Molecular Framework for the Activation of RNA-dependent Protein Kinase

McKenna, Sean Andrew; Lindhout, Darrin A.; Kim, Insil; Liu, Corey W.; Gelev, Vladimir; Wagner, Gerhard; Puglisi, Joseph D.

doi:10.1074/jbc.m700301200

Cited by 59 publications

(91 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…With the experimentally determined hydrodynamic parameters, we were unable to fit the acquired scattering data to any other complex stoichiometry. These results are consistent with previous NMR experiments in which the same complex stoichiometry is observed McKenna et al 2007). It is important to stress that this study has been focused on a bound conformation of the PKR-TAR complex, and no comment can be made on whether this complex represents an active conformation.…”

Section: Discussionsupporting

confidence: 81%

“…S2). The model also suggests that the C-terminal end of the protein (dsRBM2) may be oriented such that the 80-residue interdomain linker and kinase domain are not directly affected by interaction with the RNA; similar observations have been made previously (McKenna et al 2007). The RNA sequesters the tandem dsRBMs entirely, which would preclude the RNA from interacting with another molecule of PKR or an interaction between the dsRBMs and kinase domains from a single PKR molecule.…”

Section: Discussionsupporting

confidence: 52%

“…The dsRNA binding and kinase domains are joined by a third region, an 80-amino-acid flexible interdomain linker implicated in PKR self-association (Lemaire et al 2005;VanOudenhove et al 2009). Previous studies have suggested significant flexibility in the two linkers (one between the dsRBMs, the other between the dsRBMs and kinase domains), allowing for two distinct conformations to be observed: an extended "open" conformation where dsRBMs and kinase domains are not in contact, and a collapsed "closed" conformation (Carpick et al 1997;Gabel et al 2006; Lemaire et al 2006;McKenna et al 2007;VanOudenhove et al 2009). Together, the data suggest a direct communication between domains that allows propagation of the activation signal from the dsRNA binding to the kinase domain via the interdomain linker.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Recognition of viral RNA stem–loops by the tandem double-stranded RNA binding domains of PKR

Dzananovic

Patel

Deo

et al. 2013

RNA

Self Cite

View full text Add to dashboard Cite

In humans, the double-stranded RNA (dsRNA)-activated protein kinase (PKR) is expressed in late stages of the innate immune response to viral infection by the interferon pathway. PKR consists of tandem dsRNA binding motifs (dsRBMs) connected via a flexible linker to a Ser/Thr kinase domain. Upon interaction with viral dsRNA, PKR is converted into a catalytically active enzyme capable of phosphorylating a number of target proteins that often results in host cell translational repression. A number of high-resolution structural studies involving individual dsRBMs from proteins other than PKR have highlighted the key features required for interaction with perfectly duplexed RNA substrates. However, viral dsRNA molecules are highly structured and often contain deviations from perfect A-form RNA helices. By use of small-angle X-ray scattering (SAXS), we present solution conformations of the tandem dsRBMs of PKR in complex with two imperfectly base-paired viral dsRNA stemloops; HIV-1 TAR and adenovirus VA I -AS. Both individual components and complexes were purified by size exclusion chromatography and characterized by dynamic light scattering at multiple concentrations to ensure monodispersity. SAXS ab initio solution conformations of the individual components and RNA-protein complexes were determined and highlight the potential of PKR to interact with both stem and loop regions of the RNA. Excellent agreement between experimental and model-based hydrodynamic parameter determination heightens our confidence in the obtained models. Taken together, these data support and provide a framework for the existing biochemical data regarding the tolerance of imperfectly base-paired viral dsRNA by PKR.

show abstract

Section: Discussionsupporting

confidence: 81%

Section: Discussionsupporting

confidence: 52%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Recognition of viral RNA stem–loops by the tandem double-stranded RNA binding domains of PKR

Dzananovic

Patel

Deo

et al. 2013

RNA

Self Cite

View full text Add to dashboard Cite

show abstract

“…RNA binding induces a conformational change that relieves the autoinhibition; however, the validity of this mechanism is still being debated (35). Others have recently reported that dimerization of PKR is required for signal transduction (36). Similarly, multimerization of RIG-I is likely to play an important role in activation of the signaling functions (18).…”

Section: Discussionmentioning

confidence: 99%

Essential Role of the N-terminal Domain in the Regulation of RIG-I ATPase Activity

