Isolation of a retroviruslike sequence from the TL locus of the C57BL/10 murine major histocompatibility complex.

Two retroviruslike sequences have been isolated from the TL locus of the major histocompatibility complex of C57BL/10 mice. One sequence (TLev2) hybridizes only with probes derived from the pol region of the murine leukemia provirus AKR; the other sequence (TLev1) hybridizes with gag, pol, and env AKR region probes. This 9-kilobase endogenous, TL region-associated virus (TLev1) has been further characterized. The TLev1 genome has been shown to contain murine leukemia virus-related sequences bounded by retroviruslike, VL30 long terminal repeats. Hybridization of TLev1-derived probes to mouse genomic digests reveals multiple copies which show distinct patterns compared with those observed with murine leukemia virus probes. The study of TLev1 may prove significant with respect to the interaction of retroviral sequences within the genome, expression of genes within the TL locus, and polymorphisms within the major histocompatibility complex.     

Identification of a protease gene of human T-cell leukemia virus type I (HTLV-I) and its structural comparison

We determined the nucleotide sequence of a region between the gag and pol genes of a replication-competent proviral clone of a human T-cell leukemia virus type I (HTLV-I) from MT-2 cells. This region overlapping the gag and pol genes contains an open reading frame with a different phase from others. The deduced amino acid sequences show significant homology with the known protease gene of other retroviruses, and harbors highly conserved amino acid sequences that are well conserved in other retroviral protease domains. These results indicate that this open reading frame encodes a HTLV-I protease.     

Murine mammary tumor virus pol-related sequences in human DNA: characterization and sequence comparison with the complete murine mammary tumor virus pol gene.

Sequences in the human genome with homology to the murine mammary tumor virus (MMTV) pol gene were isolated from a human phage library. Ten clones with extensive pol homology were shown to define five separate loci. These loci share common sequences immediately adjacent to the pol-like segments and, in addition, contain a related repeat element which bounds this region. This organization is suggestive of a proviral structure. We estimate that the human genome contains 30 to 40 copies of these pol-related sequences. The pol region of one of the cloned segments (HM16) and the complete MMTV pol gene were sequenced and compared. The nucleotide homology between these pol sequences is 52% and is concentrated in the terminal regions. The MMTV pol gene contains a single long open reading frame encoding 899 amino acids and is demarcated from the partially overlapping putative gag gene by termination codons and a shift in translational reading frame. The pol sequence of HM16 is multiply terminated but does contain open reading frames which encode 370, 105, and 112 amino acid residues in separate reading frames. We deduced a composite pol protein sequence for HM16 by aligning it to the MMTV pol gene and then compared these sequences with other retroviral pol protein sequences. Conserved sequences occur in both the amino and carboxyl regions which lie within the polymerase and endonuclease domains of pol, respectively.     

Provirus of M7 baboon endogenous virus: nucleotide sequence of the gag-pol region

A 3,023-base nucleotide sequence of the M7 baboon endogenous virus genome, spanning the 5' noncoding region as well as the entire gag gene and part of the pol gene, is reported. Within the 562-base 5' noncoding region, a 21-base sequence complementary to the OH terminus of tRNApro is located immediately downstream from the long terminal repeat. Amino acid sequences were deduced from the 1,596 nucleotides comprising the gag gene, and the four structural gag polypeptides, p12, p15, p30, and p10, appeared to be coded contiguously. Only one termination codon interrupted the M7 gag and pol genes. The data suggest that 55 additional amino acids may be attached to the NH2 terminus of the gag precursor protein. However, such a sequence was not detected in virions or in virus-infected cells. With the exception of the p15 region, nucleotide and amino acid sequences of the gag and pol regions of M7 virus exhibited strong homologies to those of Moloney leukemia virus.     

Nucleotide sequence of a full-length human endogenous retroviral segment.

The nucleotide sequence of a full-length (8.8-kilobase) endogenous C-type human retroviral DNA (clone 4-1) is presented and compared with that of Moloney murine leukemia virus (MoMuLV) DNA. Colinearity of deduced amino acids of clone 4-1 with MoMuLV in the gag and pol regions was clearly evident, and overall amino acid homology in these regions was about 40%. Identification of the putative N terminus of gag and p30, the gag-pol junction, and the C terminus of pol could be established on the basis of sequence homology with MoMuLV. Unique characteristics of the endogenous human retroviral DNA included a tRNA Glu primer binding site separated from the 5' long terminal repeat by a pentanucleotide and a putative env sequence which does not appear to overlap the C terminus of pol and has virtually no homology with the env gene of known infectious retroviruses. Clone 4-1 represents a defective prototype of a human C-type retrovirus which integrated into the germ line some time in the distant past.     

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.     

Pseudoknot-dependent read-through of retroviral gag termination codons: importance of sequences in the spacer and loop 2.

