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Mechanism of white matter damage caused by virus infection
International Congress Series 1251 (2003) 139 – 147
Mechanism of white matter damage caused by virus infection Shingo Semba, Hirofumi Sawa, Kazuo Nagashima * Laboratory of Molecular and Cellular Pathology, Hokkaido University School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo 060-8638, Japan Received 13 December 2002; accepted 3 February 2003
Abstract White matter can be damaged by numerous causes resulting in various neurological deficits. The investigation of white matter damage associated with viruses can be simplified by studying JC virus infection of the central nervous system, which is known as progressive multifocal leukoencephalopathy (PML). JC virus capsid protein VP1 recognizes sialylated molecules of cell membrane and enters into various cells including neural and non-neural cells of humans, monkeys and rodents. The incorporated virus particle is transported along a clathrin pathway to the nucleus where the coat is removed. One of the cell-specific transcriptional factors, p75, may promote viral transcription, and the viral proteins are then transcribed. These findings may clarify the mechanism of viral neurotropism and provide basic knowledge for therapy of neurological disease. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Viral neurotropism; JC virus; Viral receptor; Transcriptional factor
1. Introduction The mechanism of white matter diseases associated with viral infection is difficult to study if an immune reaction has occurred. To avoid this complexity, we have chosen JC virus infection of the central nervous system (CNS) which is thought to cause demyelination by direct viral attack on oligodendrocyte. JC virus infects oligodendrocyte, affects myelination, and causes the demyelinating disease, ‘‘progressive multifocal leukoencephalopathy (PML)’’. However, the mechanism by which JC virus selectively infects
* Corresponding author. Tel.: +81-11-706-5052; fax: +81-11-706-7806. E-mail address: [email protected] (K. Nagashima). 0531-5131/03 D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0531-5131(03)00105-5
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human oligodendrocyte remains unknown. In this article, we would like to focus on the mechanism of viral neurotropism or viral affinity to certain cells. In general, there are several steps in the process by which viruses replicate in cells, beginning with viral attachment to the cell membrane, incorporation into cytoplasm, transport to the nucleus, entry into the nucleus and viral replication, and resulting in the virus spreading to other cells. Initial attachment of the virus to cells is restricted by specific receptors, which will be discussed first. Next, the mechanism of JCV entry and transport is presented, and finally, we will describe the nuclear factors responsible for JCV replication.
2. Viral neurotropism and receptors The major virus receptors established to date are listed in Table 1 [1– 10]. Poliovirus mainly affects human motor neurons and causes poliomyelitis. Taking advantage of the fact that the virus specifically infects primates, the poliovirus receptor has been isolated, and its structural similarity to immunoglobulin established [1]. The receptor is widely expressed in human tissues other than motor neurons, and hence, the receptor is not associated with neurotropism. However, when the poliovirus infected a transgenic mouse expressing the poliovirus receptor, viral replication occurred in the motor neuron, causing paralysis similar to human disease [11]. These results indicate that poliovirus neurotropism is not only defined by the membrane receptor but also by other factors relating to a post-infectional regulation. The measles virus receptor CD46 has been used for Edmonston strain in experimental examination, but the wild type measles virus has not been examined [2]. A receptor for wild type measles virus, however, has recently been identified as a signaling lymphocyteactivation molecule (SLAM, CDw150) [3]. The results indicate that the virus receptors are different for each strain of the same virus group. By contrast, herpes simplex virus (HSV) and cytomegalovirus (CMV) are two different viruses sharing the same molecule of heparin sulfate as receptor [5,6], but HSV affects neuron, glia and endothelium, causing necrotizing encephalitis, while CMV usually affects astrocytes in the subependymal layer. This difference may depend on the mechanism of post-internalized pathways. The receptor for human immunodeficiency virus type I (HIV-I) was first identified as CD4 [8], but later Table 1 Receptors for selected neurotropic viruses Family
Species
Receptor
References
Picornaviridae Myxoviridae
Poliovirus Measles virus
Poliovirus receptor CD46 (Edmonston strain) SLAM (clinical sample) Sialic acid Heparan sulfate Heparan sulfate Gangliosides CD4 Chemokine receptor Sialic acid
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
Rhabdoviridae Retroviridae
Influenza A, B HSV-1 CMV Rabies virus HIV-1
Polyomaviridae
JC virus
Herpesviridae
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a human chemokine receptor was also identified as a co-receptor [9]. HIV-1 can enter cells expressing these two receptor molecules, but so far has not produced viral progenies, further necessitating human cyclin T [12]. However, the mouse cell expressing all these molecules has still not resulted in HIV-I proliferation. Thus, we conclude that several additional molecules may be required for HIV-I proliferation for rodent cells.
