Circular RNAs as emerging regulators in COVID-19 pathogenesis and progression

Abstract

Coronavirus disease 2019 (COVID-19), an infectious acute respiratory disease caused by a newly emerging RNA virus, is a still-growing pandemic that has caused more than 6 million deaths globally and has seriously threatened the lives and health of people across the world. Currently, several drugs have been used in the clinical treatment of COVID-19, such as small molecules, neutralizing antibodies, and monoclonal antibodies. In addition, several vaccines have been used to prevent the spread of the pandemic, such as adenovirus vector vaccines, inactivated vaccines, recombinant subunit vaccines, and nucleic acid vaccines. However, the efficacy of vaccines and the onset of adverse reactions vary among individuals. Accumulating evidence has demonstrated that circular RNAs (circRNAs) are crucial regulators of viral infections and antiviral immune responses and are heavily involved in COVID-19 pathologies. During novel coronavirus infection, circRNAs not only directly affect the transcription process and interfere with viral replication but also indirectly regulate biological processes, including virus-host receptor binding and the immune response. Consequently, understanding the expression and function of circRNAs during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection will provide novel insights into the development of circRNA-based methods. In this review, we summarize recent progress on the roles and underlying mechanisms of circRNAs that regulate the inflammatory response, viral replication, immune evasion, and cytokines induced by SARS-CoV-2 infection, and thus highlighting the diagnostic and therapeutic challenges in the treatment of COVID-19 and future research directions.

Keywords: COVID-19; biological regulator; circRNAs; inflammatory response; vaccine.

Figure 1

Cell entry mechanism and life cycle of SARS-CoV-2. SARS-CoV-2 virions consist of structural proteins, including spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. When in contact with host cells, the S protein of SARS-CoV-2 specifically interacts with cellular receptors [such as angiotensin-converting enzyme 2 (ACE2)] and host factors [such as the cell surface serine protease TMPRSS2 and major endocytosis regulator AP2-related protein kinase 1 (AAK1)] to promote viral uptake and fusion at the cellular or endosomal membranes (37, 38). Following entry, viral genomic RNA is released into the cytoplasm and translated into polypeptides, which are subsequently hydrolysed and cotranslationally cleaved by proteases to generate nonstructural proteins (nsps). nsps further form RNA-dependent RNA polymerase (RdRP) complexes in the endoplasmic reticulum. Subsequently, the RdRP complex is involved in the transcription and RNA replication of the - sense subgenome and the + sense subgenome. Translation of the −sense and +sense subgenomes further enables the synthesis of structural and accessory proteins at the endoplasmic reticulum membrane. At the same time, the nucleocapsid buds into an ER-Golgi intermediate compartment (ERGIC) filled with S, E, and M proteins. Finally, virions are secreted from infected cells via exocytosis. As a result, MHC class I contributes towards antiviral immunity by facilitating the presentation of viral antigens to CD8 cytotoxic T cells. Moreover, the ability of antigen-presenting cells to capture external antigens and present them as peptide fragments, loaded on MHC class II molecules, which can transduce signals required for B-cell activation, to CD4+ T cells is a crucial part of the adaptive immune response (–47). In addition, the RNA released by the virus is captured and recognized by the pattern recognition receptor Toll-like receptor (TLR) located on the endosomal membrane. Subsequently, TLR activates and induces further self-immunity of cells, resulting in the formation of protein complexes, the migration of transcription factors (TFs) to the nucleus, and the expression of proinflammatory cytokines (48, 49).

Figure 2

CircRNAs function as miRNA sponges to influence viral replication. (A) A quintuple ceRNA network exists in SARS-CoV-1 infection that includes one miRNA (MMU-miR-124-3p), one lncRNA (Gm26917), one TF (Stat2), one mRNA (Ddx58) and two circRNAs (Ppp1r10, C330019G07Rik). They form a closed 3-node miRNA feed-forward loop and a 4-node ceRNA network, respectively. Upregulation of Ddx58 leads to reprogramming of miRNA splicing events, resulting in downregulation of miRNA expression. Meanwhile, the helicase domain of the RIG-I/Ddx58 receptor can interact with SARS-CoV-1 nonstructural protein 13 (NSP13) to initiate the viral life cycle. Furthermore, Ppp1r10 and C330019G07Rik act as sponges for miR-124-3p, inhibiting miR-124-3p expression, which in turn impedes Ddx58 degradation and further inhibits SARS-CoV-1 replication (65). (B) circ_0067985 derived from the FNDC3B gene and circ_0006275 derived from the CNOT1 gene serve as miR-127 and miR-2392 sponges, respectively, to regulate the downstream expression of MAP3K9, MYO15B, SPOCK1, MEF2C, USP15 and ZBTB11. Of these, the upstream regulator of the MAPK pathway, MAP3K9, further regulates the downstream ERK/MAPK pathway to inhibit MERS-CoV replication (18).

