Comparative study of papovavirus DNA: BKV(MM), BKV(WT) and SV40.

Extensive physical mapping revealed that approximately 90% of the genomes of BKV(prototype, WT) and BKV (MM strain) are identical or closely related. Nucleotide sequences of the non-homologous regions and a large portion of the homologous regions have been determined for both genomes. The coding sequence of small t antigen of BKV(MM) is 216 nucleotides shorter than that of BKV(WT), even though no differences in biological function of the t antigen was observed. Both genomes contain three similar sets of 44-61 base-pair repeated sequences. However, the DNA sequence of the tandem repeats is totally different between BKV (human cell as host) and SV40 (monkey cell as host). On the other hand, the region between the N-terminus of the T antigen genes and the origin of replication is dominated by a similar set of palindromic sequences in BKV and SV40 DNA. There is also extensive homology between the regions which code for proteins in BKV and SV40, suggesting a close evolutionary relationship.     

Arrangement of the genome of the human papovavirus RF virus.

DNA from plaque-purified RF virus, a variant of BK virus, was found to contain two species of molecules. Hybridization of each DNA species to the fragments of BK virus DNA revealed that one species had a deletion corresponding to at least 50% of the late region and the other had a deletion corresponding to at least 40% of the early region of BK virus DNA. Analysis by cleavage of each RF virus DNA species with restriction endonucleases EcoRI, HindIII, AvaII, and PvuII, when compared with BK virus DNA, revealed that the size and number of fragments were different. These results suggest the loss of some restriction sites and the appearance of new sites, probably as a result of base changes in each RF virus DNA species. Furthermore, analysis of the restriction map of each DNA molecule revealed in insertion(s) in both DNA species.     

Uniform representation of the human papovavirus BK genome in transformed hamster cells.

The DNA of three cloned lines of hamster kidney cells transformed by human papovavirus BK DNA was examined by reassociation kinetics for viral sequences and found to contain 2.7 to 5.3 equivalents of viral DNA per diploid genome. In the one line examined with the four R-HindIII fragments of the human papovavirus BK genome, the entire viral genome was uniformly represented.     

Nucleotide sequence of the DNA replication origin for human papovavirus BKV: sequence and structural homology with SV40.

DNA and RNA sequencing techniques were used to obtain the sequence surrounding the origin of DNA replication for human papovavirus BKV. The structure is characterized by a true palindrome of 17 residues followed by two sets of symmetrical sequences and a stretch of 20 AT residues. Within the two symmetrical sequences is a segment containing a strong purine bias, 23 of 26 nucleotides. These structures are similar, if not identical, to those found in the region of the SV40 replication, origin. Within the homologous DNA segments, 60-80% of the BKV and SV40 nucleotides are the same. The remarkable similarity of BKV and SV40 sequences containing the origins of DNA replication would appear to confirm our previous suggestion of an evolutionary relationship between the two genomes. In addition, topological similarities between these sequences suggest the possibility of certain structural requirements for bidirectional replication origins in these superhelical DNAs.     

Characterization of the genome of the murine papovavirus K.

The DNA genome of the murine papovavirus K virus (KV) was characterized and compared with the genome of polyoma virus. A physical map of the KV genome was constructed by analysis of the size of DNA fragments generated by sequential cleavage with combinations of restriction endonucleases. By using one of the three EcoRI sites in the KV genome as the 0 map position, the KV physical map was then oriented to the polyoma virus genome. Of 42 restriction sites mapped within the KV genome, 7 were localized within 0.01 map unit of their respective sites in the polyoma virus genome; an eighth site mapped within 0.02 map unit. KV replication was examined and found to be bidirectional, initiating at approximately 0.70 map unit. This corresponds well to the origin of replication within the polyoma virus genome and further supports the orientation of the KV physical map.     

Organization of viral genome in a T antigen-negative hamster tumor induced by human papovavirus BK.

We analyzed by blot hybridization the state and structure of the viral DNA in an exceptional BK virus-induced hamster tumor (choroid plexus papilloma Vn-324) that contains about one copy of the BK virus genome per cell, but no intranuclear T antigen as assayed by indirect immunofluorescence. The BK viral DNA was found to be integrated into cellular DNA at a site in the middle of the early region of the viral genome (between 0.32 and 0.41 map units). The structure of the inserted viral DNA shows that it cannot encode a full-size large T antigen, but may encode small T antigen and an N-terminal portion of large T antigen.     

Characterization of human papovavirus BK DNA.