3. JC virus receptor and entry JC virus is a DNA virus, the nucleic acid in a double-stranded circular form, consisting of 5130 bp [13]. As an early protein, it encodes small t and large T proteins by splicing, and these proteins are known to be important for DNA replication. As a late protein, it encodes VP1, VP2/3 and agnoprotein. VP1 and VP2/3 constitute a viral capsid structure and is a structural protein. The viral regulatory region functions as a promoter and enhancer, and it is particularly important to define cell tropism. To investigate the early viral attachment of JC virus infection, we have analyzed the susceptibility of 16 different cell lines, which include human, monkey and mouse cells derived from neural and non-neural tissues (Table 2). Among these cell lines, only IMR-32 is known to be particularly susceptible for viral replication, while SH-EP and COS-7 cells are slightly susceptible. By using a semi-quantitative PCR assay of cellular DNAs for detection of JC virus DNA, it was shown that within 10 min following incubation with the JC virus to IMR-32 cells, the viral genome was detected, and the signal intensity of the PCR products increased in a time-dependent manner, while the signal intensity of h-globin remained constant (Fig. 1, upper left panel). As with the results obtained in the human neural cells, JC virus genome was detected in cytoplasmic and nuclear portions of U-138MG Table 2 List of analyzed cell lines and their origins Cell type
Origin
IMR-32 SH-EP 293T
neuroblastoma, human neuroblastoma, human kidney cells, expressing SV40 large T antigen, human kidney cells, human SV40 transformed kidney cells, monkey kidney cells, monkey glioblastoma, human glioblastoma, human hepatoblastoma, human oligodendrocyte, human melanoma, human lung carcinoma, human cervix carcinoma, human colon carcinoma, human neuroblastoma, mouse neuroblastoma, mouse
HEK293 COS-7 CV-1 U-138 MG U-87MG Hep G2 OL MeWo A-549 HeLa SW480 Neuro-2a NIE-115
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Fig. 1. Results of a PCR-based JC virus entry assay of cellular DNAs for the detection of JC virus DNA. Upper left panel: Detection of the viral genome 10 min after incubation with the JC virus to IMR-32 cells. Note that the signal intensity increased in a time-dependent manner, while that of h-globin remained constant. Upper right panel: Detectable level standard of JC virus; 0.001 hemagglutination units and 103 copies of JC virus DNA. Lower panel: Presence of JC virus DNA in a permissive cell (IMR-32) as well as non-permissive cells (U-138MG, HeLa, and SW480). Detection of JC virus DNA in both cytoplasmic and nuclear fractions in a timedependent manner. C: cytoplasma; N: nucleus; JCT: JC virus genome targeted to large T region; COI: cytochrome oxidase I used for PCR internal positive control of cytoplasmic DNA (mitochondria); h-globin: positive control of nuclear DNA.
glioblastoma cells, HeLa cells and SW480 colonic cells, all of which are non-susceptible to JC virus infection (Fig. 2 lower panel). These results demonstrated that the JC virus receptor is widely expressed in various cells [14]. In order to obtain direct evidence of viral entry, we performed in situ hybridization. Immediately after virus inoculation, no signals were detected. At 10 min after the inoculation, however, the viral DNA was detected inside the cells of not only susceptible cell line SH-EP, but also non-permissive cell lines A-549 or HeLa [14]. This clearly confirmed the results of PCR experiments. As for the viral factors for membrane attachment, we investigated the function of viral capsid protein VP1. When we incubated JC virus with anti-VP1 antibody, the virus could not enter various cell types. In contrast, transferrin can enter the cell even after treatment with VP1-antibody. The results indicate that viral capsid protein VP1 is used for cellular attachment and entry into the cell. A similar inhibition of viral entry was observed when we treated cells with sialidase [10]. Hence, cell attachment is initiated by interaction of the viral capsid protein VP1 with cellular sialylated molecules [10].
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Fig. 2. Electromobility shift assay (EMSA) and competitive assay using JC virus regulatory sequences and nuclear lysates of various cells. Left panel: A part of DNA sequences selected for binding sites for transcriptional factors. Right panel: Confirmation of the specific binding site of #3 sequence (control) by reaction with abundant cold DNA probes. Inhibition was found only by #3 probes, but not by #1 or #5.
To identify the widely distributed membrane molecules for the virus entry, we developed virus-like particles (VLP), a recombinant virus particle, and performed an overlay assay using thin layer chromatography. VLP was shown to bind sialoglycoproteins, which depended on a2 – 3-linked sialic acids and N-linked sugar chains. We generated several neoglycoproteins by attaching various terminal sialic acids to albumin. The neoglycoproteins containing the terminal a2 –6-linked sialic acid had highest affinity with VLP, and inhibited hemagglutination activity of VLP and JCV, and attachment of VLP to cells. Furthermore, the neoglycoprotein and ganglioside, GT1b, inhibited JCV infection in the susceptible cell line IMR-32. These results suggest that the oligosaccharides of glycolipids and glycoproteins function as JCV receptor [15].
