Loss of integrated viral DNA sequences in polyomatransformed cells is associated with an active viral A function.

Rat cells transformed by polyoma virus contain, in addition to integrated viral DNA, a small number of nonintegrated viral DNA molecules. The free viral DNA originates from the integrated form through a spontaneous induction of viral DNA replication in a minority of the cell population. Its presence is under the control of the viral A locus. To determine whether the induction of free viral DNA replication was accompanied by a loss of integrated viral DNA molecules in a phenomenon similar to the "curing" of lysogenic bacteria, we selected for revertants arising in the transformed rat populations and determined whether these cells had lost integrated viral genomes. We further investigated whether the viral A function was necessary for "curing" by determining the frequency of cured cells in populations of rat cells transformed by the ts-a mutant of polyoma virus following propagation at the permissive or nonpermissive temperature. A large proportion of the revertants isolated were negative or weakly positive when assayed by immunofluorescence for polyoma T antigen and were unable to produce infectious virus upon fusion with permissive mouse cells. The T antigen-negative, virus rescue-negative clones can be retransformed by superinfection and appear to have lost a considerable proportion of integrated viral DNA sequences. Restriction enzyme analysis of the integrated viral DNA sequences shows that the parental transformed lines contain tandem repeats of integrated viral molecules, and that this tandem arrangement is generally lost in the cured derivatives. While cells transformed by wild-type virus undergo "curing" with about the same frequency at 33 degrees or 39 degrees C, cells transformed by the ts-a mutant contain a much higher frequency of cured cells after propagation at 33 degrees than at 39 degrees C. Our results indicate that in polyoma-transformed rat cells, loss of integrated viral DNA can occur at a rather high rate, producing (at least in some cases) cells which have reverted partially or completely to a normal phenotype. Loss of integrated viral DNA is never total and appears to involve an excision event. The polyoma A function (large T antigen) is necessary for such excision to occur. In the absence of a functional A gene product, the association of the viral DNA with the host DNA appears to be very stable.     

Inducible permissive cells transformed by a temperature-sensitive polyoma virus: superinfection does not allow excision of the resident viral genome.

After exposure of mouse embryo cells to the early temperature-sensitive mutant tsP155 of polyoma virus (Py), a transformed cell line (Cyp line) that can be readily induced to synthesize Py by transfer to 33 degrees C was isolated at 39 degrees C (7). Virus production and synthesis of free viral DNA occurring after temperature shiftdown or superinfection with wild-type Py or both were studied in several clonal isolates of the Cyp cell line. Measurements of virus yields indicated that, although some could be induced more effectively than others, all cell clones behaved as highly permissive when subjected to superinfection. We analyzed the origin of free viral DNA accumulating in the superinfected cultures, taking advantage of (i) the unique physical properties of the low-molecular-weight DNA which, in the case of one of the Cyp clones, accumulates during temperature shiftdown, and (ii) the differences between resident and superinfecting viral genomes in their susceptibilities towards restriction endonucleases. At 33 degrees C, both viral genomes were found to accumulate in all clones studied whereas in the case of the clones with lower inducibility, the replication of the resident genome appeared to be enhanced by superinfection. At 39 degrees C, however, accumulation of the superinfecting genome was not accompanied by that of the resident genome, unless it had already been initiated before superinfection. These findings demonstrate that, when routinely cultivated at 39 degrees C, Cyp cells contain few viral DNA molecules readily available for autonomous replication and that, upon transfer to 33 degrees C, therefore, excision must first take place before the resident genome can accumulate as free viral DNA. Our findings also suggest that, unlike the P155 gene product provided by the resident viral genome upon induction, the allelic gene product supplied by the superinfecting genome may be less effective in triggering excision than in promoting replication.     

The physical state of herpes simplex virus DNA in infected human cells.

Treatment of herpes simplex virus type 1 (HSV-1)-infected human embryo lung (HEL) cells with phosphonoacetic acid (PAA) resulted in complete inhibition of HSV DNA replication. DNA was extracted from PAA-treated HEL cells infected with HSV-1 and centrifuged in a neutral CsCl density gradient. The HSV DNA sequences in the nuclei of PAA treated cells at 24 hr post infection banded at the same density as free HSV DNA (1.725 g/cm3), but a significant amount of viral DNA sequences were detected in the regions of cell DNA (1.700 g/cm3) as well as in the intermediate fractions as determined by hybridization with 3H HSV complementary RNA. The viral DNA sequences of lower deisntiy did not change in density by recentrifugation in a CsCl density gradient, but did change to the density of free viral DNA after treatment with EcoR1 restriction endonuclease. When the DNA from the nuclei of PAA treated cells was analyzed in an alkaline glycerol gradient, more than 95% of the viral DNA sequences were found in the free viral DNA fractions. Since the viral and cellular hybrid DNA represented approximately 33% of the total viral DNA sequences, it is concluded that some of the HSV DNA sequences in PAA treated, infected cells are associated with cell DNA by alkali-labile bonds.     

