Transposable elements have been routinely used for genetic manipulation in lower organisms, including generating transgenic animals and insertional mutagenesis. In contrast, the usage of transposons in mice and other vertebrate systems is still limited due to the lack of an efficient transposition system. We have tested the ability of piggyBac (PB), a DNA transposon from the cabbage looper moth Trichoplusia ni, to transpose in mammalian systems. We show that PB elements carrying multiple genes can efficiently transpose in human and mouse cell lines and also in mice. PB permits the expression of the marker genes it carried. During germline transposition, PB could excise precisely from original insertion sites and transpose into the mouse genome at diverse locations, preferably transcription units. These data provide a first and critical step toward a highly efficient transposon system for a variety of genetic manipulations including transgenesis and insertional mutagenesis in mice and other vertebrates.

Summary Intracellular RIG-I-like receptors (RLRs, including RIG-I, MDA-5, and LGP-2) recognize viral RNAs as pathogen-associated molecular patterns (PAMPs) and initiate an antiviral immune response. To understand the molecular basis of this process, we determined the crystal structure of RIG-I in complex with double-stranded RNA. The dsRNA is sheathed within a network of protein domains that include a conserved “helicase” domain (regions HEL1 and HEL2), a specialized insertion domain (HEL2i), and a C-terminal regulatory domain (CTD). A V-shaped pincer connects HEL2 and the CTD by gripping an α-helical shaft that extends from HEL1. In this way, the pincer coordinates functions of all the domains and couples RNA binding with ATP hydrolysis. RIG-I falls within the Dicer-RIG-I clade of super family 2 of helicases and this structure reveals complex interplay between motor domains, accessory mechanical domains and RNA that has implications for understanding the nanomechanical function this protein family and other ATPases more broadly.

Defining the functional determinants for RNA surveillance by RIG-IThis study shows that HEL2i-domain mediated scanning allows RIG-I to sense the length of RNA targets, and a short RNA duplex that binds one RIG-I molecule can stimulate ATPase activity and interferon response.

Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein essential for efficient in vivo splicing of all group I and II introns and activation of mRNA translation. Catalysis of intron splicing by Mss116 is coupled to its ATPase activity. Knowledge of the kinetic pathway(s) and biochemical intermediates populated during RNA-stimulated Mss116 ATPase is fundamental for defining how Mss116 ATP utilization is linked to in vivo function. We therefore measured the rate and equilibrium constants underlying Mss116 ATP utilization and nucleotide-linked RNA binding. RNA accelerates the Mss116 steady-state ATPase ~7-fold by promoting rate-limiting ATP hydrolysis, such that Pi release becomes (partially) rate-limiting. RNA binding displays strong thermodynamic coupling to the chemical states of the Mss116-bound nucleotide such that Mss116 with bound ADP-Pi binds RNA more strongly than with bound ADP or in the absence of nucleotide. The predominant biochemical intermediate populated during in vivo steady-state cycling is the strong RNA binding, Mss116-ADP-Pi state. Strong RNA binding allows Mss116 to fulfill its biological role in stabilization of group II intron folding intermediates. ATPase cycling allows for transient population of the weak RNA binding, ADP state of Mss116 and linked dissociation from RNA, which is required for the final stages of intron folding. In cases where Mss116 functions as a helicase, the data collectively favor a model in which ATP hydrolysis promotes a weak-to-strong RNA binding transition that disrupts stable RNA duplexes. The subsequent strong-to-weak RNA binding transition associated with Pi release dissociates RNA-Mss116 complexes, regenerating free Mss116.

The type I interferon (IFN) signaling pathway restricts infection of many divergent families of RNA and DNA viruses by inducing hundreds of IFN-stimulated genes (ISGs), some of which have direct antiviral activity. We screened 813 short hairpin RNA (shRNA) constructs targeting 245 human ISGs using a flow cytometry approach to identify genes that modulated infection of West Nile virus (WNV) in IFN-β-treated human cells. Thirty ISGs with inhibitory effects against WNV were identified, including several novel genes that had antiviral activity against related and unrelated positive-strand RNA viruses. We also defined one ISG, activating signal cointegrator complex 3 (ASCC3), which functioned as a negative regulator of the host defense response. Silencing of ASCC3 resulted in upregulation of multiple antiviral ISGs, which correlated with inhibition of infection of several positive-strand RNA viruses. Reciprocally, ectopic expression of human ASCC3 or mouse Ascc3 resulted in downregulation of ISGs and increased viral infection. Mechanism-of-action and RNA sequencing studies revealed that ASCC3 functions to modulate ISG expression in an IRF-3- and IRF-7-dependent manner. Compared to prior ectopic ISG expression studies, our shRNA screen identified novel ISGs that restrict infection of WNV and other viruses and defined a new counterregulatory ISG, ASCC3, which tempers cell-intrinsic immunity.

