The L2 Minor Capsid Protein of Human Papillomavirus Type 16 Interacts with a Network of Nuclear Import Receptors

Abstract

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...

Bovine Papillomavirus Type 1 Infection Is Mediated by SNARE Syntaxin 18

“…In addition, there is evidence that upon cell binding, a conformational change allows for the exposure of hidden L2 residues that can serve as neutralizing epitopes (41,46,52). These latter experiments support the hypothesis that L2 is in part mediating the process of infection after viral binding to the cell surface, including the translocation of the encapsidated genome to the nucleus (1,11,18).Recent improvements in the process to generate PV particles have made it possible to study the process of viral entry and infection in detail (4, 40). These pseudovirions are capable of encapsidating plasmid DNA containing transgenes and full papillomavirus genomes.…”

Karyopherin β3: A new cellular target for the HPV-16 E5 oncoprotein

“…KNβ3 also binds to and mediates nuclear transport of some viral proteins, including the L2 protein of HPV-16 and HPV-11. This interaction occurs via L2 nuclear localization signals, one in the N-terminus and one in the C-terminus [32,33]. We show that the 16E5-KNβ3 interaction primarily is mediated by 10 amino acids at the C-terminus of 16E5, since a C-terminal 16E5 (−10) deletion mutant is largely defective for binding KNβ3.…”

DNA Virus Replication Compartments

“…It is likely that, upon infection, genome transport toward the nucleus is regulated by the L2 capsid protein via interaction with microtubules (266). Since cell division is required for nuclear genome translocation and expression (267), the viral genome may enter the cell nucleus only during mitotic membrane breakdown.…”