Viroporin activity of the JC polyomavirus is regulated by interactions with the adaptor protein complex 3 Tadaki Suzukia,b, Yasuko Orbaa, Yoshinori Makinoa, Yuki Okadac, Yuji Sundend, Hideki Hasegawab,e, William W. Halle,f, and Hirofumi Sawaa,e,g,1 aDivision of Molecular Pathobiology, Research Center for Zoonosis Control, Hokkaido University, Sapporo 001-0020, Japan; bDepartment of Pathology, National Institute of Infectious Diseases, Musashimurayama 208-0011, Japan; cLaboratory of Molecular Cellular Pathology, Hokkaido University School of Medicine, Sapporo 060-8638, Japan; dLaboratory of Comparative Pathology, Hokkaido University School of Veterinary Medicine, Sapporo 060-0818, Japan; eGlobal Virus Network, Baltimore, MD 21201; fCentre for Research in Infectious Diseases, University College Dublin, Dublin 4, Ireland; and gGlobal Centers of Excellence Program for Zoonosis Control, Hokkaido University, Sapporo 001-0020, Japan Edited* by Robert C. Gallo, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, and approved October 3, 2013 (received for review June 20, 2013) Viroporins, which are encoded by a wide range of animal viruses, In the present study, we have investigated the role of cellular oligomerize in host cell membranes and form hydrophilic pores proteins in the viroporin activity of the JCV agnoprotein. We that can disrupt a number of physiological properties of the cell. demonstrated an interaction of the agnoprotein with the δ sub- Little is known about the relationship between host cell proteins unit of adaptor protein complex 3 (AP-3) (AP3D) and showed – fi and viroporin activity. The human JC polyomavirus (JCV) is the that this interaction prevents AP-3 mediated vesicular traf cking causative agent of progressive multifocal leukoencephalopathy. and blocks the targeted transport of the agnoprotein for lysosomal The JCV-encoded agnoprotein, which is essential for viral replica- degradation. Ultimately, this results in the transport of agnoprotein tion, has been shown to act as a viroporin. Here we demonstrate to the cell surface, thus facilitating viroporin activity. fi that the JCV agnoprotein speci cally interacts with adaptor pro- Results tein complex 3 through its δ subunit. This interaction interrupts – fi adaptor protein complex 3–mediated vesicular trafficking with Agnoprotein Impairs AP-3 Mediated Intracellular Vesicular Traf cking. To investigate if host protein(s) are associated with the viroporin suppression of the targeting of the protein to the lysosomal deg- activity of agnoprotein, we performed a yeast two-hybrid screen radation pathway and instead permits the transport of agnopro- using the N-terminal 24 amino acid (a.a.) residues as bait, which tein to the cell surface with resulting membrane permeabilization. fi included the RK residues. Among 29 clones, 7 clones encoding The ndings demonstrate a previously undescribed paradigm in partial sequences of AP3D were identified, and the shortest frag- – virus host interactions allowing the host to regulate viroporin ac- ment among these encoded the 548–850 a.a. residues of AP3D tivity and suggest that the viroporins of other viruses may also be isoform 2 (Fig. 1A). The agnoprotein–AP3D interaction was con- highly regulated by specific interactions with host cell proteins. firmed using several GST-AP3D recombinant proteins (AP3D- Y27, AP3D-Y27N, and AP3D-Y27C) produced in Escherichia coli. pathogen–host cell interaction | intracellular vesicular trafficking No interactions were seen with agnoprotein with GST alone or with GST-AP3D-Y27N. In contrast, interactions were seen with C polyomavirus (JCV) is a member of the family of poly- GST-AP3D-Y27 and GST-AP3D-Y27C (Fig. 1B and Fig. S1A), Jomaviruses and the etiologic agent of progressive multifocal suggesting that the C-terminal 206 a.a. of the AP3D hinge re- leukoencephalopathy, a neurological disorder associated with gion is important in WT binding but not the RK8AA mutant. The interaction was also confirmed in mammalian cells using destructive infection of oligodendrocytes (1). The genome of JCV immunoprecipitation methods (Fig. 1 C and D). GST pull- consists of three functional regions: an early coding region, a late down assays using the minimum binding site of AP3D (AP3D- coding region, and a transcriptional control region. The early coding region encodes the large and small T antigens, and the late coding Significance region encodes VP1, VP2, VP3, and agnoprotein (2). Recently,wehaveshownthatJCVagnoproteinactsasaviro- porin and promotes virus replication by increasing membrane Viroporins are small, hydrophobic viral proteins that form permeabilization, which facilitates the release of progeny virions. pores on host cell membranes, and their expression can