RNA m5C modifications are catalyzed by RNA m5C methyltransferases, with S-adenosyl-l-methionine (SAM) as a methyl donor for methylation modification at the 5th carbon atom of cytosine (32). Various m5C-specific methyltransferases have been identified and characterized, including members of the NOL1/NOP2/SUN domain (NSUN) family of proteins (consisting of seven members in humans, NSUN1-7) and the DNA methyltransferase (DNMT) homologue DNMT2 (33). Within the NSUN family, NSUN2 is one of the most characterized members and a major m5C “writer” (34). m5C methylation has been found in diverse RNA species, including tRNAs (6), rRNAs (35), mRNAs (25), vault RNAs (vtRNAs) (36), small nuclear RNAs (snRNAs) (22), small nucleolar RNAs (snoRNAs) (37), microRNAs (miRNAs) (38), and viral RNAs (27). m5C modification, a common methylation modification on eukaryotic mRNAs, is more likely to occur in GC dibase-enriched regions, such as near the translation initiation site/start codon of the coding regions (CDS) (19).

The identified m5C-modifying demethylases include the Ten-eleven translocation (TET) protein family (TET1-3) and ALKBH1 (AlkB Homolog 1, Histone H2A Dioxygenase). TETs are α-ketoglutarate- and Fe²⁺-dependent dioxygenases responsible for catalyzing m5C demethylation and generating 5-hydroxymethylcytosine (hm5C) (39). In contrast to TETs, ALKBH1 catalyzes the 1^st step of m5C demethylation by converting m5C to 5-hydroxymethylcytosine (hm5C) and further oxidizing 5hmC to 5-formylcytosine (f5C). ALKBH1 has also been reported to be mainly involved in regulating the demethylation of tRNAs, including mt-tRNA Leu and mt-tRNA Met (9, 40).

The identified m5C methylation recognition proteins of eukaryotic mRNAs include Aly/REF export factor (ALYREF) (41), Fragile X mental retardation protein (FMRP) (42), Y-box binding protein (YBX)1, and YBX2 (43). ALYREF and FMRP are predominantly present in the nucleus, whereas YBX1 and YBX2 are in the cytoplasm (34). Accumulating studies have provided evidence for the tight association between these recognition proteins and m5C modifications. For example, ALYREF specifically recognizes m5C modifications on mRNAs to facilitate their export from the nucleus through lysine K171 (44). YBX1 recognizes m5C modifications on mRNAs through an indole ring of W65 within its cold-shock domain to maintain its stability (26). YBX2 binds with m5C-modified mRNAs through the W100 of the cold-shock domain to facilitate liquid-liquid phase separation (43). FMRP participates in the TET1-mediated R-loop demethylation via the recruitment of DNMT2, ensuring the effective repair of DNA damage (42).

Over the past two decades, there has emerged 3 zoonotic coronaviruses: SARS-CoV, middle east respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. These coronaviruses spilled over from animal repositories into humans. Similar to other coronaviruses, SARS-CoV-2 is an enveloped, positive sense, and single-stranded RNA virus with a genome of about 30,000 nucleotides (45). Four major structural proteins [spike (S), nucleocapsid (N), membrane (M), and envelope (E) proteins] are encoded by the genome. Noticeably, a helical ribonucleoprotein complex is formed when the genomic RNA binds with the N protein (46) ( Figure 2A ). Accumulating studies have demonstrated the presence of SARS-CoV-2 viral protein and nucleic acid substances in myocardial cells of COVID-19 patients, even in patients without clinical symptoms of cardiac involvement (47–52). This indicates that SARS-CoV-2 infection can occur in myocardial cells. SARS-CoV-2 can infect myocardial cells through direct and indirect pathways.

