5 research outputs found
Recombinant viral RdRps can initiate RNA synthesis from circular templates
The crystal structure of the recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) revealed extensive interactions between the fingers and the thumb subdomains, resulting in a closed conformation with an established template channel that should specifically accept single-stranded templates. We made circularized RNA templates and found that they were efficiently used by the HCV RdRp to synthesize product RNAs that are significantly longer than the template, suggesting that RdRp could exist in an open conformation prior to template binding. RNA synthesis using circular RNA templates had properties similar to those previously documented for linear RNA, including a need for higher GTP concentration for initiation, usage of GTP analogs, sensitivity to salt, and involvement of active-site residues for product formation. Some products were resistant to challenge with the template competitor heparin, indicating that the elongation complexes remain bound to template and are competent for RNA synthesis. Other products were not elongated in the presence of heparin, indicating that the elongation complex was terminated. Lastly, recombinant RdRps from two other flaviviruses and from the Pseudomonas phage φ6 also could use circular RNA templates for RNA-dependent RNA synthesis, although the φ6 RdRp could only use circular RNAs made from the 3′-terminal sequence of the φ6 genome
The RIG-I-like Receptor LGP2 Recognizes the Termini of Double-stranded RNA*S⃞
The RIG-I-like receptors (RLRs), RIG-I and MDA5, recognize single-stranded
RNA with 5′ triphosphates and double-stranded RNA (dsRNA) to initiate
innate antiviral immune responses. LGP2, a homolog of RIG-I and MDA5 that
lacks signaling capability, regulates the signaling of the RLRs. To establish
the structural basis of dsRNA recognition by the RLRs, we have determined the
2.0-Å resolution crystal structure of human LGP2 C-terminal domain bound
to an 8-bp dsRNA. Two LGP2 C-terminal domain molecules bind to the termini of
dsRNA with minimal contacts between the protein molecules. Gel filtration
chromatography and analytical ultracentrifugation demonstrated that LGP2 binds
blunt-ended dsRNA of different lengths, forming complexes with 2:1
stoichiometry. dsRNA with protruding termini bind LGP2 and RIG-I weakly and do
not stimulate the activation of RIG-I efficiently in cells. Surprisingly,
full-length LGP2 containing mutations that abolish dsRNA binding retained the
ability to inhibit RIG-I signaling
Structure and Function of LGP2, a DEX(D/H) Helicase That Regulates the Innate Immunity Response*S⃞
RNA recognition receptors are important for detection of and response to
viral infections. RIG-I and MDA5 are cytoplasmic DEX(D/H) helicase
proteins that can induce signaling in response to RNA ligands, including those
from viral infections. LGP2, a homolog of RIG-I and MDA5 without the caspase
recruitment domain required for signaling, plays an important role in
modulating signaling by MDA5 and RIG-I, presumably through heterocomplex
formation and/or by serving as a sink for RNAs. Here we demonstrate that LGP2
can be coexpressed with RIG-I to inhibit activation of the NF-κB
reporter expression and that LGP2 protein produced in insect cells can bind
both single- and double-stranded RNA (dsRNA), with higher affinity and
cooperativity for dsRNA. Electron microscopy and image reconstruction were
used to determine the shape of the LGP2 monomer in the absence of dsRNA and of
the dimer complexed to a 27-bp dsRNA. LGP2 has striking structural similarity
to the helicase domain of the superfamily 2 DNA helicase, Hef
Identification of Arabidopsis rat Mutants
Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various “reverse genetic” approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants