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Biomedical subjects

W J Atwood

Publications and source records attributed to W J Atwood.

At least 19 recordsLinked to original sources
PubMed2006

The human polyomaviruses.

The Polyomavirus family includes two members, BK virus (BKV) and JC virus (JCV), that naturally infect humans. These viruses are widely distributed among the population worldwide. Primary infection occurs in early childhood and remains for life clinically unapparent in immunocompetent individuals. In the context of severe immunosuppression and other predisposing factors BKV and JCV may reactivate and cause serious illnesses known as Polyomavirus-induced nephropathy and progressive multifocal leukoencephalopathy, respectively. Here we briefly examine the biological and physical characteristics and the lifecycle, namely receptor(s) interaction, mode of entry, intracellular trafficking, viral transcription and replication, and progeny assembly of these two human Polyomaviruses. We also provide an overview of the clinical manifestation of Polyomavirus-induced disorders in affected individuals and discuss the potential involvement of BKV and JCV in human cancer.

BK Virus↗
PubMed2006

Update on BK virus entry and intracellular trafficking.

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 alpha(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.

Animals↗
PubMed2006

Invasion of host cells by JC virus identifies a novel role for caveolae in endosomal sorting of noncaveolar ligands.

Invasion of glial cells by the human polyomavirus, JC virus (JCV), leads to a rapidly progressing and uniformly fatal demyelinating disease known as progressive multifocal leukoencephalopathy. The endocytic trafficking steps used by JCV to invade cells and initiate infection are not known. We demonstrated that JCV infection was inhibited by dominant defective and constitutively active Rab5-GTPase mutants that acted at distinct steps in endosomal sorting. We also found that labeled JCV colocalized with labeled cholera toxin B and with caveolin-1 (cav-1) on early endosomes following internalization by clathrin-dependent endocytosis. JCV entry and infection were both inhibited by dominant defective mutants of eps15 and Rab5-GTPase. Expression of a dominant-negative scaffolding mutant of cav-1 did not inhibit entry or infection by JCV. A single-cell knockdown experiment using cav-1 shRNA did not inhibit JCV entry but interfered with a downstream trafficking event important for infection. These data show that JCV enters cells by clathrin-dependent endocytosis, is transported immediately to early endosomes, and is then sorted to a caveolin-1-positive endosomal compartment. This latter step is dependent on Rab5-GTPase, cholesterol, caveolin-1, and pH. This is the first example of a ligand that enters cells by clathrin-dependent endocytosis and is then sorted from early endosomes to caveosomes, indicating that caveolae-derived vesicles play a more important role than previously realized in sorting cargo from early endosomes.

Biomarkers↗
PubMed2006

JC virus minor capsid proteins Vp2 and Vp3 are essential for virus propagation.

Virus-encoded capsid proteins play a major role in the life cycles of all viruses. The JC virus capsid is composed of 72 pentamers of the major capsid protein Vp1, with one of the minor coat proteins Vp2 or Vp3 in the center of each pentamer. Vp3 is identical to two-thirds of Vp2, and these proteins share a DNA binding domain, a nuclear localization signal, and a Vp1-interacting domain. We demonstrate here that both the minor proteins and the myristylation site on Vp2 are essential for the viral life cycle, including the proper packaging of its genome.

Base Sequence↗
PubMed2001

HIV type 1 infection of human astrocytes is restricted by inefficient viral entry.

The mechanism by which HIV infects astrocytes is not known. We used the simian virus 40 (SV40)-transformed human astrocyte cell line, SVG-A, to investigate HIV infection of astrocytes. We previously reported that SVG-A cells are susceptible to low levels of CD4/CXCR4-independent infection by an X4 strain of HIV-1. Infection was greatly increased when the prototypical X4 receptors, CD4 and CXCR4, were expressed on the SVG-A cells (SVGCD4-X4). These data suggest that HIV-1 enters astrocytes by a novel mechanism that is inefficient compared with CD4/CXCR4-mediated entry. In this article, we report high levels of early viral gene expression in both SVG-A and SVGCD4-X4 cells once the HIV entry pathway is circumvented. These data indicate that HIV-1 infection of SVG-A cells is restricted by inefficient viral entry rather than by post-entry events. As we were unable to detect infection of nontransformed primary astrocytes, we investigated whether SV40 transformation affects the susceptibility of astrocytes to HIV infection. To study this, we transformed primary fetal and adult astrocytes with the same origin-defective SV40 mutant that was used to transform the SVG-A cell line. We found that SV40 transformation did not alter the susceptibility of astrocytes to HIV infection. Furthermore, high levels of early viral gene expression were detected in these cells once the HIV entry process was by-passed. Taken together, the results of these studies indicate that HIV infection of human astrocytes is restricted by inefficient viral entry.

