HIF-1α levels in EBV-infected human cancer cell lines (Kondo et al., 2006). These mechanisms cause the abnormal accumulation of HIF-1α under normoxic conditions. HIF-1α, a master regulator of metabolism, drives angiogenesis, cell proliferation, and invasion by activating glycolytic pathways.

Aerobic glycolysis hyperactivates LDH-A, accelerating the conversion of pyruvate to lactate even under oxygen-sufficient conditions. Lactate accumulation triggers multiple vicious cycles. (1) Lactate acidification activates matrix metalloproteinases (MMPs), promoting tumor invasion (Hirschhaeuser et al., 2011; Cardeal et al., 2012). (2) Immunosuppression inhibits T-cell/dendritic cell function and synergizes with viral proteins to facilitate immune evasion (Fischer et al., 2007). (3) Angiogenesis synergistically promotes neovascularization via the HIF-1α/vascular endothelial growth factor (VEGF) axis (EBV-dominant) and direct lactate induction by VEGF/TGF-β (HPV-dominant) (Fischer et al., 2007; Polet and Feron, 2013). (4) Enhanced migration: lactate and EBV-LMP1 promote cell migration (Dawson et al., 2008; Liu et al., 2012).

Immune surveillance is a well-established characteristic of tumor cells (Hanahan and Weinberg, 2011). At the innate immune level, general virology studies show that HPV–EBV jointly suppresses the Toll-like receptor (TLR) pathways to facilitate immune evasion. TLRs are specialized pattern recognition receptors that play a crucial role in initiating antiviral immune responses (Rakoff-Nahoum and Medzhitov, 2009). HPV E6/E7 proteins significantly downregulate TLR2/3/5/7/9 expression (Hasan et al., 2013; Cigno et al., 2020), whereas EBV targets and inhibits TLR2/9 function through lytic proteins (Zhang et al., 2024), such as BGLF5 and BALF1 (Reiser et al., 2011). Furthermore, HPV-mediated TLR9 downregulation may significantly impair the ability of the host to detect EBV, and the association between TLR9 downregulation and increased EBV infection risk further demonstrates the synergistic promotion of immune evasion by HPV–EBV coinfection (Jabłońska et al., 2020).

In the innate immune system that defends against pathogens, the cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) signaling pathway can be activated by abnormal intracellular double-stranded DNA (dsDNA) signals to exert anti-inflammatory or pro-tumor effects (Tufail et al., 2025). Insights from viral immunology suggest that coinfection with HPV and EBV may amplify the pro-tumor activity of this pathway, aiding cancer cells in evading immune surveillance (Figure 3) (Zhang and Zhang, 2025). Specifically, E1 and E5 proteins of HPV16 suppress interferon regulatory factor 3 (IRF3) function, reducing type I interferon (IFN-1) production (Li et al., 2025; Scott et al., 2020). HPV E6 directly binds IRF3 to decrease IFN-α/β/γ/κ generation (Lee et al., 2001), while E1/E6/E7 broadly interferes with RIG-I expression, diminishing IFN-stimulated gene (ISG) production (Lee et al., 2001; Li et al., 2025). Recent studies have revealed that the NLRX1 sequence of HPV16 E7 degrades STING, disrupting the cGAS-STING pathway (Luo et al., 2020). EBV, via its capsid protein BRLF1, reduces IRF3/7 expression to inhibit IFN-β production (Bentz et al., 2010). The early protein BFRF1 suppresses IRF3 phosphorylation, dimerization, and nuclear translocation, thereby inhibiting interferon-β (IFN-β) generation (Wang et al., 2020). The envelope protein LF2 (BILF4) blocks IRF3 activation, reduces IFN-α and IFN-β production (Wang et al., 2009; Wu et al., 2009), and uses miR-BART16-5p to degrade the ISG co-activator CREB-binding protein (CBP), thereby downregulating IFN-α (Hooykaas et al., 2017). The miR-BART6-3p targets the RIG-1 receptor to reduce IFN-β production (Lu et al., 2017). The EBV membrane protein BSRF 1 inhibits I-κB kinase (IKK) complex activation by blocking IKKα/IKKβ heterodimer formation, thereby interfering with IFN-β production and promoting viral replication (Chen et al., 2025). Coinfection with these two viruses may synergistically target the core IRF3 pathway and downstream ISGs, jointly establishing an IFN-suppressed immune environment that promotes tumor cell growth.