Gee

Chua

Gevorkyan

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

Retinoic acid-inducible gene I (RIG-I) is a cytosolic receptor that recognizes viral RNA and activates the interferon-mediated innate antiviral response. To understand the mechanism of signal activation at the receptor level, we cloned, expressed, and purified human RIG-I containing the two caspase activation and recruitment domains (CARDs) followed by the C-terminal helicase domain. We found that recombinant RIG-I is a functional protein that interacts with double-stranded RNA with substantially higher affinity as compared with single-stranded RNA structures unless they contain a 5-triphosphate group. Viral RNA binding to RIG-I stimulates the velocity of ATP hydrolysis by 33-fold, which at the cellular level translates into a 43-fold increase of interferon-␤ expression. In contrast, the isolated ATPase/helicase domain is constitutively activated while also retaining its RNA ligand binding properties. These results support the recent model by which RIG-I signaling is autoinhibited in the absence of RNA by intra-molecular interactions between the CARDs and the C terminus. Based on pH profile and metal ion dependence experiments, we propose that the active site of RIG-I cannot efficiently accommodate divalent cations under the RNA-free repressed conformation. Overall, these results show a direct correlation between RNA binding and ATPase enzymatic function leading to signal transduction and suggest that a tight control of ATPase activity by the CARDs prevents RIG-I signaling in the absence of viral RNA.Pathogen recognition receptors constitute one of the first lines of human host cell defense against viral infections. Among them, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) helicases are specialized sensors of viral nucleic acid. RIG-I was shown to be involved in the recognition of Sendai virus, Hepatitis C virus (HCV), 2 and vesicular stomatitis virus, whereas MDA-5 mediates the antiviral response to picornaviruses (1-3). Both RIG-I and MDA-5 are modular proteins of ϳ920 residues that contain an ATPase/helicase domain for RNA binding and two caspase activation and recruitment domains (CARDs) located in tandem at the N-terminal end of the protein (4). The CARDs, by way of homotypic interactions, are required for the signaling function between RIG-I and the newly discovered interferon-␤ promoter stimulator-1 (IPS-1), also called Cardif, MAVS, and VISA (for review see Ref. 5). The latter serves as an intermediate to the cascade leading to the activation of NF-B and IRF-3/7, which results in the production of antiviral cytokines such as type-1 interferons (6 -9). The receptor function of RIG-I is non-redundant and therefore essential to coordinate the signaling cascade leading to antiviral response (4), as confirmed by knock-out studies (10). Notably, the Huh7.5 cell line of hepatocytes is particularly permissive to HCV and vesicular stomatitis virus infection as the result of a defective RIG-I protein bearing a single mutation in its first CARD (11); the antiviral sig...

show abstract

“…The PKR and RNase L genes are both key components of the host response to virus infection linked to the interferon response (31,58,68). Activation of PKR from an inactive monomeric molecule involves dimerization mediated by double-stranded RNA (dsRNA), followed by autophosphorylation (46,47), to produce the active molecule. One key activity of PKR is to phosphorylate and inactivate the translation factor eIF2␣ (57,79).…”

mentioning

confidence: 99%

Roles of Vaccinia Virus Genes E3L and K3L and Host Genes PKR and RNase L during Intratracheal Infection of C57BL/6 Mice

Rice

Turner

Embury

et al. 2011

J Virol

View full text Add to dashboard Cite

The importance of the 2-5 oligoadenylate synthetase (OAS)/RNase L and double-stranded RNA (dsRNA)-dependent protein kinase (PKR) pathways in host interferon induction resulting from virus infection in response to dsRNA has been well documented. In poxvirus infections, the interactions between the vaccinia virus (VV) genes E3L and K3L, which target RNase L and PKR, respectively, serve to prevent the induction of the dsRNA-dependent induced interferon response in cell culture. To determine the importance of these host genes in controlling VV infections, mouse single-gene knockouts of RNase L and PKR and double-knockout mice were studied following intratracheal infection with VV, VV⌬K3L, or VV⌬E3L. VV caused lethal disease in all mouse strains. The single-knockout animals were more susceptible than wild-type animals, while the RNase L ؊/؊ PKR ؊/؊ mice were the most susceptible. VV⌬E3L infections of wild-type mice were asymptomatic, demonstrating that E3L plays a critical role in controlling the host immune response. RNase L ؊/؊ mice showed no disease, whereas 20% of the PKR ؊/؊ mice succumbed at a dose of 10 8 PFU. Lethal disease was routinely observed in RNase L ؊/؊ PKR ؊/؊ mice inoculated with 10 8 PFU of VV⌬E3L, with a distinct pathology. VV⌬K3L infections exhibited no differences in virulence among any of the mouse constructs, suggesting that PKR is not the exclusive target of K3L. Surprisingly, VV⌬K3L did not disseminate to other tissues from the lung. Hence, the cause of death in this model is respiratory disease. These results also suggest that an unanticipated role of the K3L gene is to facilitate virus dissemination.Small-animal (mouse) models to study poxvirus pathogenesis have been employed for decades and have provided invaluable insight into host-virus interactions and mechanistic insight as to how individual viral genes influence the host response. Infection of mice by various routes continues to be productive for study of a number of orthopoxviruses, including vaccinia virus (VV), ectromelia virus, cowpox virus, and rabbitpox virus (23, 24, 44, 45, 48-50, 67, 75, 77). The general purpose of these infected mouse studies is to provide surrogate models for human smallpox virus infection and host response to evaluate antiviral drug efficacy and the effectiveness of various smallpox vaccines. Advantages of mouse models include their relatively low cost, small size of the animals, and the fact that there are many mouse mutant strains available for genes that control responses to infection.A number of routes for the initiation of poxvirus infections in mice have been studied, including intranasal (i.n.), intratracheal (i.t.), and intradermal (i.d.) (ear pinnae, footpad) routes and scarification. Other routes, such as the intracranial (i.c.) or intraperitoneal (i.p.) route, are rarely used today because they do not effectively reproduce "natural" infection conditions. Arguably, the most relevant routes of infection in terms of human disease are the i.n. and i.t. routes, because smallpox is transmitted via th...

show abstract

Molecular Framework for the Activation of RNA-dependent Protein Kinase

Cited by 59 publications

References 32 publications

Recognition of viral RNA stem–loops by the tandem double-stranded RNA binding domains of PKR

Recognition of viral RNA stem–loops by the tandem double-stranded RNA binding domains of PKR

Essential Role of the N-terminal Domain in the Regulation of RIG-I ATPase Activity

Roles of Vaccinia Virus Genes E3L and K3L and Host Genes PKR and RNase L during Intratracheal Infection of C57BL/6 Mice

Contact Info

Product

Resources

About