Viral replication centers and the DNA damage response in JC virus-infected cells.

JCV is a human polyomavirus (PyV) that establishes a persistent infection in its host. Current immunomodulatory therapies, such as Natalizumab for multiple sclerosis, can result in JCV reactivation, leading to the debilitating brain disease progressive multifocal leukoencephalopathy (PML). JCV is among the viruses that recruit and modulate the host DNA damage response (DDR) to replicate its genome. We have identified host proteins recruited to the nuclear sites of JC viral DNA (vDNA) replication using three cell types susceptible to infection in vitro. Using confocal microscopy, we found that JCV recruited a similar repertoire of host DDR proteins to these replication sites previously observed for other PyVs. Electron tomography of JCV "virus factories" showed structural features like those described for murine PyV. These results confirm and extend previous observations for PyVs to JCV emphasizing a similar replication strategy among members of this virus family.

Recombinant VLP-Z of JC Polyomavirus: A Novel Vector for Targeting Gene Delivery.

Virus-like particle (VLP) of JC polyomavirus (JCPyV) is capable of packaging and delivering exogenous DNA into human cells and can be used for mediating therapeutic gene expression. However, many human cells express the JCPyV receptor. Thus, wild-type VLP can transduce a wide range of human cells nonspecifically. This study tested the feasibility of using a modified VLP with a IgG binding domain (Z domain) of protein A in its capsid for targeted gene delivery. The Z domain of protein A isolated from the membrane of Staphylococcus aureus was inserted into the NH3-terminus of VP1, the major JCPyV capsular protein. The recombinant VLP-Z was produced using Escherichia coli. Electron-microscopic analysis showed that VLP-Z has a viral-like structure. A hemagglutination test showed that VLP-Z has hemagglutination activity. VP(1) was detected in the nuclei of HeLa cells by immunostaining after VLP-Z inoculation, suggesting that VLP-Z is viable and can enter the cell nucleus. Inoculating HeLa cells with pEGFP-N(1) plasmid packaged in VLP-Z has resulted in enhanced green fluorescent protein expression in the cells. In addition, a binding assay showed that VLP-Z was able to bind IgG through the interaction of the Z domain in VLP-Z and IgG. These data suggest that VLP-Z could be armed with cell-specific antibody and be used to deliver therapeutic genes to target cells.

UVC Inactivation of dsDNA and ssRNA Viruses in Water: UV Fluences and a qPCR-Based Approach to Evaluate Decay on Viral Infectivity.

Disinfection by low-pressure monochromatic ultraviolet (UVC) radiation (253.7 nm) became an important technique to sanitize drinking water and also wastewater in tertiary treatments. In order to prevent the transmission of waterborne viral diseases, the analysis of the disinfection kinetics and the quantification of infectious viral pathogens and indicators are highly relevant and need to be addressed. The families Adenoviridae and Polyomaviridae comprise human and animal pathogenic viruses that have been also proposed as indicators of fecal contamination in water and as Microbial Source Tracking tools. While it has been previously suggested that dsDNA viruses may be highly resistant to UVC radiation compared to other viruses or bacteria, no information is available on the stability of polyomavirus toward UV irradiation. Here, the inactivation of dsDNA (HAdV2 and JCPyV) and ssRNA (MS2 bacteriophage) viruses was analyzed at increasing UVC fluences. A minor decay of 2-logs was achieved for both infectious JC polyomaviruses (JCPyV) and human adenoviruses 2 (HAdV2) exposed to a UVC fluence of 1,400 J/m(2), while a decay of 4-log was observed for MS2 bacteriophages (ssRNA). The present study reveals the high UVC resistance of dsDNA viruses, and the UV fluences needed to efficiently inactivate JCPyV and HAdV2 are predicted. Furthermore, we show that in conjunction with appropriate mathematical models, qPCR data may be used to accurately estimate virus infectivity.

