The Intracellular DNA Sensor IFI16 Gene Acts as Restriction Factor for Human Cytomegalovirus Replication

Human interferon (IFN)-inducible IFI16 protein, an innate immune sensor of intracellular DNA, modulates various cell functions, however, its role in regulating virus growth remains unresolved. Here, we adopt two approaches to investigate whether IFI16 exerts pro- and/or anti-viral actions. First, the IFI16 gene was silenced using specific small interfering RNAs (siRNA) in human embryo lung fibroblasts (HELF) and replication of DNA and RNA viruses evaluated. IFI16-knockdown resulted in enhanced replication of Herpesviruses, in particular, Human Cytomegalovirus (HCMV). Consistent with this, HELF transduction with a dominant negative form of IFI16 lacking the PYRIN domain (PYD) enhanced the replication of HCMV. Second, HCMV replication was compared between HELFs overexpressing either the IFI16 gene or the LacZ gene. IFI16 overexpression decreased both virus yield and viral DNA copy number. Early and late, but not immediate-early, mRNAs and proteins were strongly down-regulated, thus IFI16 may exert its antiviral effect by impairing viral DNA synthesis. Constructs with the luciferase reporter gene driven by deleted or site-specific mutated forms of the HCMV DNA polymerase (UL54) promoter demonstrated that the inverted repeat element 1 (IR-1), located between −54 and −43 relative to the transcription start site, is the target of IFI16 suppression. Indeed, electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated that suppression of the UL54 promoter is mediated by IFI16-induced blocking of Sp1-like factors. Consistent with these results, deletion of the putative Sp1 responsive element from the HCMV UL44 promoter also relieved IFI16 suppression. Together, these data implicate IFI16 as a novel restriction factor against HCMV replication and provide new insight into the physiological functions of the IFN-inducible gene IFI16 as a viral restriction factor

Quaternary structure of Dictyostelium discoideum nucleoside diphosphate kinase counteracts the tendency of monomers to form a molten globule

Multimeric enzymes that lose their quaternary structure often cease to be catalytically competent. In these cases, conformational stability depends on contacts between subunits, and minor mutations affecting the surface of the monomers may affect overall stability. This effect may be sensitive to pH, temperature, or solvent composition. We investigated the role of oligomeric structure in protein stability by heat and chemical denaturation of hexameric nucleoside diphosphate kinase from Dictyostelium discoideum and its P105G mutant over a wide range of pH. The wild-type enzyme has been reported to unfold without prior dissociation into monomers, whereas monomer unfolding follows dissociation for the P105G mutant (Giartosio et al. (1996) J. Biol. Chem. 271, 17845-51). We show here that these features are also preserved at alkaline pH, with the wild-type enzyme always hexameric at room temperature whereas the mutant dissociates into monomers at pH g10. In acidic conditions (pH e6), even in the absence of denaturant, the predominant species for both proteins is an intermediate monomeric form with the characteristics of a molten globule: disordered tertiary native structure but preserved secondary structure. Monomers therefore seem to have a low intrinsic stability, which is overcome by the conformational organization in the oligomeric structure