Mutation of amino acids within the gibbon ape leukemia virus (GALV) receptor differentially affects feline leukemia virus subgroup B, simian sarcoma-associated virus, and GALV infections.

The three type C retroviruses, gibbon ape leukemia virus (GALV), simian sarcoma-associated virus (SSAV), and feline leukemia virus subgroup B (FeLV-B), infect human cells by interacting with the same cell surface receptor, GLVR1. Using LacZ retroviral pseudotypes and murine cells transfected with mutant GLVR1 expression vectors, we show that the same 9-amino-acid region of human GLVR1 is critical for infection by the three viruses. Rat cells were not susceptible to infection by LacZ (FeLV-B) pseudotypes because of a block at the receptor level. We found multiple amino acid differences from human GLVR1 in the 9-amino-acid critical region of rat GLVR1. Expression of a human-rat chimeric GLVR1 in murine cells demonstrated that rat GLVR1 could function as a receptor for GALV and SSAV but not for FeLV-B. Substitution of human GLVR1 amino acids in the critical region of rat GLVR1 identified three amino acids as responsible for resistance to FeLV-B infection; two of these affect SSAV infection, but none affects GALV infection.     

Simian Sarcoma-Associated Virus Fails To Infect Chinese Hamster Cells despite the Presence of Functional Gibbon Ape Leukemia Virus Receptors

We have sequenced the envelope genes from each of the five members of the gibbon ape leukemia virus (GALV) family of type C retroviruses. Four of the GALVs, including GALV strain SEATO (GALV-S), were originally isolated from gibbon apes, whereas the fifth member of this family, simian sarcoma-associated virus (SSAV), was isolated from a woolly monkey and shares 78% amino acid identity with GALV-S. To determine whether these viruses have identical host ranges, we evaluated the susceptibility of several cell lines to either GALV-S or SSAV infection. GALV-S and SSAV have the same host range with the exception of Chinese hamster lung E36 cells, which are susceptible to GALV-S but not SSAV. We used retroviral vectors that differ only in their envelope composition (e.g., they contain either SSAV or GALV-S envelope protein) to show that the envelope of SSAV restricts entry into E36 cells. Although unable to infect E36 cells, SSAV infects GALV-resistant murine cells expressing the E36-derived viral receptor, HaPit2. These results suggest that the receptors present on E36 cells function for SSAV. We have constructed several vectors containing GALV-S/SSAV chimeric envelope proteins to map the region of the SSAV envelope that blocks infection of E36 cells. Vectors bearing chimeric envelopes comprised of the N-terminal region of the GALV-S SU protein and the C-terminal region of SSAV infect E36 cells, whereas vectors containing the N-terminal portion of the SSAV SU protein and C-terminal portion of GALV-S fail to infect E36 cells. This finding indicates that the region of the SSAV envelope protein responsible for restricting SSAV infection of E36 cells lies within its amino-terminal region.     

Feline leukemia virus subgroup B uses the same cell surface receptor as gibbon ape leukemia virus.

Pseudotypes of gibbon ape leukemia virus/simian sarcoma-associated virus (GALV/SSAV) and feline leukemia virus subgroup B (FeLV-B) have been constructed by rescuing a Moloney murine leukemia virus vector genome with wild-type GALV/SSAV or FeLV-B. The resulting recombinant viruses utilized core and envelope proteins from the wild-type virus and conferred resistance to growth in L-histidinol upon infected cells by virtue of the HisD gene encoded by the vector genome. They displayed the host range specificity of the rescuing viruses and could be neutralized by virus-specific antisera. Receptor cross-interference was observed when the GALV/SSAV or FeLV-B pseudotypes were used to superinfect cells productively infected with either GALV/SSAV or FeLV-B. Although murine cells are resistant to FeLV-B infection, murine cells expressing the human gene for the GALV/SSAV receptor became susceptible to FeLV-B infection. Therefore GALV/SSAV and FeLV-B utilize the same cell surface receptor.     

Deletions of specific regions of the simian sarcoma-associated virus genome are found in defective viruses and in the simian sarcoma virus.

Extrachromosomal DNA was isolated from tissue culture cells that were acutely infected with simian sarcoma virus (SSV) and its associated helper (simian sarcoma-associated virus [SSAV]). Two sizes of closed circular viral genomic DNA intermediates were isolated, cleaved at the single EcoRI site, and ligated to the Charon 21A phage lambda vector. Cloned molecules of the larger size all represented the full-length (9.0-kilobase [kb]) SSAV molecule. A heterogeneous group of clones was derived from the smaller DNA circles. These included the SSV genome and SSAV deletion mutants. When two SSV clones were compared with the helper, they contained the following three characteristic deletions: (i) a 250-base pair deletion in the gag gene about 1.0 kb from the 5' end of the genome; (ii) a 1.85-kb deletion in the pol gene; and (iii) a 1.9-kb deletion at the 3' end, which included part of the env gene. This latter deletion was the site of the onc gene substitution. Six other clones of the smaller molecules represented the following variants of the SSAV genome: (i) two clones of the entire genome containing only one long terminal repeat unit; (ii) one clone with the 1.85-kb deletion of the pol gene observed in SSSV; and (iii) three clones having a deletion of the 3' end of the SSAV genome. In each of the latter clones, the 5' border of the deletion was indistinguishable from the 5' border of the onc substitution in SSV. The fidelity of genetic deletions observed suggested that certain regions of the SSAV genome were deleted at a high frequency. In certain cases, these deletions may have been accompanied by a substitution of cellular sequences to generate SSV.     

