Fig 1: Interplay between cellular responses to viral and host dsRNAs, the OAS-RNase L pathway, and antagonism by 2-5A-degrading enzymes. (A) OAS1–3 are IFN-induced dsRNA sensors. Once activated, they synthesize the antiviral substance 2′,5′-oligoadenylate (2-5A) from ATP. 2-5A binds inactive monomeric RNase L, inducing active RNase L dimers, which in turn degrade viral and host single-stranded RNAs. The balance between 2-5A accumulation by OAS enzymes and its degradation by host and viral enzymes determines cell and virus fate and inflammatory responses. (B) Domain structures of viral and cellular 2′,5′-PEs and human PDE12 (an endonuclease/exonuclease/phosphatase [EEP] family member). Features of full-length MERS-CoV NS4b, MHV NS2, RVA SA11 VP3, and Mus musculus AKAP7γ proteins, including a nuclear localization sequence (NLS) and catalytic 2′,5′-PE domains, are compared (modified from reference 24). Positions of conserved histidines within the catalytic domain of 2′,5′-PEs are shown. PKA-RII-α-BD, binding domain for regulatory subunit II (RII) of cAMP-dependent protein kinase A (27). Guanylyltransferase (Gtase) and methyltransferase (Mtase) domains are also shown (25, 29). The mitochondrial-matrix-targeting peptide (MTP) and the catalytic EEP domain of PDE12 are shown (55). Domains shown are not drawn to scale.
Fig 2: Mechanism of 2′-5′-p3A3 degradation by the 2′,5′-PE (subfamily of the 2H-PE superfamily) and EEP (endonuclease/exonuclease/phosphatase) families. MERS-CoV NS4b, MHV-CoV NS2, RVA VP3, and mammalian mouse AKAP7 from the 2′,5′-PE subfamily cleave 2′,5′-p3A3 and leave 2′,3′ >p groups on the 5′ products, while human PDE12, an EEP family member, degrades 2′,5′-p3A3 to yield ATP and AMP.
Fig 3: Specific cleavage of trimer 2-5A (2′,5′-p3A3) by viral and cellular 2′,5′-PEs. (A to F) HPLC analysis of intact 2′,5′-p3A3 (A), followed by its cleavage with either the viral 2′,5′-PE MERS-NS4b (B), MHV NS2 (C), or RVA VP3-CTD (D) or the host 2′,5′-PE muAKAP7 (E) or human PDE12 (F). Purified 2′,5′-p3A3 (200 μM) was incubated with 1 μM the indicated proteins at 30°C for 1 h. (G to K) HPLC analysis of catalytically inactive mutants of these enzymes incubated with 2′,5′-p3A3 under similar conditions for MERS-NS4bH182R (G), MHV NS2H126R (H), RVA VP3-CTDH718A (I), muAKAP7H93A;H185R (J), and human PDE12E351A (K). Experiments performed at least three times produced similar 2′,5′-p3A3 degradation patterns for each 2′,5′-PE. Arrows indicate elution times of the standards ATP, AMP, and adenosine (Ado). Peaks shown in gray were determined from experiments done in Fig. 5 and 6. AU, arbitrary units.
Fig 4: Specific cleavage of trimer 2-5A (2′,5′-p3A3) by viral and cellular 2′,5′-PEs. (A to F) HPLC analysis of intact 2′,5′-p3A3 (A), followed by its cleavage with either the viral 2′,5′-PE MERS-NS4b (B), MHV NS2 (C), or RVA VP3-CTD (D) or the host 2′,5′-PE muAKAP7 (E) or human PDE12 (F). Purified 2′,5′-p3A3 (200 μM) was incubated with 1 μM the indicated proteins at 30°C for 1 h. (G to K) HPLC analysis of catalytically inactive mutants of these enzymes incubated with 2′,5′-p3A3 under similar conditions for MERS-NS4bH182R (G), MHV NS2H126R (H), RVA VP3-CTDH718A (I), muAKAP7H93A;H185R (J), and human PDE12E351A (K). Experiments performed at least three times produced similar 2′,5′-p3A3 degradation patterns for each 2′,5′-PE. Arrows indicate elution times of the standards ATP, AMP, and adenosine (Ado). Peaks shown in gray were determined from experiments done in Fig. 5 and 6. AU, arbitrary units.
Fig 5: Interplay between cellular responses to viral and host dsRNAs, the OAS-RNase L pathway, and antagonism by 2-5A-degrading enzymes. (A) OAS1–3 are IFN-induced dsRNA sensors. Once activated, they synthesize the antiviral substance 2′,5′-oligoadenylate (2-5A) from ATP. 2-5A binds inactive monomeric RNase L, inducing active RNase L dimers, which in turn degrade viral and host single-stranded RNAs. The balance between 2-5A accumulation by OAS enzymes and its degradation by host and viral enzymes determines cell and virus fate and inflammatory responses. (B) Domain structures of viral and cellular 2′,5′-PEs and human PDE12 (an endonuclease/exonuclease/phosphatase [EEP] family member). Features of full-length MERS-CoV NS4b, MHV NS2, RVA SA11 VP3, and Mus musculus AKAP7γ proteins, including a nuclear localization sequence (NLS) and catalytic 2′,5′-PE domains, are compared (modified from reference 24). Positions of conserved histidines within the catalytic domain of 2′,5′-PEs are shown. PKA-RII-α-BD, binding domain for regulatory subunit II (RII) of cAMP-dependent protein kinase A (27). Guanylyltransferase (Gtase) and methyltransferase (Mtase) domains are also shown (25, 29). The mitochondrial-matrix-targeting peptide (MTP) and the catalytic EEP domain of PDE12 are shown (55). Domains shown are not drawn to scale.
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