SARS-CoV-2 typically induces upper and lower respiratory tract infections, often accompanied by fever, cough, and loss of smell and taste. Most infections are mild, and up to 20-40% of patients are asymptomatic. However, severe and fatal conditions may also occur in some patients, such as systemic inflammation, tissue damage, acute respiratory distress syndrome, thromboembolic complications, cardiac injury, and/or cytokine storms (60) ( Figure 2B ). Based on current research advances, the mechanisms of myocardial injury in patients with SARS-CoV-2 can be summarized into the following 3 main points:

The innate immune response is the first barrier of the organism against invasion of exogenous microorganisms in the course of long-term evolution. Pattern recognition receptors (PRRs), an integral part of the host’s innate immune system, are essential for body resistance to pathogenic microbial infections. During viral entry and replication, the innate immune system can sense viral components. For example, once entering the cytoplasm, SARS-CoV-2 is thought to follow the same pathway as the other CoVs; the coronavirus genomic RNA is translated into two large polyproteins (pp1a and pp1ab), encoding 16 non-structural proteins (NSPs). These proteins promote the formation of viral replication-transcription complexes that can produce antisense negative strand templates from viral RNA (74). After that, NSP3 and NSP4 reorganize membranes originating from the endoplasmic reticulum (ER) and Golgi apparatus to generate double-membrane vesicles (DMV) that separate viral replication and transcription from host sensor detection (74–76). The S, E, and M proteins of the SARS-CoV-2 virus are exposed to host cell surface sensors during their binding, and host cytoplasmic sensors can detect viral proteins and nucleic acids before they are segregated by the NSP (76).

Innate immune cells [including macrophages, monocytes, dendritic cells (DCs), neutrophils, and innate lymphocytes (ILCs) such as natural killer (NK) cells] are equipped with a set of PRRs that can recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) to induce inflammatory signaling pathways and immune responses (77, 78). Toll-like receptors (TLRs) (79), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) (80), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) (81), C-type lectin receptors (82) and Absent in melanoma 2 (AIM2)-like receptors are five main PRR families. Signaling through these receptors in innate immune cells in response to pathogens, PAMP or DAMP, triggers the production of inflammatory cytokines and chemokines while inducing cell death to clear the infected cells (77, 78). To date, several PRRs, especially TLRs, have been shown to activate their signaling pathways in response to SARS-CoV-2-induced myocarditis.

SARS-CoV-2 infection triggers the complex and sophisticated response of the host’s innate immune system, in which multiple signaling pathways and molecules are involved in a synergistic manner ( Figure 3 ). In the human respiratory tract, TLRs are key receptors in the innate immune system that are extensively distributed throughout the respiratory tract. Different TLRs show heterogeneous expressions in innate immune cells. For example, TLR3 is abundant in NK cells, whereas TLR4 is common in macrophages (83). TLRs trigger inflammatory cytokine production by transducing signals mainly through two key adaptor molecules: myeloid differentiation primary response gene 88 (MyD88) and TIR domain-containing adaptor-inducing interferon-β (TRIF) (84, 85). TLR4 can specifically bind with MyD88 or TRIF and signaling to activate molecules [such as nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and interferon regulatory factor (IRF)], thereby transcriptionally activating various pro-inflammatory cytokines (including TNF, IL-6, and IL-1) (86).

TLR2 has been extensively documented to play roles in SARS-CoV-2 infection. For example, in macrophages of TLR2-deficient mice and TLR2 inhibitors-treated humans, SARS-CoV-2-mediated proinflammatory signaling pathways and cytokine production were attenuated after stimulation with SARS-CoV-2 E protein (87). Moreover, there is experimental evidence for the in vivo induction of TLR2-dependent inflammation by the SARS-CoV-2 E protein. However, further studies are still needed to determine whether TLR2 binds directly to E proteins or other SARS-CoV-2 ligands. Regarding TLR3, it has been shown that the TLR3 signaling pathway plays a protective role against SARS-CoV-2 in the organism (79, 88). Nevertheless, little is known about the precise role of TLR3 in SARS-CoV-2 infection. Although several genetic analyses have pointed out an association between congenital TLR3 defect and disease severity, further validation is needed. In a clinical study of myocarditis, TLR1-TLR10 mRNA can be detected in normal peripheral blood T cells, but only TLR2-TLR5 and TLR9 expression could be detected at the protein level. However, in patients with myocarditis, TLR1, TLR2, and TLR7 are overexpressed at the mRNA level (89). These findings imply that TLRs might be implicated in the pathogenesis of myocarditis. Additionally, the activation of TLRs can induce the production of inflammatory factors [including IFN-γ, IL-6, and tumor growth factor (TGF)-β], stimulate the differentiation of natural cells into Th1 cells, and then aggravate the immune response as well as the pathophysiological process of myocarditis. TLR overactivation, T-cell-mediated myocardial cytotoxicity, and cytokine overproduction may be important factors in SARS-CoV-2-induced myocarditis (90).