JC virus inclusions in progressive multifocal leukoencephalopathy: scaffolding promyelocytic leukemia nuclear bodies grow with cell cycle transition through an S-to-G2-like state in enlarging oligodendrocyte nuclei.

In progressive multifocal leukoencephalopathy, JC virus-infected oligodendroglia display 2 distinct patterns of intranuclear viral inclusions: full inclusions in which progeny virions are present throughout enlarged nuclei and dot-shaped inclusions in which virions are clustered in subnuclear domains termed "promyelocytic leukemia nuclear bodies" (PML-NBs). Promyelocytic leukemia nuclear bodies may serve a scaffolding role in viral progeny production. We analyzed the formation process of intranuclear viral inclusions by morphometry and assessed PML-NB alterations in the brains of 2 patients with progressive multifocal leukoencephalopathy. By immunohistochemistry, proliferating cell nuclear antigen was most frequently detected in smaller nuclei; cyclin A was detected in larger nuclei. This suggests an S-to-G2 cell cycle transition in infected cells associated with nuclear enlargement. Sizes of PML-NBs were variable, but they were usually either small speckles 200 to 400 nm in diameter or distinct spherical shells with a diameter of 1 µm or more. By confocal microscopy, JC virus capsid proteins were associated with both small and large PML-NBs, but disruption of large PML-NBs was observed by ground-state depletion fluorescence nanoscopy. Clusters of progeny virions were also detected by electron microscopy. Our data suggest that, in progressive multifocal leukoencephalopathy, JC virus produces progeny virions in enlarging oligodendrocyte nuclei in association with growing PML-NBs and with cell cycle transition through an S-to-G2-like state.

JC virus intranuclear inclusions associated with PML-NBs: analysis by electron microscopy and structured illumination microscopy.

Progressive multifocal leukoencephalopathy is a fatal demyelinating disorder caused by JC virus infection. JC virus was recently found to target promyelocytic leukemia nuclear bodies (PML-NBs), punctuate domains in the nuclei. Thus, the virus progenies cluster in dots as intranuclear inclusions (ie, as dot-shaped inclusions). In the present study, both the viral major and minor capsid proteins were expressed from polycistronic expression vectors with a powerful promoter, and formation into virus-like particles (VLPs) was examined by electron microscopy. When the upstream regulatory sequence including the agnogene (nt 275 to 490) was present, capsid protein expression was suppressed, but numerous VLPs were efficiently formed with restricted accumulation to PML-NBs. VLPs were uniform, and the cells were severely degraded. In contrast, when the 5' terminus of the agnogene (nt 275 to 409; 135 bp) was deleted, capsid protein expression was markedly enhanced, but VLPs were more randomly produced in the nucleus outside of PML-NBs. VLPs were pleomorphic, and cell degradation was minimal. JC virus association with PML-NBs was confirmed in human brain tissues by structured illumination microscopy. PML-NBs were shaped in spherical shells, with viral capsid proteins circumscribing the surface. These findings indicate that PML-NBs are intranuclear locations for pathogenic JC virus proliferation. Either the agnogene or its product likely supports efficient progeny production at PML-NBs, leading to subsequent degeneration of host glial cells.

Gold nanoparticle arrangement on viral particles through carbohydrate recognition: a non-cross-linking approach to optical virus detection.

We propose a new approach to optical virus detection based on the spatial assembly of gold nanoparticles on the surface of viruses. Since JC virus-like particles (VLPs) comprise a repeating viral capsid protein that binds to sialic acid, the conjugation of sialic acid-linked Au particles with VLPs enables the spatial arrangement of Au particles on the VLP surface. This structure produced a red shift in the absorption spectrum due to plasmon coupling between adjacent Au particles, leading to the construction of an optical virus detection system. Our system depends not on the simple cross-linking of VLPs and Au particles, but on an ordered Au structure covering the entire surface of the VLPs and can be applied to various virus detection systems using the inherent ligand recognition of animal viruses.

