The baboon endogenous virus genome. II. Provirus sequence variations in baboon cell DNA.

Restriction analysis of the approximately 100 integrated baboon endogenous virus (BaEV) proviruses in baboon cells and tissues has revealed two major sequence variations, both in the gag gene region of the genome. One, a 150 nucleotide pair insert, is present in a small proportion of the proviral DNAs and some baboons, but is present in the majority of the proviral DNAs of other baboons. The second, a Bam HI recognition sequence located 2.25 kb from the proviral 5' end, is missing or modified in approximately one-half of the integrated genomes. We consider the possibility that accumulation of proviruses not containing the 0.15 kb insert is correlated with viral activation and expression since it is this form that is a replication intermediate in freshly infected permissive cells. It is evident from these initial studies that the organization of the multiple BaEV proviruses in baboon DNA has undergone modification during evolution.     

Complete nucleotide sequence of simian endogenous type D retrovirus with intact genome organization: evidence for ancestry to simian retrovirus and baboon endogenous virus.

A complete endogenous type D viral genome has been isolated from a baboon genomic library. The provirus, simian endogenous retrovirus (SERV), is 8,393 nucleotides long and contains two long terminal repeats and complete genes for gag, pro, pol, and env. The primer binding site is complementary to tRNA(Lys)3, like in lentiviruses. The env GP70 protein is highly homologous to that of baboon endogenous virus (BaEV). PCR analysis of primate DNA showed that related proviral sequences are present in Old World monkeys of the subfamily Cercopithecinae but not in apes and humans. Analysis of virus and host sequences indicated that the proviral genomes were inherited from a common ancestor. Comparison of the evolution of BaEV, exogenous simian retrovirus types 1 to 3 (SRV1 to SRV3), and SERV suggests that SERV is ancestral to both BaEV and the SRVs.     

Expression of baboon endogenous virus in exogenously infected baboon cells.

Strains of low-passage, fetal diploid, baboon (Papio cynocephalus) fibroblasts were susceptible to exogenous infection with three independent isolates of baboon endogenous virus, as measured by an immunofluorescence assay specific for viral p28. Infectivity of the M7 strain of baboon endogenous virus for baboon cells of fetal skin muscle origin was equivalent to that for human and dog cells in that similar, linear, single-hit titration patterns were obtained. The assay for supernatant RNA-dependent DNA polymerase, however, showed that baboon cells produced only low levels of virus after infection compared with the production by heterologous cells. The results showed that baboon endogenous virus was capable of penetrating baboon cells and that viral genes were expressed in infected cells. Replication of complete infectious virus was restricted, however, indicating that in this primate system homologous cells differentially regulated the expression of viral genes.     

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.     

Purification and characterization of baboon endogenous virus DNA polymerase.

An RNA-directed DNA polymerase was purified from baboon endogenous type-C virus by successive column chromatography on DEAE cellulose, phosphocellulose and hydroxyapatite. The purified DNA polymerase has a molecular weight of 68 000, a pH optimum of 8.0, a Mn2+ optimum of 1 mM, and a KCl optimum of 40 mM. The purified enzyme transcribes heteropolymeric regions of viral 60--70 S RNA isolated from different type-C viruses. The purified enzyme is immunologically related to a similarly purified polymerase from the cat endogenous type-C virus RD114.     

Structure of the baboon endogenous virus genome: cloning of circular virus DNA in bacteriophage lambda.

Linear, small and large circular forms of unintegrated viral DNAs were detected in Hirt supernatant fraction of human cultured cells infected with baboon endogenous virus M7. The circular M7 DNAs were cloned in bacteriophage lambda, Charon 28. Seventeen independent clones were isolated and analyzed by restriction endonuclease mapping. Nine clones were carrying a viral sequence of 8.6 kilobase pairs (kb) with two tandem repeats of 0.6 kb, which correspond to the large circular form of the unintegrated M7 DNA. Eight other clones had the viral insert of 8.0 kb, i. e., the small circular form, and were deleted one of the repeated sequences. The repeated sequences correspond to the long terminal repeats of 0.6 kb, located at both ends of the linear M7 DNA of 8.6 kb. One of the recombinants of the large circular M7 DNA had an inversion of 2.5 kb. One end of the inverted sequence was near the terminus of the long terminal repeats and the other in the gag gene region. The inversion seems to be occurred by integration of a viral DNA within itself during early periods of infection. The mechanism of the processes leading to integration is discussed from the structure of these unintegrated M7 DNAs as the precursors.     

The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencing.

We have determined the complete primary structure (8031 base pairs) of an infectious clone of cauliflower mosaic virus strain CM1841. The sequence was obtained using the strategy of cloning shotgun restriction fragments in the sequencing vector M13mp7. Comparison of the CM1841 sequence with that published for another caMV strain (Strasbourg) reveals 4.4% changes, mostly nucleotide substitutions with a few small insertions and deletions. The six open reading frames in the sequence of the Strasbourg isolate are also present in CM1841.     

