Cloned human polyomavirus JC DNA can transform human amnion cells.

The genome of the human polyomavirus JC (Mad-1 strain) was molecularly cloned in Escherichia coli by using the plasmid vector pBR322. Recombinant DNA molecules were constructed with the entire JC genome inserted either at its unique EcoRI site at 0.0 map units or at its unique BamHI site at 0.51 map units. Viral DNA from each of these recombinant plasmids was capable of transforming human amnion cells, and cell lines established from transformed foci were positive for JC tumor antigen as assayed by indirect immunofluorescence.     

A cell-free system for the replication fo bacteriophage M-13 duplex DNA.

Cell-free extracts from M-13 am5 infected Escherichia coli cells which are highly concentrated on cellophane membrane disks replicate efficiently endogenous M-13 duplex DNA. If the reaction is carried out in the presence of bromodeoxyuridine triphosphate, the majority of the label is found in two classes of hybrid DNA molecules in which either the viral or the complementary strand is newly synthesized. A minor portion of the label is incorporated into fully synthetic duplex DNA. DNA synthesis requires ATP and is inhibited by nalidixic acid, novobiocin, and arabinosylnucleoside triphosphates. Rifampicin blocks preferentially the synthesis of molecules with labeled complementary strands. A similar effect is observed upon addition of the helix-destabilising M-13 gene V protein. In contrast, addition of E. coli helix-destabilising protein (Eco HD-protein) stimulates the synthesis of both types of hybrid DNA molecules as well as the formation of fully synthetic duplex DNA.     

Site-specific and temporally controlled initiation of DNA replication in a human cell-free system

We have recently established a cell-free system from human cells that initiates semi-conservative DNA replication in nuclei isolated from cells which are synchronised in late G₁ phase of the cell division cycle. We now investigate origin specificity of initiation using this system. New DNA replication foci are established upon incubation of late G₁ phase nuclei in a cytosolic extract from proliferating human cells. The intranuclear sites of replication foci initiated in vitro coincide with the sites of earliest replicating DNA sequences, where DNA replication had been initiated in these nuclei in vivo upon entry into S phase of the previous cell cycle. In contrast, intranuclear sites that replicate later in S phase in vivo do not initiate in vitro. DNA replication initiates in this cell-free system site-specifically at the lamin B2 DNA replication origin, which is also activated in vivo upon release of mimosine-arrested late G₁ phase cells into early S phase. In contrast, in the later replicating ribosomal DNA locus (rDNA) we neither detected replicating rDNA in the human in vitro initiation system nor upon entry of intact mimosine-arrested cells into S phase in vivo. As a control, replicating rDNA was detected in vivo after progression into mid S phase. These data indicate that early origin activity is faithfully recapitulated in the in vitro system and that late origins are not activated under these conditions, suggesting that early and late origins may be subject to different mechanisms of control.     

Human polyomavirus JC virus genome.

The complete DNA sequence of the human JC virus, which was found to consist of 5,130 nucleotide pairs, is presented. The amino acid sequence of six proteins could be deduced: the early, nonstructural proteins, large T and small t antigens; the late capsid proteins, VP1, VP2, and VP3; and the agnogene product encoded within the late leader sequence, called the agnoprotein in simian virus 40. The extent of homology between JC virus DNA and the genomes of simian virus 40 (69%) and BK virus (75%) confirmed the close evolutionary relationship of these three polyomaviruses. The sequences showing the greatest divergence in these viral DNAs occurred within the tandem repeats located to the late side of the replication origins.     

JC human papovavirus replication in human amnion cells.

JC human papovavirus was found to replicate in primary human amnion cells. The virus has undergone eight passages in amnion cells and was identified by serological methods as JC virus. By restriction endonuclease analysis of the viral DNA, the fragments observed were identical to those previously reported for the prototype strain.     

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.     

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.     

A role for single-stranded templates in cell-free adeno-associated virus DNA replication

Assays have been described in which duplex adeno-associated virus (AAV) DNA can be replicated in HeLa cell extracts with exogenous AAV Rep protein. These assays appear to mimic the AAV DNA replication that occurs in the cell, including the ability of extracts from adenovirus (Ad)-infected cells to replicate duplex AAV DNA templates more efficiently than extracts from uninfected cells can. We showed previously that the Ad-infected extract was able to support a more processive replication than the uninfected extract. When the Ad single-stranded DNA binding protein (Ad-DBP) was added to an uninfected extract, DNA replication became processive. Based on a strand displacement replication model, we hypothesized that the Ad-DBP was stabilizing the displaced single-stranded DNA during strand displacement replication. In this report, we show that in Ad-infected extracts most of the newly replicated duplex DNA is converted into a single-stranded form shortly after synthesis. Using the results of assays for the replication of single-stranded AAV DNA, we show that these single-stranded molecules serve as templates for additional replication. In addition, we identify a class of molecules which are likely to be intermediates of replication on single-stranded templates. We discuss a possible role for replication of single-stranded molecules in the infected cell.     

