The normally benign human polyomavirus JCPyV can cause the fatal neurodegenerative disease Progressive Multifocal Leukoencephalopathy (PML). It is a common virus that establishes an asymptomatic persistent infection of the kidneys. Normal immune surveillance is thought to keep the virus in check and individuals are only at risk for disease when immunocompromised due to an underlying illness such as HIV/AIDS, or are undergoing treatment with immunomodulatory therapies. Once considered a rare disease, PML is becoming more clinically relevant as more diseases are treated with a growing number of immunomodulators such as natalizumab and rituximab. Patients on these therapies routinely have their blood tested for the presence of anti-JCPyV antibodies and the drug treatment is halted if the patient tests positive. Currently, there are no effective treatments against JCPyV or PML. This often leads physicians to switch their patients from long-term immunomodulatory therapy to less effective treatments in order to decrease the risk of developing PML. By better understanding how the virus normally infects cells and how this changes during disease, we can identify ways to inhibit the virus and allow for patients to remain on the most effective therapies for their underlying condition.
JCPyV is a small virus with three structural proteins that make up the capsid and enclose the approximately 5000 bp genome. Despite the simplicity of this virus, its lifecycle, how it invades the brain, and the factors responsible for the development of PML are not entirely known. Our lab is interested in understanding virus-receptor interactions, entry and nuclear trafficking in order to develop drugs that block these steps in the viral lifecycle. The interaction of viruses with the cell surface is a critical step in infection, spread and pathogenesis of disease. With our collaborators, we have solved the crystal structure to understand how JCPyV interacts with sialic acid-containing receptors. We know that wild type JCPyV (Mad-1 strain) recognizes LSTc with the highest affinity but that it also binds to GM1, GD1b, GM2, and GD1a at lower affinities (1). A mutant form of JCPyV isolated from kidney (WT3 strain) recognizes these same structures but has higher affinity for GM1 than does the Mad-1 strain (2). It has been unclear whether capsid differences between virus isolated from kidneys versus virus isolated from the brains of PML patients play a role in disease progression. Experiments designed to ascertain whether PML associated mutations cause the virus to behave differently than wild type virus are difficult to perform as these mutant viruses do not spread in culture.
To circumvent these challenges, we adapted a reporter based-pseudovirus system in which the viral JCPyV capsid packages a reporter plasmid (3). Pseudoviruses have qualities that make them superior to virus for certain aspects of researching viral entry. Primarily, they are faster, cheaper and easier to both grow and assay than virus and can be used for a multitude of studies due to the ease of detection. In addition, they provide the means to investigate viral mutants that do not grow in culture. I demonstrated that these pseudoviruses mimic entry and trafficking of infectious virus (4). We have used psuedoviruses to confirm the essential role LSTc plays in initial receptor engagement and subsequent infection (1). The use of pseudoviruses has allowed us to study naturally occurring mutant viruses found in PML patients and show that these mutations inhibit engagement of the viral receptor on glial cells and render the virus non-infectious (5). Other labs using this system have recently provided evidence that these PML mutations may promote disease by allowing the virus to escape immunodetection (6).
While most of my research is focused on JCPyV, other members of the Polyomavirus family also cause disease in humans (7-9). The similarities between these viruses indicate that the conserved aspects are universally important for completing the viral lifecycle. Our system allowed us to examine the role of one characteristic of all polyomavirus capsids during infection. We determined that the viral capsid five-fold pore is necessary for uncoating in the endoplasmic reticulum (10). By exchanging the JCPyV structural proteins for those of other polyomaviruses, we can broaden our research potential. This method has recently allowed us to begin the initial studies into the newly discovered human polyomavirus TSPyV, which does not yet have an established cell culture system (11).
Currently I lead the Pseudovirus and Virus Production Core of a program project grant and am in charge of providing research material to three multidisciplinary laboratories studying early events in the virus lifecycle. In addition to our own studies, we collaborate with a lab using genetic approaches including high-throughput genetic screens to identify cellular factors that are critical for JCPyV invasion of cells and another laboratory that focuses on the structural aspects of the virus receptor interaction. Progress on these projects will require reagents that I will design and supply. I am responsible for the large-scale growth of wild type and mutant forms of JCPyV and JC pseudoviruses, as well as other polyomaviruses. I also design and provide the appropriate biological tools which can be used study previously unknown aspects of the viral lifecycle.
In addition to my main focus as Core Leader, I collaborate on understanding other aspects of the polyomavirus lifecycle. I have helped uncover the role autophagy plays in BK polyomavirus entry (12). I am also collaborating with another colleague to clarify the role of serotonin receptors in JCPyV infection using a CRISPR/Cas9 model to knock out these receptors in kidney and glial cells (13, 14). Furthermore, I continue to work with a chemistry group to improve inhibitors of JCPyV retrograde transport (15, 16). Our future plans include working with our collaborators to identify other proteins which interact with the virus using a FLAG tag pull-down pseudovirus and virus system (17), and investigating the role of newly identified sorting motifs in JCPyV infection (18).
