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

Abstract

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.

Role of nucleotide-binding oligomerization domain 1 (NOD1) and its variants in human cytomegalovirus control in vitro and in vivo

“…A cytoplasmic dsDNA sensor, ZBP1, activates IRF3 on infection, and its overexpression inhibits HCMV replication (7). IFN-inducible protein IFI16 modestly inhibits HCMV by blocking Sp1-mediated transcription of HCMV-encoded UL54 and UL44, which are involved in viral DNA synthesis (8).…”

Distinct Roles for Human Cytomegalovirus Immediate Early Proteins IE1 and IE2 in the Transcriptional Regulation of MICA and PVR/CD155 Expression

“…Recombinant adenoviral vectors (AdV) encoding HCMV IE2 (AdV-IE2) and Escherichia coli b-galactosidase (AdV-LacZ) have been previously described (55,56). AdV-IE72 (AdV-IE1) was provided by Dr. Timothy F. Kowalik (University of Massachusetts Medical School, Worcester, MA) (27).…”

“…AdV-IE72 (AdV-IE1) was provided by Dr. Timothy F. Kowalik (University of Massachusetts Medical School, Worcester, MA) (27). Recombinant AdV stocks were generated, purified, and titrated as previously described (27,55,56). For adenoviral transduction, HFFs were infected at ∼80-90% confluence at an MOI of 4 PFU/cell in DMEM without FCS for 2 h at 37˚C.…”

The emerging role of human PYHIN proteins in innate immunity: Implications for health and disease

“…By regulating transcription factors, IFI16 can also act as a restriction factor during human cytomegalovirus (HCMV) infection. It does so by interacting with the transcription factor Sp1, displacing it from promoters, thereby leading to a decrease in HCMV DNA synthesis, a decrease in viral gene expression and inhibition of viral replication [25]. Furthermore, IFI16 has recently been shown to be required for gene induction in response to innate immune RNA and DNA detection, since IFI16 is required for optimal RNA polymerase II occupancy on IFN-a and Immune Stimulated Gene (ISG) promoters [26].…”