Astrocytes↗
PubMed2001

CD4/CXCR4-independent infection of human astrocytes by a T-tropic strain of HIV-1.

HIV-1 establishes a low-level persistent infection in astrocytes. In this study, we studied the susceptibility of a human astrocyte cell line (SVG-A) to infection with luciferase expressing reporter viruses pseudotyped with envelopes derived from five isolates of HIV-1. SVG-A cells were susceptible to infection by a T-cell tropic isolate and the infection was both CD4 and CXCR4 independent. These data confirm the susceptibility of astrocytes to infection with T-tropic strains of HIV-1 and suggest a novel mechanism by which T-tropic strains of HIV can infect cells.

AIDS Dementia Complex↗
PubMed2001

Virus receptors in the human central nervous system.

The initial event in the life cycle of a virus is its interaction with receptors present on the surface of a cell. Understanding these interactions is important to our understanding of viral tropism, spread, and pathogenesis. This is particularly true of viruses that target the central nervous system as these viruses must maintain a tropism for both the nervous system and for peripheral organs that allow for viral replication and spread to new susceptible hosts. These viruses therefore interact with a diverse set of cells and tissues, interactions that are likely mediated by both common and unique receptors present on each target tissue. In addition, physiological changes in the host can lead to increased or decreased expression of virus receptors, which influence virus trafficking, spread, and tissue specific pathology. This review will focus on the relatively few virus receptor systems that have been described in some level of detail for viruses that target the human central nervous system.

Central Nervous System↗
PubMed2001

A combination of low-dose chlorpromazine and neutralizing antibodies inhibits the spread of JC virus (JCV) in a tissue culture model: implications for prophylactic and therapeutic treatment of progressive multifocal leukencephalopathy.

The human polyomavirus, JCV, is the etiologic agent of a fatal central nervous system (CNS) demyelinating disease known as progressive multifocal leukoencephalopathy (PML). PML occurs predominantly in immunosuppressed patients and remains an intractable complication in AIDS. To date, there are no effective therapies to treat PML. We previously demonstrated that the neuroleptic drug, chlorpromazine, inhibits the endocytic pathway used by JCV to infect glial cells. In this paper, we demonstrate that nontoxic doses of chlorpromazine are effective at inhibiting JCV multiplication and spread in a tissue culture model. The clinical efficacy of this drug or related compounds in treating PML has not been evaluated.

Antibodies, Viral↗
PubMed2000

JC virus enters human glial cells by clathrin-dependent receptor-mediated endocytosis.

The human polyomavirus JC virus (JCV) is the etiologic agent of a fatal central nervous system (CNS) demyelinating disease known as progressive multifocal leukoencephalopathy (PML). PML occurs predominantly in immunosuppressed patients and has increased dramatically as a result of the AIDS pandemic. The major target cell of JCV infection and lytic replication in the CNS is the oligodendrocyte. The mechanisms by which JCV initiates and establishes infection of these glial cells are not understood. The initial interaction between JCV and glial cells involves virus binding to N-linked glycoproteins containing terminal alpha(2-6)-linked sialic acids. The subsequent steps of entry and targeting of the viral genome to the nucleus have not been described. In this report, we compare the kinetics and mechanisms of infectious entry of JCV into human glial cells with that of the related polyomavirus, simian virus 40 (SV40). We demonstrate that JCV, unlike SV40, enters glial cells by receptor-mediated clathrin-dependent endocytosis.

Cell Line↗
PubMed2000

JC virus binds to primary human glial cells, tonsillar stromal cells, and B-lymphocytes, but not to T lymphocytes.

The human polyomavirus, JCV, is the etiological agent of the fatal central nervous system demyelinating disease, progressive multifocal leukoencephalopathy (PML). In PML patients, JC Virus (JCV) can be detected in glial cells in the central nervous system (CNS); in B-lymphocytes in the peripheral blood, bone marrow, spleen, and tonsil; and in tonsillar stromal cells. In vitro, JCV infects glial cells, tonsillar stromal cells, and to a limited extent B-lymphocytes. The presence or absence of as yet unidentified cell type specific transcription factors contributes to the restricted tropism of JCV for these cell types. However, several studies indicate that cell surface receptors may also contribute to the limited host range of JCV. To examine this latter possibility we measured the binding of purified JCV virions to primary cultures of glial cells, tonsillar stromal cells, peripheral blood lymphocytes, and to several established cell lines. Our results demonstrate that JCV binds to primary glial cells, stromal cells, and B cells, but does not bind to primary T cells. In contrast, JCV bound to all cell lines tested, including the Namalwa B cell line and the Jurkat T cell line. These data are novel and demonstrate that JCV selectively interacts with cells in vivo that are known to be susceptible to infection. This selectivity appears to be lost when one examines virus binding to a variety of human, monkey, or mouse tumor cell lines. We next examined the susceptibility of primary peripheral blood lymphocytes and the Namalwa B cell line to infection with JCV. Our results demonstrate that the majority of infectious JCV virions remain cell surface associated and do not efficiently establish infection of B cells. This may explain the in vivo observation that JCV DNA is frequently detected in association with lymphocytes by PCR but that JCV mRNA is rarely detected in association with lymphocytes by reverse transcriptase PCR. These results also confirm previous data regarding the association of JCV with human B cells in vivo and support the hypothesis that B cells may be involved in trafficking of JCV to the CNS.