1. Stroh LJ, Maginnis MS, Blaum BS, Nelson CD, Neu U, Gee GV, O'Hara BA, Motamedi N, DiMaio D, Atwood WJ, Stehle T. 2015. The Greater Affinity of JC Polyomavirus Capsid for alpha2,6-Linked Lactoseries Tetrasaccharide c than for Other Sialylated Glycans Is a Major Determinant of Infectivity. J Virol 89:6364-6375.
2. Gorelik L, Reid C, Testa M, Brickelmaier M, Bossolasco S, Pazzi A, Bestetti A, Carmillo P, Wilson E, McAuliffe M, Tonkin C, Carulli JP, Lugovskoy A, Lazzarin A, Sunyaev S, Simon K, Cinque P. 2011. Progressive multifocal leukoencephalopathy (PML) development is associated with mutations in JC virus capsid protein VP1 that change its receptor specificity. J Infect Dis 204:103-114.
3. Tolstov YL, Pastrana DV, Feng H, Becker JC, Jenkins FJ, Moschos S, Chang Y, Buck CB, Moore PS. 2009. Human Merkel cell polyomavirus infection II. MCV is a common human infection that can be detected by conformational capsid epitope immunoassays. Int J Cancer 125:1250-1256.
4. Gee GV, O'Hara BA, Derdowski A, Atwood WJ. 2013. Pseudovirus mimics cell entry and trafficking of the human polyomavirus JCPyV. Virus Res 178:281-286.
5. Maginnis MS, Stroh LJ, Gee GV, O'Hara BA, Derdowski A, Stehle T, Atwood WJ. 2013. Progressive multifocal leukoencephalopathy-associated mutations in the JC polyomavirus capsid disrupt lactoseries tetrasaccharide c binding. MBio 4:e00247-00213.
6. Ray U, Cinque P, Gerevini S, Longo V, Lazzarin A, Schippling S, Martin R, Buck CB, Pastrana DV. 2015. JC polyomavirus mutants escape antibody-mediated neutralization. Sci Transl Med 7:306ra151.
7. Gardner SD, Field AM, Coleman DV, Hulme B. 1971. New human papovavirus (B.K.) isolated from urine after renal transplantation. Lancet 1:1253-1257.
8. Feng H, Shuda M, Chang Y, Moore PS. 2008. Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science 319:1096-1100.
9. van der Meijden E, Janssens RW, Lauber C, Bouwes Bavinck JN, Gorbalenya AE, Feltkamp MC. 2010. Discovery of a new human polyomavirus associated with trichodysplasia spinulosa in an immunocompromized patient. PLoS Pathog 6:e1001024.
10. Nelson CD, Stroh LJ, Gee GV, O'Hara BA, Stehle T, Atwood WJ. 2015. Modulation of a pore in the capsid of JC polyomavirus reduces infectivity and prevents exposure of the minor capsid proteins. J Virol 89:3910-3921.
11. Stroh LJ, Gee GV, Blaum BS, Dugan AS, Feltkamp MC, Atwood WJ, Stehle T. 2015. Trichodysplasia spinulosa-Associated Polyomavirus Uses a Displaced Binding Site on VP1 to Engage Sialylated Glycolipids. PLoS Pathog 11:e1005112.
12. Bouley SJ, Maginnis MS, Derdowski A, Gee GV, O'Hara BA, Nelson CD, Bara AM, Atwood WJ, Dugan AS. 2014. Host cell autophagy promotes BK virus infection. Virology 456-457:87-95.
13. Assetta B, Maginnis MS, Gracia Ahufinger I, Haley SA, Gee GV, Nelson CD, O'Hara BA, Allen Ramdial SA, Atwood WJ. 2013. 5-HT2 receptors facilitate JC polyomavirus entry. J Virol 87:13490-13498.
14. Sanjana NE, Shalem O, Zhang F. 2014. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11:783-784.
15. Nelson CD, Carney DW, Derdowski A, Lipovsky A, Gee GV, O'Hara B, Williard P, DiMaio D, Sello JK, Atwood WJ. 2013. A retrograde trafficking inhibitor of ricin and Shiga-like toxins inhibits infection of cells by human and monkey polyomaviruses. MBio 4:e00729-00713.
16. Carney DW, Nelson CD, Ferris BD, Stevens JP, Lipovsky A, Kazakov T, DiMaio D, Atwood WJ, Sello JK. 2014. Structural optimization of a retrograde trafficking inhibitor that protects cells from infections by human polyoma- and papillomaviruses. Bioorg Med Chem 22:4836-4847.
17. Einhauer A, Jungbauer A. 2001. The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J Biochem Biophys Methods 49:455-465.
18. Feng Z, Hensley L, McKnight KL, Hu F, Madden V, Ping L, Jeong SH, Walker C, Lanford RE, Lemon SM. 2013. A pathogenic picornavirus acquires an envelope by hijacking cellular membranes. Nature 496:367-371.