Interleukin-10 (IL-10) is primarily known for its anti-inflammatory properties and plays a crucial role in preventing excessive immune activation and maintaining immune homeostasis. In an immunohistochemical analysis of 133 laryngeal carcinoma specimens, IL-10 was identified as a prognostic risk factor for laryngeal cancer (Sun et al., 2018). Coinfection with HPV and EBV could synergistically amplify the pro-neoplastic effects of IL-10 (Figure 4). Single HPV and EBV infections, as well as coinfection with both viruses, promote IL-10 secretion by specific subsets of regulatory T cells, B cells, macrophages, monocytes, dendritic cells (DCs), and T helper cells (Chen et al., 2024). IL-10 binds to its receptor (IL-10R) to form a heterodimer (Berti et al., 2017). This activation stimulates the Janus kinase/signal transducer and activator of transcription 3 (JAK-STAT3) signaling cascade, and the activated STAT3 pathway further promotes IL-10 secretion through positive feedback (Huynh et al., 2017). Phosphorylated STAT3 recruits and activates myeloid-derived suppressor cells (MDSCs), which suppress T-cell responses. Additionally, it induces VEGF production and promotes tumor angiogenesis. Upon activation, JAK-STAT3 translocates to the nucleus and mediates the transcription of IL-10-immunosuppressive target genes. Furthermore, it enhances PD-L1 expression on tumor cell surfaces. This weakens T-cell function and facilitates tumor cell escape from immune surveillance via interactions with PD-1 on the T cells. The EBV BCRF1-encoded viral IL-10 (vIL-10) is a homologue of IL-10 (Stewart and Rooney, 1992). In animal studies, enhanced expression of vIL-10 in mice increased acute pathogenicity. However, it does not affect viral latency or reactivation during acute infection, and it plays a specific role in expanding the permissive host cell population, promoting the initial survival and spread of the virus within the infected cells (Lindquester et al., 2014). Furthermore, vIL-10 suppresses co-stimulatory molecules, uses gp42 to block cross-presentation, and disrupts the IFN signaling pathway via BZLF1, thereby promoting immune evasion (Salek-Ardakani et al., 2002; Jochum et al., 2012; Gujer et al., 2019).

Additionally, in the presence of IL-10, tumor-associated macrophages (TAMs) have been observed to shift to the M2 phenotype in HPV- and EBV-infected cells (Shivarudrappa et al., 2024; Chen et al., 2024; Xiang et al., 2024). TAM-M2 cells reduce the release of pro-inflammatory cytokines and growth factors, thereby providing an immunosuppressive environment conducive to tumor progression (Chen et al., 2019, 2024). Within the tumor microenvironment (TME), IL-10 also suppresses antigen-presenting cell maturation and activity, limits the ability of DCs to present tumor antigens, and impedes adaptive immune responses to facilitate immune evasion (Rallis et al., 2021; Mirlekar, 2022), reducing the production of pro-inflammatory cytokines such as IFN-γ and TNF-α (Bruchhage et al., 2018). IL-10 weakens the effector functions of cytotoxic T lymphocytes. Conversely, the immunosuppressive environment promotes IL-10 secretion, limiting its ability to control tumor growth (Groux et al., 1998; Li et al., 2020).

In adaptive immune evasion, mechanisms documented in viral oncology reveal that both mechanisms may synergistically reduce antigen presentation: HR-HPV inhibits human leukocyte antigen class I (HLA-I) transport to the Golgi apparatus via E5 and suppresses the major histocompatibility complex class I (MHC-I) heavy-chain promoter activity through E7, thereby inhibiting gene transcription, lowering HLA-I expression levels, and diminishing CD8⁺ T-cell responses (Georgopoulos et al., 2000; Jemon et al., 2016; de Freitas et al., 2017). The EBV nucleoprotein EBNA1 inhibits MHC-I-derived antigen peptide presentation (Levitskaya et al., 1995). The BGLF5 gene product, initially identified as an alkaline nucleic acid exonuclease, suppresses de novo protein synthesis, leading to loss of cell surface HLA-I molecules (Rowe et al., 2007). BNLF2a blocks the transporter associated with antigen processing (TAP) peptide transport (Horst et al., 2009). BILF1 may downregulate MHC-I molecules by enhancing endocytic degradation, thereby weakening CD8⁺ T-cell responses (Gujer et al., 2019; Singh et al., 2025). EBV disrupts CD4⁺ T-cell activation by impeding MHC-II/peptide-T-cell receptor (TCR) interactions via the gp42/gH/gL complex (Ressing et al., 2003). Cells coinfected with HPV and EBV could, in theory, synergistically create a low MHC-I and MHC-II environment for tumor cells, suppressing the detection of cells harboring latent and replicating viruses by CD8⁺ and CD4⁺ T lymphocytes.

The multifaceted synergy between HPV and EBV, as detailed in the preceding sections, is underpinned by a critical functional sequence that defines the proposed “two-hit” model. It is crucial to emphasize that the latent infection state of EBV in our model is a specific adaptation to the aberrant and differentiation-deficient epithelial microenvironment reshaped by preceding HPV infection. The first hit, mediated by HPV, extends beyond the canonical inactivation of p53 and pRb. Its fundamental role is to reprogram the epithelial niche, disrupting differentiation and innate immune sensing, thereby establishing a cellular microenvironment permissive to viral persistence. This altered microenvironment effectively facilitates the establishment of EBV latency, setting the stage for sustained coexistence (Makielski et al., 2016; Guidry et al., 2019). Within this preconditioned landscape, the second hit is executed by EBV. Latent proteins such as LMP1 engage in extensive molecular cross talk with persistent HPV oncoproteins, resulting in amplified oncogenic perturbations in DNA damage response, cellular metabolism, and immune evasion pathways (Shimabuku et al., 2014). Thus, the model emphasizes that HPV-led microenvironmental remodeling is a pivotal precursor event, enabling and potentiating the subsequent collaborative strike from EBV. While definitive proof of infection order and the exact sequence of molecular events in LSCC in vivo remains challenging, this functional logic, drawn from extrapolated data, provides a coherent framework for understanding the pronounced oncogenic synergy observed in coinfected lesions.