Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase alpha-primase.

Host species specificity of the polyomaviruses simian virus 40 (SV40) and mouse polyomavirus (PyV) has been shown to be determined by the host DNA polymerase alpha-primase complex involved in the initiation of both viral and host DNA replication. Here we demonstrate that DNA replication of the related human pathogenic polyomavirus JC virus (JCV) can be supported in vitro by DNA polymerase alpha-primase of either human or murine origin indicating that the mechanism of its strict species specificity differs from that of SV40 and PyV. Our results indicate that this may be due to differences in the interaction of JCV and SV40 large T antigens with the DNA replication initiation complex.     

Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication.

We recently described a soluble cell-free system derived from monkey cells that is capable of replicating exogenous plasmid DNA molecules containing the simian virus 40 (SV40) origin of replication (J.J. Li, and T.J. Kelly, Proc. Natl. Acad. Sci. U.S.A. 81:6973-6977, 1984). Replication in the system is completely dependent upon the addition of the SV40 large T antigen. In this report we describe additional properties of the in vitro replication reaction. Extracts prepared from cells of several nonsimian species were tested for the ability to support origin-dependent replication in the presence of T antigen. The activities of extracts derived from human cell lines HeLa and 293 were approximately the same as those of monkey cell extracts. Chinese hamster ovary cell extracts also supported SV40 DNA replication in vitro, but the extent of replication was approximately 1% of that observed with human or monkey cell extracts. No replication activity was detectable in extracts derived from BALB/3T3 mouse cells. The ability of these extracts to support replication in vitro closely parallels the ability of the same cells to support replication in vivo. We also examined the ability of various DNA molecules containing sequences homologous to the SV40 origin to serve as templates in the cell-free system. Plasmids containing the origins of human papovaviruses BKV and JCV replicated with an efficiency 10 to 20% of that of plasmids containing the SV40 origin. Plasmids containing Alu repeat sequences (BLUR8) did not support detectable DNA replication in vitro. Circular DNA molecules were found to be the best templates for DNA replication in the cell-free system; however, linear DNA molecules containing the SV40 origin also replicated to a significant extent (10 to 20% of circular molecules). Finally, electron microscopy of replication intermediates demonstrated that the initiation of DNA synthesis in vivo takes place at a unique site corresponding to the in vivo origin and that replication is bidirectional. These findings provide further evidence that replication in the cell-free system faithfully mimics SV40 DNA replication in vivo.     

Large T Antigen Promotes JC Virus Replication in G2-arrested Cells by Inducing ATM- and ATR-mediated G2 Checkpoint Signaling

Large T antigen (TAg) of the human polyomavirus JC virus (JCV) possesses DNA binding and helicase activities, which, together with various cellular proteins, are required for replication of the viral genome. We now show that JCV-infected cells expressing TAg accumulate in the G₂ phase of the cell cycle as a result of the activation of ATM- and ATR-mediated G₂ checkpoint pathways. Transient transfection of cells with a TAg expression vector also induced G₂ checkpoint signaling and G₂ arrest. Analysis of TAg mutants with different subnuclear localizations suggested that the association of TAg with cellular DNA contributes to the induction of G₂ arrest. Abrogation of G₂ arrest by inhibition of ATM and ATR, Chk1, and Wee1 suppressed JCV genome replication. In addition, abrogation of the G₂-M transition by Cdc2 depletion disabled Wee1 depletion-induced suppression of JCV genome replication, suggesting that JCV replication is facilitated by G₂ arrest resulting from G₂ checkpoint signaling. Moreover, inhibition of ATM and ATR by caffeine suppressed JCV production. The observation that oligodendrocytes productively infected with JCV in vivo also undergo G₂ arrest suggests that G₂ checkpoint inhibitors such as caffeine are potential therapeutic agents for JCV infection.     

The DNA-binding properties of polyomavirus large T antigen are altered by ATP and other nucleotides.

We have examined the influence of ATP on the DNA-binding properties of polyomavirus large T antigen (Py TAg). Utilizing nitrocellulose filter binding, DNase I footprinting, and gel mobility shift assays, we observed that ATP increased Py TAg binding to DNA fragments containing either all Py TAg-binding sites (whole origin) or those sites within (core origin) or adjacent to (early) the origin of replication. Even nonspecific binding to DNA fragments lacking Py TAg-binding sites was increased somewhat by ATP. Binding to the core origin was increased to a greater extent than binding to other DNA fragments tested. Gel band mobility shift assays revealed that ATP increased the production of core origin-specific Py TAg-DNA complexes of high molecular weight. ATP stimulation depended on the presence of MgCl2. Other nucleotides and nonhydrolyzable ATP analogs also increased Py TAg binding to the core origin but to various degrees: ATP, dATP, 5'-adenylyl imidodiphosphate (AMPPNP) greater than 5'-adenylyl methylenediphosphate (AMPPCP) greater than dCTP greater than UTP greater than TTP. GTP and dGTP did not increase DNA binding by Py TAg. The rates of association and disassociation of Py TAg with all the DNA fragments were altered by the presence of ATP. DNase I footprinting showed that ATP extensively extended the region protected within the core origin and also produced a distinctive DNase I-hypersensitive site on the late strand at nucleotides 5255 to 5262 (TTACTATG).     