4. JC virus trafficking and uncoating In the cytoplasm, there are two pathways for molecular transport: the caveolin pathway and the clathrin pathway [16]. Chlorpromazine is an inhibitor of the clathrin pathway. When virus-infected cells were treated with chlorpromazine, the JC virus signal disappeared in the nuclear fraction. Similarly, after treatment with chlorpromazine, both VLP and transferrin were not found in the cytoplasm, but remained on the cell membrane. The results suggest that the virus particles are transported in the cytoplasm using a clathrinmediated endocytosis pathway to the nucleus [14].
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To examine the site where the viral coat is removed after infection, we applied DNase I treatment. In this experiment, naked viral DNA can be digested with DNase I, while coated viral DNA is resistant. JC virus DNA was resistant to DNase I both in the cytoplasm and nucleus, while naked DNA of plasmid pBRMad1 used for positive control disappeared after DNase I treatment. However, when the virus particles were disrupted by hypertonic solution, such as dithiothreitol (DTT), JC virus DNA became undetectable. From these experiments, it is shown that JC virus enters into the nucleus as an uncoated particle [14]. Subsequently, we examined whether the uncoated virus can replicate in the nuclei in the various cells. Out of 16 cell lines examined (Table 2), only IMR-32 cells provided a replication site for the virus, and the virus proliferation could be recorded from 20 days onward. These data suggest that the virus can enter the various cell lines, but the virus can replicate only in the human neural cell line IMR-32 [14].
5. Nuclear transcriptional factors The next question is how does virus replication commence in the nuclei in the IMR-32 cells. Cell-specific transcriptional factors are believed to regulate the specificity of JC virus infection in oligodendrocytes of the PML brain. The binding regions of such transcriptional factors are located in the regulatory region of JC virus [17], and are completely different from those of other polyomaviruses including SV40 and BK virus [13]. The regulatory region of the prototype JC virus consists of the 98-bp tandem repeat. However, the regulatory regions are variable, and for further analyses, we chose the shortest DNA sequences isolated from the JCV producing cell line, JCI cells, which is a sequence approximately 290 bp long. We divided the 290-bp region into nine sub-fragments each with a length of 40 bp. These nine sub-fragments labeled by 32P were incubated with nuclear lysates from the JCV permissive cell line IMR-32 and the non-permissive cell line HeLa, A549, SW480, and HepG2 cells, and the mixtures were subjected to an electromobility shift assay (EMSA) (Fig. 2, left panel). Among the shifted bands, the expected band should be found only in the nuclear lysate of IMR-32, but not in the nuclear lysate of non-neural cells. One of the candidates was found in the lysates hybridized with the #3 probe, and designated as #3-binding protein. In order to confirm the DNA binding specificity of the #3-binding protein, we performed a competitive assay using abundant cold DNA probes. After the reaction with the cold #3 probe, the protein – DNA complex reduced in intensity, indicating that the binding was inhibited, while the reaction with, for example, #1 or #5 probe was unchanged (Fig. 2, right panel). This means that the protein specifically bound to the #3 sequences of the JC virus regulatory region is found only in the virus permissive cell. These results suggest that #3 binding protein is one of the candidates for a specific transcriptional factor. In the previous reports, one of the transcriptional factors Oct-6 can bind the #3 sequences [18,19], but our EMSA assay failed to confirm its binding to #3 sequences. Using IMR-32 and HeLa nuclear lysates, two kinds of new proteins hybridized with #3 DNA were exclusively detected in IMR-32 cells after the mono-Q column purification at the concentration of 0.40 and 0.55 M of
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NaCl. Further UV cross-linking analysis revealed that these proteins consist of heterodimers. Finally, one of the molecules has been identified as a protein of 75 kDa, and we are presently attempting to establish this protein as a transcriptional factor by applying various functional assays.
6. JC virus life cycle In most viruses, neurotropism and the subsequent cellular tropism do not involve membrane receptors, but are determined by various unknown intracellular molecules. JC virus infects human oligodendrocyte causing demyelination. The virus has been shown to enter various types of cell, initiated by attachment of the viral capside protein VP1 to cellular membrane proteins of sialylated glycoproteins. The cytoplasmic viral particles are conveyed to the nucleus through a clathrin-dependent pathway. Although the question how the virus particles enter the nucleus requires further clarification, the viral coat may be removed and viral DNA exposed in the nucleus. However, the virus can proliferate exclusively in the nuclei of oligodendrocytes. The possible nuclear factors, which are essential for JC virus replication, have been identified. Using such transcriptional factors, the viral gene is transcribed and the viral proteins are translated with viral proteins being expressed in the nucleus and cytoplasm [20] (Fig. 3).
Fig. 3. Schematic illustration of JC virus life cycle, showing from the viral entry, trafficking and uncoating to transcription.