Promyelocytic leukemia nuclear bodies provide a scaffold for human polyomavirus JC replication and are disrupted after development of viral inclusions in progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy is a fatal demyelinating disorder due to human polyomavirus JC infection in which there are viral inclusions in enlarged nuclei of infected oligodendrocytes. We report that the pathogenesis of this disease is associated with distinct subnuclear structures known as promyelocytic leukemia nuclear bodies (PML-NBs). Postmortem brain tissues from 5 patients with the disease were examined. Affected cells with enlarged nuclei contained distinct dot-like subnuclear PML-NBs that were immunopositive for PML protein and nuclear body protein Sp100. Major and minor viral capsid proteins and proliferating cell nuclear antigen, an essential component for DNA replication, colocalized with PML-NBs. By in situ hybridization, viral genomic DNA showed dot-like nuclear accumulation, and by electron microscopy, virus-like structures clustered in subnuclear domains, indicating that PML-NBs are the site of viral DNA replication and capsid assembly. Molecules involved in the ubiquitin proteosome pathway (i.e. ubiquitin and small ubiquitin-like modifier 1) did not accumulate in the nuclei with viral inclusions, indicating that cell degeneration may not be dependent on this pathway. When viral progeny production was advanced, PML-NBs were disrupted. These data suggest that: 1) PML-NBs allow for efficient viral propagation by providing scaffolds, 2) disruption of PML-NBs is independent of the ubiquitin-proteasome pathway, and 3) this disruption probably heralds oligodendrocyte degeneration and the resulting demyelination.

Progressive multifocal leukoencephalopathy developing in advanced pulmonal sarcoidosis.

Coincidence of pulmonal sarcoidosis and progressive multifocal leukoencephalopathy (PML) rarely occurs. So far an entire course has been recorded in only very few cases. We demonstrate the case of a 49-year-old male developing an infratentorial localized PML in the setting of advanced pulmonal sarcoidosis. PML was not included in the diagnostic considerations in the first instance. Regarding the diagnosis of pulmonal sarcoidosis proved by lung biopsy, the neurological impairment was first thought to be due to a neurosarcoidosis. But magnetic resonance tomography (MRI) clearly showed a demyelination process in the cerebellum. Because of the inconsistency of the radiological findings with a neurosarcoidosis the diagnosis of an acute disseminated encephalomyelitis (ADEM) was favoured. Therefore, the patient was initially treated with corticosteroids. Because of increasing deterioration further diagnostic testings were performed. In the cerebrospinal fluid (CSF) as well as in the paraffin-embedded tissue of a stereotactical brain biopsy JCV-DNA was successfully demonstrated by PCR. Cidofovir was administered. The progression of the disease could not be influenced. The patient died 5 months after the first neurological symptoms. This report stresses the diagnostic difficulties considering patients with sarcoidosis and neurological symptoms.

[Development of intranuclear inclusions of human polyomavirus JC. Capsid proteins are assembled into virions at the PML nuclear bodies].

Human polyomavirus JC (JCV) is a causative agent for progressive multifocal leukoencephalopathy, a fatal demyelinating disorder. The viruses form intranuclear viral inclusions in infected oligodendrocytes. The outer capsid of JCV is thought to be composed of 360 molecules of major capsid protein VP1, and minor capsid proteins VP2 and VP3 in an appropriate ratio. However, the regulatory mechanisms of gene expression for the capsid proteins, their nuclear transport, and formation of viral inclusions are not well understood. We have recently clarified the following regarding the mechanism underlying JCV virion assembly; (i) major and minor capsid proteins are synthesized from messenger RNAs, the expression ratio of which is determined by alternative splicing, (ii) messenger RNAs for the major and minor capsid proteins are polycistronic, and their translation occurs downstream of the regulatory protein, agnoprotein, (iii) major and minor capsid proteins are translocated to the nucleus in a cooperative manner and accumulate at the dot-shaped intranuclear structures called promyelocytic leukemia nuclear bodies (PML-NBs), (iv) efficient viral replication can occur at the PML-NBs, where capsid assembly is likely to be associated with viral DNA replication. PML-NBs are the sites for expression of important nuclear functions for the host cells. The finding that the target of JCV infection is the PML-NB may contribute greatly to our understanding of the mechanism underlying cellular degeneration, which occurs after the formation of intranuclear viral inclusions.

