Aisling S. Dugan

An N-Linked Glycoprotein with α(2,3)-Linked Sialic Acid Is a Receptor for BK Virus

ABSTRACT BK virus (BKV) is a common human polyomavirus infecting >80% of the population worldwide. Infection with BKV is asymptomatic, but reactivation in renal transplant recipients can lead to polyomavirus-associated nephropathy. In this report, we show that enzymatic removal of α(2,3)-linked sialic acid from cells inhibited BKV infection. Reconstitution of asialo cells with α(2,3)-specific sialyltransferase restored susceptibility to infection. Inhibition of N-linked glycosylation with tunicamycin reduced infection, but inhibition of O-linked glycosylation did not. An O-linked-specific α(2,3)-sialyltransferase was unable to restore infection in asialo cells. Taken together, these data indicate that an N-linked glycoprotein containing α(2,3)-linked sialic acid is a critical component of the cellular receptor for BKV.

IL-1α Signaling Initiates the Inflammatory Response to Virulent Legionella pneumophila In Vivo

Legionella pneumophila is an intracellular bacterial pathogen that is the cause of a severe pneumonia in humans called Legionnaires’ disease. A key feature of L. pneumophila pathogenesis is the rapid influx of neutrophils into the lungs, which occurs in response to signaling via the IL-1R. Two distinct cytokines, IL-1α and IL-1β, can stimulate the type I IL-1R. IL-1β is produced upon activation of cytosolic sensors called inflammasomes that detect L. pneumophila in vitro and in vivo. Surprisingly, we find no essential role for IL-1β in neutrophil recruitment to the lungs in response to L. pneumophila. Instead, we show that IL-1α is a critical initiator of neutrophil recruitment to the lungs of L. pneumophila–infected mice. We find that neutrophil recruitment in response to virulent L. pneumophila requires the production of IL-1α specifically by hematopoietic cells. In contrast to IL-1β, the innate signaling pathways that lead to the production of IL-1α in response to L. pneumophila remain poorly defined. In particular, although we confirm a role for inflammasomes for initiation of IL-1β signaling in vivo, we find no essential role for inflammasomes in production of IL-1α. Instead, we propose that a novel host pathway, perhaps involving inhibition of host protein synthesis, is responsible for IL-1α production in response to virulent L. pneumophila. Our results establish IL-1α as a critical initiator of the inflammatory response to L. pneumophila in vivo and point to an important role for IL-1α in providing an alternative to inflammasome-mediated immune responses in vivo.

Human α-Defensins Inhibit BK Virus Infection by Aggregating Virions and Blocking Binding to Host Cells

BK virus (BKV) is a polyomavirus that establishes a lifelong persistence in most humans and is a major impediment to success of kidney grafts. The function of the innate immune system in BKV infection and pathology has not been investigated. Here we examine the role of antimicrobial defensins in BKV infection of Vero cells. Our data show that α-defensin human neutrophil protein 1 (HNP1) and human α-defensin 5 (HD5) inhibit BKV infection by targeting an early event in the viral lifecycle. HD5 treatment of BKV reduced viral attachment to cells, whereas cellular treatment with HD5 did not. Colocalization studies indicated that HD5 interacts directly with BKV. Ultrastructural analysis revealed HD5-induced aggregation of virions. HD5 also inhibited infection of cells by other related polyomaviruses. This is the first study to demonstrate polyomavirus sensitivity to defensins. We also show a novel mechanism whereby HD5 binds to BKV leading to aggregation of virion particles preventing normal virus binding to the cell surface and uptake into cells.

Direct Correlation between Sialic Acid Binding and Infection of Cells by Two Human Polyomaviruses (JC Virus and BK Virus)

ABSTRACT For the human polyomaviruses JC virus (JCV) and BK virus (BKV), the first step to a successful infection involves binding to sialic acid moieties located on the surfaces of host cells. By stripping and then reconstituting specific sialic acid linkages on host cells, we show that JCV uses both α(2,3)-linked and α(2,6)-linked sialic acids on N-linked glycoproteins to infect cells. For both JCV and BKV, the sialic acid linkages required for cell surface binding directly correlate with the linkages required for infection. In addition to sialic acid linkage data, these data suggest that the third sugar from the carbohydrate chain terminus is important for virus binding and infection.

Update on BK virus entry and intracellular trafficking

Abstract: BK virus (BKV) is a small, non‐enveloped, double‐stranded DNA virus and a member of the Polyomaviridae family. As the recently recognized etiologic agent of polyomavirus‐associated nephropathy, the events involved in BKV invasion of host cells are an important area of study. Using cell culture models, the mechanism by which BKV infects permissive hosts to gain access to the replication machinery within these cells is beginning to unfold. BKV uses an N‐linked glycoprotein containing an α(2,3)‐linked sialic acid as a receptor. After this initial attachment, BKV enters cells through caveolae‐mediated endocytosis. Intracellular trafficking via cellular cytoskeletal components follows this relatively slow and cholesterol‐dependent internalization. BKV must reach the nucleus for viral transcription and replication to occur. Elucidating the steps of the early viral lifecycle would provide clues to help explain the infectious spread and pathology of this human pathogen.

