Abstract During entry, the archetype polyomavirus SV40 traffics from the plasma membrane to the late endosome (LE). From the LE, SV40 is targeted to the endoplasmic reticulum (ER) where it penetrates the ER membrane to reach the cytosol and then the nucleus to cause infection. How SV40 targets from the LE to the ER remains mysterious. This application hypothesizes that SV40 in fact exploits a unique subcellular structure called the membrane contact site formed between the LE and ER to gain entry to the ER from the LE. Accordingly, the objective of this R21 proposal is to elucidate, at the molecular level, how the MCS between the LE and ER is hijacked to enable successful entry leading to productive SV40 infection.