HPV16 entry requires dynein for minus-end transport and utilizes kinesin Kif11 for plus-end transport along microtubules during mitosis.

Human papillomaviruses (HPVs) travel from the trans-Golgi network (TGN) to the condensed (mitotic) chromosomes during mitosis. Partially uncoated HPV capsids utilize a unique vesicular structure for trafficking and nuclear import, which is directed by the minor capsid protein L2. However, it is still unknown which precise factors facilitate post-TGN HPV trafficking to the nucleus. Herein, we analyzed HPV16-infected mitotic cells using high-resolution microscopy, coupled with motor protein inhibition, to further elaborate on post-TGN trafficking by tracking the location and/or quantification of EdU-labeled HPV pseudogenomes on microtubules, certain kinesins, and mitotic chromosomes. We also adapted a knocksideways approach to determine if L2 and Kif11 interact in infected cells. We visualized dynein co-localization with HPV pseudogenomes along mitotic microtubules and measured HPV pseudogenome accumulation after short-term dynein inhibition. Additional inhibitor studies implicated a specific kinesin, Kif11, as participating in HPV pseudogenome delivery to the nucleus. Short-term inhibition of Kif11 decreased HPV pseudogenome accumulation at mitotic chromatin. In addition, Kif11, along with kinesins Kif18a and Kif25, were in proximity to L2 during infection. While we were unable to determine a direct interaction between L2 and Kif11, we were able to show via knocksideways approach that relocalization of exogenous Kif11 decreased HPV pseudogenome accumulation to the mitotic chromatin. Our data support a model whereby HPV16 utilizes dynein for minus-end trafficking along mitotic microtubules and utilizes Kif11 for plus-end movement in the late stage of viral entry. IMPORTANCE: Human papillomaviruses (HPV) utilize a unique vesicular structure to shield their genomes from detection during trafficking from the trans-Golgi network (TGN) to the nucleus during mitosis. The exact cellular factors responsible for trafficking these HPV genome containing vesicles along mitotic microtubules via the L2 minor protein remain unknown. We show via high-resolution microscopy that pharmacological inhibition of dynein and the kinesin Kif11 significantly decreases HPV pseudogenome accumulation on mitotic chromatin. Several kinesins were detected in proximity to incoming HPV pseudogenomes. Finally, using a novel knocksideways approach, we show reduced HPV pseudogenome accumulation on mitotic chromatin upon Kif11 relocalization to the mitochondria. Herein, our data suggest HPV utilizes minus- and plus-end mediated trafficking along mitotic microtubules to complete its genome trafficking to the nucleus.