Introduction of sequences encoding functional human adenosine deaminase into mouse cells using a retroviral shuttle system

A retroviral packaging system was used to generate a murine virus carrying sequences encoding human adenosine deaminase (ADA). To this end, human ADA cDNA was inserted into the retroviral shuttle vector pZIP-NeoSV(X)1. This vector provides all of the cis-acting sequences necessary for the efficient packaging and transmission of the viral genome as well as a selectable gene for G418 resistance. Transfection of this recombinant plasmid into cells that provide essential virus products (psi-2 cells) yielded cell lines that stably produced virions carrying the coding sequence of human ADA. We have used these virions to infect NIH3T3 cells, which after 48 h synthesized catalytically active human ADA. Furthermore, G418-resistant cell lines were obtained from the virus-infected NIH3T3 cells that stably produced the human ADA enzyme.     

Automated recognition of retroviral sequences in genomic data--RetroTector

Eukaryotic genomes contain many endogenous retroviral sequences (ERVs). ERVs are often severely mutated, therefore difficult to detect. A platform independent (Java) program package, RetroTector (ReTe), was constructed. It has three basic modules: (i) detection of candidate long terminal repeats (LTRs), (ii) detection of chains of conserved retroviral motifs fulfilling distance constraints and (iii) attempted reconstruction of original retroviral protein sequences, combining alignment, codon statistics and properties of protein ends. Other features are prediction of additional open reading frames, automated database collection, graphical presentation and automatic classification. ReTe favors elements >1000-bp long due to its dependence on order of and distances between retroviral fragments. It detects single or low-copy-number elements. ReTe assigned a 'retroviral' score of 890-2827 to 10 exogenous retroviruses from seven genera, and accurately predicted their genes. In a simulated model, ReTe was robust against mutational decay. The human genome was analyzed in 1-2 days on a LINUX cluster. Retroviral sequences were detected in divergent vertebrate genomes. Most ReTe detected chains were coincident with Repeatmasker output and the HERVd database. ReTe did not report most of the evolutionary old HERV-L related and MalR sequences, and is not yet tailored for single LTR detection. Nevertheless, ReTe rationally detects and annotates many retroviral sequences.     

The distributions of "new" and "old" Alu sequences in the human genome: the solution of a "mystery"

The distribution in the human genome of the largest family of mobile elements, the Alu sequences, has been investigated for the past 30 years, and the vast majority of Alu sequences were shown to have the highest density in GC-rich isochores. Ten years ago, it was discovered, however, that the small "youngest" (most recently transposed) Alu families had a strikingly different distribution compared with the "old" families. This raised the question as to how this change took place in evolution. We solved what was considered to be a "mystery" by 1) revisiting our previous results on the integration and stability of retroviral sequences, and 2) assessing the densities of acceptor sites TTTT/AA in isochore families. We could conclude 1) that the open state of chromatin structure plays a crucial role in allowing not only the initial integration of retroviral sequences but also that of the youngest Alu sequences, and 2) that the distribution of old Alus can be explained as due to Alu sequences being unstable in the GC-poor isochores but stable in the compositionally matching GC-rich isochores, again in line with what happens in the case of retroviral sequences.     

Identification of paralogous HERV-K LTRs on human chromosomes 3, 4, 7 and 11 in regions containing clusters of olfactory receptor genes

A locus harboring a human endogenous retroviral LTR (long terminal repeat) was mapped on the short arm of human chromosome 7 (7p22), and its evolutionary history was investigated. Sequences of two human genome fragments that were homologous to the LTR-flanking sequences were found in human genome databases: (1) an LTR-containing DNA fragment from region 3p13 of the human genome, which includes clusters of olfactory receptor genes and pseudogenes; and (2) a fragment of region 21q22.1 lacking LTR sequences. PCR analysis demonstrated that LTRs with highly homologous flanking sequences could be found in the genomes of human, chimp, gorilla, and orangutan, but were absent from the genomes of gibbon and New World monkeys. A PCR assay with a primer set corresponding to the sequence from human Chr 3 allowed us to detect LTR-containing paralogous sequences on human chromosomes 3, 4, 7, and 11. The divergence times for the LTR-flanking sequences on chromosomes 3 and 7, and the paralogous sequence on chromosome 21, were evaluated and used to reconstruct the order of duplication events and retroviral insertions. (1) An initial duplication event that occurred 14-17 Mya and before LTR insertion - produced two loci, one corresponding to that located on Chr 21, while the second was the ancestor of the loci on chromosomes 3 and 7. (2) Insertion of the LTR (most probably as a provirus) into this ancestral locus took place 13 Mya. (3) Duplication of the LTR-containing ancestral locus occurred 11 Mya, forming the paralogous modern loci on Chr 3 and 7.     

