To enhance the selectivity of HSV-1 for infecting epithelial-derived malignancies while minimizing the risk of infection in healthy somatic cells and preventing uncontrolled spread of HSV-1 to normal somatic cells, numerous research groups knockout the γ134.5 gene. The diploid gene γ134.5 located within the inverted terminal repeats flanking the long unique sequence of HSV-1 DNA is classified as a gamma-late or “leaky late” gene. It encodes ICP34.5, a neurovirulent protein. ICP34.5 consists of 263 amino acids organized into three main domains: the N-terminal domain, the linker region, and the C-terminal domain. Those domains are responsible for binding host proteins essential for both viral replication and immune evasion (26–29).

The principal function attributed to ICP34.5 involves enhancing viral propagation across peripheral tissues alongside central nervous systems, contributing significantly toward HSV, and inducing neurovirulence via various mechanisms including protein phosphatase I (PPI) dephosphorylating eIF2α, thus preventing shutoff from host protein synthesis while enabling continuous production (11, 30, 31). Furthermore, ICP34.5 converts proliferating cell nuclear antigen (PCNA) from repair mode back toward a replicative state, crucially initiating HSV replication (32).

Moreover, ICP34.5 inhibits antiviral signaling pathways ensuring persistent infections. ICP34.5 disrupts retinoic-acid-inducible gene I (RIG-I) signaling preventing interaction between RIG-I and mitochondrial antiviral signaling protein (MAVS), a pivotal adaptor inhibiting downstream activation IRF3 and subsequent IFN production (33). Stimulator interferon genes (STING) is another important player during antiviral responses where N-terminal domain binds/inactivates STING, thereby diminishing IRF3 activation/IFN secretion (34).

Additionally, ICP34.5 impedes autophagic processes through Beclin binding interactions specifically targeting Beclin-1 (Atg6) (35). Such engagement hinders this vital cellular defense mechanism allowing enhanced pathogenesis while blocking class II antigen presentation, further augmenting HSV virulence (36, 37).

Lastly, ICP34.5 also disrupts NF-kB activation suppressing dendritic maturation and ultimately impairing effective adaptive immunity against infection. The N-terminal domain of ICP34.5 interacts with IKKα/β, components of the IκB kinase complex, while its C-terminal domain recruits PP1α. This interaction leads to the dephosphorylation of IκB kinase, preventing the activation of NF-κB, a transcription factor that regulates genes involved in immune responses, inflammation, and cell survival (38, 39).

Through these combined mechanisms, ICP34.5 serves as a critical factor in HSV-1 pathogenesis by supporting viral replication, evading multiple immune pathways, and altering host cellular functions. However, after silent gene γ134.5, the oncolytic efficacy of HSV-1 in malignant tumors of neurological origin (e.g., glioblastoma and neurofibroma) has decreased. In clinical treatment, it is necessary to choose the oncolytic HSV-1 with silent gene γ134.5 according to the tissue source of the tumor.

To reduce immune evasion and subsequent uncontrolled viral infection after the injection of oncolytic HSV-1 into patients, gene US11 was selectively silenced. It can not only reduce the immune evasion of oncolytic HSV virus immunocompromised patients but also decrease the replication and spread of the virus in healthy cells. The US11 protein is a small basic phosphoprotein with a molecular weight of approximately 18 kDa. Its coding region extends from the ATG codon at residue 12,641 to the TAG stop codon at residue 12,158, resulting in a protein mass of 17,756 Da. The carboxy-terminal half contains several arginine-X-proline (R-X-P) repeats that confer RNA-binding capability (40). These repeats also harbor nucleolar import and nuclear export signals that facilitate localization within both nucleus and cytoplasm as required. Encoded by the late γ2 gene, US11 is expressed during later stages of HSV infection and performs several crucial functions enhancing HSV-1 survival within host cells (41).

Inhibition of protein kinase R (PKR) pathway alongside support for viral protein synthesis are primary functions attributed to US11. The PKR pathway becomes activated upon binding double-stranded RNA (dsRNA), leading to phosphorylation of eIF2α, an event typically halting protein synthesis as part of an antiviral response. US11 exhibits high affinity for dsRNA, allowing it to sequester this molecule away from PKR (42, 43). By obstructing PKR activation through this mechanism, US11 prevents eIF2α phosphorylation, thus sustaining viral protein synthesis. Furthermore, when expressed early during infection, US11 can partially compensate for ICP34.5’s function inhibiting eIF2α phosphorylation. This redundancy enables HSV-1 to maintain ongoing translation even if ICP34.5 is absent. However, both proteins are generally necessary for full resistance against type I interferon (IFN) responses (43).

Additionally, US11 modulates various host antiviral pathways facilitating escape from immune responses by HSV-1. During late phases when levels of dsRNA peak, US11 binds/sequesters dsRNA effectively preventing MDA5/RIG-I activations, which subsequently suppress IRF3 activity along with interferons production (44, 45). Such inhibition impedes induction of ISGs establishment, hence compromising antiviral states among infected cells. Another important mechanism involves oligoadenylate synthetase (OAS) pathway activated via dsRNA binding where US11 inhibits OAS activity, blocking RNase L, thereby aiding virus evade degradation while preserving infectivity (46).