Figure 3

Immune response involving circRNAs in SARS-CoV-2 infection and the potential mechanism of circRNAs in inflammation. During virus infection, nuclear factor 90 (NF90) and its 110 (NF110) isoform produced by interleukin-enhanced binding factor 3 (ILF3) bind to viral mRNA to inhibit virus replication through two pathways: transport from the nucleus to the cytoplasm and decoupling from the circRNA-protein complex (CircRNPs) in the cytoplasm. Among them, the transport of NF90/NF110 from the nucleus to the cytoplasm can reduce the expression of circRNAs. In contrast, the binding of NF90/NF110 to dsRNA formed during pre-mRNA processing can not only stabilize the RNA duplex but also promote reverse splicing to form circRNA (57). 2’-5’ Oligoadenylate (2’-5’A) is generated by the combination of 2’-5’ oligoadenylate synthase (OAS) and viral genome dsRNA and plays an antiviral effect by significantly increasing the activity of RNase L to degrade viral RNA and interfere with viral protein synthesis. Endogenous circRNAs often form incomplete RNA duplexes and act as inhibitors of PKR activation by dsRNAs associated with innate immunity. Meanwhile, circRNAs can be globally degraded by the endonuclease RNase L to activate the PKR antiviral pathway (77). In addition, parental genes enriched for differentially expressed circRNAs in signalling pathways that are significantly regulated upon SARS-CoV-2 infection are associated with multiple antiviral signalling pathways (16, 74, 78). The generation of a cytokine storm is related to the overproduction of proinflammatory cytokines mediated by circRNAs. In macrophages, circ_09505 acts as a sponge of miR-6089 through the IκBα/NFκB signalling pathway, on the one hand, promoting the expression of AKT1 in macrophages and the activation of NF-κB, and on the other hand, promoting the production of the proinflammatory cytokines TNF-α, IL-6 and IL-12 (79). Furthermore, circ_0044073 in HUVSMCs and HUVECs functions as a miR-107 sponge to downregulate the expression levels of target mRNAs, while activation of the JAK/STAT signalling pathway enhanced the expression of the downstream proteins Bcl2 and c-myc. Moreover, circ_0044073 significantly upregulated the levels of the proinflammatory cytokines IL-1β, IL-6 and TNF-α (80). In addition, the occurrence of a cytokine storm disrupts the balance of proinflammatory and anti-inflammatory mechanisms, thereby invading the patient’s nervous system (81, 82).

Figure 4

CircRNAs regulate immune evasion of SARS-CoV-2. During SARS-CoV-2 proliferation, genomic RNAs are recognized by TLR receptors and pattern recognition receptors (RLRs) and subsequently activate immune responses. The proviral action of the immune factor ISG15 leads to the generation of an autocrine loop, which amplifies and prolongs autocrine signalling and ultimately induces viral drug resistance (89, 90). Furthermore, the S protein of SARS-CoV-2 can prevent the virus from being cleared by immune factors, and mutation of the S protein will further enhance the ability of the virus to evade immune responses (91, 92). In addition, the expression of viral nsp14 can upregulate the levels of circRNAs related to innate immunity, thereby inhibiting viral replication and immune evasion (93). Likewise, viral circRNAs may be involved in the mechanism of genome recombination, resulting in mutation of virions leading to immune evasion. There are various types of gene fusions in the circRNA genome of SARS-CoV-2, which may cause the virus to mutate and evade immunity.

Figure 5

Potential application of circRNAs in the treatment of COVID-19. CircRNAs are a new class of regulatory factors that mediate host–virus interactions. The identification and isolation of exosome-associated circRNAs, virus-encoded circRNAs, and significantly DE circRNAs after SARS-CoV-2 infection may be helpful in the diagnosis of COVID-19. In addition, circRNAs may serve as potential therapeutic targets against COVID-19 by indirectly regulating the expression of host receptors, such as ACE2 and AXL, that bind to SARS-CoV-2; HIF-1α and other signalling pathways related to the immune response; and multiple signalling pathways related to SARS-CoV-2 replication. Additionally, vaccines based on circRNAs and antisense circRNAs have shown initial effectiveness in preventing and inhibiting SARS-CoV-2. The coloured arrows in the figure are only for the convenience of differentiation and have no special meaning.