Gene targeting with retroviral vectors: recombination by gene conversion into regions of nonhomology.

We have designed and constructed integration-defective retroviral vectors to explore their potential for gene targeting in mammalian cells. Two nonoverlapping deletion mutants of the bacterial neomycin resistance (neo) gene were used to detect homologous recombination events between viral and chromosomal sequences. Stable neo gene correction events were selected at a frequency of approximately 1 G418r cell per 3 x 10(6) infected cells. Analysis of the functional neo gene in independent targeted cell clones indicated that unintegrated retroviral linear DNA recombined with the target by gene conversion for variable distances into regions of nonhomology. In addition, transient neo gene correction events which were associated with the complete loss of the chromosomal target sequences were observed. These results demonstrated that retroviral vectors can recombine with homologous chromosomal sequences in rodent and human cells.     

A New Family of Retroviral Long Terminal Repeat Elements in the Human Genome Identified by Their Homologies to an Element 5′ to the Spider Monkey Haptoglobin Gene

A new family of retroviral long terminal repeats that we name Spm-LTR has been identified as a result of DNA sequence comparisons between the entire GenBank databank and an element, SPHP, located 5′ to the haptoglobin gene of spider monkeys. The 18 human Spm-LTR sequences so identified fall into three subtypes. There is no sequence similarity between Spm-LTR elements and any endogenous retroviral LTR sequences previously reported except for general features that define LTRs. However, a previously described repeated sequence (MER-4) forms a portion of the Spm-LTR sequence.     

Isolation of a transformation-defective mutant of the McDonough strain of feline sarcoma virus exhibiting tyrosine kinase activity in vitro but not in vivo.

NRK cells transformed by the McDonough strain of feline sarcoma virus (SM-FeSV) were mutagenized by the use of 5'-azacytidine. Four cell lines expressing different transformation-defective phenotypes were isolated. Superinfection of these cell lines with simian sarcoma-associated virus (SSAV) led in three instances to the recovery of transforming virus particles carrying an intact fms gene. A nonconditional transformation-defective virus, designated td26-SM-FeSV (SSAV), was isolated from one of the cell lines. NRK cells infected with this mutant contained actin cables and fibronectin networks and exhibited normal cell morphology. Such cells formed only small colonies in soft agar and exhibited a mitogenic activity similar to that of noninfected cells. Cells infected with td26-SM-FeSV (SSAV) synthesized a gag-fms fusion glycoprotein (gp180gag-fms). This polypeptide was processed in the normal manner into the intracellular gp120v-fms and a transformation-defective gp140td-v-fms which was expressed at the surface of infected cells. This species had an increased electrophoretic mobility on polyacrylamide gels compared with the molecule from wild-type virus.gp140td-v-fms had endo-beta-N-acetylglucosaminidase H-resistant carbohydrate side chains. No tyrosine kinase activity was detectable in vivo in td26-SM-FeSV (SSAV)-infected cells even when the cells were treated with sodium orthovanadate. In vitro, fms molecules from td26-SM-FeSV (SSAV)-infected cells exhibited tyrosine kinase activity as determined by autophosphorylation and phosphorylation of exogenous (poly)Glu-Tyr. At low ATP concentrations (less than 5 microM) this in vitro tyrosine kinase activity was significantly reduced compared with that of the wild-type counterpart.     

Amplification and chromosomal dispersion of human endogenous retroviral sequences.

Endogenous retroviral sequences in humans have undergone amplification events involving both viral and flanking cellular sequences. We cloned members of an amplified family of full-length endogenous retroviral sequences. Genomic blotting, employing a flanking cellular DNA probe derived from a member of this family, revealed a similar array of reactive bands in both humans and chimpanzees, indicating that an amplification event involving retroviral and associated cellular DNA sequences occurred before the evolutionary separation of these two primates. Southern analyses of restricted somatic cell hybrid DNA preparations suggested that endogenous retroviral segments are widely dispersed in the human genome and that amplification and dispersion events may be linked.     

Mutational analysis of the human T-cell leukemia virus type I trans-acting rex gene product.

Expression of the human T-cell leukemia virus type I (HTLV-I) rex gene is a prerequisite for the expression of the retroviral structural proteins. We have generated internal deletion mutants of this 27-kDa nucleolar trans-acting gene product to define functional domains in the Rex protein. The phenotype of the various mutant proteins was tested on the homologous HTLV-I rex response element sequence and the heterologous human immunodeficiency virus type 1 (HIV-1) rev response element sequence. Our results indicate that a region between amino acid residues 55 and 132 in the 189-amino-acid Rex protein is required for Rex-mediated trans activation on both retroviral response element sequences. In addition, substitution of the Rex nuclear localization signal by a sequence of the HIV-1 rev gene product targets the Rex protein to the correct subcellular compartment required for Rex function.