Retroviruses whose gag and pol genes are in the same reading frame depend upon approximately 5% read-through of the gag UAG termination codon to make the gag-pol polyprotein. For murine leukemia virus, this read-through is dependent on a pseudoknot located eight nucleotides 3' of the UAG. Other retroviruses whose gag and pol genes are in the same frame can potentially form similar pseudoknots 3' of their UAG codons. Beyond the similar secondary structures, there is strong sequence conservation in the spacer region and in loop 2 of the pseudoknots. The detrimental effects of substitutions of several of these conserved spacer and loop 2 nucleotides in the murine leukemia virus sequence show their importance for the read-through process. The importance of specific nucleotides in loop 2 of the pseudoknot contrasts with the flexibility of sequence in loop 2 of the most intensively studied frameshift-promoting pseudoknot which occurs in infectious bronchitis virus. Two nucleotides in loop 2 of the murine leukemia virus pseudoknot, which were shown to be important by mutagenic analysis, display hypersensitivity to the single-strand specific nuclease, S1. They are likely to be particularly accessible or are in an unusually reactive conformation.     

Nucleotide sequence of AKV murine leukemia virus.

AKV is an endogenous, ecotropic murine leukemia virus that serves as one of the parents of the recombinant; oncogenic mink cell focus-forming viruses that arise in preleukemic AKR mice. I report the 8,374-nucleotide-long sequence of AKV, as determined from the infectious molecular clone AKR-623. The 5'-leader sequence of AKV extends to nucleotide 639, after which lies a long open reading frame encoding the gag and pol gene products. The reading frame is interrupted by a single amber codon separating the gag and pol genes. The pol gene overlaps the env gene within the 3' region of the AKV genome. The nucleotide sequence of the 5' region of AKV reveals the following features. (i) The 5'-leader sequence lacks any AUG codon to initiate translation of gPr80gag, suggesting that gPr80gag is not required for the replication of AKV. (ii) A short portion of the leader region diverges in sequence from the closely related Moloney murine leukemia virus and appears to be related to a sequence highly repeated in eucaryotic genomes. (iii) As in Moloney murine leukemia virus, there is a potential RNA secondary structure flanking the amber codon that separates the gag and pol genes. This structure might function as a regulatory protein binding site that controls the relative levels of synthesis of the gag and pol precursors. The nucleotide sequence of the 3' region of AKV is compared with sequences reported previously from both infectious and noninfectious molecular clones of AKV.     

Complete nucleotide sequence and genome organization of a Drosophila transposable genetic element, 297

The complete nucleotide sequence of 297, a Drosophila copia-like transposable element, was determined and compared with those of other similar Drosophila elements and mammalian retrovirus proviruses. It was found that 297 contains three long open reading frames, comparable in sizes and locations with gag, pol, and env genes in the proviruses of replication-competent retroviruses in vertebrates. The first and second open reading frames of 297 exhibit sequence homologies to gag and pol, respectively, of Moloney murine leukaemia virus. In particular, as with 17.6, another Drosophila copia-like element, the second open reading frame of 297 was shown to be very similar in its entire organization to the retroviral pol gene and to consist of three enzymatic domains. By contrast, no appreciable homology was found between the third open reading frame of 297 and the retroviral env gene. It is also suggested that 297 and 17.6 are a peculiar pair of copia-like elements recently diverged from a common progenitor.     

Nucleotide sequence analysis establishes the role of endogenous murine leukemia virus DNA segments in formation of recombinant mink cell focus-forming murine leukemia viruses.

The sequence of 363 nucleotides near the 3' end of the pol gene and 564 nucleotides from the 5' terminus of the env gene in an endogenous murine leukemia viral (MuLV) DNA segment, cloned from AKR/J mouse DNA and designated as A-12, was obtained. For comparison, the nucleotide sequence in an analogous portion of AKR mink cell focus-forming (MCF) 247 MuLV provirus was also determined. Sequence features unique to MCF247 MuLV DNA in the 3' pol and 5' env regions were identified by comparison with nucleotide sequences in analogous regions of NFS -Th-1 xenotropic and AKR ecotropic MuLV proviruses. These included (i) an insertion of 12 base pairs encoding four amino acids located 60 base pairs from the 3' terminus of the pol gene and immediately preceding the env gene, (ii) the deletion of 12 base pairs (encoding four amino acids) and the insertion of 3 base pairs (encoding one amino acid) in the 5' portion of the env gene, and (iii) single base substitutions resulting in 2 MCF247 -specific amino acids in the 3' pol and 23 in the 5' env regions. Nucleotide sequence comparison involving the 3' pol and 5' env regions of AKR MCF247 , NFS xenotropic, and AKR ecotropic MuLV proviruses with the cloned endogenous MuLV DNA indicated that MCF247 proviral DNA sequences were conserved in the cloned endogenous MuLV proviral segment. In fact, total nucleotide sequence identity existed between the endogenous MuLV DNA and the MCF247 MuLV provirus in the 3' portion of the pol gene. In the 5' env region, only 4 of 564 nucleotides were different, resulting in three amino acid changes between AKR MCF247 MuLV DNA and the endogenous MuLV DNA present in clone A-12. In addition, nucleotide sequence comparison indicated that Moloney-and Friend-MCF MuLVs were also highly related in the 3' pol and 5' env regions to the cloned endogenous MuLV DNA. These results establish the role of endogenous MuLV DNA segments in generation of recombinant MCF viruses.     