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The genes, which govern developmental processes of oligodendrocytes such as lineage commitment and cell type specification, have been identified by research in developmental neurobiology [21,22]. By contrast, our approach to identify nuclear factors from the study of JC virus is likely to result in the identification of further new molecules of the human brain. Acknowledgements This work has been supported by CREST (Core Research for Evolutional Science and Technology), Japan Science and Technology (JST). References [1] C.L. Mendelsohn, E. Wimmer, V.R. Racaniello, Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily, Cell 56 (1989) 855 – 865. [2] R.E. Dorig, A. Marcil, A. Chopra, C.D. Richardson, The human CD46 molecule is a receptor for measles virus (Edmonston strain), Cell 75 (1993) 295 – 305. [3] H. Tatsuo, N. Ono, K. Tanaka, Y. Yanagi, SLAM (CDw150) is a cellular receptor for measles virus, Nature 406 (2000) 893 – 897. [4] W. Weis, J.H. Brown, S. Cusack, J.C. Paulson, J.J. Skehel, D.C. Wiley, Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid, Nature 333 (1988) 426 – 431. [5] D. WuDunn, P.G. Spear, Initial interaction of herpes simplex virus with cells is binding to heparan sulfate, J. Virol. 63 (1989) 52 – 58. [6] T. Compton, D.M. Nowlin, N.R. Cooper, Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate, Virology 193 (1993) 834 – 841. [7] F. Superti, B. Hauttecoeur, M.J. Morelec, P. Goldoni, B. Bizzini, H. Tsiang, Involvement of gangliosides in rabies virus infection, J. Gen. Virol. 67 (1986) 47 – 56. [8] D. Klatzmann, E. Champagne, S. Chamaret, J. Gruest, D. Guetard, T. Hercend, J.C. Gluckman, L. Montagnier, T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV, Nature 312 (1984) 767 – 768. [9] H. Deng, R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. Di Marzio, S. Marmon, R.E. Sutton, C.M. Hill, C.B. Davis, S.C. Peiper, T.J. Schall, D.R. Littman, N.R. Landau, Identification of a major coreceptor for primary isolates of HIV-1, Nature 381 (1996) 661 – 666. [10] C.K. Liu, G. Wei, W.J. Atwood, Infection of glial cells by the human polyomavirus JC is mediated by an Nlinked glycoprotein containing terminal a(2 – 6)-linked sialic acids, J. Virol. 72 (1998) 4643 – 4649. [11] S. Koike, C. Taya, T. Kurata, S. Abe, I. Ise, H. Yonekawa, A. Nomoto, Transgenic mice susceptible to poliovirus, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 951 – 955. [12] P. Wei, M.E. Garber, S.M. Fang, W.H. Fischer, K.A. Jones, A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA, Cell 92 (1998) 451 – 462. [13] R.J. Frisque, G.L. Bream, M.T. Cannella, Human polyomavirus JC virus genome, J. Virol. 51 (1984) 458 – 469. [14] S. Suzuki, H. Sawa, R. Komagome, Y. Orba, M. Yamada, Y. Okada, Y. Ishida, H. Nishihara, S. Tanaka, K. Nagashima, Broad distribution of the JC virus receptor contrasts with a marked cellular restriction of virus replication, Virology 286 (2001) 100 – 112. [15] R. Komagome, H. Sawa, T. Suzuki, Y. Suzuki, S. Tanaka, W.J. Atwood, K. Nagashima, Oligosaccharides as receptors for JC virus, J. Virol. 76 (2002) 12992 – 13000. [16] S. Kenney, V. Natarajan, D. Strike, G. Khoury, N.P. Salzman, JC virus enhancer – promoter active in human brain cells, Science 226 (1984) 1337 – 1339.
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[17] M.T. Pho, A. Ashok, W.J. Atwood, JC virus enters human glial cells by clathrin-dependent receptormediated endocytosis, J. Virol. 74 (2000) 2288 – 2292. [18] Y. Hara, A.C. Rovescalli, Y. Kim, M. Nirenberg, Structure and evolution of four POU domain genes expressed in mouse brain, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 3280 – 3284. [19] M. Wegner, D.W. Drolet, M.G. Rosenfeld, Regulation of JC virus by the POU-domain transcription factor Tst-1: implications for progressive multifocal leukoencephalopathy, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 4743 – 4747. [20] Y. Okada, H. Sawa, S. Endo, Y. Orba, T. Umemura, H. Nishihara, A.C. Stan, S. Tanaka, K. Nagashima, Expression of JC virus (JCV) agnoprotein in progressive multifocal leukoencephalopathy (PML) brain, Acta Neuropathol. 104 (2002) 130 – 136 (in press). [21] M. Wegner, Expression of transcription factors during oligodendroglial development, Microsc. Res. Tech. 52 (2001) 746 – 752. [22] J. Grinspan, Cells and signaling in oligodendrocyte development, J. Neuropathol. Exp. Neurol. 61 (2002) 297 – 306.
