Using evo­lu­tion­ary genomics, tran­scrip­tomics, and pro­tein inter­ac­tion data, we iden­ti­fied gam­ma-inter­fer­on-inducible pro­tein 16 (IFI16) as poten­tial anti­retro­vi­ral restric­tion fac­tor. In the first fund­ing peri­od of this SPP, we per­formed thor­ough func­tion­al and muta­tion­al analy­ses of IFI16. We found that IFI16 restricts HIV‑1 inde­pen­dent­ly of immune sens­ing by seques­ter­ing the tran­scrip­tion fac­tor Sp1 and inhibit­ing viral gene expres­sion. Anti­retro­vi­ral activ­i­ty and Sp1 inter­ac­tion require the N‑terminal pyrin domain and nuclear local­iza­tion of IFI16, but not the HIN domains involved in DNA bind­ing. Inter­est­ing­ly, we found that high­ly preva­lent sub­type C strains that are respon­si­ble for about half of all HIV‑1 infec­tions world­wide are less depen­dent on Sp1 and capa­ble of evad­ing IFI16-medi­at­ed restric­tion. In addi­tion, our results sug­gest that IFI16 plays a role in the main­te­nance of HIV‑1 laten­cy and the con­trol of the LINE 1 retro­trans­po­son. IFI16 is only one of five human PYHIN pro­teins and our data sug­gest that two oth­er mem­bers of this fam­i­ly (MNDA and IFIX) share the antivi­ral activ­i­ty of IFI16. Final­ly, exper­i­ments in knock­out mice indi­cate that PYHIN pro­teins restrict retro­vi­ral spread in vivo, inde­pen­dent­ly of effects on inter­fer­on pro­duc­tion. Alto­geth­er, our results sup­port an impor­tant role of PYHIN pro­teins in the innate immune defense against retro­vi­ral pathogens. How­ev­er, many ques­tions regard­ing the under­ly­ing mech­a­nisms and the rel­e­vance of PYHIN pro­teins for virus restric­tion remain to be addressed. In the 2nd fund­ing peri­od, we will deter­mine the exact mech­a­nism of IFI16-medi­at­ed inhi­bi­tion of Sp1 and ana­lyze the con­se­quences for Sp1-dri­ven cel­lu­lar gene expres­sion. Anoth­er goal will be to clar­i­fy, how sub­type C HIV‑1 strains evade IFI16 inhi­bi­tion and to elu­ci­date the role of IFI16 and Sp1 inhi­bi­tion in HIV laten­cy. In addi­tion, we will exam­ine the effects of human PYHIN pro­teins on endoge­nous retroele­ments. Final­ly, we will per­form col­lab­o­ra­tive stud­ies to deter­mine the rel­e­vance of PYHIN pro­teins for retro­vi­ral restric­tion in vivo. Our results will clar­i­fy whether PYHIN pro­teins play key roles in tran­scrip­tion­al silenc­ing of exoge­nous retro­virus­es and endoge­nous retroelements.