Structural Based Analyses of the JC Virus T-Antigen F258L Mutant Provides Evidence for DNA Dependent Conformational Changes in the C-Termini of Polyomavirus Origin Binding Domains

The replication of human polyomavirus JCV, which causes Progressive Multifocal Leukoencephalopathy, is initiated by the virally encoded T-antigen (T-ag). The structure of the JC virus T-ag origin-binding domain (OBD) was recently solved by X-ray crystallography. This structure revealed that the OBD contains a C-terminal pocket, and that residues from the multifunctional A1 and B2 motifs situated on a neighboring OBD molecule dock into the pocket. Related studies established that a mutation in a pocket residue (F258L) rendered JCV T-ag unable to support JCV DNA replication. To establish why this mutation inactivated JCV T-ag, we have solved the structure of the F258L JCV T-ag OBD mutant. Based on this structure, it is concluded that the structural consequences of the F258L mutation are limited to the pocket region. Further analyses, utilizing the available polyomavirus OBD structures, indicate that the F258 region is highly dynamic and that the relative positions of F258 are governed by DNA binding. The possible functional consequences of the DNA dependent rearrangements, including promotion of OBD cycling at the replication fork, are discussed.     

Species specificity of simian virus 40 DNA replication in vitro requires multiple functions of human DNA polymerase alpha

Human cell extracts support the replication of SV40 DNA, whereas mouse cell extracts do not. Species specificity is determined at the level of initiation of DNA replication, and it was previously found that this requires the large subunit, p180, of DNA polymerase alpha-primase to be of human origin. Furthermore, a functional interaction between SV40 large T antigen (TAg) and p180 is essential for viral DNA replication. In this study we determined that the N-terminal regions of human p180, which contain the TAg-binding sites, can be replaced with those of murine origin without losing the ability to support SV40 DNA replication in vitro. The same substitutions do not prevent SV40 TAg from stimulating the activity of DNA polymerase alpha-primase on single-stranded DNA in the presence of replication protein A. Furthermore, biophysical studies show that the interactions of human and murine DNA polymerase alpha-primase with SV40 TAg are of a similar magnitude. These studies strongly suggest that requirement of SV40 DNA replication for human DNA polymerase alpha depends neither on the TAg-binding site being of human origin nor on the strength of the binary interaction between SV40 TAg and DNA polymerase alpha-primase but rather on sequences in the C-terminal region of human p180.     

Identification of critical elements within the JC virus DNA replication origin.

The T antigen of JC virus (JCV) does not interact productively with the simian virus 40 (SV40) origin of replication. In contrast, the SV40 T antigen does drive replication from the JCV origin as well as from its own. The basis for this restricted interaction was investigated by analyzing the structure of the JCV replication origin. The replication activities of JCV-SV40 hybrid origin plasmids were tested in cells constitutively producing either the JCV or SV40 T antigen. Results indicated that a region of the JCV origin critical for interaction with the JCV T antigen was positioned to the late side of the central palindrome of the putative core origin. A mutational analysis of this region indicated that the sequence of the A + T-rich tract was primarily responsible for determining the efficiency with which JCV can initiate replication from its origin. The tandemly repeated pentameric sequence AGGGA located proximal to the A + T-rich tract in the JCV enhancer element was found to stimulate JCV, but not SV40, T antigen-mediated replication. The effect on replication of other elements within the JCV enhancer was also dependent on the T antigen employed for initiation. A plasmid containing the replication origin of prototype BK virus was unable to replicate in cells containing JCV T antigen, again indicating the inflexibility of the JCV T antigen in interacting with heterologous origins.     

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.     