3T3 Cells↗
PubMed1998

Infection of glial cells by the human polyomavirus JC is mediated by an N-linked glycoprotein containing terminal alpha(2-6)-linked sialic acids.

The human JC polyomavirus (JCV) is the etiologic agent of the fatal central nervous system (CNS) demyelinating disease progressive multifocal leukoencephalopathy (PML). PML typically occurs in immunosuppressed patients and is the direct result of JCV infection of oligodendrocytes. The initial event in infection of cells by JCV is attachment of the virus to receptors present on the surface of a susceptible cell. Our laboratory has been studying this critical event in the life cycle of JCV, and we have found that JCV binds to a limited number of cell surface receptors on human glial cells that are not shared by the related polyomavirus simian virus 40 (C. K. Liu, A. P. Hope, and W. J. Atwood, J. Neurovirol. 4:49-58, 1998). To further characterize specific JCV receptors on human glial cells, we tested specific neuraminidases, proteases, and phospholipases for the ability to inhibit JCV binding to and infection of glial cells. Several of the enzymes tested were capable of inhibiting virus binding to cells, but only neuraminidase was capable of inhibiting infection. The ability of neuraminidase to inhibit infection correlated with its ability to remove both alpha(2-3)- and alpha(2-6)-linked sialic acids from glial cells. A recombinant neuraminidase that specifically removes the alpha(2-3) linkage of sialic acid had no effect on virus binding or infection. A competition assay between virus and sialic acid-specific lectins that recognize either the alpha(2-3) or the alpha(2-6) linkage revealed that JCV preferentially interacts with alpha(2-6)-linked sialic acids on glial cells. Treatment of glial cells with tunicamycin, but not with benzyl N-acetyl-alpha-D-galactosaminide, inhibited infection by JCV, indicating that the sialylated JCV receptor is an N-linked glycoprotein. As sialic acid containing glycoproteins play a fundamental role in mediating many virus-cell and cell-cell recognition processes, it will be of interest to determine what role these receptors play in the pathogenesis of PML.

Acetylgalactosamine↗
PubMed1998

The human polyomavirus, JCV, does not share receptor specificity with SV40 on human glial cells.

The initial event in the life cycle of a virus is its interaction with specific receptors present on the surface of a cell. Understanding these interactions is important to our understanding of viral tropism and tissue specific pathology associated with viral disease. The human polyomavirus, JCV, is the etiological agent of the fatal central nervous system (CNS) demyelinating disease, progressive multifocal leukoencephalopathy (PML). PML is the direct result of JCV infection of oligodendrocytes, the myelin producing cell in the CNS. In vivo, JCV can be detected in oligodendrocytes, astrocytes, lymphoid tissue, and peripheral blood of PML patients. In vitro, JCV infects human glial cells, tonsilar stromal cells, and, to a limited extent, human B lymphocytes. The initial step in infection of cells by JCV is at the level of attachment and entry. A specific cell surface receptor for JCV on human glial cells has not been identified. To begin to understand the nature of JCV receptors on human glial cells, large quantities of a previously characterized hybrid JC virus (Mad-1/SVEdelta) were purified. A direct virus binding assay demonstrated that these highly purified and labeled JCV virions bound to a finite number of cellular receptors on human glial cells. A competitive virus binding assay demonstrated that an excess of unlabeled JCV competed with labeled JCV more efficiently than did an excess of purified SV40. Furthermore, anti-class I antibodies which inhibited infection of glial cells by SV40 had no significant effect on infection by JCV. These results imply that JCV does not share receptor specificity with the related polyomavirus, SV40.

Animals↗
PubMed1996

JC virus infection of hematopoietic progenitor cells, primary B lymphocytes, and tonsillar stromal cells: implications for viral latency.