The DNA of the BK virus (BKV) human papovavirus was found to be heterogeneous, consisting of at least four discrete species of DNA. Only the largest of these four species, BKV DNA (i), which has a molecular weight calculated to be 96% that of simian virus 40 (SV40) DNA, was infectious. Homogeneous preparations of BKV DNA were obtained, however, from virions purified after low multiplicity infections of human embryonic kidney cells. BKV DNA (i) was shown to contain a single R-Eco RI and four R-Hind cleavage sites. The R-Eco RI site was localized in the largest R-Hind cleavage fragment. Radiolabeled BKV DNA reassociated slightly faster than SV40 DNA; 20 to 30% polynucleotide sequence homology was demonstrated between the genomes of BKV and SV40 when the reaction was monitored by chromatography on hydroxyapatite.     

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.     

Genome analysis of MG virus, a human papovavirus.

The single late 26S mRNA of Semliki Forest virus (SFV) directs the synthesis of the four viral structural proteins, C, E3, E2, and E1, and the recently described nonstructural protein, 6K. We report here partial NH2-terminal amino acid sequences of the SFV polypeptides E3 and 6K and of p62, the precursor to E3 and E2. In addition, were have determined a partial NH2-terminal sequence of the Sindbis virus homolog of 6K, the 4.2K protein. p62 and E3 of SFV have identical NH2-terminal amino acid sequences. Comparison of the partial NH2-terminal sequences of 6K of SFV and 4.2K of Sindbis virus with the deduced amino acid sequence encoded by the 26S mRNA of each virus reveals that the genes for these peptides are located in each case between those for E2 and E1. The order of the genes on the 26S mRNA of the alphaviruses is therefore 5'-C-E3-E2-6K-E1-3'. We discuss two mechanisms by which the nascent viral glycoproteins may be inserted into the membrane of the endoplasmic reticulum.     

Biological properties and physical map of the genome of a new papovavirus, HD virus.

The superhelical DNA of the HD papovavirus is heterogeneous and consists of two discrete size classes with molecular weights of 3.45 X 10(6) and 3.25 X 10(6). Both size classes of DNA are encapsidated into HD virion particles. Their relative intracellular amounts differ, depending on the cell system. Vero-76 carrier cultures in which HD virus was detected contain both size classes of DNA, with the larger molecules prevailing by a factor of 10. Five clonal lines derived from Vero-76 cell cultures contain exclusively the larger DNA. On the other hand, after cocultivation of Vero-76 with CV-1 cells for several passages, minicircular DNA is accumulated such that both size classes are synthesized in equal amounts. Any of the originally viral DNA-producing cell lines may, upon subcultivation, cease yielding virus. The RITA cell line of Cercopithecus aethiops origin is the only cell line among numerous ones tested which upon infection permits the establishment of a one-step growth cycle. However, between 6 and 8 days after infection, viral DNA synthesis is discontinued, and a persistent viral infection cannot be established. Physical maps of the genomes were constructed, and it could be shown that the smaller, minicircular DNA had originated from the larger DNA as the result of a deletion. The sequences missing in the minicircular DNA are confined to the relative map position 0.15 to 0.21.     

The early region of human papovavirus JC induces dysmyelination in transgenic mice

Transgenic mice containing the early region of human papovavirus JC were produced. Some of these mice exhibited a shaking disorder similar to the previously described mutant mice jimpy or quaking. Neuropathological analysis indicated a dysmyelination in the central nervous system, but not the peripheral nervous system. A high level of JCV T-antigen mRNA was present in the brains of the mice exhibiting the myelin disorder. JC virus is associated in humans with a degenerative demyelinating disease: progressive multifocal leukoencephalopathy. The JCV-containing transgenic mice may therefore provide an animal model for studying this disease.     

The DNA sequence of the human cytomegalovirus genome.

In the first part of this article we review what has been learnt from the analysis of the sequence of HCMV. A summary of this information is presented in the form of an updated map of the viral genome. HCMV is representative of a major lineage of herpesviruses distinct from previously sequenced members of this viral family and demonstrates striking differences in genetic content and organization. The virus encodes approximately 200 genes, including nine gene families, a large number of glycoprotein genes, and homologues of the human HLA class I and G protein-coupled receptor genes. The HCMV sequence thus provides a sound basis for future molecular studies of this highly complex eukaryotic virus. The second part discusses the practical rate of DNA sequencing as deduced from this and other studies. The 229 kilobase pair DNA genome of human cytomegalovirus (HCMV) strain AD169 is the largest contiguous sequence determined to date, and as such provides a realistic benchmark for assessing the practical rate of DNA sequencing as opposed to theoretical calculations which are usually much greater. The sequence was determined manually and we assess the impact of new developments in DNA sequencing.