Novel Endogenous Type C Retrovirus in Baboons: Complete Sequence, Providing Evidence for Baboon Endogenous Virus gag-pol Ancestry

A complete endogenous type C viral genome has been isolated from a baboon genomic library. The provirus, Papio cynocephalus endogenous retrovirus (PcEV), is 8,572 nucleotides long, and 38 to 59 proviral copies per baboon genome are found. The PcEV provirus possesses the typical simple retroviral gene organization, including two long terminal repeats and genes encoding gag, pol, and env proteins. The open reading frames for gag-pol and env are complete but have premature stop codons or frameshift mutations. The primer binding site of PcEV is complementary to tRNAGly. The gag and pol genes of PcEV are closely related to those of the baboon endogenous virus (BaEV). The env coding region of PcEV is related to the env genes of type C retroviruses. This suggests that PcEV is one of the ancestors of BaEV contributing the type C gag-pol genome fragment to the type C/D recombinant virus BaEV. Earlier it was shown that another endogenous type D virus (simian endogenous retrovirus) provided the env gene for BaEV (A. C. van der Kuyl et al., J. Virol. 71:3666-3676, 1997).     

Human retroviral sequences on the Y chromosome.

Novel endogenous human retroviral sequences were cloned by low-stringency hybridization, using the pol gene of endogenous human retrovirus 51-1. One clone, lambda NP-2, contained gag, pol, env, and long terminal repeat sequences related to the corresponding portions of clone 51-1 and the closely related full-length endogenous human retrovirus 4-1. The sequence of the env gene of NP-2 was 73% homologous to that of 4-1. Genomic Southern blots of male and female DNAs showed that NP-2 is located on the Y chromosome and that the Y chromosome also contains one other sequence closely related to the env and 3' flanking regions of NP-2. Conservation of flanking DNA suggests that the second Y chromosome copy of the NP-2 env sequence arose by gene duplication rather than provirus insertion.     

Molecular analysis of several classes of endogenous feline leukemia virus elements

Five recombinant DNA clones of endogenous feline leukemia virus-related DNA sequences were isolated by screening a lambda phage genomic library of cat placental DNA with a probe specific to the gag-pol region of infectious feline leukemia virus. The clones containing retroviral long terminal repeat-like sequences demonstrated the existence of different size classes of endogenous elements in the cat genome, including those of nearly full length in which the gag region is heterogeneous but all of pol and most of env are highly conserved. Other size classes included elements with major deletions in gag or pol. A genomic DNA analysis suggested that the majority of endogenous elements were close to full length in size and that the highly truncated sequences which we described previously (Soe et al., J. Virol. 46:829-840, 1983) represented only a subset of the elements present. A restriction analysis of genomic DNA suggested a high degree of conservation in pol and the 5' portion of env among the various endogenous sequences present in the cat genome. We also found by using DNA transfection that while all of the endogenous clones were noninfectious, there was differential expression of the elements which we examined. These findings correlate with the subgenomic expression of endogenous feline leukemia virus sequences in cat placental tissue.     

Molecular cloning of proviral DNA and structural analysis of the transduced myc oncogene of avian oncovirus CMII.

Molecularly cloned proviral DNA of avian oncogenic retrovirus CMII was isolated by screening a genomic library of a CMII-transformed quail cell line with a myc-specific probe. On a 10.4-kilobase EcoRI fragment, the cloned DNA contained 4.4 kilobases of CMII proviral sequences extending from the 5' long terminal repeat to the EcoRI site within the partial (delta) complement of the env gene. The gene order of CMII proviral DNA is 5'-delta gag-v-myc-delta pol-delta env-3'. All three structural genes are partially deleted: the gag gene at the 3' end, the env gene at the 5' end, and the pol gene at both ends. The delta gag (0.83 kilobases)-v-myc (1.50 kilobases) sequences encode the p90gag-myc transforming protein of CMII. In comparison with the p110gag-myc protein of acute leukemia virus MC29, p90gag-myc lacks amino acids corresponding to additional 516 bases of gag sequences and 12 bases of 5' v-myc sequences present in the MC29 genome. Nucleotide sequence analysis of CMII proviral DNA at the delta gag-v-myc and the v-myc-delta pol junctions revealed significant homologies between avian retroviral structural genes and the cellular oncogene c-myc precisely at the positions corresponding to the gene junctions in CMII. Furthermore, the delta gag-v-myc junction in CMII corresponds to sequence elements in gag and C-myc that are possible splicing signals. The data suggest that transduction of cellular oncogenes may involve RNA splicing and recombination with homologous sequences on retroviral vectors. Different sequence elements of both the retroviral vectors and the c-myc gene recombined during genesis of highly oncogenic retroviruses CMII, MC29, or MH2.