Nucleotide sequence and structural organization of the 3'-terminal region of potato virus M

The sequence of 2630 3'-terminal nucleotides has been determined for the genomic RNA of potato virus M (PVM), a type member of the carlavirus group. Analysis of this nucleotide sequence revealed five open reading frames coding for proteins of mol. wt. 25, 12, 7, 34 and 11 kDa (in 5'----3' direction). The PVM genome organization has been shown to be basically analogous to that of potexviruses, except that the latter at their 3' end lack the gene encoding 11K protein. Amino acid sequence comparison between the PVM 34K protein and the coat proteins of potexviruses has shown a high extent of homology in the C-terminal amino acids. Homology has also been found between the 25, 12 and 7K proteins encoded by PVM RNA and corresponding potexvirus proteins. All this gave grounds for suggestion that carlaviruses and potexviruses belong to the same subgroup of phytoviruses.     

Genomic structure of human polyoma virus JC: nucleotide sequence of the region containing replication origin and small-T-antigen gene

The nucleotide sequence of the region of human polyoma virus JC DNA between 0.5 and 0.7 map units from a unique EcoRI cleavage site was determined and compared with those of the corresponding regions of another human polyoma virus, BK, and simian virus 40 DNAs. Within this region consisting of 945 base pairs, we located the origin of DNA replication near 0.7 map units, the entire coding region for small T antigen, and the splice junctions for large-T-antigen mRNA. The deduced amino acid sequences for small T antigen and the part of large T antigen markedly resembled those of polyoma virus BK and simian virus 40. The results strongly suggest that polyoma virus JC has the same organization of early genome as polyoma virus BK and simian virus 40 on the physical map, with the EcoRI site as a reference point.     

Cell receptors for baboon endogenous virus recognized by monoclonal antibodies.

Hybridomas from mice immunized with baboon endogenous virus (BaEV) from A204(M7) cells produced several antiviral monoclonal antibodies and, in addition, antibodies D-12 and E-4, which appeared to be virus specific because they reacted with BaEV but not with Mason-Pfizer virus or RD-114 virus. However, they also bound to human virus-free cells, and they did not recognize BaEV from bat or canine host cells. Cell membrane targets for these antibodies comigrated with an 18,000-dalton protein, which may contain specific determinants of BaEV receptors since antibody masking of these cell sites prevented BaEV but not Mason-Pfizer virus or RD-114 virus adsorption. However, RD-114 virus interfered with BaEV adsorption. Thus, the two viral receptors must be adjacent, but the antibody D-12 and E-4 targets are not within the active site of RD-114 virus receptor. Conversely, cell coating with BaEV from bat or canine hosts inhibited antibody D-12 binding. Noncultivated human lymphocytes and cells from fetal organs bound much less antibody D-12 than did cells from established cell lines, with a correlation between amounts of antibody D-12 acceptor sites and BaEV receptors. Thus, in vivo, BaEV infection of human cells may be inefficient. In vitro, antibody D-12 treatment of chronically infected A204(M7) cells caused intracellular accumulation of viral proteins and decreased virus release, with no such effect on RD-114 virus-producing cells. Canine cells bound antibody D-12 only if coated with BaEV from A204(M7) cells, indicating that the human determinant coadsorbed with the virions to animal cells. Possibly, determinants of cell receptors participate in BaEV maturation and become associated with the virions.     

The nucleotide sequence of the genes encoded in early region 2b of human adenovirus type 7

The nucleotide sequence of a cloned DNA segment encoding the early region 2b from the group B human adenovirus Ad7 has been determined. When compared to Ad2, a group C adenovirus, these sequences were found to be approx. 80% homologous within the l-strand gene-coding regions. Most changes are transitions or transversions, although several deletions/insertions also occur within the N-terminal domain of one of the coding regions. The substantial nucleotide homology results in a high degree of amino acid conservation in the predicted polypeptides encoded by the early region 2b genes. Two major open reading frames, corresponding to the Mr 87000 and Mr 140000 polypeptides of Ad2, are found in the l strand of Ad7 between genome coordinates 28.5 to 23.1 and 13.8, respectively. The r strand of the DNA in this region encodes the three leader segments joined to the 5' end of the most late viral mRNAs, and also encodes the i-leader segment found between the second and third leaders on some mRNAs. The positions of the donor and acceptor splice sites of the three leaders are conserved and can be identified by homology to Ad2. Only two of the unidentified open reading frames (URF) in Ad2 (Gingeras et al., J. Biol. Chem., in press) can be found in Ad7. URF1, encoding an Mr 13500 polypeptide at genome coordinate 17, is predominantly conserved in nucleotide and amino acid sequence, but contains one half as many arginine amino acids as does URF1 of Ad2. URF2, encoding an Mr 13600 protein which lies within the i-leader region, is not well conserved in either nucleotide or amino acid sequence.