[ZKKL] Academic Presentation | Latency, Recurrent Infection, and Prevention & Treatment of Herpes Simplex Virus

HSV is highly infectious, capable of causing a wide range of prevalent diseases and readily establishing latent infections. HSV‑1 and HSV‑2 can remain latent for life in the trigeminal ganglion and the sacral ganglia, respectively. Kesan et al. have defined HSV‑1 latency as follows: (1) the virus persists in the host without eliciting any overt clinical symptoms; (2) latently infected HSV can be reactivated, resulting in the production of infectious viral particles; (3) during latency, the virus does not express any replication‑related genes but accumulates large amounts of latency‑associated transcripts (LATs) within the host cell nucleus; and (4) the virus fails to express relevant antigens, yet its genome remains detectable in the host. This definition characterizes the phenotypic features of HSV‑1 latency across four distinct dimensions and aligns with the characteristics of HSV‑2 latency. Although advances in detection technologies have revealed low‑level expression of replication‑related genes such as ICP0 and the TK kinase during latent infection, researchers attribute this to the dynamic equilibrium between latency and reactivation. The primary mechanisms underlying HSV‑2 latency include: (1) immune evasion following viral infection; (2) suppression and regulation of immediate‑early protein expression by LAT genes; (3) interactions between the viral genome and host cells, encompassing inhibition of cellular protein and RNA synthesis, selective translation of HSV mRNA, and modulation of the cell cycle and apoptosis; (4) microRNA‑mediated regulatory processes encoded by the viral genome; and (5) CTCF‑dependent regulation of latent reactivation through specific binding sites on the HSV genome.

2.1 Mechanisms by which HSV evades immune surveillance DNA and RNA viruses of different species can all evade immune surveillance and persist as long-term parasites within their hosts. The virus’s success in evading…

Immune evasion is one of the major causes of chronic herpesvirus infection. Its mechanisms primarily encompass the following six aspects.

2.1.1 Restrictive Expression of Viral Genes Almost all viruses exploit this mechanism to varying degrees to evade surveillance by the host immune system, with herpesviruses and certain retroviruses being particularly prominent. Once herpes simplex virus establishes latency within neurons, transcription of virtually all viral genes—except for a single one—is shut down, leaving the infected neuron virtually devoid of any detectable viral components. Extensive research has demonstrated that LAT possesses the ability to suppress viral gene expression.

2.1.2 Exploitation of the host’s immune-privileged sites A small number of human tissues and organs are sites where immune cells are barred from entering, enjoying an “immune privilege.” Consequently, viruses that gain access to these immune‑privileged niches can temporarily evade immune surveillance without triggering local inflammatory responses. In the central nervous system (CNS), the blood–brain barrier restricts lymphocyte entry, making it difficult for T lymphocytes to recognize CNS antigens. As a result, the CNS serves as a reservoir for chronic infection and latency of several viruses, including HSV.

2.1.3 Mutation of Viral Antigens HSV can also evade host immune surveillance through genetic mutations. When the virus undergoes mutation, the antigens it encodes likewise change. Variations in surface antigens may enable mutant strains to temporarily escape neutralization or opsonization by pre-existing antibodies, thereby gaining a selective advantage. Cytotoxic T lymphocytes (CTLs) and helper T cells (Th cells) play crucial roles in eradicating and controlling chronic viral infections, as they recognize T‑cell epitopes presented by viral proteins via MHC molecules. If genetic mutations during viral replication happen to alter the original T‑cell epitopes—such that the resulting peptide either fails to bind to the host’s MHC molecules or, even when bound, is no longer recognized by T cells—the mutant strain can transiently avoid CTL and Th cell–mediated immunity, acquiring a replicative advantage absent in the parental strain. This phenomenon has been demonstrated in HSV, hepatitis B virus, and Epstein–Barr virus.

2.1.4 Interference with Antigen Presentation by Host Cells Lipid antigens presented by CD1d molecules on the surface of antigen-presenting cells can elicit an immune response from NK cells, which is crucial for the host’s defense against viral antigens. Liu et al. found that HSV‑1 infection suppresses the expression of CD1d molecules on antigen‑presenting cells. This suppression does not result from HSV‑mediated inhibition of CD1d synthesis; rather, it arises from the redistribution of endocytosed CD1d molecules to the lysosomal membrane, thereby preventing their re‑emergence on the cell surface. It may also be due to HSV‑induced blockade of the trafficking of newly synthesized CD1d to the cell surface. Furthermore, studies have shown that the HSV‑encoded protein ICP47 can bind to the antigen‑presentation‑related transporters TAP‑1/2, thereby inhibiting the translocation of viral peptides into the endoplasmic reticulum and their subsequent loading onto nascent MHC class I molecules, ultimately impeding their presentation to CD8+ T cells.