The JC virus-like particle overlay assay.

JC virus (JCV) belongs to the family of double-stranded DNA polyomaviruses and in humans causes a demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy (PML). It has been reported that sialic acids play a pivotal role in hemagglutination of red blood cells and entry into host cells of JCV and that JCV can enter a wide variety of cell types and localize to the nuclei. The outer shell of the JCV virion comprises the major capsid protein VP1, and a virus-like particle (VLP) consisting of recombinant VP1 made from Escherichia coli exhibit a virion-like structure and physiological functions (cellular attachment and intracytoplasmic trafficking) similar to those of JCV virions. To examine the mechanism of cell attachment of JCV, an overlay assay using a VLP has been developed, revealing that sialoglycoproteins, including alpha1 acid-glycoprotein, fetuin, and transferrin receptor bind with VLP. In addition, VLPs bind to glycolipids, such as lactosylceramide and gangliosides including GM3, GD2, GD3, GD1b, GT1b, and GQ1b, and VLP weakly bind to GD1a. In this section, detailed procedures for the synthesis of VLP from E. coli and VLP overlay assay are described.

Molecular cloning and expression of major structural protein VP1 of the human polyomavirus JC virus: formation of virus-like particles useful for immunological and therapeutic studies.

The major structural viral protein, VP1, of the human polyomavirus JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), was expressed by using recombinant baculoviruses. Recombinant VP1 formed virus-like particles (VLP) with the typical morphology of empty JCV capsids. Purified VP1 VLP bind to SVG, B, and T cells, as well as to monkey kidney cells. After binding, VP1 VLP were also internalized with high efficiency and transported to the nucleus. Immunization studies revealed these particles as highly immunogenic when administered with adjuvant, while immunization without adjuvant induced no immune response. VP1 VLP hyperimmune serum inhibits binding to SVG cells and neutralizes natural JCV. Furthermore, the potential of VP1 VLP as an efficient transporter system for gene therapy was demonstrated. Exogenous DNA could be efficiently packaged into VP1 VLP, and the packaged DNA was transferred into COS-7 cells as shown by the expression of a marker gene. Thus, VP1 VLP are useful for PML vaccine development and represent a potential new transporter system for human gene therapy.

Immunohistochemical detection of JC virus in nontumorous renal tissue of a patient with renal cancer but without progressive multifocal leukoencephalopathy.

We performed immunohistochemical staining on the nontumorous renal tissue of 45 patients with renal cancer but without progressive multifocal encephalopathy using JCV-specific antibody. For one patient we found positive staining of the nuclei of the renal collecting ducts. Immunoelectron microscopic examination of the positive cell nuclei revealed electron-dense polyomavirus-like particles.

Fine needle aspiration biopsy of progressive multifocal leukoencephalopathy in a patient with AIDS. A case report.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is one of the most common opportunistic infections, with a range of 4-7% in acquired immunodeficiency syndrome (AIDS) patients. Clinical diagnosis is often difficult, and the specific pathologic agent requires cytologic and pathologic confirmation. CASE: A 38-year-old, Haitian male was admitted with a new-onset seizure disorder. On computed tomography (CT), there were right frontoparietal cortex, right external capsule and right basal ganglia lucencies. Fine needle aspiration biopsy (FNAB) of the radiolucent area revealed foci of white matter demyelination and a few eosinophilic inclusions in oligodendrocytes plus abnormal giant astrocytes. Ultrastructurally, JC virions were observed in the nuclei and cytoplasm of the oligodendrocytes. CONCLUSION: Diagnostic cranial CT-guided FNAB, with cytologic and histologic studies, is extremely valuable in evaluating the nature of central nervous system demyelinated and space-occupying lesions in AIDS.

