Topography of the Human Papillomavirus Minor Capsid Protein L2 during Vesicular Trafficking of Infectious Entry

Abstract

The human papillomavirus (HPV) capsid is composed of the major capsid protein L1 and the minor capsid protein L2. During entry, the HPV capsid undergoes numerous conformational changes that result in endosomal uptake and subsequent trafficking of the L2 protein in complex with the viral DNA to the trans-Golgi network. To facilitate this transport, the L2 protein harbors a number of putative motifs that, if capable of direct interaction, would interact with cytosolic host cell factors. These data imply that a portion of L2 becomes cytosolic during infection. Using a low concentration of digitonin to selectively permeabilize the plasma membrane of infected cells, we mapped the topography of the L2 protein during infection. We observed that epitopes within amino acid residues 64 to 81 and 163 to 170 and a C-terminal tag of HPV16 L2 are exposed on the cytosolic side of intracellular membranes, whereas an epitope within residues 20 to 38, which are upstream of a putative transmembrane region, is luminal. Corroborating these findings, we also found that L2 protein is sensitive to trypsin digestion during infection. These data demonstrate that the majority of the L2 protein becomes accessible on the cytosolic side of intracellular membranes in order to interact with cytosolic factors to facilitate vesicular trafficking. IMPORTANCEIn order to complete infectious entry, nonenveloped viruses have to pass cellular membranes. This is often achieved through the viral capsid protein associating with or integrating into intracellular membrane. Here, we determine the topography of HPV L2 protein in the endocytic vesicular compartment, suggesting that L2 becomes a transmembrane protein with a short luminal portion and with the majority facing the cytosolic side for interaction with host cell transport factors. Human papillomaviruses (HPVs) are nonenveloped DNA tumor viruses that infect the skin and mucosal epithelial cells. Infection by HPVs can induce hyper-proliferative lesions of the tissues, whereupon high-risk HPVs can progress to malignant tumors. Of the high-risk HPVs, 70% of cervical cancer can be attributed to HPVs 16 and 18 (1). These HPVs are associated with a variety of anogenital and oral carcinomas. Despite the success of the prophylactic vaccine against these high-risk HPVs, HPV-associated cancers will continue to be a global health burden in the future due to low vaccination rates and lack of therapeutic effect (2). Therefore, uncovering the conserved steps of how HPVs establish infection can be used to find antiviral targets that may be less virus restrictive than the current vaccines.The HPV capsid is composed of 360 molecules of the major capsid protein, L1, and up to 72 molecules of the minor capsid protein, L2 (3-6). Much of our advancement in understanding HPV entry can be attributed to the efficient generation of pseudovirions. With respect to cellular entry, pseudovirions are considered to be indistinguishable from native virions but encapsidate a reporter plasmid (pseudogenome) in place of the HP...

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

“…Regardless, our data suggest that the L2 protein is important for directing the HPV-containing membranous structures toward the nucleus during mitosis. It was recently demonstrated that the L2 protein becomes transmembranous to engage numerous cytoplasmic factors required for trafficking to the TGN and possibly the ER, with some 30 N-terminal residues being luminal (28,32). We have shown that L2 assumes a similar topography during mitosis, suggesting that HPV-harboring vesicles bud out of the TGN or the ER during the onset of mitosis.…”

“…As reported previously, the L2-specific antibodies show a high level of background even in uninfected cells, along with a low signal intensity (32). In addition, the antibodies are large macromolecules of ∼150 kDa and have limited accessibility for proteins present in larger complexes.…”

“…HaCaT cells were infected with HPV16 pseudovirus for 24 h and then selectively permeabilized using a low concentration of digitonin (DIG) that permeabilizes the plasma membrane and early endosomes but not TGN, Golgi, or endoplasmic reticulum (ER) (32). We observed that the epitope comprising residues 18-35 as well as L1 was still…”

“…Using differential permeabilization and epitope-mapped monoclonal antibodies (Fig. 1A), we recently demonstrated that the L2 protein assumes a transmembranous configuration in the TGN where the putative transmembrane domain (TM; residues 45-65) separates the luminal epitope (residues 18-35) from epitopes located C-terminally of the TM domain and facing the cytosol (32). In contrast, L1 protein was found exclusively in the luminal compartment of the TGN.…”

“…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).…”