2.1.5 Inhibition of TCR Signal Transduction T lymphocytes are a critical component of the immune response against HSV infection. Yang et al. demonstrated that HSV inactivates T cells by inhibiting TCR signal transduction. This inhibition of TCR signaling occurs both at the T cell activation‑inducing step within the TCR signaling cascade and at downstream steps, including suppression of calcium influx and multiple MAPK (mitogen‑activated protein kinase) pathways. HSV‑induced T cell inactivation results in reduced phosphorylation of tyrosine residues on the T cell activation‑inducing complex, whereas such tyrosine phosphorylation is essential for TCR signal transduction.

2.1.6 Disruption of the functions of immune effector molecules Interferons constitute the host’s first line of defense against viral infection. Extensive research has demonstrated that interferons comprise a family of soluble proteins that mediate antiviral effects, regulate cell growth, and modulate the activation of immune responses. The intrinsic antiviral activity of interferons is both highly effective and rapid. Consequently, many viruses evade host immune surveillance by inhibiting interferon synthesis or downstream antiviral pathways. Davis et al. found that HSV-encoded ICP34.5 plays a critical role in enabling the virus to escape the host’s innate immune response. ICP34.5 dephosphorylates eIF2α—a translation initiation factor phosphorylated by PKR during the antiviral response—thereby reducing the expression of cytokines such as interferon‑β. Mogensen reported that HSV‑1 promotes the expression of the viral regulatory protein VP16, which suppresses the expression of pro‑inflammatory cytokines by destabilizing IL‑6 mRNA. This suppression is not mediated directly by VP16 but depends on the viral immediate‑early genes encoding ICP4 (an infected‑cell protein) and ICP27. In the early stages of HSV infection, these proteins act in concert with VP16…

The mechanism of dependence is elucidated. The HSV envelope glycoprotein gC can bind to complement C3b, thereby protecting the virus from neutralization by complement components. Experiments demonstrate that wild-type HSV strains retain full infectivity in the presence of complement, whereas mutant strains lacking gC are neutralized by complement even in the absence of antibodies.

2.2 The role of the LAT gene in HSV latency and reactivation

The LAT family comprises major LATs—consisting of 2.0 kb, 1.5 kb, and 1.45 kb transcripts—and a non‑major LAT, which is 8.3 kb in length. These RNA molecules exist in both linear and circular forms but are activated through distinct folding patterns. The non‑major LAT is an 8.3‑kb transcript that, upon splicing, yields a 2.0‑kb LAT; further removal of approximately 500 bp generates either a 1.5‑kb or a 1.45‑kb LAT. LATs are thought to play a critical role in the efficient establishment of latency. Moreover, deletion of small segments within the LAT promoter region has been shown to cause a sharp reduction in the number of latently infected cells, underscoring that LAT transcription is essential for establishing latent infection. LATs exhibit multifunctional properties and contribute to both the establishment and maintenance of HSV latency. Currently, the primary approach to investigating LAT’s role in latent infection involves the use of deletion mutants.

2.2.1 The Role of LAT in Establishing Latent Infection LAT is considered to play a crucial role in the efficient establishment of latency; even the deletion of small fragments within the LAT promoter sequence can lead to a sharp reduction in the number of latently infected cells, suggesting that LAT transcription is essential for establishing latent infection.

2.2.2 The role of LAT in maintaining latent infection The mechanisms that maintain HSV latency remain unclear. Two competing hypotheses have been proposed: one posits that, during latency, the 2.0-kb LAT transcript suppresses ICP0 protein expression via an antisense RNA–mediated pathway, thereby blocking viral replication; the other suggests that, in the latent state, a non‑canonical LAT transcript inhibits ICP4 protein expression through an antisense RNA mechanism, thus preventing viral replication and delaying the transition to the lytic phase. Studies have also revealed the presence of several mRNAs encoding lytic viral genes during latency; however, their low transcriptional levels make them difficult to detect. Chen et al. demonstrated that, in LAT‑mutant strains during latency, the transcripts of the immediate‑early gene ICP4 and the early gene TK were expressed at levels several-fold higher than those observed in wild‑type virus. Further investigations showed that ICP4 downregulates the expression of the RNA‑binding protein GRSF1 by binding to the precursor of miR‑101, hsa‑mir‑101‑2, thereby suppressing HSV‑1 replication.

2.2.3 The Role of LAT in Viral Recurrence During the reactivation phase, early expression of the LAT gene may play a crucial role in transitioning lytic genes from a silent to an active state. In the latent phase, ICP4 mRNA is continuously expressed in the ganglia, albeit at very low levels. Consequently, the appearance of ICP0 mRNA and its upregulation serve as indicators that the virus has entered the lytic replication cycle. Experimental data show that, in mice carrying the LAT gene, the HSV‑1 viral DNA load is threefold higher than in LAT‑deficient mice, and its activation and expression are associated with CD8, PD‑1, and Tim‑3.

