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Bhushan Lokesh Panigrahi R. Rashmi Bhat S. Amruta Dharmaiah Srisathiyanarayanan Mathur R. N. Murthy Handanahal S. Savithri 《PloS one》2010,5(3)
Groundnut bud necrosis virus (GBNV), a member of genus Tospovirus in the family Bunyaviridae, infects a large number of leguminosae and solanaceae plants in India. With a view to elucidate the function of nonstructural protein, NSs encoded by the small RNA genome (S RNA), the NSs protein of GBNV- tomato (Karnataka) [1] was over-expressed in E. coli and purified by Ni-NTA chromatography. The purified rNSs protein exhibited an RNA stimulated NTPase activity. Further, this activity was metal ion dependent and was inhibited by adenosine 5′ (β, γ imido) triphosphate, an ATP analog. The rNSs could also hydrolyze dATP. Interestingly, in addition to the NTPase and dATPase activities, the rNSs exhibited ATP independent 5′ RNA/DNA phosphatase activity that was completely inhibited by AMP. The 5′ α phosphate could be removed from ssDNA, ssRNA, dsDNA and dsRNA thus confirming that rNSs has a novel 5′ α phosphatase activity. K189A mutation in the Walker motif A (GxxxxGKT) resulted in complete loss of ATPase activity, but the 5′ phosphatase activity was unaffected. On the other hand, D159A mutation in the Walker motif B (DExx) resulted in partial loss of both the activities. These results demonstrate for the first time that NSs is a bifunctional enzyme, which could participate in viral movement, replication or in suppression of the host defense mechanism. 相似文献
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Xiaojun Li C. T. Ranjith-Kumar Monica T. Brooks S. Dharmaiah Andrew B. Herr Cheng Kao Pingwei Li 《The Journal of biological chemistry》2009,284(20):13881-13891
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.The innate immune response is the first line of defense against invading
pathogens; it is the ubiquitous system of defense against microbial infections
(1). Toll-like receptors
(TLRs)3 and RIG-I
(retinoic acid-inducible gene
1)-like receptors (RLRs) play key roles in innate immune response
toward viral infection
(2-5).
Toll-like receptors TLR3, TLR7, and TLR8 sense viral RNA released in the
endosome following phagocytosis of the pathogens
(6). RIG-I-like receptors RIG-I
and MDA5 detect viral RNA from replicating viruses in infected cells
(3,
7,
8). Stimulation of these
receptors leads to the induction of type I interferons (IFNs) and other
proinflammatory cytokines, conferring antiviral activity to the host cells and
activating the acquired immune responses
(4,
9).RIG-I discriminates between viral and host RNA through specific recognition
of the uncapped 5′-triphosphate of single-stranded RNA (5′ ppp
ssRNA) generated by viral RNA polymerases
(10,
11). In addition, RIG-I also
recognizes double-stranded RNA generated during RNA virus replication
(7,
12). Transfection of cells
with synthetic double-stranded RNA stimulates the activation of RIG-I
(13,
14). Synthetic dsRNA mimics,
such as polyinosinic-polycytidylic acid (poly(I·C)), can activate MDA5
when introduced into the cytoplasm of cells. Digestion of poly(I·C)
with RNase III transforms poly(I·C) from a ligand for MDA5 into a
ligand for RIG-I, suggesting that MDA5 recognizes long dsRNA, whereas RIG-I
recognizes short dsRNA (15).
Studies of RIG-I and MDA5 knock-out mice confirmed the essential roles of
these receptors in antiviral immune responses and demonstrated that they sense
different sets of RNA viruses
(12,
16).RIG-I and MDA5 contain two caspase recruiting domains (CARDs) at their N
termini, a DEX(D/H) box RNA helicase domain, and a C-terminal
regulatory or repressor domain (CTD). The helicase domain and the CTD are
responsible for viral RNA binding, whereas the CARDs are required for
signaling (3,
8). The current model of RIG-I
activation suggests that under resting conditions RIG-I is in a suppressed
conformation, and viral RNA binding triggers a conformation change that leads
to the exposure of the CARDs for the recruitment of the downstream protein
IPS-1 (also known as MAVS, Cardif, or VISA)
(14,
17). Limited proteolysis of
the RIG-I·dsRNA complex showed that RIG-I residues 792-925 of the CTD
are involved in dsRNA and 5′ ppp ssRNA binding
(14). The CTD of RIG-I
overlaps with the C terminus of the previously identified repressor domain
(18). The structures of RIG-I
and LGP2 (laboratory of genetics and
physiology 2) CTD in isolation have been determined by
x-ray crystallography and NMR spectroscopy
(14,
19,
20). A large, positively
charged surface on RIG-I recognizes the 5′ triphosphate group of viral
ssRNA (14,
19). RNA binding studies by
titrating RIG-I CTD with dsRNA and 5′ ppp ssRNA suggested that
overlapping sets of residues on this charged surface are involved in RNA
binding (14). Mutagenesis of
several positively charged residues on this surface either reduces or disrupts
RNA binding by RIG-I, and these mutations also affect the induction of
IFN-β in vivo
(14,
19). However, the exact nature
of how the RLRs recognize viral RNA and how RNA binding activates these
receptors remains to be established.LGP2 is a homolog of RIG-I and MDA5 that lacks the CARDs and thus has no
signaling capability (21,
22). The expression of LGP2 is
inducible by dsRNA or IFN treatment as well as virus infection
(21). Overexpression of LGP2
inhibits Sendai virus and Newcastle disease virus signaling
(21). When coexpressed with
RIG-I, LGP2 can inhibit RIG-I signaling through the interaction of its CTD
with the CARD and the helicase domain of RIG-I
(18). LGP2 could suppress
RIG-I signaling by three possible ways
(23): 1) binding RNA with high
affinity, thereby sequestering RNA ligands from RIG-I; 2) interacting directly
with RIG-I to block the assembly of the signaling complex; and 3) competing
with IKKi (IκB kinase ε) in the NF-κB signaling pathway for a
common binding site on IPS-1. To elucidate the structural basis of dsRNA
recognition by the RLRs, we have crystallized human LGP2 CTD (residues
541-678) bound to an 8-bp double-stranded RNA and determined the structure of
the complex at 2.0 Å resolution. The structure revealed that LGP2 CTD
binds to the termini of dsRNA. Mutagenesis and functional studies showed that
dsRNA binding is likely not required for the inhibition of RIG-I signaling by
LGP2. 相似文献
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