Nuclei act as independent and integrated units of replication in a Xenopus cell-free DNA replication system.

We have used a novel approach to investigate the control of initiation of replication of sperm nuclei in a Xenopus cell-free extract. Nascent DNA was labelled with biotin by supplementing the extract with biotin-11-dUTP, and isolated nuclei were then probed with fluorescein-conjugated streptavidin. Flow cytometry was used to measure the biotin content of individual nuclei and their total DNA content. This showed that incorporation of the biotinylated precursor increases linearly with DNA content. Haploid sperm nuclei replicate fully to reach the diploid DNA content over 2-6 h in the extract. Synthesis stops once the diploid DNA content is reached. Different nuclei enter S phase at different times over greater than 1.5 h, although they share the same cytoplasmic environment. Nuclei reach their maximum rates of synthesis soon after entry into S phase and some replicate fully in less than 0.5 h, resembling the rates of replication observed in the intact egg. These results indicate that initiations are coordinated within each nucleus such that the nucleus is the fundamental unit of replication in the cell-free system.     

CPT11 prevents virus replication in JCI cells persistently infected with JC polyomavirus

JC polyomavirus (JCPyV) is the causative agent of the demyelinating disease of the central nervous system known as progressive multifocal leukoencephalopathy (PML), which occurs in immunocompromised patients. Moreover, patients treated with natalizumab for multiple sclerosis or Crohn disease can develop PML, which is then termed natalizumab‐related PML. Because few drugs are currently available for treating PML, many antiviral agents are being investigated. It has been demonstrated that the topoisomerase I inhibitors topotecan and β‐lapachone have inhibitory effects on JCPyV replication in IMR‐32 cells. However, both of these drugs have marginal inhibitory effects on virus propagation in JC1 cells according to RT‐PCR analysis. In the present study, the inhibitory effect of another topoisomerase I inhibitor, 7‐ethy‐10‐[4‐(1‐piperidino)‐1‐piperidino] carbonyloxy camptothecin (CPT11), was assessed by investigating viral replication, propagation, and viral protein 1 (VP1) production in cultured cells. JCPyV replication was assayed using real‐time PCR combined with Dpn I treatment in IMR‐32 cells transfected with JCPyV DNA. It was found that JCPyV replicates less in IMR‐32 cells treated with CPT11 than in untreated cells. Moreover, CPT11 treatment of JCI cells persistently infected with JCPyV led to a dose‐dependent reduction in JCPyV DNA and VP1 production. Additionally, the inhibitory effect of CPT11 was found to be stronger than those of topotecan and β‐lapachone. These findings suggest that CPT11 may be a potential anti‐JCPyV agent that could be used to treat PML.

     

DNA replication in cell-free extracts from Drosophila melanogaster.

We have developed an efficient in vitro replication system from 0-2 h Drosophila melanogaster embryos. Demembranated Xenopus sperm DNA when incubated in such an extract first becomes enclosed in a nucleus-like structure with a nuclear envelope and a karyoskeleton. It then undergoes one round of semiconservative replication--this replication appears completely dependent on nuclear formation. Up to 30% of input DNA is nucleated in one reaction. Efficient nuclear formation and replication are dependent on a cold treatment step, prior to disruption of the embryos. They also depend on the age of the embryos used. Extracts from older embryos (0-5 h) are capable of nuclear formation, although at a much reduced efficiency, and repair synthesis, but seem to have lost the ability to initiate DNA replication. In addition to replicating sperm DNA this system appears capable of carrying out semi-conservative replication on some plasmids. However, it cannot use these to trigger nuclear formation; replication is only seen if the plasmids are coincubated with sperm DNA. The in vitro formed nuclei have not been observed to trigger nuclear envelope breakdown and entry into mitosis. However, they can re-replicate the DNA if the nuclei are permeabilized. This system should be a useful complement to the previously isolated Xenopus in vitro replication system. In addition the amenability of Drosophila to genetic study should open up new approaches not previously possible with Xenopus.     

Genetic analysis of the enhancer requirements for polyomavirus DNA replication in mice.