Recently, multiple episodes of myocarditis have been observed following vaccination with the mRNA SARS-CoV-2, especially in young men. The proposed explanations for this phenomenon involve mechanisms such as molecular mimicry, overreaction of the immune system to the mRNA, and dysregulation of the immune system (91, 92). Altogether, upon viral entry into the myocardium, the recognition of TLRs stimulates the activation of NF-kB and the release of pro-inflammatory cytokines (such as IL-1, IL-6, IL-12, TNF-α, and IFN-γ). The release of these cytokines further attracts immune cells to eradicate viral pathogens. However, sustained release of pro-inflammatory cytokines and immune cells may also cause myocardial cell damage and subsequent myocardial dysfunction (4). This cascade of pathophysiologic processes may work together in SARS-CoV-2-induced myocarditis. However, in-depth studies are still needed to explore the exact mechanisms.

Additionally, the IFN pathway is triggered once single-stranded RNA produced by viral infection is sensed by intracellular RLRs [such as RIG-I and melanoma differentiation-associated protein 5 (MDA5)]. IFN production and release stimulate the downstream signaling through the IFN receptor, inducing the production of various interferon-stimulated genes (ISGs) with antiviral functions. These ISGs [including lymphocyte antigen 6 family member E (Ly6E), members of the IFIT family, and bone marrow stromal cell antigen 2 (BST2)] can limit viral replication (93). However, the fine-tuning of a type I interferon (IFN-I) is critical as both over-activation and under-activation of IFN signaling can be deleterious to the host. Additionally, the cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) signaling pathway is activated when cytoplasmic DNA is detected, which is critical for limiting the replication of DNA and RNA viruses after infection. However, the SARS-CoV-2 accessory proteins (ORF3a and 3CL) can antagonize the cGAS-STING signaling and inhibit antiviral immune responses (94, 95). Overall, SARS-CoV-2 infection triggers the activation of several innate immune signaling pathways, including TLR, RLR, NLR, and cGAS-STING; these pathways exert synergistic effects, which is critical to the host resistance to viral infection ( Figure 3 ). However, more comprehensive studies are still needed to investigate the detailed mechanisms and inter-regulation of each signaling pathway in SARS-CoV-2-induced myocarditis.

RNA m5C methylation is an important form of RNA modification that has garnered significant interest since its first discovery in 1975 in the intracellular 26S RNA of Sindbis virus (96). It has been shown that m5C methylation is widely present in multiple viruses, such as murine leukemia virus (MLV), human immunodeficiency virus type 1 (HIV-1), and SARS-CoV. In MLV infections, NSUN2 can modulate m5C modification, which in turn affects gene expression and viral replication (97, 98). Similarly, in HIV-1 infection, DNMT2 and NSUN2 co-regulate RNA m5C modification, affecting multiple stages of viral replication (99–101). For EBV infection, NSUN2 catalyzes the m5C modification of EBER1, which impacts its metabolism and defense against the virus (102). Flaviviruses (103), hepatitis C virus (HCV) (104), Dengue Virus (DENV) (105), Zika Virus (ZIKV) (103) and others regulated by NSUN2; the methyl donor inhibitor A9145 has been found to inhibit the replication of DENV and ZIKV (105). It has been reported that the knockdown of NSUN2 does not significantly alter the m5C methylation of viral RNAs, but reduces the replication and gene expression of a variety of viruses (RSV, VSV, hMPV, SeV, and HSV) (22). In 2020, Kim et al. have selected SARS-CoV-2-infected Vero cells for their study; they uncovered the transcriptome and epitranscriptome profiles of SARS-CoV-2 for the first time using nanopore direct RNA sequencing and nanosphere DNA sequencing. However, further experiments are necessary to ascertain whether 6 (located on C atoms) of the 41 modification sites present on SARS-CoV-2 RNA include m5C modification (106). Anming Huang et al. have recently re–examined the m5C modification sites reported in HIV and MLV and analyzed the m5C methylome of SARS–CoV–2. With a rigorous bisulfite sequencing protocol and accurate data analytics though, no evidence of m5C has been found in these viruses (107). These results suggest that m5C methylation may indirectly affect SARS-CoV-2 infection.