Insights into the Initiation of JC Virus DNA Replication Derived from the Crystal Structure of the T-Antigen Origin Binding Domain

JC virus is a member of the Polyomavirus family of DNA tumor viruses and the causative agent of progressive multifocal leukoencephalopathy (PML). PML is a disease that occurs primarily in people who are immunocompromised and is usually fatal. As with other Polyomavirus family members, the replication of JC virus (JCV) DNA is dependent upon the virally encoded protein T-antigen. To further our understanding of JCV replication, we have determined the crystal structure of the origin-binding domain (OBD) of JCV T-antigen. This structure provides the first molecular understanding of JCV T-ag replication functions; for example, it suggests how the JCV T-ag OBD site-specifically binds to the major groove of GAGGC sequences in the origin. Furthermore, these studies suggest how the JCV OBDs interact during subsequent oligomerization events. We also report that the OBD contains a novel “pocket”; which sequesters the A1 & B2 loops of neighboring molecules. Mutagenesis of a residue in the pocket associated with the JCV T-ag OBD interfered with viral replication. Finally, we report that relative to the SV40 OBD, the surface of the JCV OBD contains one hemisphere that is highly conserved and one that is highly variable.     

Molecular biology and immunoregulation of human neurotropic JC virus in CNS

The human polyomavirus, JC virus (JCV), provides an excellent model system to investigate the reciprocal interaction of the immune and nervous systems. Infection with JCV occurs during childhood and the virus remains in the latent state with no apparent clinical symptoms. However, under immunosuppressed conditions, the virus enters the lytic cycle and upon cytolytic destruction of glial cells, causes the fatal demyelinating disease of the central nervous system (CNS), named progressive multifocal leukoencephalopathy (PML). In this short review, we discuss the molecular pathogenesis of PML by highlighting the role of the immune system in modulating JCV gene activation and replication, and the latency/reactivation of this virus upon immunosuppression. Further, due to the higher incidence of PML among AIDS patients, we further elaborate on the cross-talk between JCV and HIV-1 by direct and indirect pathways that lead to enhanced expression of the JCV genome.     

High-level polyomavirus JC viruria following long-term steroid therapy

C virus is a highly seroprevalent ubiquitous polyomavirus which is acquired at an early age through respiratory or oral route, Thereafter JCV establishes persistent, but mainly asymptomatic, infections in various tissues, including the genitourinary tract and brain. In individuals with altered immunity, viral replication can occur leading to serious organ diseases. In contrast to polyomavirus BK, that is found infrequently in the urine of healthy adults, JC viruria occurs universally. Herein we describe a case of an otherwise immunocompetent patient who presented a very high-level asymptomatic polyomavirus JC viruria following long-term steroid therapy.     

Derivation and characterization of POJ cells, transformed human fetal glial cells that retain their permissivity for JC virus.

The study of the medically important polyomavirus JC virus is limited to only a few laboratories, primarily because the permissive cell system most often used, primary human fetal glial cells, is difficult to obtain and propagate. We have introduced mutations at the origin of DNA replication of JC virus and transformed glial cells with the replication-defective genomes. Although normal glial cell cultures rapidly lose their permissivity for the virus after subculture, the transformed cells (designated POJ) had a greatly expanded life span and remained permissive for JC virus even after 30 passages in vitro. POJ cells constitutively express a functional T protein that complements the replication defect of lethal early-region mutations in JC virus. We expect that these cells will greatly facilitate the study of this human virus.     

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.     

Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells.

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.     

JC virus large T-antigen and IGF-I signaling system merge to affect DNA repair and genomic integrity

The progression of cancer is often associated with genomic instability, which may develop as a result of compromised defense mechanisms responsible for the maintenance of chromosomal integrity. These include defects in telomere preservation, chromosomal segregation, and DNA repair. In this review, we discuss molecular interactions between viral and cellular signaling components, which interfere with DNA repair mechanisms, and possibly contribute to the development of a mutagenic phenotype. Our studies indicate that large T-antigen from the human polyomavirus JC (JCV T-antigen) inhibits homologous recombination directed DNA repair (HRR)-causing accumulation of mutations in the affected cells (JCP 2005, in press). Surprisingly, T-antigen does not operate directly, but utilizes insulin receptor substrate 1 (IRS-1), which is the major signaling molecule for insulin-like growth factor I receptor (IGF-IR). Following T-antigen-mediated nuclear translocation, IRS-1 binds Rad51 at the site of damaged DNA. This T-antigen-mediated inhibition of HRR does not function in cells lacking IRS-1, and can be reproduced in the absence of T-antigen by IRS-1 with an artificial nuclear localization signal. The interplay described between the IGF-IR signaling system and JCV T-antigen in the process of DNA repair could be relevant, since nearly 90% of the human population is seropositive for JC virus, JCV T-antigen transforms cells in vitro, is tumorigenic in experimental animals, and the presence of JC virus has been shown in an increasing number of biopsies of human cancer.