Assembly of JC virus-like particles in COS7 cells.

JC virus lacks an appropriate cell line to support virus replication. The establishment of a JC pseudovirus assembly system could play an alternative role for a virus culture system. COS7 cells and a transfer vector, pcDL-SR alpha 296, were used to express JC viral structural genes. VP231-SR alpha, which encodes VP2/VP3 and VP1, but lacks 137 bp of the 5'-terminus of agnogene, showed both efficient nuclear migration and quantitative expression of the major capsid protein VP1. JC pseudovirus assembly was observed in the nucleus of VP231-SR alpha transfected cells. Evidence of JC pseudovirus assembly is presented. The further utilization of this system, which includes a study for the viral morphogenesis, serological diagnosis, as well as the potential application for gene transfer vector, is discussed.

[Assembly of JC virus pseudovirus particles by recombinant DNA in COS7 cells].

JS virus lacks an appropriate cell line to support virus replication. The establishment of a JC pseudovirus assembly system would play an alternative role for a virus culture system. COS7 cells and a transfer vector, pcDL-SR alpha 296 were used to express JC viral structural genes. VP231-SR alpha, which encodes VP2/VP3 and VP1, but lacks 137bp of the 5'-terminus of agnogene, showed both efficient nuclear migration and quantitative expression of the major capsid protein VP1. JC pseudovirus assembly was observed in the nucleus of VP231-SR alpha transfected cells. In this study, evidence for JC pseudovirus assembly is presented. The further utilization of this system, which includes a study for the viral morphogenesis and serological diagnosis, are discussed.

[Analysis of JC pseudotype virus generated by expression of VP231-SR alpha in COS7 cells].

In order to investigate those viruses which are difficult to propagate in tissue culture, it is necessary to develop artificial systems which will fascilliate molecular analysis. One approach has been the development of pseudotype viruses. JC virus (JCV), the etiological agent of progressive multifocal leukoencephalopathy (PML), which is particularly difficult to grow in culture, has been artificially generated as a pseudotype virus (JCPV) by transfecting and expressing the genes coding the viral coat proteins (VP1, VP2, VP3) in COS7 cells under control of the SR alpha promoter. We have analyzed the morphology, function and molecular characteristics of the JCPV. Electron microscopic examination of purified JCPV disclosed a virus structure identical to that of wild-type JCV. In addition, viral DNA could be detected by Southern blot hybridization. The infectivity of JCPV for COS7 cells was demonstrated by PCR, and localization of virus in the nuclei of infected cells was confirmed by in situ PCR. On the basis of these results, it is clear that JCPV is composed of artificial recombinant virus particles possessing infectivity, and that this may be useful not only for studies of viral replication but also perhaps as a vehicle for gene transfer.

Propagation of JC virus in human neuroblastoma cell line IMR-32.

JC virus (JCV), the causative agent of a human demyelinating disease, progressive multifocal leukoencephalopathy, has a very narrow host range. Cells permissive for infection by JCV have been essentially limited to primary human fetal glial cells, which are difficult to obtain and maintain. In pilot studies, it was found that JCV can multiply in an established cell line of human neuroblastoma. JCV strains Mad-1 and Tokyo-1 were inoculated, respectively, into two cell lines, IMR-32 (neuroblastoma) and A-172 (glioblastoma). Viral infection with cytopathic effect was observed only in IMR-32 cells, and the most efficient viral proliferation was obtained in cells cultured in medium containing 2% fetal calf serum (FCS). Both Mad-1 and Tokyo-1 strains propagated well, with the former being more efficient than the latter. Viral replication was confirmed by immunofluorescence, electron microscopy, and a hemagglutination assay. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis of the purified virus revealed the characteristic JCV protein profile. Thus, IMR-32 cells have been found to be permissive for JCV, which should provide a useful system for further studies of virus proliferation and viral tissue tropism.