In a recent study, Wang et al. have reported that SARS-CoV-2 infection significantly lowered NSUN2 mRNA levels in Caco-2 cells. Through transcriptome sequencing of RNA isolated from bronchoalveolar lavage fluid (BALF) of two SARS-CoV-2 patients, NSUN2 mRNA level is found decreased in SARS-CoV-2 patients compared with that in healthy individuals. In comparison to the uninfected group, the infected mice with the SARS-CoV-2 WT strain or the K18-hACE2 KI mouse model with BA.1 omicron variant-activated innate immune response show significantly lower endogenous NSUN2 mRNA levels (103). These results could be also observed in the SARS-CoV-2-infected Caco-2 cell model and SARS-CoV-2 patients, suggesting that NSUN2 plays an important regulatory role in SARS-CoV-2 infection. These findings not only offer important clues to the molecular mechanisms of m5C methylation in SARS-CoV-2 infection, but also highlight the ability of the virus to regulate gene expression in host cells. Future studies are expected to disclose the effects of these modifications on viral replication and host immune responses, opening up new avenues for the development of antiviral therapeutic strategies.

RNA m5C modification is crucial in the antiviral innate immune response of host cells (103) ( Figure 3 ). Kariko et al. have reported that m5C-modified RNA would eliminate the activity of signaling activated by TLR3, TLR7, and TLR8. Compared with unmodified RNA, m5C-modified RNA can inhibit the expression of cytokines and DC activation markers. This indicates that m5C modification may inhibit the potential of RNA in DC activation, and lead to a weakened immune response (108). Moreover, a previous study has shown that NSUN5-mediated m5C modification of GPX4 can activate the cGAS-STING signaling (109). NSUN2, as one of the most reported “writers” of m5C modifications, is crucial in maintaining tRNA structure and regulating oxidative stress. Notably, this modification decreases the m5C level of tRNAs and the production of short and noncoding RNAs (tRF-Gln-CTG-026), which can affect the cellular translational process (110). VtRNAs formed by m5C modification can regulate gene expression, which can also be utilized by viruses to suppress the antiviral immune response of host cells (12, 14). For instance, the influenza A virus can inhibit the activation of the antiviral protein PKR through high expression of VtRNA, thereby promoting viral replication. NSUN2 may also affect the viral genome and host immune response through the regulation of RNA metabolic processing (111, 112).

NSUN2 negatively regulates IFN-I responses during multiple viral infections, including SARS-CoV-2 infection (103). During infection by RNA viruses (RSV, VSV, hMPV, and SeV) and DNA viruses (HSV), NSUN2 alters m5C levels and either directly or indirectly regulates the RIG-I signaling pathway-mediated IFN-I response through downregulating levels of specific ncRNAs (particularly RPPH1 and 7SL RNAs), thereby enhancing the antiviral response (22). In addition, NSUN2 specifically mediates m5C methylation of IRF3 mRNA and speeds up its degradation, resulting in lower levels of IRF3 and downstream IFN-β (103). NSUN2 knockout or knockdown can enhance the expression of IFN-I and downstream ISGs during various viral infections in vitro (103). In vivo, the NSUN2+/- mice show more significantly enhanced antiviral innate response than NSUN2+/+ mice. The identified highly m5C-methylated cytosines exhibit higher IRF3 mRNA levels in cells. However, SeV, VSV, HSV-1, or ZIKV infections result in reduced endogenous NSUN2 levels. In particular, SARS-CoV-2 infection (including the WT strain and the BA.1 omicron variant) also reduces the endogenous levels of NSUN2 in SARS-CoV-2 patients and K18-hACE2 KI mice, which further upregulates the levels of IFN-1 and downstream ISGs (103). These findings emphasize the significance of NSUN2 in the regulation of the host antiviral immune response. Nevertheless, there have been no published investigations on m5C modifications and IFN upstream signaling pathways. Hence, future research is anticipated to provide insights into the specific effects of these m5C modifications on viral replication and host immune responses, thus laying a solid foundation for antiviral therapeutic strategies.