Genetic heterogeneity of the human papovaviruses BK and JC

We have examined the structure and infectivity of BKV and JCV genomes from prototype strains after cell culture passage and of BKV genomes from primary isolates. Genomic structures were determined by restriction endonuclease analysis of molecularly cloned DNA. Infectivity was determined by transfection of the cloned genomes into urine-derived epithelial cells and assaying for viral proteins and virus production. Prototype BKV DNA, which was cloned after 14 passages in three different cell lines, contained no alterations in restriction enzyme sites and was infectious. In contrast, prototype JCV acquired changes in the late region of the genome during passage in cell culture and the cloned DNA was not infectious. Urine-derived cells were used to isolate virus from the urine of two renal transplant patients and one asymptomatic individual. The genome of the virus isolated from the normal individual was indistinguishable from prototype BKV except for a 60-base pair deletion, which was localized between 0.62 and 0.72 map units. Two isolates from transplant patients differed from each other and from prototype BKV at a number of restriction enzyme cleavage sites located in the early region and were infectious. Genomes containing deletions from 100 to 600 base pairs were also cloned but were not infectious.     

The role of DNA methylation in virus replication: inhibition of frog virus 3 replication by 5-azacytidine

Frog virus 3 (FV3) DNA is the most highly methylated DNA of any known DNA virus; about 20% of the cytosine residues in FV3 DNA are methylated (D. Willis and A. Granoff, 1980, Virology 107, 250-257). To understand the role of DNA methylation in virus replication, we have examined the effect of 5-azacytidine, a drug that inhibits DNA methylation. 5-Azacytidine (10 microM) reduced the production of infectious FV3 by 100-fold or more and inhibited methylation of viral DNA by about 80%. Inhibition of DNA methylation did not affect viral gene expression since there was no detectable inhibition of virus-specific RNA or protein synthesis in 5-azacytidine-treated cells. In contrast, the size of the replicating DNA measured under completely denaturing conditions, was much smaller than that found during infection in the absence of drug. These results suggest that the undermethylated DNA was susceptible to endodeoxyribonuclease(s). Additionally, electron microscopic examination of FV3-infected, 5-azacytidine-treated cells revealed that preformed capsids remained empty or were only partially filled with viral DNA. Based on these data, it is suggested that methylation of DNA protects it from endonucleolytic cleavage and that the integrity of genomic DNA is required for its proper packaging into virions.     

Differential association with cellular substructures of pseudorabies virus DNA during early and late phases of replication

Pseudorabies virus DNA synthesis can be divided into two phases, early and late, which can be distinguished from each other on the basis of the structures of the replicating DNA. The two types of replicating virus DNA can also be distinguished from each other on the basis of the cellular substructures with which each is associated. Analysis by electron microscopic autoradiography showed that during the first round of replication, nascent virus DNA was found in the vicinity of the nuclear membrane; during later rounds of replication the nascent virus DNA was located centrally within the nucleus. The degree of association of virus DNA synthesized at early and late phases with the nuclear matrix fractions also differed; a larger proportion of late than of early nascent virus DNA was associated with this fraction. While nascent cellular DNA only was associated in significant amounts with the nuclear matrix fraction, a large part (up to 40%) of all the virus DNA remained associated with this fraction. However, no retention of specific virus proteins in this fraction was observed. Except for two virus proteins, which were preferentially extracted from the nuclear matrix, approximately 20% of all virus proteins remained in the nuclear matrix fraction. The large proportion of virus DNA associated with the nuclear fraction indicated that virus DNA may be intimately associated with some proteins. Indeed, protease-treated, "purified" DNA preparations contained two proteins (15K and 10K) with histone-like properties which were protected by the DNA from deproteinization, probably by virtue of being at the center of the concatemeric tangles of virus DNA. It is possible that these proteins play a role in anchoring virus DNA to the nuclear matrices.     

Genome-scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features

In metazoans, thousands of DNA replication origins (Oris) are activated at each cell cycle. Their genomic organization and their genetic nature remain elusive. Here, we characterized Oris by nascent strand (NS) purification and a genome-wide analysis in Drosophila and mouse cells. We show that in both species most CpG islands (CGI) contain Oris, although methylation is nearly absent in Drosophila, indicating that this epigenetic mark is not crucial for defining the activated origin. Initiation of DNA synthesis starts at the borders of CGI, resulting in a striking bimodal distribution of NS, suggestive of a dual initiation event. Oris contain a unique nucleotide skew around NS peaks, characterized by G/T and C/A overrepresentation at the 5' and 3' of Ori sites, respectively. Repeated GC-rich elements were detected, which are good predictors of Oris, suggesting that common sequence features are part of metazoan Oris. In the heterochromatic chromosome 4 of Drosophila, Oris correlated with HP1 binding sites. At the chromosome level, regions rich in Oris are early replicating, whereas Ori-poor regions are late replicating. The genome-wide analysis was coupled with a DNA combing analysis to unravel the organization of Oris. The results indicate that Oris are in a large excess, but their activation does not occur at random. They are organized in groups of site-specific but flexible origins that define replicons, where a single origin is activated in each replicon. This organization provides both site specificity and Ori firing flexibility in each replicon, allowing possible adaptation to environmental cues and cell fates.     

