Products of the porcine group C rotavirus NSP3 gene bind specifically to double-stranded RNA and inhibit activation of the interferon-induced protein kinase PKR

Langland, Jeffrey; Pettiford, Sherrie M.; Jiang, Baoming; Jacobs, Bertram L.

doi:10.1128/jvi.68.6.3821-3829.1994

Cited by 86 publications

(32 citation statements)

References 43 publications

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“…A potential explanation for the absence of a VP3 CTD from some rotavirus species is that they evolved additional mechanisms to target similar innate antiviral pathways. For example, RVC strains, which lack a VP3 CTD, encode an 8-kDa dsRNA-binding protein that inhibits PKR activation (43). RVH viruses also encode a small protein with homology to dsRNA-binding proteins (44).…”

Section: Discussionmentioning

confidence: 99%

Structural Basis for 2′-5′-Oligoadenylate Binding and Enzyme Activity of a Viral RNase L Antagonist

Ogden

Jha

et al. 2015

J Virol

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Synthesis of 2=-5=-oligoadenylates (2-5A) by oligoadenylate synthetase (OAS) is an important innate cellular response that limits viral replication by activating the latent cellular RNase, RNase L, to degrade single-stranded RNA. Some rotaviruses and coronaviruses antagonize the OAS/RNase L pathway through the activity of an encoded 2H phosphoesterase domain that cleaves 2-5A. These viral 2H phosphoesterases are phylogenetically related to the cellular A kinase anchoring protein 7 (AKAP7) and share a core structure and an active site that contains two well-defined H⌽(S/T)⌽ (where ⌽ is a hydrophobic residue) motifs, but their mechanism of substrate binding is unknown. Here, we report the structures of a viral 2H phosphoesterase, the C-terminal domain (CTD) of the group A rotavirus (RVA) VP3 protein, both alone and in complex with 2-5A. The domain forms a compact fold, with a concave ␤-sheet that contains the catalytic cleft, but it lacks two ␣-helical regions and two ␤-strands observed in AKAP7 and other 2H phosphoesterases. The cocrystal structure shows significant conformational changes in the R loop upon ligand binding. Bioinformatics and biochemical analyses reveal that conserved residues and residues required for catalytic activity and substrate binding comprise the catalytic motifs and a region on one side of the binding cleft. We demonstrate that the VP3 CTD of group B rotavirus, but not that of group G, cleaves 2-5A. These findings suggest that the VP3 CTD is a streamlined version of a 2H phosphoesterase with a ligand-binding mechanism that is shared among 2H phosphodiesterases that cleave 2-5A. IMPORTANCEThe C-terminal domain (CTD) of rotavirus VP3 is a 2H phosphoesterase that cleaves 2=-5=-oligoadenylates (2-5A), potent activators of an important innate cellular antiviral pathway. 2H phosphoesterase superfamily proteins contain two conserved catalytic motifs and a proposed core structure. Here, we present structures of a viral 2H phosphoesterase, the rotavirus VP3 CTD, alone and in complex with its substrate, 2-5A. The domain lacks two ␣-helical regions and ␤-strands present in other 2H phosphoesterases. A loop of the protein undergoes significant structural changes upon substrate binding. Together with our bioinformatics and biochemical findings, the crystal structures suggest that the RVA VP3 CTD domain is a streamlined version of a cellular enzyme that shares a ligand-binding mechanism with other 2H phosphodiesterases that cleave 2-5A but differs from those of 2H phosphodiesterases that cleave other substrates. These findings may aid in the future design of antivirals targeting viral phosphodiesterases with cleavage specificity for 2-5A. O ne of the earliest steps in the battle between a virus and its host is the detection of pathogen-associated molecular patterns by cellular sensors. For RNA viruses, these patterns may be found in RNA products synthesized during viral transcription and replication. A number of cytoplasmic cellular molecules have been described that sense foreign RNA and resp...