Papovavirus detection by electron microscopy in the brain of an elderly patient without overt progressive multifocal leukoencephalopathy.

Virions resembling papovavirus were demonstrated in glial cells in the brain of an aged patient without overt progressive multifocal leukoencephalopathy. The patient was not in a severely immunocompromised state. On histological examination, only a few tiny incomplete necrotic foci were found in the subcortical area. These foci were widely dispersed. Rare, swollen oligodendroglial cells and astrocytes in which papovavirus capsid protein (VP-1) was demonstrated immunohistochemically were present around the foci. The two typical types of virus particles i.e. 35 to 40 nm round particles and elongated particles, were observed in the nuclei of the swollen glial cells. The latter were in the minority. Distinct crystals were also found in the nuclei. The centre-to-centre distance of the particles in the crystals, about 40 nm, and the electron-opaque spots of the round-shaped virions and of the elongated particles, were indicative of structural subunits of papovavirus capsids. This case provides further evidence that papovavirus, possibly JC virus, may be reactivated in the brains of aged patients who are not in an immunocompromised state.

Ultrastructural studies in the lytic phase of progressive multifocal leukoencephalopathy in AIDS patients.

Brain fragments from eight cases (four autopsies and four biopsies) of patients with acquired immune deficiency syndrome (AIDS) with JC virus (JCV) lytic infections were examined ultrastructurally. Particular efforts were made to look for virions and their subcellular distribution in cells not usually involved by papovavirus infection. The cellular and subcellular distribution of virions was investigated with emphasis on cell types not normally associated with papovavirus infection. The pattern of JCV infection was as follows: 1) oligodendrocytes; nucleus only, 7 cases; cytoplasm only, no cases; 2) astrocytes (normal and "bizarre"); nucleus and cytoplasm, two cases; cytoplasm only, four cases; 3) macrophages; nucleus and cytoplasm, one case; cytoplasm only, four cases; and 4) neurons; nucleus and cytoplasm, two cases; cytoplasm only, three cases. Perivascular, endothelial, ependymal, and microglial cells were never infected. Our ultrastructural data indicate that cell types other than oligodendrocytes can be involved productively by JCV in the lytic phase of progressive multifocal leukoencephalopathy (PML) in AIDS patients. Neuronal cells, especially, can be infected productively by the JCV, and this should be considered in clinical interpretation of cortical symptoms and signs in suspected or proven cases of PML.

JC virus and simian virus 40 enhancers and transforming proteins: role in determining tissue specificity and pathogenicity in transgenic mice.

When introduced into the germ line of mice, the simian virus 40 (SV40) T antigen under the control of its own transcriptional enhancer and promoter selectively induced tumors in the choroid plexus as well as thymic hyperplasia and kidney pathology. In contrast, the JC virus (JCV) T antigen under the control of its own regulatory sequences induced hypomyelination of the central nervous system and tumors of neural origin. Since SV40 and JCV have extensive sequence homology, except for their transcriptional control regions, these observations suggest but do not prove that, although the diseases induced by the two viruses, are consequences of the transforming gene, they are determined predominantly by the respective viral enhancers and promoters. To test this hypothesis, the regulatory regions of the two viruses were exchanged, and transgenic mice were derived with either chimeric construct. Like wild-type JCV, the construct containing the SV40 T antigen under the control of the JCV regulatory region induced hypomyelination of the central nervous system and neural tumors. Surprisingly, mice with this construct also developed choroid plexus carcinomas. Like the wild-type SV40 transgenic mice, mice with the JCV T antigen under the control of the SV40 enhancer and promoter developed choroid plexus tumors and renal pathology. Unexpectedly, they also had hyperplasia of the thyroid follicular cells. These findings not only provide direct evidence for the specificity of the respective viral regulatory region but also, more importantly, show that the transforming genes play a critical role in determining viral pathogenesis.