The late promoter of the human papovavirus BK is contained within the early promoter enhancer region

We have analyzed the sequences necessary for late promoter function and mapped the late mRNA start sites of the human papovavirus BK (prototype). Our results show that, under both replicating and nonreplicating conditions, the BKV late promoter is contained within the same region defined as the enhancer for the early promoter. This region consists of three 68-bp repeats (the middle one of which has an 18-bp deletion) and a 66-bp region containing an enhancer element denoted as c, located to the late side of the 68-bp repeats. Deletions within the early enhancer domain indicate that elements of the late promoter are found throughout the entire region.     

The pattern of replication at a human telomeric region (16p13.3): its relationship to chromosome structure and gene expression.

We have studied replication throughout 325 kb of the telomeric region of a human chromosome (16p13.3) and related the findings to various aspects of chromosome structure and function (DNA sequence organization, nuclease-hypersensitive sites, nuclear matrix attachment sites, patterns of methylation and gene expression). The GC-rich isochore lying adjacent to the telomere, which contains the alpha-globin locus and many widely expressed genes, replicates early in the cell cycle regardless of the pattern of gene expression. In subtelomeric DNA, replication occurs later in the cell cycle and the most telomeric region (20 kb) is late replicating. Juxtaposition of early replicating DNA next to the telomere causes it to replicate later in S-phase. Analysis of the timing of replication in chromosomes with deletions, or in transgenes containing various segments of this telomeric region, suggests that there are no critical origins or zones that initiate replication, rather the pattern of replication appears to be related to the underlying chromatin structure which may restrict or facilitate access to multiple, redundant origins. These results contrast with the pattern of replication at the human beta-globin locus and this may similarly reflect the different chromosomal environments containing these gene clusters.     

Unique DNA methylome profiles in CpG island methylator phenotype colon cancers

A subset of colorectal cancers was postulated to have the CpG island methylator phenotype (CIMP), a higher propensity for CpG island DNA methylation. The validity of CIMP, its molecular basis, and its prognostic value remain highly controversial. Using MBD-isolated genome sequencing, we mapped and compared genome-wide DNA methylation profiles of normal, non-CIMP, and CIMP colon specimens. Multidimensional scaling analysis revealed that each specimen could be clearly classified as normal, non-CIMP, and CIMP, thus signifying that these three groups have distinctly different global methylation patterns. We discovered 3780 sites in various genomic contexts that were hypermethylated in both non-CIMP and CIMP colon cancers when compared with normal colon. An additional 2026 sites were found to be hypermethylated in CIMP tumors only; and importantly, 80% of these sites were located in CpG islands. These data demonstrate on a genome-wide level that the additional hypermethylation seen in CIMP tumors occurs almost exclusively at CpG islands and support definitively that these tumors were appropriately named. When these sites were examined more closely, we found that 25% were adjacent to sites that were also hypermethylated in non-CIMP tumors. Thus, CIMP is also characterized by more extensive methylation of sites that are already prone to be hypermethylated in colon cancer. These observations indicate that CIMP tumors have specific defects in controlling both DNA methylation seeding and spreading and serve as an important first step in delineating molecular mechanisms that control these processes.     

Site and sequence specific DNA methylation in the neurofibromatosis (NF1) gene includes C5839T: the site of the recurrent substitution mutation in exon 31

CpG dinucleotides provide hotspots for transitional mutations in a variety of genes, some leading to genetic diseases in humans. Although this phenomenon is attributed to cytosine methylation at such sites, direct and specific observations of CpG methylation at the sites of recurrent mutations are lacking. We have used a bisulfite genomic sequencing method to analyze DNA methylation within three representative exons from the neurofibromatosis type 1 (NF1) gene, well recognized for its high frequency of spontaneous mutations. We observed that the cytosine methylation within NF1 exons 28, 29, and 31 is restricted to CpG dinucleotides, including the CpG dinucleotide present at the site of the recurrent NF1 mutation (C5839T; also referred to as R1947X). At several sites, clone-specific methylation differences were also observed. Our results provide experimental evidence for the hypothesis that methylatable CpGs in the NF1 gene contribute to spontaneous germline mutations associated with this gene, by showing that DNA methylation does occur at all CpGs contained within these representative NF1 exons. As well, the DNA methylation seen at the common mutation site in exon 31 may explain why this site is frequently mutated. Methylation-dependent mutagenesis may also provide a basis for some somatic (second hit) mutations which disable the normal allele and result in the development of NF1 associated symptoms.      

Genotypes of human polyomaviruses in urine samples of pregnant women in Taiwan

The viral DNA of human polyomaviruses JC virus (JCV) and BK virus (BKV) was detected by the polymerase chain reaction (PCR) in urine samples from 31 pregnant women in Taiwan. A pair of appropriate primers amplified both JCV and BKV DNA of the regulatory region simultaneously in PCR. An oligonucleotide probe homologous to both JCV and BKV regulatory region was used subsequently to detect the viral DNA by Southern blotting after PCR amplification. Approximately 36% of the examined urine samples were human polyomavirus positive. The genotypes of JCV and BKV were determined by DNA sequencing of their regulatory regions. Besides CY archetype, a new strain (Taiwan-1) of JCV with a pentanucleotide (GGGAA) deletion and a new strain (Taichung-1) of BKV with two nucleotide alterations within the regulatory region were found in the urine samples. Eight of the examined samples were JCV infected, one was BKV infected, and two were JCV and BKV mix-infected. The JCV positive individuals were infected by CY archetype and Taiwan-1 strain equally. However, Taichung-1 strain was the only BKV strain found in the BKV positive individuals. © 1996 Wiley-Liss, Inc.