State of the viral DNA in rat cells transformed by polyma virus. II. Identification of the cells containing nonintegrated viral DNA and the effect of viral mutations.

F2408 rat cells transformed by polyoma virus contained integrated and nonintegrated viral DNA. The presence of nonintegrated viral DNA is under control of the A early viral function. Polyoma ts-a-transformed rat cells lose the free viral DNA when growth at the nonpermissive temperature (40 degrees C), but they reexpress it 1 to 3 days after they are shifted back to the permissive temperature. In contrast, rat cells transformed by a late viral mutant, ts-8, contain free viral DNA at both permissive and nonpermissive temperatures. Treatment of the transformed rat cells with mitomycin C produces a large increase in the quantity of free viral DNA and some production of infectious virus. Experiments of in situ hybridization, with 3H-labeled polyoma complementary RNA as a probe, show that only a minority (approximately 0.1%) of the transformed cells contain nonintegrated viral DNA at any given time. These results suggest that the presence of free viral DNA in polyoma-transformed rat cells is caused by a spontaneous induction of viral DNA replication, occurring with low but constant probability in the transformed cell population, and that the free viral DNA molecules originate from the integrated ones, probably through a phenomenon of excision and limited replication.     

An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

To identify specific cellular factors which could be required during the synthesis of retroviral DNA, we have studied the replication of murine leukemia virus in mouse cells temperature sensitive for cell DNA synthesis (M. L. Slater and H. L. Ozer, Cell 7:289-295, 1976) and in several of their revertants. This mutation has previously been mapped on the X chromosome. We found that a short incubation of mutant cells at a nonpermissive temperature (39 degrees C) during the early part of the virus cycle (between 0- to 20-h postinfection) greatly inhibited virus production. This effect was not observed in revertant or wild-type cells. Molecular studies by the Southern transfer procedure of the unintegrated viral DNA synthesized in these cells at a permissive (33 degrees C) or nonpermissive temperature revealed that the levels of linear double-stranded viral DNA (8.8 kilobase pairs) were nearly identical in mutant or revertant cells incubated at 33 or 39 degrees C. However, the levels of two species of supercoiled viral DNA (with one or two long terminal repeats) were significantly lower in mutant cells incubated at 39 degrees C than in mutant cells incubated at 33 degrees C or in revertant cells incubated at 39 degrees C. Pulse-chase experiments showed that linear viral DNA made at 39 degrees C could not be converted into supercoiled viral DNA in mutant cells after a shift down to 33 degrees C. In contrast, such conversion was observed in revertant cells. Restriction endonuclease analysis did not detect differences in the structure of linear viral DNA made at 39 degrees C in mutant cells as compared to linear viral DNA isolated from the same cells at 33 degrees C. However, linear viral DNA made at 39 degrees C in mutant cells was poorly infectious in transfection assays. Taken together, these results strongly suggest that this X-linked gene, affecting mouse cell DNA synthesis, is operating in the early phase of murine leukemia virus replication. It seems to affect the level of production of unintegrated linear viral DNA only slightly while greatly reducing the infectivity of these molecules. In contrast, the accumulation of supercoiled viral DNA and subsequent progeny virus production are greatly reduced. Our pulse-chase experiments suggest that the apparent, but not yet identified, defect in linear viral DNA molecules might be responsible for their subsequent impaired circularization.     

