Using nonstringent annealing conditions and a 2.75-kilobase segment of cloned African green monkey DNA that specifically hybridizes to the proviruses of AKR'ecotropic murine leukemia virus (MuLV) and baboon endogenous virus (BaEV) as a probe, we detected related sequences in three different preparations of human brain DNA fragments. The blot-hybridization pattern obtained with cleaved human DNA was similar to that previously reported for the interaction of MuLV cDNA and cleaved mouse DNA and suggested the presence ofnumerous copies of retrovirus-related sequences in the human genome. The labeled 2.75-kilobase fragment derived from cloned monkey DNA was used to screen a human DNA library in Charon 4A. One clone obtained hybridized to three contiguous MuLV-and BaEV-reactive fragments of the cloned monkey DNA and to multiple fragments of human DNA including a prominent 1.0-kilobase EcoRI fragment also present in the clone.In the mouse, certain endogenous type C proviruses have been shown to be vertically transmitted (1-3), have been mapped to specific chromosomal loci (4-6), and have the potential ofbeing expressed as infectious ecotropic and xenotropic (7) murine leukemia viruses (MuLV). Comparison ofresults from nucleic acid hybridization experiments, which indicate the existence of numerous copies of endogenous mouse proviruses (8-10), with those derived from genetic andvirus isolation studies, which point to a few defined inducible loci in the genomes of certain inbred mouse strains (4-6, 11), strongly suggests that the majority of the MuLV-reactive sequences in mouse DNA consist ofincomplete viral DNA segments whose function is unknown.Type C endogenous retroviruses have also been recovered from several different Old World monkeys including the baboon (12), stump-tail macaque (13), rhesus (14), and colobus (15 We recently. reported the molecular cloning of a 17-kilobase (kb) segment from African green monkey (AGM) DNA which had nearly 5 kb of homology with AKR ecotropic MuLV DNA (21). In the present manuscript we show that the internal organization of the endogenous type C AGM and baboon provi:-ruses, including 8 of 10 restriction enzyme cleavage sites, hasbeen highly conserved. Furthermore, by using a radiolabeled. 2.75-kb BamHI fragment ofthe cloned ACM-DNA, which specifically hybridizes to a similarly sized BamHI fragment (22) of the BaEV provirus, hybridization to several bands in three different restricted preparations of human DNA was observed. Based on our ability to detect type C virus-related sequences in human DNA, we cloned an 11-kb segment from human DNA which hybridizes to AGM and mouse proviral DNAs.MATERIALS AND METHODS Preparation and Cleavage ofCellular DNA. High molecular weight AGM and rhesus monkey liver DNAs were purified from fresh tissue as described (23). Baboon cellular-DNA was prepared from a cell line (CP 21) established from primary skin fibroblasts. DNA was isolated from three human brain specimens as outlined (24). Cellular DNAs were digested with restriction enzymes, ele...

A specific probe for detecting ecotropic murine leukemia virus sequences was constructed by cloning a 500-base-pair DNA segment, corresponding to a portion of the env region of the AKR ecotropic virus, in a pBR322/Escherichia coli K-12 host/vector system. This probe was used to screen the cellular DNAs of six inbred strains of mice for the presence of ecotropic retroviral DNA sequences by the Southern blot hybridization procedure. Three copies of ecotropic viral DNA were detected in AKR/N (a high-ecotropic virus strain) and two were found in BALB/c (a low-ecotropic virus strain) DNAs. As expected, no sequences reactive with this probe were found in NFS mouse DNA (a virus-negative strain). However, cellular DNA sequences that reacted strongly with the ecotropicsecific DNA probe were detected in certain NZB, C57L, and 129 mice (all virus-negative strains). In contrast to the reactive sequences in AKR and BALB/c, the reactions were chiefly associated with EcoRI segments that were subgenomic in size. The chromosomal DNA of inbred and feral mice contains multiple copies of sequences reactive with murine leukemia virus (MuLV) nucleic acid probes (1-7). Among these sequences are complete, potentially infectious genomes of ecotropic and xenotropic viruses (8), the complete genetic information of amphotropic and additional xenotropic viruses that apparently cannot be expressed as infectious viruses in inbred mice (ref. 9; unpublished data), proviral DNA that is expressed only in the form of viral antigens (10-12), and probably many subgenomic viral DNA segments that are not expressed.Because of their abundance in mouse chromosomal DNA and the extensive polynucleotide sequence homology between large portions of the genomes of the various classes of MuLV, little is known about the molecular organization of the endogenous proviral DNA sequences of the particular MuLV types. Although absorption of labeled MuLV cDNA with purified viral RNA from a heterologous MuLV decreases the crossreaction with endogenous sequences (6, 7), such probes still hybridize to a significant extent to related proviruses present in mouse DNA. Clearly, the preparation of nucleic probes that recognize type-specific regions of proviral DNA would offer great advantages.The molecular cloning of AKR ecotropic MuLV (AKV) DNA (13) has made it possible to generate such a probe. We have subcloned, in Escherichia colt K-12 with a pBR322 vector, a portion of the AKV env gene region that is specific for ecotropic viruses in the operational sense that it does not react either with cellular DNA prepared from the prototype ecotropic-negative mouse strain NFS/N or with genomic DNA of a xenotropic MuLV.We describe here the construction and characterization of the recombinant plasmid, illustrate the strategy developed for using the cloned DNA to identify endogenous ecotropic proviruses, and report the presence of a unique set of nucleotide sequences reactive with this probe in the DNA of several mouse strains that, like NFS/N, are biologically negative for...