Identification of Amino Acid Residues in BK Virus VP1 That Are Critical for Viability and Growth

ABSTRACT BK virus (BKV) is a ubiquitous pathogen that establishes a persistent infection in the urinary tract of 80% of the human population. Like other polyomaviruses, the major capsid protein of BKV, virion protein 1 (VP1), is critical for host cell receptor recognition and for proper virion assembly. BKV uses a carbohydrate complex containing α(2,3)-linked sialic acid attached to glycoprotein and glycolipid motifs as a cellular receptor. To determine the amino acids important for BKV binding to the sialic acid portion of the complex, we generated a series of 17 point mutations in VP1 and scored them for viral growth. The first set of mutants behaved identically to wild-type virus, suggesting that these amino acids were not critical for virus propagation. Another group of VP1 mutants rendered the virus nonviable. These mutations failed to protect viral DNA from DNase I digestion, indicating a role for these domains in capsid assembly and/or packaging of DNA. A third group of VP1 mutations packaged DNA similarly to the wild type but failed to propagate. The initial burst size of these mutations was similar to that of the wild type, indicating that there is no defect in the lytic release of the mutated virions. Binding experiments revealed that a subset of the BKV mutants were unable to attach to their host cells. These motifs are likely important for sialic acid recognition. We next mapped these mutations onto a model of BKV VP1 to provide atomic insight into the role of these sites in the binding of sialic acid to VP1.

The amoebal MAP kinase response to Legionella pneumophila is regulated by DupA.

The amoeba Dictyostelium discoideum can support replication of Legionella pneumophila. Here we identify the dupA gene, encoding a putative tyrosine kinase/dual-specificity phosphatase, in a screen for D. discoideum mutants altered in allowing L. pneumophila intracellular replication. Inactivation of dupA resulted in depressed L. pneumophila growth and sustained hyperphosphorylation of the amoebal MAP kinase ERK1, consistent with loss of a phosphatase activity. Bacterial challenge of wild-type amoebae induced dupA expression and resulted in transiently increased ERK1 phosphorylation, suggesting that dupA and ERK1 are part of a response to bacteria. Indeed, over 500 of the genes misregulated in the dupA(-) mutant were regulated in response to L. pneumophila infection, including some thought to have immune-like functions. MAP kinase phosphatases are known to be highly upregulated in macrophages challenged with L. pneumophila. Thus, DupA may regulate a MAP kinase response to bacteria that is conserved from amoebae to mammals.

The role of sialic acid in human polyomavirus infections.

JC virus (JCV) and BK virus (BKV) are human polyomaviruses that infect approximately 85% of the population worldwide [1,2]. JCV is the underlying cause of the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML), a condition resulting from JCV induced lytic destruction of myelin producing oligodendrocytes in the brain [3]. BKV infection of kidneys in renal transplant recipients results in a gradual loss of graft function known as polyomavirus associated nephropathy (PVN) [4]. Following the identification of these viruses as the etiological agents of disease, there has been greater interest in understanding the basic biology of these human pathogens [5,6]. Recent advances in the field have shown that viral entry of both JCV and BKV is dependent on the ability to interact with sialic acid. This review focuses on what is known about the human polyomaviruses and the role that sialic acid plays in determining viral tropism.

Modulation of PML protein expression regulates JCV infection.

JC virus (JCV) is a human polyomavirus that infects the majority of the human population worldwide. It is responsible for the fatal demyelinating disease Progressive Multifocal Leukoencephalopathy. JCV binds to cells using the serotonin receptor 5-HT(2A)R and alpha(2-6)- or alpha(2-3)-linked sialic acid. It enters cells using clathrin-dependent endocytosis and traffics to the early endosome and possibly to the endoplasmic reticulum. Viral DNA is then delivered to the nucleus where transcription, replication, and assembly of progeny occur. We found that the early regulatory protein large T antigen accumulates in microdomains in the nucleus adjacent to ND-10 or PML domains. This observation prompted us to explore the role of these domains in JCV infection. We found that a reduction of nuclear PML enhanced virus infection and that an increase in nuclear PML reduced infection. Infection with JCV did not directly modulate nuclear levels of PML but our data indicate that a host response involving interferon beta is likely to restrict virus infection by increasing nuclear PML.

Transcriptional Regulation of BK Virus by Nuclear Factor of Activated T Cells

ABSTRACT The human polyomavirus BK virus (BKV) is a common virus for which 80 to 90% of the adult population is seropositive. BKV reactivation in immunosuppressed patients or renal transplant patients is the primary cause of polyomavirus-associated nephropathy (PVN). Using the Dunlop strain of BKV, we found that nuclear factor of activated T cells (NFAT) plays an important regulatory role in BKV infection. Luciferase reporter assays and chromatin immunoprecipitation assays demonstrated that NFAT4 bound to the viral promoter and regulated viral transcription and infection. The mutational analysis of the NFAT binding sites demonstrated complex functional interactions between NFAT, c-fos, c-jun, and the p65 subunit of NF-κB that together influence promoter activity and viral growth. These data indicate that NFAT is required for BKV infection and is involved in a complex regulatory network that both positively and negatively influences promoter activity and viral infection.