Mechanism of white matter damage caused by virus infection Shingo Semba, Hirofumi Sawa, Kazuo Nagashima * Laboratory of Molecular and Cellular Pathology, Hokkaido University School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo 060-8638, Japan Received 13 December 2002; accepted 3 February 2003
Abstract White matter can be damaged by numerous causes resulting in various neurological deficits. The investigation of white matter damage associated with viruses can be simplified by studying JC virus infection of the central nervous system, which is known as progressive multifocal leukoencephalopathy (PML). JC virus capsid protein VP1 recognizes sialylated molecules of cell membrane and enters into various cells including neural and non-neural cells of humans, monkeys and rodents. The incorporated virus particle is transported along a clathrin pathway to the nucleus where the coat is removed. One of the cell-specific transcriptional factors, p75, may promote viral transcription, and the viral proteins are then transcribed. These findings may clarify the mechanism of viral neurotropism and provide basic knowledge for therapy of neurological disease. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Viral neurotropism; JC virus; Viral receptor; Transcriptional factor
1. Introduction The mechanism of white matter diseases associated with viral infection is difficult to study if an immune reaction has occurred. To avoid this complexity, we have chosen JC virus infection of the central nervous system (CNS) which is thought to cause demyelination by direct viral attack on oligodendrocyte. JC virus infects oligodendrocyte, affects myelination, and causes the demyelinating disease, ‘‘progressive multifocal leukoencephalopathy (PML)’’. However, the mechanism by which JC virus selectively infects
* Corresponding author. Tel.: +81-11-706-5052; fax: +81-11-706-7806. E-mail address: [email protected] (K. Nagashima). 0531-5131/03 D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0531-5131(03)00105-5
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human oligodendrocyte remains unknown. In this article, we would like to focus on the mechanism of viral neurotropism or viral affinity to certain cells. In general, there are several steps in the process by which viruses replicate in cells, beginning with viral attachment to the cell membrane, incorporation into cytoplasm, transport to the nucleus, entry into the nucleus and viral replication, and resulting in the virus spreading to other cells. Initial attachment of the virus to cells is restricted by specific receptors, which will be discussed first. Next, the mechanism of JCV entry and transport is presented, and finally, we will describe the nuclear factors responsible for JCV replication.
2. Viral neurotropism and receptors The major virus receptors established to date are listed in Table 1 [1– 10]. Poliovirus mainly affects human motor neurons and causes poliomyelitis. Taking advantage of the fact that the virus specifically infects primates, the poliovirus receptor has been isolated, and its structural similarity to immunoglobulin established [1]. The receptor is widely expressed in human tissues other than motor neurons, and hence, the receptor is not associated with neurotropism. However, when the poliovirus infected a transgenic mouse expressing the poliovirus receptor, viral replication occurred in the motor neuron, causing paralysis similar to human disease [11]. These results indicate that poliovirus neurotropism is not only defined by the membrane receptor but also by other factors relating to a post-infectional regulation. The measles virus receptor CD46 has been used for Edmonston strain in experimental examination, but the wild type measles virus has not been examined [2]. A receptor for wild type measles virus, however, has recently been identified as a signaling lymphocyteactivation molecule (SLAM, CDw150) [3]. The results indicate that the virus receptors are different for each strain of the same virus group. By contrast, herpes simplex virus (HSV) and cytomegalovirus (CMV) are two different viruses sharing the same molecule of heparin sulfate as receptor [5,6], but HSV affects neuron, glia and endothelium, causing necrotizing encephalitis, while CMV usually affects astrocytes in the subependymal layer. This difference may depend on the mechanism of post-internalized pathways. The receptor for human immunodeficiency virus type I (HIV-I) was first identified as CD4 [8], but later Table 1 Receptors for selected neurotropic viruses Family
Species
Receptor
References
Picornaviridae Myxoviridae
Poliovirus Measles virus
Poliovirus receptor CD46 (Edmonston strain) SLAM (clinical sample) Sialic acid Heparan sulfate Heparan sulfate Gangliosides CD4 Chemokine receptor Sialic acid
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
Rhabdoviridae Retroviridae
Influenza A, B HSV-1 CMV Rabies virus HIV-1
Polyomaviridae
JC virus
Herpesviridae
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a human chemokine receptor was also identified as a co-receptor [9]. HIV-1 can enter cells expressing these two receptor molecules, but so far has not produced viral progenies, further necessitating human cyclin T [12]. However, the mouse cell expressing all these molecules has still not resulted in HIV-I proliferation. Thus, we conclude that several additional molecules may be required for HIV-I proliferation for rodent cells.