Integration and excision of SV40 DNA from the chromosome of a transformed cell

The single insertion of SV40 DNA present in the genome of the 14B line of transformed rat cells has been cloned in procaryotic vectors. Analysis of the clones reveals a complex arrangement of viral sequences in which a small tract of DNA is inverted with respect to the major insertion. The nucleotide sequences at the two junctions show sharp transitions between cellular and viral sequences. The sequences which flank the viral insertion have been used as probes to clone the corresponding genomic sequences from the DNA of untransformed rat cells. Analysis of the structure of these clones shows that a rearrangement of cellular sequences has occurred, presumably as a consequence of integration. When 14B cells are fused with uninfected simian cells a heterogeneous set of low molecular weight superhelical DNAs containing viral sequences is generated. These have been cloned in procaryotic vectors and their structures have been analyzed. All of them span the origin of SV40 DNA replication and are colinear with various segments of the integrated viral DNA and its flanking sequences. The shorter molecules contain part of the integrated viral genome and cellular sequences from one side of the insertion. They were therefore generated by recombination between the viral DNA and its flanking cellular sequences. The longer molecules contain cellular sequences from both sides of the insertion as well as an entire copy of the integrated viral DNA. They were therefore generated by recombination between the flanking cellular sequences. These results argue strongly against the involvement of specific excision enzymes, and rather are discussed in terms of a model involving replication of the integrated viral DNA followed by recombination for release of integrated viral sequences.     

Variations in integration site of avian oncornaviruses in different hosts.

We examined the integration site of avian oncornaviruses in the genome of different hosts with respect to the repetitive frequency of the cellular DNA sequences adjacent to the integrated proviral DNA. The following systems were studied: avian sarcoma virus (B-77) and avian leukosis virus (Rous-associated virus-61) in cultured duck embryonic cells and B-77 in cultured mouse 3T3 cells. These systems represent different host responses to viral infection, i.e., one in which both cellular transformation and viral replication occur (B-77-infected duck cells), one in which viral replication, but not transformation, occurs (Rous-associated virus-61-infected duck cells), and one in which transformation, but not viral replication, occurs (B-77-infected 3T3 cells). Two sequential hybridizations were used. First, large denatured DNA fragments (2.8 X 10(6) daltons) were reassociated to different C0t (mole-seconds per liter) values. Next, DNA remaining single stranded at different C0t values was isolated by hydroxylapatite column chromatography, immobilized on nitrocellulose filters, and hybridized with an excess of 3H-labeled 35S viral RNA to titrate the concentration of proviral DNA. Results show that B-77 sarcoma virus and Rous-associated virus-61 integrate in the unique region of duck DNA, whereas B-77 proviral DNA is associated with both repeated and unique host DNA sequences in transformed mouse 3T3 cells.     

A plasmid cloning system utilizing replication and packaging functions of the filamentous bacteriophage fd

DNA cloning vectors were developed which utilize the replication origin (ori) of bacteriophage fd for their propagation. These vectors depend on the expression of viral gene 2 that was inserted into phage lambda, which in turn was integrated into the host genome. The constitutive expression of gene 2 in the host cells is sufficient for the propagation of at least 100 pfd plasmids per cell. In addition to the fd ori, the pfd vectors carry various antibiotic-resistance genes and unique restriction sites. Some of these vectors have no homologies to commonly used pBR plasmids or to lambda DNA. The nucleotide sequence of the vectors can be deduced from published sequences. Large DNA inserts can be stably propagated in pfd vectors; these are more stable than similar DNA fragments cloned in intact genomes of filamentous bacteriophage. Inclusion of phage sequences required for efficient phage packaging and infection with a helper phage resulted in formation of phage particles containing single-stranded plasmid genomes. Growth at 42 degrees C without selective pressure results in loss of pfd plasmids.     

Sequences flanking the pentanucleotide T-antigen binding sites in the polyomavirus core origin help determine selectivity of DNA replication.

Replication of the genomes of the polyomaviruses requires two virus-specified elements, the cis-acting origin of DNA replication, with its auxiliary DNA elements, and the trans-acting viral large tumor antigen (T antigen). Appropriate interactions between them initiate the assembly of a replication complex which, together with cellular proteins, is responsible for primer synthesis and DNA chain elongation. The organization of cis-acting elements within the origins of the polyomaviruses which replicate in mammalian cells is conserved; however, these origins are sufficiently distinct that the T antigen of one virus may function inefficiently or not at all to initiate replication at the origin of another virus. We have studied the basis for such replication selectivity between the murine polyomavirus T antigen and the primate lymphotropic polyomavirus origin. The murine polyomavirus T antigen is capable of carrying out the early steps of the assembly of an initiation complex at the lymphotropic papovavirus origin, including binding to and deformation of origin sequences in vitro. However, the T antigen inefficiently unwinds the origin, and unwinding is influenced by sequences flanking the T antigen pentanucleotide binding sites on the late side of the viral core origin. These same sequences contribute to the replication selectivity observed in vivo and in vitro, suggesting that the inefficient unwinding is the cause of the replication defect. These observations suggest a mechanism by which origins of DNA replication can evolve replication selectivity and by which the function of diverse cellular origins might be temporally activated during the S phase of the eukaryotic cell cycle.     