Moreover, US11 plays pivotal roles regulating cell survival pathways ultimately promoting enhanced replication through prolongation of lifespans among infected hosts. Within nuclei, US11 interacts with homeodomain-interacting protein kinase HIPK2 responding stress signals including those arising from ER-regulating cycle progression/pro-apoptotic signaling (47). By antagonizing growth-arrest-induced HIPK2, HSV-1-infected cells evade apoptosis, continuing to facilitate virion propagation (48).

Through multifaceted functionalities, US11 facilitates HSV-1 replication by preventing translational shutoff, inhibiting immunological signaling and obstructing pro-apoptotic response. By suppressing activations across PKR, OAS, MDA5, and RIG-I enable HSV-1 to evade defenses and sustain syntheses, thus augmenting survivability and pathogenicity. After the silencing of US11, the therapeutic effect of a single injection of oncolytic HSV-1 may be transient. Throughout the course of treating malignant tumors, multiple injections of oncolytic HSV-1 are required, and continuous monitoring of tumor growth is necessary to evaluate whether to administer oncolytic HSV-1 again.

One of the immune evasion mechanisms of HSV-1 is to inhibit antigen presentation by binding to TAP, thereby preventing cytotoxic T cells from recognizing infected cells. The protein encoded by the US12 gene is key to binding with TAP. The US12 gene (ICP47) spans residues 12,972 to 12,708 and encodes the immediate-early protein ICP47, which consists of 88 amino acids. Similar to US1 at the opposite end of the Us region, both the promoter region of ICP47 and a significant portion of its 5′-non-coding mRNA are situated within the terminal repeat (TR) sequences of HSV-1 (26). ICP47 plays a pivotal role in HSV-1’s immune evasion strategy through various mechanisms and polymorphic functions during different stages of infection. It exhibits high-affinity binding to the transporter associated with antigen presentation (TAP). By occupying TAP’s substrate-binding site, ICP47 inhibits viral peptide loading onto MHC class I molecules for presentation on cell surfaces to CD8+ T cells, effectively blocking cytotoxic T lymphocyte (CTL) recognition of infected cells and enabling HSV-1 to evade immune detection (14, 49).

The function of ICP47 is polymorphic as infection progresses. During early infection stages, it may impede RNA splicing, thus limiting host and viral gene expression in a tightly regulated manner. In later stages, however, ICP47 appears to facilitate viral mRNA export from the nucleus into the cytoplasm, thereby supporting efficient viral replication (50).

Deletion of US12 has been shown to enhance HSV-1’s oncolytic potential and tumor-cell-killing ability alongside a stronger immune response. This deletion places US11 under immediate-early promoter control while enhancing replication efficiency in tumor cells for HSV-1 strains lacking ICP34.5, suggesting promising applications for oncolytic virotherapy (51, 52).

In summary, when ICP47 is deleted from its genome context, it can serve as an effective tool to reduce immune evasion in immunodeficient environments and tumor cells.

The UL39 gene is situated within the Unique Long Region of the HSV-1 genome and plays a critical role in viral replication and in modulating physiological processes within host cells (53). To reduce the spread of oncolytic HSV-1 proliferation within tumors, gene UL39 is selected as the candidate gene to be silenced. Unlike certain other HSV-1 genes, UL39 does not generate repetitive sequences with adjacent regions of the viral genome, rendering it structurally distinct. This gene is expressed early during the HSV-1 replication cycle, prior to the entry of the viral genome into the host cell nucleus (54, 55). Its initial translation depends on transcription and translation mechanisms within host cells, enabling HSV-1 to swiftly produce essential proteins for sustained infection (55).

ICP6, which is encoded by the UL39 gene and serves as the large subunit of ribonucleotide reductase, is vital for converting ribonucleotides into deoxyribonucleotides necessary for DNA synthesis in viruses (56). Additionally, ICP6 can phosphorylate eIF2α, a key initiation factor, thereby suppressing host protein synthesis and favoring production of viral proteins over cellular functions. This mechanism facilitates enhanced viral replication within infected hosts (57).

Another significant function attributed to ICP6 involves its modulation of programmed cell death (PCD) processes in infected cells through its receptor-interacting protein-homotypic interaction motif (RHIM) (54). The RHIM domain prevents necroptosis by obstructing RIPK1-RIPK3 complex formation (receptor-interacting protein kinases 1 and 3) in human cells (58). Furthermore, it promotes aggregation of RIPK1 that subsequently undergoes degradation via aggrephagy, further diminishing necroptotic activity (11). It also inhibits RIPK1/RIPK3-dependent necroptosis in human cells. Beyond preventing necroptosis, ICP6 additionally suppresses apoptosis by directly binding to and inhibiting caspase-8. This dual inhibition strategy allows HSV-1 to circumvent major apoptotic pathways while promoting both survival and proliferation within host environments (53).