Human DNA contains many copies of endogenous retroviral sequences. Characterization of molecular clones of these structures reveals the existence of two related families. One family consists of full-length (8.8 kilobases) proviral structures, with typical long terminal repeates (LTR's). The other family consists of structures, which contain only 4.1 kilobases of gag-pol sequences, bounded by a tandem array of imperfect repeats 72 to 76 base pairs in length. Typical LTR sequences that exist as solitary elements in the genome were cloned and characterized.

The full-length sequence of simian foamy virus serotype 2 (SFVmcy-2), isolated from a Taiwanese macaque, was determined. SFVmcy-2 was highly related to SFV serotype 1 (SFVmcy-1), an isolate from the same species, except in the putative receptor binding domain (RBD) in env, which contained novel sequences related to SFV serotype 3 (SFVagm-3), isolated from an African green monkey. The results identify a potential region of neutralization in SFVs and demonstrate recombination between genetically divergent foamy viruses. Simian foamy viruses (SFVs) belong in the Spumavirus genus of the Spumaretrovirinae subfamily of Retroviridae and are widespread in all nonhuman primates (NHPs) (1, 2). SFVs have been isolated from various tissues of different NHPs and were originally designated based upon neutralization serotyping (3). Original foamy virus isolates were designated SFV serotype 1, SFV serotype 2, and SFV serotype 3 (SFV-1, SFV-2, and SFV-3, respectively). These original monkey isolates were renamed to indicate the species of isolation (4): SFV-1, which was isolated from a Taiwanese macaque (Formosan Rock macaque or Macaca cyclopis [mcy]) (5, 6), was designated SFVmac, and SFV-3, which was isolated from an African green monkey (AGM; Chlorocebus aethiops) (7), was designated SFVagm. To distinguish SFVmac/SFV-1 from SFV-2, which was also isolated from M. cyclopis (5), in this paper we have designated these SFVmac viruses SFVmcy-1 and SFVmcy-2, respectively, and the SFVagm virus SFVagm-3 to distinguish it from other AGM isolates. Early results of virus isolation or antibody detection based upon neutralization serotyping showed that, although macaques generally harbor SFVs of serotype 1, viruses with serotype 2 were also present in some cases. In fact, SFVs of both serotype 1 and serotype 2 were isolated from different organs within the same monkey as well as from a single organ (8). AGMs were found to be typically infected with SFVs of serotype 2 and serotype 3, but SFV of serotype 1 was also seen (7-9). More recent molecular studies have also shown that although SFVs generally circulate within a host species, interspecies transmission can occur (10, 11).The biological properties of SFVmcy-1 and SFVagm-3 have been well studied, and whole-genome sequences for both viruses have been determined (6, 12, 13). Our previous studies using a variety of cell lines from different species showed that SFVmcy-2 has a broad host range but different kinetics of replication in different cell lines (14). In this study, we have determined the complete nucleotide sequence of SFVmcy-2 and have determined its genetic relatedness to SFVmcy-1 and SFVagm-3 as well as to other SFVs.The genomic sequence and structure of SFVmcy-2 was determined using virus acquired from the American Type Culture Collection (Manassas, VA) (SFV type 2, catalogue number VR 277, FV-34, lot 4D, 91-10). Full-length SFVmcy-2 sequences were obtained by assembly of cloned DNAs obtained by restriction enzyme digestion (DNA 447) or PCR amplication (DNA 448 and DNA 549) of h...