Replication and recombination in adenovirus-infected cells are temporally and functionally related.

We have studied the temporal and functional relationships between DNA replication and recombination in adenovirus-infected cells by using Southern blot hybridization to detect recombinant products among intracellular viral genomes. The data show that recombination can be detected soon after DNA replication has commenced and that the proportion of recombinant products increases thereafter. To determine the functional relationship between DNA replication and recombination, replication was blocked with the protein synthesis inhibitor anisomycin, the replication inhibitor cytosine arabinoside, and conditionally lethal mutations in either the virus-specified DNA-binding protein or the DNA polymerase. All treatments that directly or indirectly blocked DNA replication caused a delay in the appearance of recombinant products and a marked decline in their abundance relative to products of parental genotype. These data strongly suggest that DNA replication and recombination are interrelated, either because both processes share functions or because DNA structures produced by replication are suitable substrates for recombination. In addition, we have shown that some recombination function(s) is intrinsically thermolabile at 40.9 degrees C, even in wild-type crosses, since the appearance of recombinant products is delayed and their extent is reduced compared with that from crosses performed at 39.9 degrees C.     

An autonomous replicating element within the KSHV genome

Members of the herpesviridae family including Kaposi's sarcoma-associated herpesvirus (KSHV) persist latently in their hosts and harbor their genomes as closed circular episomes. Propagation of the KSHV genome into new daughter cells requires replication of the episome once every cell division and is considered critically dependent on expression of the virus encoded latency-associated nuclear antigen (LANA). This study demonstrates a LANA-independent mechanism of KSHV latent DNA replication. A cis-acting DNA element within a discreet KSHV genomic region termed the long unique region (LUR) can initiate and support replication of plasmids lacking LANA-binding sequences or a eukaryotic replication origin. The human cellular replication machinery proteins ORC2 and MCM3 associated with the LUR element and depletion of cellular ORC2 abolished replication of the plasmids indicating that recruitment of the host cellular replication machinery is important for LUR-dependent replication. Thus, KSHV can initiate replication of its genome independent of any trans-acting viral factors.     

The papillomavirus E8-E2C protein represses DNA replication from extrachromosomal origins

Carcinogenic DNA viruses such as high-risk human papillomaviruses (HPV) and Epstein-Barr-Virus (EBV) replicate during persistent infections as low-copy-number plasmids. EBV DNA replication is restricted by host cell replication licensing mechanisms. In contrast, copy number control of HPV genomes is not under cellular control but involves the viral sequence-specific DNA-binding E2 activator and E8-E2C repressor proteins. Analysis of HPV31 mutant genomes revealed that residues outside of the DNA-binding/dimerization domain of E8-E2C limit viral DNA replication, indicating that binding site competition or heterodimerization among E2 and E8-E2C proteins does not contribute to copy number control. Domain swap experiments demonstrated that the amino-terminal 21 amino acids of E8-E2C represent a novel, transferable DNA replication repressor domain, whose activity requires conserved lysine and tryptophan residues. Furthermore, E8-E2C (1-21)-GAL4 fusion proteins inhibited the replication of the plasmid origin of replication of EBV, suggesting that E8-E2C functions as a general replication repressor of extrachromosomal origins. This finding could be important for the development of novel therapies against persistent DNA tumor virus infections.     

Tracking adenovirus genomes identifies morphologically distinct late DNA replication compartments

In adenoviral virions, the genome is organized into a chromatin‐like structure by viral basic core proteins. Consequently viral DNAs must be replicated, chromatinized and packed into progeny virions in infected cells. Although viral DNA replication centers can be visualized by virtue of viral and cellular factors, the spatiotemporal regulation of viral genomes during subsequent steps remains to be elucidated. In this study, we used imaging analyses to examine the fate of adenoviral genomes and to track newly replicated viral DNA as well as replication‐related factors. We show de novo formation of a subnuclear domain, which we termed Virus‐induced Post‐Replication (ViPR) body, that emerges concomitantly with or immediately after disintegration of initial replication centers. Using a nucleoside analogue, we show that viral genomes continue being synthesized in morphologically distinct replication compartments at the periphery of ViPR bodies and are then transported inward. In addition, we identified a nucleolar protein Mybbp1a as a molecular marker for ViPR bodies, which specifically associated with viral core protein VII. In conclusion, our work demonstrates the formation of previously uncharacterized viral DNA replication compartments specific for late phases of infection that produce progeny viral genomes accumulating in ViPR bodies.