2.2.4 LAT exerts anti-apoptotic functions by encoding microRNAs MicroRNAs (miRNAs) are a newly discovered class of small, single-stranded, non-coding RNAs that regulate gene expression and serve as critical factors in modulating RNA stability and translational efficiency in animals, plants, and viruses. Both HSV‑1 and HSV‑2 LATS encode multiple functional miRNAs, with their expression levels during latency markedly higher than during acute infection. For HSV‑1, Cui et al., using computational modeling and sequence alignment with miRNA databases, identified 24 candidate miRNA pairs and 13 miRNA precursors. In 2010, Igor Jura et al. confirmed, via high-throughput sequencing, 17 miRNAs expressed by HSV‑2; to date, the miRNA database MiRBase has cataloged 18 such miRNAs. Recent research from our group has demonstrated that the miRNA miR‑H4‑5p encoded by HSV‑2 LATs can target host cell genes CDKN2A and CDKL2, reducing their mRNA and protein levels, thereby counteracting ActD‑induced apoptosis and promoting entry into the S phase of the cell cycle, thus enhancing cellular proliferation. Meanwhile, miR‑H3 also exerts anti‑ActD‑induced apoptotic effects but does not significantly influence the cell cycle; its precise molecular mechanisms remain unclear. These findings indicate that HSV‑2 miRNAs regulate both viral and host gene expression, mediating RNA interference to suppress neuronal apoptosis and sustain latent viral persistence. By up- or downregulating miRNA expression, it is possible to modulate the expression of target genes for therapeutic or intervention purposes. As a novel class of drug‑targetable molecules, miRNAs have increasingly attracted attention across disciplines. New therapeutic strategies for HSV‑2 may emerge by targeting miRNAs to specifically inhibit or disrupt HSV‑2 replication. Although these approaches are currently at the experimental stage, there is reason to believe that small‑molecule miRNA therapeutics could become a promising avenue for treating latent HSV infections.

2. 3 The role of CTCF in HSV latency and reactivation

CTCF comprises 727 amino acid residues; its middle domain (M‑domain), consisting of 312 residues, adopts 11 consecutive zinc‑finger motifs. The N‑terminal (N‑domain) and C‑terminal (C‑domain) regions contain 265 and 150 residues, respectively. CTCF is a ubiquitously expressed, highly evolutionarily conserved 11‑zinc‑finger DNA‑binding protein that engages target DNA sequences of approximately 50 base pairs via its zinc fingers, forming diverse CTCF–DNA complexes. These complexes regulate a wide array of functions across different genes, including transcriptional repression and activation, hormone‑regulated gene silencing, methylation‑dependent chromatin insulation, as well as roles in eukaryotic histone acetylation, genomic imprinting, and heterochromatin formation. Moreover, through self‑association into multimers or interactions with other proteins, and by co‑localizing with subnuclear structures, CTCF contributes to the establishment and maintenance of specific chromatin architectures, thereby playing a pivotal role in epigenetic regulation. CTCF serves as a central node in regulatory networks governing cell growth, proliferation, and development. A clear correlation exists between the extent of CTCF mRNA and protein downregulation and the degree of inhibition of cellular growth, underscoring the critical importance of normal CTCF expression for cell proliferation. Many insulator elements harbor numerous CTCF binding sites; DNA methylation at these sites can block CTCF binding and thereby suppress its function. CTCF is also the only transcription factor experimentally shown to mediate the enhancer‑blocking activity of distinct insulators. As a core component of higher‑order chromatin architecture, CTCF’s functions are highly conserved from Drosophila to humans, encompassing even certain human pathogens such as EBV, KSHV, and HSV. CTCF can induce epigenetic alterations, and model viruses have been employed to investigate how higher‑order chromatin structures respond to external perturbations.

Studies have shown that the HSV‑1 genome harbors numerous CTCF‑specific binding sites, particularly within the LAT, ICP0, and ICP4 regions. During latent HSV‑1 infection, CTCF protein is enriched at insulator elements, and in wild‑type latently infected HSV‑1, CTCF dissociates from its binding sites upon activation. The CTCF‑binding sequences upstream of ICP0 and ICP4 constitute canonical insulators that can attenuate the enhancer activity of LAT. Overall, during both latency and reactivation, the strength of CTCF–DNA interactions is dynamically regulated to modulate HSV‑1 gene expression. These findings underscore the critical role of epigenetic regulation in the switch between latency and reactivation of HSV‑1. Within the 2.0‑kb intron of HSV‑1 LAT, numerous CTCF‑binding sites are present, and CTCF can regulate virus‑replication‑related gene expression by binding to and releasing from these sites, thereby influencing viral latency and recurrence. Our group’s latest research represents the first systematic investigation of CTCF binding on HSV‑2; we have identified CTCF‑binding sites in the 3′ end of HSV‑2 LAT and within its intron, which exert a repressive effect on promoter activity and may play a pivotal role in HSV‑2 latency. This work opens new avenues for exploring the mechanisms underlying HSV‑2 latency and recurrence, as well as for developing novel therapeutic strategies.