Inactivation of ICP6 through fusion with LacZ results in restricted virus propagation primarily among dividing cells, particularly tumor cells capable of supplying deoxyribonucleotides via endogenous pathways (59). This tumor-specific characteristic exhibited by mutated forms of ICP6 positions HSV-1 variants makes them promising candidates for oncolytic therapies. Although silencing the gene UL39 can limit the proliferation of oncolytic HSV-1 after injection, enhancing the safety of this oncolytic virus in clinical applications, the tumor-killing effect of this oncolytic virus is also restricted, requiring a larger dosage and multiple injections to achieve the desired effect.

Induction of immune cells to kill tumor cells is one of the key mechanisms of oncolytic virus anticancer. In addition to the immune activation of the viral particles themselves, the cytokine genes carried by oncolytic viruses can be synthesized and released in tumor cells, and this process also significantly improves the killing efficacy of immune cells to tumor cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine that plays critical roles in immune modulation, serving as a bridge between hematopoiesis and immune activation (60, 61). Initially identified as a growth factor that stimulates the differentiation of bone marrow progenitor cells into granulocytes and macrophages, GM-CSF also activates various signaling pathways, including JAK/STAT, MAPK, and PI3K, through JAK2 activation, thereby influencing immune functions (62–65).

GM-CSF enhances the survival, proliferation, and differentiation of myeloid lineage cells such as neutrophils, macrophages, and dendritic cells (DCs) (66). By promoting DC maturation, GM-CSF improves antigen presentation capabilities and T-cell activation (60). To enhance the phagocytic abilities of macrophages and their anti-tumor activities, GM-CSF drives the polarization of these cells from an M2 (anti-inflammatory) phenotype to an M1 (pro-inflammatory) phenotype (67). Furthermore, GM-CSF strengthens immune recognition of cancer cell neoantigens by fostering antigen, presenting cell generation, and elevating major histocompatibility complex (MHC) expression, thereby reinforcing the overall immune response against tumors (23, 68).

Oncolytic viruses (OVs) armed with GM-CSF lead to localized cytokine expression within the tumor microenvironment while enhancing tumor cell susceptibility to viral infection by driving these cells into the cell cycle. This effectively converts “cold” tumors characterized by low immune activity into “hot” tumors exhibiting high levels of immune activity (69). Additionally, GM-CSF-armed OVs promote DC recruitment and maturation at tumor sites, which enhances T-cell priming and generates robust anti-tumor immune responses (70). This process can also foster long-term immunological memory resulting in sustained anti-tumor effects. The first oncolytic HSV-1 armed with GM-CSF, talimogene laherparepvec (T-VEC), demonstrated significant anti-tumor efficacy leading to FDA and EMA approval for melanoma therapy (71–73). However, excessive GM-CSF release also aggravates the systemic symptoms, such as fatigue and elevated body temperature. More attention is paid to the inflammatory status of patients during treatment.

To enhance the tumor resistance of NK cells and cytotoxic T lymphocytes, interleukin-12 (IL-12) was selected as a candidate gene for the insertion of oncolytic HSV-1. Its ability to reshape the tumor microenvironment while augmenting responses to checkpoint inhibitors underscores its therapeutic potential particularly when combined with other cancer immunotherapies establishing it as a formidable agent in anti-tumor immunity.

IL-12 facilitates CD4+ T-cell differentiation into Th1 cells that secrete elevated levels of interferon-gamma (IFN-γ), which subsequently activates NK cells and cytotoxic T lymphocytes (CTLs), thereby enhancing their anti-tumoral functions (74). Moreover IL-12 amplifies both growth rates and cytotoxic activities among NK cells alongside CD4+ and CD8+ T lymphocytes, resulting in increased production of perforin and granzyme B, which are key molecules essential for CTLs’ capacity to eradicate tumor cells (74, 75). Additionally, IL-12 promotes differentiation toward memory or effector T-cell phenotypes, thus improving precision persistence within targeting residual or metastatic malignant populations (76, 77).

Furthermore, IL-12 diminishes regulatory T-cell (Treg) and myeloid-derived suppressor cell (MDSC) populations within tumoral environments alleviating suppression mechanisms detrimental toward effective antitumoral responses (5, 78). It also drives macrophage polarization toward an M1 phenotype, a state characterized by pro-inflammatory properties conducive for inducing tumoricidal activity. By downregulating vascular endothelial growth factor (VEGF), IL-12 effectively reduces angiogenesis associated with tumors (25, 79).

Moreover, IL-12 sensitizes neoplasms toward checkpoint inhibitors like PD-1/PD-L1 blockade, thereby amplifying therapeutic efficacy (24, 80). With regard to promotion of tumor antigen presentation, death induced through IL-12-stimulated effectors releases TAAs further stimulating adaptive immunity assisting remaining malignant targets recognized by activated T cells (76, 77).