in- Mutational analysis of the agnoprotein has demonstrated that the crease the production of progeny virus particles. Recently, we amino acids Arg8 and Lys9 in the NH2 terminus are critical be- have shown that human JC polyomavirus agnoprotein acts as cause mutation of these RK residues (RK8AA mutant) abrogates a viroporin. We have investigated the role of host cellular viroporin function (3). Viroporins are small, nonglycosylated, proteins in the viroporin activity of the agnoprotein. We highly hydrophobic viral polypeptides which interact with cell demonstrated that an interaction of the agnoprotein with the δ membranes and increase their permeability to ions and other low- subunit of AP-3 prevents AP-3–mediated vesicular trafficking molecular weight molecules (4–6). Upon membrane insertion, and blocks the targeted transport of the agnoprotein for ly- viroporins oligomerize to create hydrophilic pores (7–11); although sosomal degradation. These findings demonstrate a previously some viroporins are not required for production of viral progeny, undescribed paradigm in virus–host interactions regulating virus their expression can significantly increase the formation of new replication and suggest that viroporins of other viruses may also virus particles (12–15). be regulated by specific interactions with host cellular proteins. Notably, some viroporins have also been shown to influence intracellular protein trafficking. Poliovirus 3A can inhibit endo- Author contributions: T.S., W.W.H., and H.S. designed research; T.S., Y. Orba, Y.M., plasmic reticulum (ER)-to-Golgi transit, resulting in the shutoff Y. Okada, Y.S., H.H., and H.S. performed research; T.S., Y. Orba, Y.M., Y. Okada, and of nascent MHC class I/peptide complex trafficking and the Y.S. contributed new reagents/analytic tools; T.S., Y. Orba, Y.M., and Y. Okada analyzed down-regulation of cytokine secretion (16, 17). Coxsackievirus data; and T.S., W.W.H., and H.S. wrote the paper. 2B, 2BC, and 3A viroporins are capable of blocking trafficking The authors declare no conflict of interest. through the Golgi complex (18, 19). Thus, although intracellular *This Direct Submission article had a prearranged editor. trafficking of proteins is important in viral infection, the relation 1To whom correspondence should be addressed. E-mail: [email protected]. fi between viroporin activity and cellular traf cking processes This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. remains poorly understood. 1073/pnas.1311457110/-/DCSupplemental. 18668–18673 | PNAS | November 12, 2013 | vol. 110 | no. 46 www.pnas.org/cgi/doi/10.1073/pnas.1311457110 Downloaded by guest on September 25, 2021 AP3D is involved in the biogenesis of lysosome-related or- ganelles (20). We investigated if agnoprotein could modify AP- 3–mediated intracellular trafficking. In AP-3 complex-deficient mammalian cells, some lysosomal membrane proteins, including Lamp-1, exhibit increased trafficking to the plasma membrane, although their steady-state distribution is still primarily lysosomal (20, 21). We examined the status of both lysosomal (Lamp-1) and nonlysosomal (CD71) integral membrane proteins by flow cytometry. Lamp-1 expression was increased on the surface of the cells transfected with agnoprotein or an AP3D-targeted siRNA compared with cells transfected with mock plasmid or a negative control siRNA. In contrast, cell surface expression levels of CD71 were unchanged (Fig. 1F). Previous studies have shown that the transport of vesicular stomatitis virus G protein (VSVG) from the trans-Golgi network (TGN) to the cell surface requires AP3D (22). The 293T cells were transfected with plasmids encoding VSVG-GFP and agno- protein and examined by confocal microscopy. In the absence of agnoprotein, VSVG-GFP was transported to the plasma mem- brane and was only minimally detected in the perinuclear region at 180 min. However, in the presence of agnoprotein, the VSVG- Fig. 1. Agnoprotein binds to AP3D and impairs AP-3–mediated vesicular GFP signal in the perinuclear region, which colocalized with the trafficking. (A) Schematic representation of the δ subunit of the human AP-3 TGN marker protein (GM130), was still evident at 180 min (Fig. complex (AP3D1 isoforms 1 and 2), showing the region encoded by the shortest 1G and Fig. S2 A and B), suggesting that transport from the TGN cDNA fragments (Y2H#27) isolated using a yeast two-hybrid assay and the to the cell surface was inhibited by agnoprotein. The transport of fragments expressed as GST fused proteins (AP3D-Y27, AP3D-Y27N, and AP3D- VSVG-GFP between the ER and the TGN, which is independent Y27C). (B) GST pull-down assay with the GST fused proteins. After incubation of AP3D, was not disrupted by agnoprotein (Fig. S2C). The in- with