The L2 minor capsid proteins enter the nucleus twice during viral infection: in the initial phase after virion disassembly and in the productive phase when, together with the L1 major capsid proteins, they assemble the replicated viral DNA into virions. In this study we investigated the interactions between the L2 protein of high-risk human papillomavirus type 16 (HPV16) and nuclear import receptors. We discovered that HPV16 L2 interacts directly with both Kap␤ 2 and Kap␤ 3 . Moreover, binding of Ran-GTP to either Kap␤ 2 or Kap␤ 3 inhibits its interaction with L2, suggesting that the Kap␤/L2 complex is import competent. In addition, we found that L2 forms a complex with the Kap␣ 2 ␤ 1 heterodimer via interaction with the Kap␣ 2 adapter. In agreement with the binding data, nuclear import of L2 in digitonin-permeabilized cells could be mediated by either Kap␣ 2 ␤ 1 heterodimers, Kap␤ 2 , or Kap␤ 3 . Mapping studies revealed that HPV16 L2 contains two nuclear localization signals (NLSs), in the N terminus (nNLS) and C terminus (cNLS), that could mediate its nuclear import. Together the data suggest that HPV16 L2 interacts via its NLSs with a network of karyopherins and can enter the nucleus via several import pathways mediated by Kap␣ 2 ␤ 1 heterodimers, Kap␤ 2 , and Kap␤ 3 .In the United States, infections caused by human papillomaviruses (HPVs) are more prevalent than all other sexually transmitted diseases combined. High-risk HPV infections are associated with more than 95% of cervical cancer, which is the second leading cause of cancer death among women. In addition, a high percentage of anal, perianal, vulvar, and penile cancers, as well as some non-melanoma skin cancers, are linked to oncogenic HPV infections. The main high-risk HPV types are HPV type 16 (HPV16), HPV18, HPV31, and HPV45, with HPV16 the most prevalent type in cervical cancers (29).HPVs are small, nonenveloped, icosahedral DNA viruses that specifically infect the basal squamous epithelial cells. The virion particles (55 to 60 nm in diameter) consist of a single molecule of 8-kb double-stranded circular DNA contained within an icosahedral capsid comprising the L1 major and L2 minor capsid proteins (7). The L1 major capsid protein forms pentamers (capsomeres), and 72 capsomeres assemble into T-7d icosahedral lattice (11). The L2 minor structural protein is at about 1/30 the abundance of L1 (21), and it interacts with the L1 pentamers (2). Expression of L1 with a vaccinia virus or baculovirus system results in the formation of virus-like particles similar to viral capsids (12, 13). During the late phase of infection, the L1 and L2 proteins are synthesized in the cytoplasm of the differentiated cells of the infected epithelium and then transported to the nucleus for assembly of the replicated viral DNA into virions. Although L1 expressed alone in mammalian cells harboring episomal DNA forms virions (26), the L2 minor capsid protein dramatically increases the efficiency of DNA encapsidation (21,24). Recent studies show that, at least for HPV33, expr...

Analysis of the interactions of low-risk human papillomavirus type 11 (HPV11) L2 with karyopherin ␤ (Kap ␤) nuclear import receptors revealed that L2 interacted with Kap ␤ 1 , Kap ␤ 2 , and Kap ␤ 3 and formed a complex with the Kap ␣ 2 ␤ 1 heterodimer. HPV11 L2 contains two nuclear localization signals (NLSs)-in the N terminus and the C terminus-that could mediate its nuclear import via a classical pathway. Each NLS was functional in vivo, and deletion of both of them abolished L2 nuclear localization. Both NLSs interacted with the viral DNA. Thus, HPV11 L2 can interact with several karyopherins and the viral DNA and may enter the nucleus via multiple pathways.Human papillomavirus (HPV) virions consist of a single molecule of 8-kb double-stranded circular DNA contained within a spherical capsid composed of 72 L1 capsomers and the L2 minor capsid protein, estimated to have 36 molecules per capsid (10, 15). Although L1 expressed alone in mammalian cells harboring episomal DNA forms virions (18), L2 expression is required for efficient encapsidation of the viral DNA (15, 17). Expression and nuclear import of L2 during the productive stage precede the expression and nuclear translocation of L1 (5). Studies with HPV virions in raft cultures have shown that L2 participates in at least two steps in the production of infectious virus (7). L2 binds to cells (9,19) and interacts with ␤-actin and tSNARE syntaxin 18 (1, 20) and also facilitates the escape of the viral genome from the endocytic compartment after viral uncoating (8). Cleavage of L2 at a furin consensus site located in the N terminus was reported to be required for infection (14). Bovine papillomavirus type 1 (BPV1) L2 termini required for infectivity can function as nuclear localization signals (NLSs) mediating nuclear import via a classical pathway, and the C-terminal NLS (cNLS) can also interact with the viral DNA (4). These results, together with the colocalization of the incoming L2 and genome in the nucleus at ND10 (3), suggest that BPV1 L2 may facilitate the nuclear localization of the genome in the initial stages of infection. In a related virus, simian virus 40, nuclear import of simian virus 40 DNA is mediated by the VP3 capsid protein via interaction with the importin heterodimer (12).Active nuclear import of proteins is mediated by import receptors of the karyopherin ␤ (Kap ␤)/importin ␤ superfamily that interact with nucleoporins at the nuclear pore complex to transport the proteins into the nucleus. Binding of nuclear RanGTP to the Kap ␤s causes dissociation of the import complexes, leading to the release of the transported cargoes inside the nucleus (6, 11). In this study we investigated the interactions of the L2 minor capsid protein of low-risk HPV11 with import receptors and viral DNA and mapped its NLSs and DNA binding sites. The His-tagged HPV11 L2 contained in the pProEX HTb plasmid vector (16) was expressed in Escherichia coli BL21-CodonPlus and purified as previously described (2). We analyzed the interactions between His-tagged HPV11 L2...