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Structures and Functions of Viral 5' Non-Coding Genomic RNA Domain-I in Group-B Enterovirus Infections - PubMed

  • ️Wed Jan 01 2020

Review

Structures and Functions of Viral 5' Non-Coding Genomic RNA Domain-I in Group-B Enterovirus Infections

Marie Glenet et al. Viruses. 2020.

Abstract

Group-B enteroviruses (EV-B) are ubiquitous naked single-stranded positive RNA viral pathogens that are responsible for common acute or persistent human infections. Their genome is composed in the 5' end by a non-coding region, which is crucial for the initiation of the viral replication and translation processes. RNA domain-I secondary structures can interact with viral or cellular proteins to form viral ribonucleoprotein (RNP) complexes regulating viral genomic replication, whereas RNA domains-II to -VII (internal ribosome entry site, IRES) are known to interact with cellular ribosomal subunits to initiate the viral translation process. Natural 5' terminally deleted viral forms lacking some genomic RNA domain-I secondary structures have been described in EV-B induced murine or human infections. Recent in vitro studies have evidenced that the loss of some viral RNP complexes in the RNA domain-I can modulate the viral replication and infectivity levels in EV-B infections. Moreover, the disruption of secondary structures of RNA domain-I could impair viral RNA sensing by RIG-I (Retinoic acid inducible gene I) or MDA5 (melanoma differentiation-associated protein 5) receptors, a way to overcome antiviral innate immune response. Overall, natural 5' terminally deleted viral genomes resulting in the loss of various structures in the RNA domain-I could be major key players of host-cell interactions driving the development of acute or persistent EV-B infections.

Keywords: 5′ terminally deleted viral forms; RNA domain-I; antiviral innate immune response; enterovirus replication; group-B enterovirus; type I interferon; viral ribonucleoprotein complexes.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1

Evolutionary relationship of human enterovirus species. Molecular phylogenetic analysis of human enteroviruses was inferred using the Neighbor-Joining method (Letunic and Bork, 2019). Phylogenetic tree was constructed using complete sequence of enteroviruses aligned by muscle method. The optimal tree with the sum of branch length = 18.13314604 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method and are in the units of the number of base substitutions per site. This analysis involved 128 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There was a total of 8522 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018). EV-A: Enterovirus A; EV-B: Enterovirus B; EV-C: Enterovirus C; EV-D: Enterovirus D; RV-A: Rhinovirus A; RV-B: Rhinovirus B; RV-C: Rhinovirus C.

Figure 2
Figure 2

Group-B enterovirus genome organization. (A) Enterovirus-B viral genome is composed of two open reading frames (ORFs) which one is a polyprotein coding for capsidic (viral proteins 1 to 4) and non-structural viral proteins such as proteases 2A, 3C, and the 3D polymerase. This long ORF is flanked by two untranslated regions (UTRs): the 5′ UTR is composed of a Cloverleaf-like secondary structure (CL, stem-loop I) responsible for the viral genomic replication separated by a spacer sequence comprising two C-rich clusters from an Internal Ribosome Entry Site element type 1 (IRES, stem-loops II to VII) which has a role in the viral translation. Nucleotide numbers are indicated below the structures. (B,C) Schematic representation of the predicted 5′ end Cloverleaf secondary structures (CL, stem-loop I) of the enteroviral positive strand RNA. The CL is composed of a stem “a” (nucleotides 2–9 with nucleotides 80–87), stem-loop “b” (nucleotides 10–34), stem-loop “c” (nucleotides 35–45), and a stem-loop “d” (nucleotides 46–79).

Figure 3
Figure 3

Viral ribonucleoprotein complexes involved in group-B enterovirus genomic replication process. (A) The enterovirus genome is single-stranded positive RNA with a 5′ non-coding region (5′ NCR) including a Cloverleaf-like structure (RNA domain-I). The genomic RNA is linked to a viral protein genome-linked (VPg) at its 5′-terminus, which acts as a primer during viral RNA synthesis. (B) Host cellular protein Poly(A) binding protein (PABP) interacts with the poly(A) tail in the viral genome 3′ end; Poly(C) Binding Protein (PCBP) 1/2 binds to the stem-loop “b” of the Cloverleaf and PCBP 2 binds to the spacer 1. Viral precursor protein 3CD interacts with stem-loop “d” of the Cloverleaf. (C) Through protein–protein interactions between PCBP 1/2 and PABP, the 5′ ends interact to form a ribonucleoprotein complex (RNP). The circularization of the viral RNA initiates the negative-strand synthesis. (D) The viral 3D polymerase synthesizes the negative strand. (E) The product of the negative-strand synthesis is a double-strand RNA complex called replication form. (F) Viral double-strand RNA complex unwinds at both ends, which enables the two Cloverleaf-like structures to interact with cellular and viral proteins. Nuclear protein, heterogeneous nuclear RNP-C (hnRNP-C), can interact with both ends of the negative-strand. Interactions of viral double-strand RNA complex with cellular and viral proteins allow maintaining a single-stranded structure at both ends of the replication form. (G) hnRNP-C could promote negative-strand circularization by oligomerization. (H) The circularization allows the initiation of the positive-strand synthesis. The 3D polymerase recruitment allows the start of positive-strand synthesis using VPg as primer. This process results in the formation of neo-synthetized positive- strands RNA and viral double-strand RNA complexes.