3. JC virus receptor and entry JC virus is a DNA virus, the nucleic acid in a double-stranded circular form, consisting of 5130 bp [13]. As an early protein, it encodes small t and large T proteins by splicing, and these proteins are known to be important for DNA replication. As a late protein, it encodes VP1, VP2/3 and agnoprotein. VP1 and VP2/3 constitute a viral capsid structure and is a structural protein. The viral regulatory region functions as a promoter and enhancer, and it is particularly important to define cell tropism. To investigate the early viral attachment of JC virus infection, we have analyzed the susceptibility of 16 different cell lines, which include human, monkey and mouse cells derived from neural and non-neural tissues (Table 2). Among these cell lines, only IMR-32 is known to be particularly susceptible for viral replication, while SH-EP and COS-7 cells are slightly susceptible. By using a semi-quantitative PCR assay of cellular DNAs for detection of JC virus DNA, it was shown that within 10 min following incubation with the JC virus to IMR-32 cells, the viral genome was detected, and the signal intensity of the PCR products increased in a time-dependent manner, while the signal intensity of h-globin remained constant (Fig. 1, upper left panel). As with the results obtained in the human neural cells, JC virus genome was detected in cytoplasmic and nuclear portions of U-138MG Table 2 List of analyzed cell lines and their origins Cell type
Origin
IMR-32 SH-EP 293T
neuroblastoma, human neuroblastoma, human kidney cells, expressing SV40 large T antigen, human kidney cells, human SV40 transformed kidney cells, monkey kidney cells, monkey glioblastoma, human glioblastoma, human hepatoblastoma, human oligodendrocyte, human melanoma, human lung carcinoma, human cervix carcinoma, human colon carcinoma, human neuroblastoma, mouse neuroblastoma, mouse
HEK293 COS-7 CV-1 U-138 MG U-87MG Hep G2 OL MeWo A-549 HeLa SW480 Neuro-2a NIE-115
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Fig. 1. Results of a PCR-based JC virus entry assay of cellular DNAs for the detection of JC virus DNA. Upper left panel: Detection of the viral genome 10 min after incubation with the JC virus to IMR-32 cells. Note that the signal intensity increased in a time-dependent manner, while that of h-globin remained constant. Upper right panel: Detectable level standard of JC virus; 0.001 hemagglutination units and 103 copies of JC virus DNA. Lower panel: Presence of JC virus DNA in a permissive cell (IMR-32) as well as non-permissive cells (U-138MG, HeLa, and SW480). Detection of JC virus DNA in both cytoplasmic and nuclear fractions in a timedependent manner. C: cytoplasma; N: nucleus; JCT: JC virus genome targeted to large T region; COI: cytochrome oxidase I used for PCR internal positive control of cytoplasmic DNA (mitochondria); h-globin: positive control of nuclear DNA.
glioblastoma cells, HeLa cells and SW480 colonic cells, all of which are non-susceptible to JC virus infection (Fig. 2 lower panel). These results demonstrated that the JC virus receptor is widely expressed in various cells [14]. In order to obtain direct evidence of viral entry, we performed in situ hybridization. Immediately after virus inoculation, no signals were detected. At 10 min after the inoculation, however, the viral DNA was detected inside the cells of not only susceptible cell line SH-EP, but also non-permissive cell lines A-549 or HeLa [14]. This clearly confirmed the results of PCR experiments. As for the viral factors for membrane attachment, we investigated the function of viral capsid protein VP1. When we incubated JC virus with anti-VP1 antibody, the virus could not enter various cell types. In contrast, transferrin can enter the cell even after treatment with VP1-antibody. The results indicate that viral capsid protein VP1 is used for cellular attachment and entry into the cell. A similar inhibition of viral entry was observed when we treated cells with sialidase [10]. Hence, cell attachment is initiated by interaction of the viral capsid protein VP1 with cellular sialylated molecules [10].
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Fig. 2. Electromobility shift assay (EMSA) and competitive assay using JC virus regulatory sequences and nuclear lysates of various cells. Left panel: A part of DNA sequences selected for binding sites for transcriptional factors. Right panel: Confirmation of the specific binding site of #3 sequence (control) by reaction with abundant cold DNA probes. Inhibition was found only by #3 probes, but not by #1 or #5.
To identify the widely distributed membrane molecules for the virus entry, we developed virus-like particles (VLP), a recombinant virus particle, and performed an overlay assay using thin layer chromatography. VLP was shown to bind sialoglycoproteins, which depended on a2 – 3-linked sialic acids and N-linked sugar chains. We generated several neoglycoproteins by attaching various terminal sialic acids to albumin. The neoglycoproteins containing the terminal a2 –6-linked sialic acid had highest affinity with VLP, and inhibited hemagglutination activity of VLP and JCV, and attachment of VLP to cells. Furthermore, the neoglycoprotein and ganglioside, GT1b, inhibited JCV infection in the susceptible cell line IMR-32. These results suggest that the oligosaccharides of glycolipids and glycoproteins function as JCV receptor [15].