In this report, we describe the first systematic analysis of the genetic requirements for polyomavirus (Py) enhancer-activated viral DNA replication during the acute phase of infection in mice. Four mutants were made which substituted XhoI sites for conserved enhancer consensus sequences (adenovirus type 5 E1A, c-fos, simian virus 40, and a glucocorticoidlike consensus sequence). Viral DNA replication in infected mouse organs was measured by DNA blot analysis. Only the loss of the glucocorticoidlike consensus sequence element significantly reduced Py DNA replication in the kidneys, the primary target organ for viral replication. The loss of the c-fos, adenovirus type 5 E1A, or simian virus 40 consensus sequences, however, expanded organ-specific viral DNA replication, relative to wild-type Py, by allowing high-level replication in the pancreas or heart or both. Analysis of Py variants selected for replication in undifferentiated embryonal carcinoma cell lines (PyF441, PyF111) showed that there was little change in levels of viral DNA replication in kidneys and other organs as compared with those in the wild-type virus. If the entire B enhancer is deleted, only low overall levels of viral replication are observed. Wild-type levels of replication in the kidneys can be reconstituted by addition of a single domain from within the A enhancer (nucleotides 5094 to 5132) to the B enhancer deletion virus, suggesting that a single domain from the A enhancer can functionally substitute for the entire B enhancer. This also indicates that the determinants for kidney-specific replication are not found in the B enhancer.     

Primary central nervous system lymphoma expressing the human neurotropic polyomavirus, JC virus, genome

B lymphocytes are known as a potential site for latency and reactivation of the human neurotropic polyomavirus, JC virus (JCV). In light of recent studies on the oncogenicity of JCV and the transforming ability of the JCV early protein, T antigen, we investigated the association of JCV with B-cell lymphomas of the central nervous system. Examination of 27 well-characterized clinical specimens by gene amplification and immunohistochemistry revealed the presence of DNA sequences corresponding to the JCV early genome and the late Agnoprotein in 22 samples and the JCV late genome encoding the viral capsid proteins in 8 samples. Expression of T antigen and that of Agnoprotein by immunohistochemistry were each detected in six specimens. No evidence of the production of viral capsid proteins was observed, ruling out productive infection of JCV in the tumor cells. The results from laser capture microdissection verified the presence of JCV T-antigen sequences in tumor cells with positive immunoreactivity to antibodies against the viral proteins T antigen and Agnoprotein. Due to previous reports demonstrating an association of the Epstein-Barr virus (EBV) with transformation of B lymphocytes, EBV DNA sequences and the EBV transforming protein, latent membrane protein 1 (LMP1), were analyzed in parallel. EBV LMP1 DNA sequences were detected in 16 of 23 samples, and LMP1 expression was detected in 16 samples, 5 of which exhibited positive immunoreactivity to JCV proteins. Double labeling demonstrated coexpression of JCV T antigen and EBV LMP1 in the same cells. The detection of the JCV genome in large numbers of B-cell lymphomas and its coexistence with EBV suggest a potential role for JCV in the pathogenesis of primary CNS lymphoma.     

Mouse DNA primase plays the principal role in determination of permissiveness for polyomavirus DNA replication.

We have investigated the species-specific replication of polyomavirus DNA in the cell-free system that was established previously (Y. Murakami, T. Eki, M. Yamada, C. Prives, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 83:6347-6351, 1986). Extracts from various species of cells supported polyomavirus DNA replication in a species-specific manner that was consistent with the host range specificity of polyomavirus; extracts prepared from mouse and hamster cells were active, whereas extracts prepared from human, monkey, and insect cells were inactive. The addition of DNA polymerase alpha-primase purified from mouse cells induced the replication of polyomavirus DNA in a cell-free system containing polyomavirus large tumor antigen and nonpermissive cell extracts, such as human and insect cell extracts. Isolated mouse DNA primase alone also induced polyomavirus DNA replication in human cell extracts but not in insect cell extracts, indicating that mouse DNA primase plays the principal role in determining permissiveness for polyomavirus DNA replication.     

DNA replication reaction in Xenopus cell-free system is suppressed by high pressure

Previously, we demonstrated that when mouse erythroleukemia cells are exposed to a pressure of 80 MPa, the cell-cycle progression of S-phase cells is retarded. To examine the effects of high pressure on DNA replication, we used a Xenopus cell-free system. From cell-cycle progression of sperm nuclei, it was found that sperm nuclei are stable to a pressure of 80 MPa, whereas egg extracts are susceptible to high pressure. Similarly, biotin-16-dUTP was incorporated into 80 MPa-treated sperm nuclei in pressure-untreated extracts, but not into naive sperm nuclei in 80 MPa-treated extracts. These results indicate that DNA replication in Xenopus cell-free system is suppressed by the susceptibility of the extracts to a pressure of 80 MPa.