Figure 4
Figure 4

Natural 5′ terminal deletions in RNA domain-I disrupt viral ribonucleoprotein complexes involved in group-B enterovirus replication activities. (A) Schematic representation of the complete predicted 5′ end Cloverleaf secondary structures (CL, stem-loop I) of the enteroviral positive-strand RNA. The CL is composed of a stem “a” (nucleotides 2–9 with nucleotides 80–87), stem-loop “b” (nucleotides 10–34), stem-loop “c” (nucleotides 35–45), and a stem-loop “d” (nucleotides 46–79). (B) Host cellular protein PABP interacts with the poly(A) tail in the viral genome 3′ end, PCBP 1/2 bind to the stem-loop “b” of the Cloverleaf and in addition, PCBP 2 binds to the spacer 1 at the end of the Cloverleaf. Viral precursor protein 3CD interacts with stem-loop “d” of the Cloverleaf. (C) Schematic representation of the 5′ terminally deleted Enterovirus RNA with 50 nucleotides deletions in Cloverleaf secondary structures. (D) 5′ terminal deletions in RNA domain-I disrupts the formation of viral ribonucleoprotein complexes: PABP interacts with the 3′ end poly(A) tail, PCBP 2 binds to the spacer 1 at the end of the Cloverleaf, and the precursor 3CD interacts with stem-loop “d” of the Cloverleaf. This loss of interactions with viral protease and cellular factor in stem-loop “b” alters EV-B replication activities.

Figure 5
Figure 5

Group-B enterovirus proteinase activities impair type 1 signaling pathway activation in infected cells. Enterovirus proteinase 3C (3Cpro) and 2Apro are mainly involved in downregulation of type I IFN, pro-inflammatory cytokines at different stages. The interacting cellular signaling molecules with different viral proteins are indicated at each level. (MDA5: melanoma-differentiation-associated protein 5, RIG-I: retinoic acid-inducible gene 1, MAVS: mitochondrial antiviral-signaling protein, TBK1: TANK-binding kinase 1, IRF3/7: Interferon Regulatory Factor 3/7, TRIF: TIR-domain-containing adapter-inducing interferon-β, TLR: toll-like receptors, TRAF6: TNF receptor-associated factor 6, TAK1: transforming growth factor-β activated kinase 1, TAB2/3: TGF-β Activated Kinase 2/3, IFNs: interferons).

Figure 6
Figure 6

Natural 5′ terminally deleted group-B enterovirus RNA forms can modulate type I interferon signaling pathway activation. (A) Full-length viral RNA is recognized by cytoplasmic sensors RIG-I or MDA5 in EV-B infections. Signaling through the adaptor protein MAVS leads to IRF3 activation and translocation to the nucleus. These molecules stimulate high level of IFN-α/β and ISG56 production for the development of effective antiviral responses to EV-B infections. (B) According to recent reports, deleted viral genomes were associated with parental complete virus in early phase group-B enterovirus infection. Host cell proteins recruited in viral RNP complexes or potentially acting as a restriction factors could provide an evolutionary advantage to the 5′ NCR truncated viral RNA forms. Deletion in RNA domain-I (5′ terminally deleted viral genomes) could impair the viral genomic RNA recognition by RLRs (RIG-I or MDA5) immune sensors during the early phase of antiviral innate immune response resulting in low level of IFN-α/β and ISG56 production. IFN: interferon; RIG-I: retinoic acid-inducible gene-I; MDA5: melanoma differentiation-associated protein 5; MAVS: mitochondria antiviral-signaling protein; IRF: Interferon Regulatory Factor 3; ISG56: interferon stimulated gene 56; 5′ NCR: 5′ non-coding region.

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