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Section: Discussionmentioning

confidence: 99%

Structural Basis for 2′-5′-Oligoadenylate Binding and Enzyme Activity of a Viral RNase L Antagonist

Ogden

Jha

et al. 2015

J Virol

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“…Having determined that MHV fails to activate or interferes with both the PKR and 2Ј-5Ј OAS pathways, it was reasonable to hypothesize that MHV might express an innate immune response antagonist. To test this idea, we made use of an approach that was previously developed and used to identify genes from other viruses that counteract the antiviral effects of IFN (6,27,34). VV⌬E3L recombinant viruses with genes of interest recombined into the E3L locus in place of the IFN antagonist are generated.…”

Section: Protein Synthesis Is Not Significantly Inhibited In Mhvinfecmentioning

confidence: 99%

Mouse Hepatitis Coronavirus A59 Nucleocapsid Protein Is a Type I Interferon Antagonist

Ye¹,

Hauns

Langland³

et al. 2007

J Virol

104

130

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The recent emergence of several new coronaviruses, including the etiological cause of severe acute respiratory syndrome, has significantly increased the importance of understanding virus-host cell interactions of this virus family. We used mouse hepatitis virus (MHV) A59 as a model to gain insight into how coronaviruses affect the type I alpha/beta interferon (IFN) system. We demonstrate that MHV is resistant to type I IFN. Protein kinase R (PKR) and the alpha subunit of eukaryotic translation initiation factor are not phosphorylated in infected cells. The RNase L activity associated with 2,5-oligoadenylate synthetase is not activated or is blocked, since cellular RNA is not degraded. These results are consistent with lack of protein translation shutoff early following infection. We used a well-established recombinant vaccinia virus (VV)-based expression system that lacks the viral IFN antagonist E3L to screen viral genes for their ability to rescue the IFN sensitivity of the mutant. The nucleocapsid (N) gene rescued VV⌬E3L from IFN sensitivity. N gene expression prevents cellular RNA degradation and partially rescues the dramatic translation shutoff characteristic of the VV⌬E3L virus. However, it does not prevent PKR phosphorylation. The results indicate that the MHV N protein is a type I IFN antagonist that likely plays a role in circumventing the innate immune response.

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“…3 can rescue E3L deleted vaccinia virus replication and IFN sensitivity (252). The porcine group C rotavirus NSP3 produces a 45 kDa protein that is cleaved into 38 and 8 kDa polypeptides (253). NSP3 protein contains a single DRBD in the extreme carboxyl terminus, which is retained in the 8 kDa protein after cleaving (253).…”

Section: Viral Drbpsmentioning

confidence: 99%

“…The porcine group C rotavirus NSP3 produces a 45 kDa protein that is cleaved into 38 and 8 kDa polypeptides (253). NSP3 protein contains a single DRBD in the extreme carboxyl terminus, which is retained in the 8 kDa protein after cleaving (253). The full-length and cleaved 8 kDa proteins have both been reported to bind dsRNA activators to prevent the autophosphorylation of PKR and are able to rescue the replication of IFN-sensitive vaccinia viruses (253).…”

Section: Viral Drbpsmentioning

confidence: 99%

The dsRNA binding protein family: critical roles, diverse cellular functions

Saunders¹,

Barber²

2003

FASEB j.

321

269

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The dsRNA binding proteins (DRBPs) comprise a growing family of eukaryotic, prokaryotic, and viral-encoded products that share a common evolutionarily conserved motif specifically facilitating interaction with dsRNA. Proteins harboring dsRNA binding domains (DRBDs) have been reported to interact with as little as 11 bp of dsRNA, an event that is independent of nucleotide sequence arrangement. More than 20 DRBPs have been identified and reportedly function in a diverse range of critically important roles in the cell. Examples include the dsRNA-dependent protein kinase PKR that functions in dsRNA signaling and host defense against virus infection and DICER, which is implicated in RNA interference (RNAi) -mediated gene silencing. Other DRBPs such as Staufen, adenosine deaminase acting on RNA (ADAR), and spermatid perinuclear RNA binding protein (SPNR) are known to play essential roles in development, translation, RNA editing, and stability. In many cases, homozygous and even heterozygous disruption of DRBPs in animal models results in embryonic lethality. These results implicate the recognition of dsRNA as an evolutionarily conserved mechanism important in the regulation of gene expression and in host defense and underscore the diversity of essential biological tasks performed by dsRNA-related processes in the cell.

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Products of the porcine group C rotavirus NSP3 gene bind specifically to double-stranded RNA and inhibit activation of the interferon-induced protein kinase PKR

Cited by 86 publications

References 43 publications

Structural Basis for 2′-5′-Oligoadenylate Binding and Enzyme Activity of a Viral RNase L Antagonist

Structural Basis for 2′-5′-Oligoadenylate Binding and Enzyme Activity of a Viral RNase L Antagonist

Mouse Hepatitis Coronavirus A59 Nucleocapsid Protein Is a Type I Interferon Antagonist

The dsRNA binding protein family: critical roles, diverse cellular functions

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