4. JC virus trafficking and uncoating In the cytoplasm, there are two pathways for molecular transport: the caveolin pathway and the clathrin pathway [16]. Chlorpromazine is an inhibitor of the clathrin pathway. When virus-infected cells were treated with chlorpromazine, the JC virus signal disappeared in the nuclear fraction. Similarly, after treatment with chlorpromazine, both VLP and transferrin were not found in the cytoplasm, but remained on the cell membrane. The results suggest that the virus particles are transported in the cytoplasm using a clathrinmediated endocytosis pathway to the nucleus [14].
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To examine the site where the viral coat is removed after infection, we applied DNase I treatment. In this experiment, naked viral DNA can be digested with DNase I, while coated viral DNA is resistant. JC virus DNA was resistant to DNase I both in the cytoplasm and nucleus, while naked DNA of plasmid pBRMad1 used for positive control disappeared after DNase I treatment. However, when the virus particles were disrupted by hypertonic solution, such as dithiothreitol (DTT), JC virus DNA became undetectable. From these experiments, it is shown that JC virus enters into the nucleus as an uncoated particle [14]. Subsequently, we examined whether the uncoated virus can replicate in the nuclei in the various cells. Out of 16 cell lines examined (Table 2), only IMR-32 cells provided a replication site for the virus, and the virus proliferation could be recorded from 20 days onward. These data suggest that the virus can enter the various cell lines, but the virus can replicate only in the human neural cell line IMR-32 [14].
5. Nuclear transcriptional factors The next question is how does virus replication commence in the nuclei in the IMR-32 cells. Cell-specific transcriptional factors are believed to regulate the specificity of JC virus infection in oligodendrocytes of the PML brain. The binding regions of such transcriptional factors are located in the regulatory region of JC virus [17], and are completely different from those of other polyomaviruses including SV40 and BK virus [13]. The regulatory region of the prototype JC virus consists of the 98-bp tandem repeat. However, the regulatory regions are variable, and for further analyses, we chose the shortest DNA sequences isolated from the JCV producing cell line, JCI cells, which is a sequence approximately 290 bp long. We divided the 290-bp region into nine sub-fragments each with a length of 40 bp. These nine sub-fragments labeled by 32P were incubated with nuclear lysates from the JCV permissive cell line IMR-32 and the non-permissive cell line HeLa, A549, SW480, and HepG2 cells, and the mixtures were subjected to an electromobility shift assay (EMSA) (Fig. 2, left panel). Among the shifted bands, the expected band should be found only in the nuclear lysate of IMR-32, but not in the nuclear lysate of non-neural cells. One of the candidates was found in the lysates hybridized with the #3 probe, and designated as #3-binding protein. In order to confirm the DNA binding specificity of the #3-binding protein, we performed a competitive assay using abundant cold DNA probes. After the reaction with the cold #3 probe, the protein – DNA complex reduced in intensity, indicating that the binding was inhibited, while the reaction with, for example, #1 or #5 probe was unchanged (Fig. 2, right panel). This means that the protein specifically bound to the #3 sequences of the JC virus regulatory region is found only in the virus permissive cell. These results suggest that #3 binding protein is one of the candidates for a specific transcriptional factor. In the previous reports, one of the transcriptional factors Oct-6 can bind the #3 sequences [18,19], but our EMSA assay failed to confirm its binding to #3 sequences. Using IMR-32 and HeLa nuclear lysates, two kinds of new proteins hybridized with #3 DNA were exclusively detected in IMR-32 cells after the mono-Q column purification at the concentration of 0.40 and 0.55 M of
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NaCl. Further UV cross-linking analysis revealed that these proteins consist of heterodimers. Finally, one of the molecules has been identified as a protein of 75 kDa, and we are presently attempting to establish this protein as a transcriptional factor by applying various functional assays.
6. JC virus life cycle In most viruses, neurotropism and the subsequent cellular tropism do not involve membrane receptors, but are determined by various unknown intracellular molecules. JC virus infects human oligodendrocyte causing demyelination. The virus has been shown to enter various types of cell, initiated by attachment of the viral capside protein VP1 to cellular membrane proteins of sialylated glycoproteins. The cytoplasmic viral particles are conveyed to the nucleus through a clathrin-dependent pathway. Although the question how the virus particles enter the nucleus requires further clarification, the viral coat may be removed and viral DNA exposed in the nucleus. However, the virus can proliferate exclusively in the nuclei of oligodendrocytes. The possible nuclear factors, which are essential for JC virus replication, have been identified. Using such transcriptional factors, the viral gene is transcribed and the viral proteins are translated with viral proteins being expressed in the nucleus and cytoplasm [20] (Fig. 3).
Fig. 3. Schematic illustration of JC virus life cycle, showing from the viral entry, trafficking and uncoating to transcription.
