A Ran-binding protein facilitates nuclear import of human papillomavirus type 16

Abstract

Human papillomaviruses (HPVs) utilize an atypical mode of nuclear import during cell entry. Residing in the Golgi apparatus until mitosis onset, a subviral complex composed of the minor capsid protein L2 and viral DNA (L2/vDNA) is imported into the nucleus after nuclear envelope breakdown by associating with mitotic chromatin. In this complex, L2 plays a crucial role in the interactions with cellular factors that enable delivery and ultimately tethering of the viral genome to mitotic chromatin. To date, the cellular proteins facilitating these steps remain unknown. Here, we addressed which cellular proteins may be required for this process. Using label-free mass spectrometry, biochemical assays, microscopy, and functional virological assays, we discovered that L2 engages a hitherto unknown protein complex of Ran-binding protein 10 (RanBP10), karyopherin alpha2 (KPNA2), and dynein light chain DYNLT3 to facilitate transport towards mitotic chromatin. Thus, our study not only identifies novel cellular interactors and mechanism that facilitate a poorly understood step in HPV entry, but also a novel cellular transport complex.

Recent Advances in Our Understanding of the Infectious Entry Pathway of Human Papillomavirus Type 16

“…However, there is no published data yet on the (proposed) plus-end movement from the MTOC to the condensed chromosomes. Approximately 50% of HPV genomes still travel to the nucleus in spite of siRNA-mediated knockdown(s) of RanBP10 and DYLN3 [87], showing that either…”

“…The basic component of the above-mentioned amphipathic peptide of HPV16 L2 protein (RKRRKR), residues 457-462, that resemble a cationic cell-penetrating peptide (CPP) [85] is required to translocate across the endosomal membrane and into the cytoplasm. The carboxyl terminus of L2 becomes sensitive to protease digestion and accessible to antibody binding in infected physically disrupted cells [84] and can be bound by cytosolic transport factors [22,23,86,87]. L2 transmembrane configuration requires acidification of the endocytic vesicle [76,84], partial uncoating of the capsid [76,82], and cyclophilin chaperone activity [19].…”

“…Furthermore, the role of dynein in infectious HPV trafficking was expanded upon as chemical inhibition of dynein decreased HPV genome import into the nucleus. HPV genome nuclear import was also decreased with knockdown of dynein light chain 3 (DYLN3) [87]. The dynein interaction with L2 has been mapped to residues 456-461 on L2 of HPV16 [83].…”

“…The dynein interaction with L2 has been mapped to residues 456-461 on L2 of HPV16 [83]. Indirect evidence suggests that plus end-directed transport along spindle microtubules is required as well, likely mediated by L2 protein [87]. Alanine substitution of arginine residues at position 302 and 305 results in loss of viral genome from spindle microtubules without affecting transport to the microtubule organizing center (MTOC) [79,84].…”

“…Alanine substitution of arginine residues at position 302 and 305 results in loss of viral genome from spindle microtubules without affecting transport to the microtubule organizing center (MTOC) [79,84]. The same L2 domain may be responsible for tethering the transport vesicles to mitotic chromosomes [87]. The steps of HPV16 viral entry, from capsid binding to vesicle-protected genome transport via L2 to the condensed chromosomes, are pictorially summarized in Figure 1.…”