SNX17 Facilitates Infection with Diverse Papillomavirus Types

Abstract

Previous studies have shown that the human papillomavirus type 16 (HPV-16) L2 capsid protein plays an essential role in viral infection, in part through its interaction with sorting nexin 17 (SNX17). We now show that this interaction between L2 and SNX17 is conserved across multiple PV types. Furthermore, we demonstrate that SNX17 is essential for infection with all PV types analyzed, indicating an evolutionarily highly conserved virus entry mechanism. Many of the more than 120 known human papillomaviruses (HPVs) cause sexually transmitted infections, with a subset of them being associated with the development of epithelial tumors and cancer (1). Infectious entry of HPVs is still poorly characterized in terms of the cellular proteins and endocytic pathways employed. It seems that some HPV types may have evolved different entry strategies (2, 3) or take advantage of cross talk between different routes of endocytosis (4,5). A recent study suggested that HPVs can usurp soluble cellular factors for interaction with a plethora of entry receptors, resulting in different routes of internalization (6). The HPV capsid, comprising the major L1 and minor L2 proteins, fulfills various roles in the establishment of a successful viral infection. L2 is critically involved in many steps of the infection process, although its precise functions in entry, intracellular trafficking, endosomal escape, and nuclear import of the viral genome have not been fully elucidated (7). While L2 is not essential for viral uncoating, it is critical for the escape of viral DNA from the endosomal compartment (8) and ultimately for aiding transport of the viral genome to the nucleus (9-11).We reported previously that the HPV-16 L2 interaction with a member of the sorting nexin family, SNX17, is essential for HPV-16 infection (12). SNX17 is localized in early endosomes and recycling tubules and is primarily involved in endosomal recycling (13,14). We showed that at least one putative SNX17 binding motif, NPxF/Y (PTB consensus binding motif) is present in L2s from diverse papillomavirus (PV) genera, indicating that this motif is evolutionarily highly conserved. A previous protein mass spectrometry analysis of HPV-11 L2 and HPV-16 L2 immunoprecipitates in HEK293 cells revealed that SNX17 is an interacting partner of both L2 proteins (12). To investigate whether SNX17 interacts with L2 proteins from diverse HPV genera, a series of pulldown assays with a panel of glutathione S-transferase (GST)-L2 proteins and endogenously derived SNX17 were performed. The results (Fig. 1A) confirmed the interaction between HPV-16 and HPV-11 L2 (genus Alpha) proteins and SNX17. The analysis was then extended to HPV-5 L2 (genus Beta), bovine papillomavirus type 1 (BPV-1) L2 (genus Delta), and cottontail rabbit papillomavirus type 1 (SfPV-1) L2 (genus Kappa). The results in Fig. 1A show interactions between these diverse L2 proteins and SNX17, although the interaction between SNX17 and SfPV L2 was consistently weaker.Previous analyses indicated that HPV-16 L2 bound SNX17 ...

A Novel PDZ Domain Interaction Mediates the Binding between Human Papillomavirus 16 L2 and Sorting Nexin 27 and Modulates Virion Trafficking

“…The plasmid construct expressing SNX17-Flag has been described previously (23). Plasmid pCA16L2, expressing wild-type (WT) HPV-16 L2 (16L2) with amino-terminal Flag and hemagglutinin (HA) tags, has been described previously (8), and the 16L2 truncation mutants used in this study were produced by the introduction of stop codons into the open reading frame of pCA16L2 using the GeneArt site-directed mutagenesis system (Invitrogen), as were the SNX17 and SNX27 C-terminal mutations and the SNX27 ⌬67-77 and ⌬96 -107 mutations.…”

“…The ultimate fate of cargos within endosomal compartments is determined by the protein complexes that are recruited into them, and the exploitation and manipulation of this system have been the evolutionary strategy by which several virus types, including papillomaviruses, gain entry into the cell nucleus. Previous studies from this laboratory have shown that human papillomavirus 16 (HPV-16) L2 binds to SNX17 and that this interaction is required for the entry of the viral genome into the nucleus (8), possibly by retention of L2 and the associated viral DNA in late endosomes in a manner analogous to the retention of P-selectin by SNX17 (11), whereas the dissociated L1 protein is sorted into a lysosomal compartment for degradation (12). The related protein sorting nexin 27 (SNX27) shares several structural features with SNX17.…”

“…While the majority of L1 is sorted into lysosomes, L2, tethered to the viral genome, is transported through the trans-Golgi network (4) and finally enters the nucleus during mitosis (5), when the nuclear membrane is dissolved, where it localizes, together with the viral genome, at promyelocytic leukemia protein (PML) nuclear bodies (6). Various interactions between L2 and cellular proteins have been shown to be important for infection; the interaction of cyclophilins with L2 has been shown to be involved in capsid disassembly and the separation of L1 from the L2/viral DNA complex in acidified endosomes (7), as has the L2 interaction with sorting nexin 17 (SNX17) (8). Additionally, the transit of vesicles to the transGolgi network and subsequent nuclear entry probably involve movement along microtubules by way of molecular motors, since L2 has been shown to interact with dynein (9), an association that is also essential for virus infection.…”

Incoming human papillomavirus type 16 genome resides in a vesicular compartment throughout mitosis

“…Host cell cyclophilins release the majority of L1 from the L2 protein, which remains in complex with the viral genome (2,23,24). A large portion of the L2 protein translocates across the endocytic membrane to engage factors, including the retromer complex, dynein, sorting nexins, and rab GTPases, that mediate transport to the trans-Golgi network (TGN) (25)(26)(27)(28)(29)(30)(31)(32)(33)). An siRNA screen has suggested that nuclear pore complexes are not required for nuclear entry, but that nuclear envelope breakdown during mitosis is necessary (34,35).…”

HPV virions hitchhike a ride on retromer complexes

“…At this time, it can also not be ruled out that a cellular L2 receptor rather than L2 itself mediates the interaction. Recently, SNX17, a sorting nexin that can directly bind to cargo, has been shown to interact with the L2 protein, thus preventing trafficking of HPV virions to lysosomes and its subsequent degradation (17). Future studies will have to establish whether SNX17 is part of the retromer complex or may actually function up-or downstream of the retromer/HPV interactions.…”