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The genes, which govern developmental processes of oligodendrocytes such as lineage commitment and cell type specification, have been identified by research in developmental neurobiology [21,22]. By contrast, our approach to identify nuclear factors from the study of JC virus is likely to result in the identification of further new molecules of the human brain. Acknowledgements This work has been supported by CREST (Core Research for Evolutional Science and Technology), Japan Science and Technology (JST). References [1] C.L. Mendelsohn, E. Wimmer, V.R. Racaniello, Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily, Cell 56 (1989) 855 – 865. [2] R.E. Dorig, A. Marcil, A. Chopra, C.D. Richardson, The human CD46 molecule is a receptor for measles virus (Edmonston strain), Cell 75 (1993) 295 – 305. [3] H. Tatsuo, N. Ono, K. Tanaka, Y. Yanagi, SLAM (CDw150) is a cellular receptor for measles virus, Nature 406 (2000) 893 – 897. [4] W. Weis, J.H. Brown, S. Cusack, J.C. Paulson, J.J. Skehel, D.C. Wiley, Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid, Nature 333 (1988) 426 – 431. [5] D. WuDunn, P.G. Spear, Initial interaction of herpes simplex virus with cells is binding to heparan sulfate, J. Virol. 63 (1989) 52 – 58. [6] T. Compton, D.M. Nowlin, N.R. Cooper, Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate, Virology 193 (1993) 834 – 841. [7] F. Superti, B. Hauttecoeur, M.J. Morelec, P. Goldoni, B. Bizzini, H. Tsiang, Involvement of gangliosides in rabies virus infection, J. Gen. Virol. 67 (1986) 47 – 56. [8] D. Klatzmann, E. Champagne, S. Chamaret, J. Gruest, D. Guetard, T. Hercend, J.C. Gluckman, L. Montagnier, T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV, Nature 312 (1984) 767 – 768. [9] H. Deng, R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. Di Marzio, S. Marmon, R.E. Sutton, C.M. Hill, C.B. Davis, S.C. Peiper, T.J. Schall, D.R. Littman, N.R. Landau, Identification of a major coreceptor for primary isolates of HIV-1, Nature 381 (1996) 661 – 666. [10] C.K. Liu, G. Wei, W.J. Atwood, Infection of glial cells by the human polyomavirus JC is mediated by an Nlinked glycoprotein containing terminal a(2 – 6)-linked sialic acids, J. Virol. 72 (1998) 4643 – 4649. [11] S. Koike, C. Taya, T. Kurata, S. Abe, I. Ise, H. Yonekawa, A. Nomoto, Transgenic mice susceptible to poliovirus, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 951 – 955. [12] P. Wei, M.E. Garber, S.M. Fang, W.H. Fischer, K.A. Jones, A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA, Cell 92 (1998) 451 – 462. [13] R.J. Frisque, G.L. Bream, M.T. Cannella, Human polyomavirus JC virus genome, J. Virol. 51 (1984) 458 – 469. [14] S. Suzuki, H. Sawa, R. Komagome, Y. Orba, M. Yamada, Y. Okada, Y. Ishida, H. Nishihara, S. Tanaka, K. Nagashima, Broad distribution of the JC virus receptor contrasts with a marked cellular restriction of virus replication, Virology 286 (2001) 100 – 112. [15] R. Komagome, H. Sawa, T. Suzuki, Y. Suzuki, S. Tanaka, W.J. Atwood, K. Nagashima, Oligosaccharides as receptors for JC virus, J. Virol. 76 (2002) 12992 – 13000. [16] S. Kenney, V. Natarajan, D. Strike, G. Khoury, N.P. Salzman, JC virus enhancer – promoter active in human brain cells, Science 226 (1984) 1337 – 1339.
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147
[17] M.T. Pho, A. Ashok, W.J. Atwood, JC virus enters human glial cells by clathrin-dependent receptormediated endocytosis, J. Virol. 74 (2000) 2288 – 2292. [18] Y. Hara, A.C. Rovescalli, Y. Kim, M. Nirenberg, Structure and evolution of four POU domain genes expressed in mouse brain, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 3280 – 3284. [19] M. Wegner, D.W. Drolet, M.G. Rosenfeld, Regulation of JC virus by the POU-domain transcription factor Tst-1: implications for progressive multifocal leukoencephalopathy, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 4743 – 4747. [20] Y. Okada, H. Sawa, S. Endo, Y. Orba, T. Umemura, H. Nishihara, A.C. Stan, S. Tanaka, K. Nagashima, Expression of JC virus (JCV) agnoprotein in progressive multifocal leukoencephalopathy (PML) brain, Acta Neuropathol. 104 (2002) 130 – 136 (in press). [21] M. Wegner, Expression of transcription factors during oligodendroglial development, Microsc. Res. Tech. 52 (2001) 746 – 752. [22] J. Grinspan, Cells and signaling in oligodendrocyte development, J. Neuropathol. Exp. Neurol. 61 (2002) 297 – 306.