The human polyomavirus JC virus (JCV) infects myelin-producing cells in the central nervous system, resulting in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV-induced PML occurs most frequently in immunosuppressed individuals, with the highest incidence in human immunodeficiency type 1-infected patients, ranging between 4 and 6% of all AIDS cases. Although JCV targets a highly specialized cell in the central nervous system, infection is widespread, with more than 80% of the human population worldwide demonstrating serum antibodies. A number of clinical and laboratory studies have now linked the pathogenesis of PML with JCV infection in lymphoid cells. For example, JCV-infected lymphocytes have been suggested as possible carriers of virus to the brain following reactivation of a latent infection in lymphoid tissues. To further define the cellular tropism associated with JCV, we have attempted to infect immune system cells, including CD34+ hematopoietic progenitor cells derived from human fetal liver, primary human B lymphocytes, and human tonsillar stromal cells. Our results demonstrate that these cell types as well as a CD34+ human cell line, KG-1a, are susceptible to JCV infection. JCV cannot, however, infect KG-1, a CD34+ cell line which differentiates into a macrophage-like cell when treated with phorbol esters. In addition, peripheral blood B lymphocytes isolated by flow cytometry from a PML patient demonstrate JCV infection. These results provide direct evidence that JCV is not strictly neurotropic but can infect CD34+ hematopoietic progenitor cells and those cells which have differentiated into a lymphocytic, but not monocytic, lineage.

B-Lymphocytes↗
PubMed1995

Evaluation of the role of cytokine activation in the multiplication of JC virus (JCV) in human fetal glial cells.

The human polyomavirus, JCV, is the etiologic agent of the fatal central nervous system demyelinating disease, progressive multifocal leukoencephalopathy. Progressive multifocal leukoencephalopathy occurs most frequently in patients with underlying immunosuppressive disorders and is the direct result of virus multiplication in oligodendrocytes, the myelin producing cell in the central nervous system. In this report we test the ability of cellular activation signals to modulate expression of the JCV genome in either transfected or infected human fetal glial cells. In addition, we analyze the binding of nuclear proteins isolated from untreated and cytokine treated human fetal glial cells to transcription factor binding sites in the JCV regulatory region. In contrast to the effects of cellular activation on the expression of the HIV-1 promoter in these cells, none of the cellular activators tested increased expression of JCV. The cytokine, TNF-alpha, increased binding of NF kappa B (p50/p65) to a JC NF kappa B site but did not modulate the binding of nuclear proteins to the overlapping NF-1/AP1 region of the JCV enhancer. When taken together these results suggest that the response of JCV to cellular activation signals may be fundamentally different from the response of HIV-1 to these signals in human fetal glial cells and that the JC NF kappa B site may not be required for JCV gene expression or multiplication in vivo.

Antibody Specificity↗
PubMed1994

An increase in p50/p65 NF-kB binding to the HIV-1 LTR is not sufficient to increase viral expression in the primary human astrocyte.

Human astrocytes can be infected with HIV-1 both in vivo and in vitro. The amount of HIV-1 p24 structural protein production is low in comparison to that of the macrophage. Several weeks following infection or transfection, however, cocultivation with uninfected lymphocytes or stimulation with the cytokines TNF-alpha and IL 1-beta will increase viral production from this cell type. In the present study we demonstrate that phorbol 12-myristate 13-acetate (PMA) also increases HIV-1 p24 production from the primary human astrocyte. Using electrophoretic mobility shift assay (EMSA) in combination with supershift studies using specific antibodies, we demonstrate that PMA, like TNF-alpha, increases the p50/p65 form of NF-kB. Furthermore we demonstrate that the protein kinase inhibitor H7 inhibits PMA- and TNF-alpha-associated increases in HIV-1 expression at a time when it has little to no inhibitory effect on the associated increases in p50/p65 NF-kB. Thus, unless p50/p65 NF-kB or its binding is affected by H7 in a manner that cannot be resolved by EMSA, an increase in this form of NF-kB is not always sufficient to increase HIV-1 expression from the astrocyte.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Stimulation of HIV type 1 gene expression and induction of NF-kappa B (p50/p65)-binding activity in tumor necrosis factor alpha-treated human fetal glial cells.

In vitro, HIV-1 infection of human fetal glial cells initiates a noncytopathic, productive infection that results in a long-term persistence during which the viral genome remains latent. The cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) reactivate HIV-1 gene expression in these cells, leading to production of infectious virus. Here we show that treatment of human fetal glial cells with TNF-alpha and IL-1 beta increase expression of the reporter gene chloramphenicol acetyltransferase (CAT) when placed under the control of the HIV-1 5' LTR. We also show that treatment of human fetal glial cells with TNF-alpha leads to increased binding of the nuclear transcription factor NF-kappa B (p50/p65) to a consensus kappa B-binding site present in the HIV-1 5'LTR. Our results suggest that TNF-alpha stimulation of HIV-1 gene expression in primary cultures of human fetal glial cells is mediated by an increase in binding of NF-kappa B (p50/p65) to the HIV-1 LTR. This is the first report documenting NF-kappa B-binding activity in primary cultures of human fetal glial cells.

Base Sequence↗