Background: RIG-I is an essential innate immune receptor that detects viral RNAs in infected cells.Results: RIG-I uses distinct subdomains to recognize specific characteristics of viral RNAs.Conclusion: The 5′-triphosphate is critical for high affinity RIG-I/RNA interaction.Significance: Characterizing the RIG-I/RNA interface is essential for understanding early stages of immune response against RNA viruses.

Endometriosis is an estrogen-dependent inflammatory disorder among reproductive-aged women associated with pelvic pain, anxiety, and depression. Pain is characterized by central sensitization; however, it is not clear if endometriosis leads to increased pain perception or if women with the disease are more sensitive to pain, increasing the detection of endometriosis. Endometriosis was induced in mice and changes in behavior including pain perception, brain electrophysiology, and gene expression were characterized. Behavioral tests revealed that mice with endometriosis were more depressed, anxious and sensitive to pain compared to sham controls. Microarray analyses confirmed by qPCR identified differential gene expression in several regions of brain in mice with endometriosis. In these mice, genes such as Gpr88, Glra3 in insula, Chrnb4, Npas4 in the hippocampus, and Lcn2 in the amygdala were upregulated while Lct, Serpina3n (insula), and Nptx2 (amygdala) were downregulated. These genes are involved in anxiety, locomotion, and pain. Patch clamp recordings in the amygdala were altered in endometriosis mice demonstrating an effect of endometriosis on brain electrophysiology. Endometriosis induced pain sensitization, anxiety, and depression by modulating brain gene expression and electrophysiology; the effect of endometriosis on the brain may underlie pain sensitization and mood disorders reported in women with the disease.

The nonstructural protein 3 (NS3) helicase/protease is an important component of the hepatitis C virus (HCV) replication complex. We hypothesized that a specific ␤-strand tethers the C terminus of the helicase domain to the protease domain, thereby maintaining HCV NS3 in a compact conformation that differs from the extended conformations observed for other Flaviviridae NS3 enzymes. To test this hypothesis, we removed the ␤-strand and explored the structural and functional attributes of the truncated NS3 protein (NS3⌬C7). Limited proteolysis, hydrodynamic, and kinetic measurements indicate that NS3⌬C7 adopts an extended conformation that contrasts with the compact form of the wild-type (WT) protein. The extended conformation of NS3⌬C7 allows the protein to quickly form functional complexes with RNA unwinding substrates. We also show that the unwinding activity of NS3⌬C7 is independent of the substrate 3-overhang length, implying that a monomeric form of the protein promotes efficient unwinding. Our findings indicate that an open, extended conformation of NS3 is required for helicase activity and represents the biologically relevant conformation of the protein during viral replication.Nonstructural protein 3 (NS3) is an essential member of the hepatitis C virus (HCV) replication complex (2, 33). It is a bifunctional enzyme that contains a serine protease domain within the N-terminal third of the protein and a nucleic acidstimulated NTPase/helicase domain within the C-terminal twothirds of the protein (40). Both enzymatic activities are critical for HCV replication. In the presence of the viral NS4A cofactor protein, the NS3 protease activity cleaves and releases downstream viral proteins from the precursor polyprotein (13). Furthermore, the NS3 protease represses the host innate immune response by cleaving cellular proteins such as TRIF and MAVS, thereby preventing a signaling cascade that leads to an antiviral cellular response (17,24,32,44,51). In addition to its protease activity, NS3 displays robust helicase activity (46). NS3 helicase activity is essential for replication of the viral RNA genome (21), potentially functioning together with the NS5B polymerase during viral replication (16). NS3 also participates in the intracellular assembly and packaging of infectious virus particles (30). Given its multiple roles throughout the viral life cycle, NS3 is an important target for antiviral drug discovery against HCV (5).The NS3 helicase (NS3hel) domain belongs to the DExH/D subgroup of DNA and RNA helicases within helicase superfamily 2 (37, 52). Members of this family contain a core helicase structure consisting of two RecA-like folds (domains 1 and 2) arranged in tandem. Together with these two RecA-like domains, NS3hel has a third domain (domain 3) that forms a single-stranded DNA/RNA binding groove (Fig. 1A) (20, 29). The NS3hel construct has been studied extensively and displays modest helicase activity in isolation. However, a unique feature of NS3 that distinguishes it from its other family members is the ...