The main objec­tive of the pro­posed project is to elu­ci­date the dif­fer­en­tial role of intact retro­vi­ral cap­sids regard­ing for­ma­tion and nuclear entry of func­tion­al repli­ca­tion com­plex­es with con­comi­tant eva­sion from innate sens­ing of the viral nucle­ic acids. These stud­ies will focus on HIV‑1 and MLV since these two virus­es exhib­it fun­da­men­tal dif­fer­ences in the rel­e­vant func­tions, and func­tion­al chimera can be con­struct­ed. With­in SPP1923, we will pro­vide our method for ana­lyz­ing pro­duc­tive repli­ca­tion com­plex­es to oth­er groups inter­est­ed; depend­ing on results, this may lead to analy­sis of fur­ther retro­virus­es in col­lab­o­ra­tion. Specif­i­cal­ly, the pro­posed project has the fol­low­ing aims: I. Com­par­a­tive char­ac­ter­i­za­tion of infec­tion, pro­duc­tive repli­ca­tion com­plex for­ma­tion, nuclear tar­get­ing and innate sens­ing for HIV‑1 (and cap­sid sta­bil­i­ty mutants there­of), MLV and chimeric HIV- 1/​MLV vari­ants in dif­fer­ent cell types. II. Iso­la­tion of bona fide repli­ca­tion com­plex­es from the cyto­plasm of (some of) these infec­tions by estab­lished and nov­el method­ol­o­gy fol­lowed by pro­teom­ic char­ac­ter­i­za­tion and analy­sis of their capac­i­ty to induce innate DNA sens­ing. III. Analy­sis of the asso­ci­a­tion of retro­vi­ral repli­ca­tion com­plex­es with known and pre­sumed host fac­tors and nucle­ic acid sens­ing mol­e­cules in the cyto­plasm of dif­fer­ent host cells infect­ed with the rel­e­vant retro­vi­ral vari­ants and apply­ing imag­ing analy­sis, prox­im­i­ty biotiny­la­tion assays and tar­get­ed dis­rup­tion of the viral capsid.