Dynein engages and disassembles cytosol-localized SV40 to promote infection.

During entry, polyomavirus (PyV) is endocytosed and sorts to the endoplasmic reticulum (ER) where it penetrates the ER membrane to reach the cytosol. From the cytosol, the virus moves to the nucleus to cause infection. How PyV is transported from the cytosol into the nucleus, a crucial infection step, is unclear. We found that upon reaching the cytosol, the archetype PyV SV40 recruits the cytoplasmic dynein motor, which disassembles the viral particle. This reaction enables the resulting disassembled virus to enter the nucleus to promote infection. Our findings reveal how a cytosolic motor can be hijacked to impart conformational changes to a viral particle, which is essential for successful infection. IMPORTANCE How a non-enveloped virus successfully traffics from the cell surface to the nucleus to cause infection remains enigmatic in many instances. In the case of the non-enveloped PyV, the viral particle is sorted from the plasma membrane to the ER and then the cytosol from where it enters the nucleus to promote infection. The molecular mechanism by which PyV reaches the nucleus from the cytosol is not entirely clear. Here we demonstrate that the prototype PyV SV40 recruits dynein upon reaching the cytosol. Importantly, this cellular motor disassembles the viral particle during cytosol-to-nuclear transport to cause infection.