Artificial insertion of a dominant gene for resistance to avian leukosis virus into the germ line of the chicken

Summary This report describes the unique biological properties of a transgenic chicken line that contains a defective avian leukosis virus (ALV) proviral insert that we call alv6. Chick embryo fibroblasts (CEF) containing this insert express subgroup A envelope glycoprotein since they yield focus-forming pseudotype virus when co-cultivated with transformed quail cells expressing envelope-defective Bryan high-liter Rous sarcoma virus (RSV). In addition, these cells display high interference to subgroup A RSV but not to subgroup B RSV infection. Chickens containing this insert are highly resistant to pathogenic subgroup A ALV infection, but show little immunological tolerance to subgroup B ALV infection. Thus we have artificially inserted a dominant gene for resistance to avian leukosis infection into the chicken germ line.     

Endogenous nonecotropic proviruses mapped with oligonucleotide probes from the long terminal repeat region

We characterized endogenous proviruses in C57BL/6J, DBA/2J, and C3H/HeJ mouse strains with oligonucleotide probes derived from long terminal repeat (LTR) sequences of three classes of nonecotropic murine leukemia virus. The segregation of proviral-host DNA junction fragments was followed in BXH and BXD recombinant inbred (RI) strain sets, and most fragments mapped readily to defined chromosomal regions. Most of the LTR fragments appear to correspond to proviruses mapped previously with oligonucleotide env region probes of the same viral class. At least 22 elements represent new proviral loci, no more than half of which may be solo LTRs, and an additional six may correspond to proviruses identified previously with less specific hybridization probes. Together with proviruses identified previously with env probes, the LTR probe-reactive elements represent the majority of endogenous murine leukemia proviruses in the mouse genome.     

The endogenous ecotropic murine retroviruses Emv-16 and Emv-17 are both capable of producing new proviral insertions in the mouse genome.

New germ line proviral insertions are acquired at a high frequency by the progeny of SWR/J-RF/J hybrid female mice that carry the endogenous ecotropic murine leukemia proviruses Emv-16 and Emv-17. The tight linkage of these RF/J strain proviral loci has prevented genetic segregation of the retroviral genomes. Hence, it is not known whether both of these proviruses are capable of giving rise to new proviral insertions. We have molecularly cloned Emv-16 and Emv-17 and have characterized them in vitro and in vivo. Restriction enzyme analysis of the recombinant clones revealed that the proviral genomes are very similar to each other and closely resemble the wild-type AKR virus. A comparison of the flanking cellular DNA suggests that the Emv-16 and Emv-17 loci did not arise by simple duplication of a viral insertion site within the RF/J genome but most likely are independent integration events. Both proviruses produce infectious virus when transfected into NIH 3T3 cells, indicating that they are nondefective retroviruses. Exogenous infection of SWR/J mice with either Emv-16 or Emv-17 leads to viremia in the host animals, and in both cases, progeny of viremic females acquire new proviral insertions. The ability of these retroviruses to generate novel retroviral integration sites in the mouse genome provides a simple method for inducing insertional mutations in mice.     

Fv-1 N- and B-tropism-specific sequences in murine leukemia virus and related endogenous proviral genomes

Oligonucleotide probes specific for the Fv-1 N- and B-tropic host range determinants of the gag p30-coding sequence were used to analyze DNA clones of various murine leukemia virus (MuLV) and endogenous MuLV-related proviral genomes and chromosomal DNA from four mouse strains. The group of DNA clones consisted of ecotropic MuLVs of known Fv-1 host range, somatically acquired ecotropic MuLV proviruses, xenotropic MuLV isolates, and endogenous nonecotropic MuLV-related proviral sequences from mouse chromosomal DNA. As expected, the prototype N-tropism determinant is carried by N-tropic viruses of several different origins. All seven endogenous nonecotropic MuLV-related proviral sequence clones derived from RFM/Un mouse chromosomal DNA, although not recognized by the N probe, showed positive hybridization with the prototype B-tropism-specific probe. The two xenotropic MuLV clones derived from infectious virus (one of BALB:virus-2 and one of AKR xenotropic virus) failed to hybridize with the N- and B-tropic oligonucleotide probes tested and with one probe specific for NB-tropic Moloney MuLV. One of two endogenous xenotropic class proviruses derived from HRS/J mouse chromosomal DNA (J. P. Stoye and J. M. Coffin, J. Virol. 61:2659-2669, 1987) also failed to hybridize to the N- and B-tropic probes, whereas the other hybridized to the B-tropic probe. In addition, analysis of mouse chromosomal DNA from four strains indicates that hybridization with the N-tropic probe correlates with the presence or absence of endogenous ecotropic MuLV provirus, whereas the B-tropic probe detects abundant copies of endogenous nonecotropic MuLV-related proviral sequences. These results suggest that the B-tropism determinant in B-tropic ecotropic MuLV may arise from recombination between N-tropic ecotropic MuLV and members of the abundant endogenous nonecotropic MuLV-related classes including a subset of endogenous xenotropic proviruses.     

Infectious and noninfectious recombinant clones of the provirus of SNV differ in cellular DNA and are apparently the same in viral DNA

Ten clones of Charon 4A containing proviruses of spleen necrosis virus, an avian retrovirus, and flanking chicken DNA sequences were isolated and characterized. Some clones gave rise to progeny with viral DNA sequences deleted or duplicated, probably as a result of crossing-over in the 600 bp terminal redundancy in viral DNA. The cellular sequences are different in each clone, indicating that all the proviruses are integrated in different sites in cellular DNA. Six clones are infectious and four are not. All the infectious molecules containing a provirus are of a similar size and are smaller than the noninfectious molecules containing a provirus. The viral DNA is not apparently different in eight clones, but two clones, one infectious and one noninfectious, lack two restriction sites each. Large changes in proviral DNA therefore do not seem responsible for the lack of infectivity of some clones. These results are consistent with the hypothesis that neighboring cellular DNA sequences control proviral expression (infectivity).     

Variation in the number of copies and in the genomic organization of ecotropic murine leukemia virus proviral sequences in sublines of AKR mice.

DNAs isolated from individual mice of four AKR sublines (AKR/J, AKR/N, AKR/Cum, and AKR/Boy) were examined by hybridization of electrophoretically separated restriction enzyme fragments to a 500-base pair, 32P-labeled probe specific for env sequences of ecotropic murine leukemia virus. Variation in the number of proviral DNA copies and in their genomic organization, as reflected by the location of restriction enzyme sites in flanking cellular sequences, was observed both between and within AKR sublines. Evidence is presented for the continual acquisition of new proviruses in the four sublines studied. The ecotropic proviral DNA copies present in the four AKR sublines can be related to their genealogy; each subline contains two or three copies of proviral DNA in common with other sublines and from one to six unique ecotropic proviruses. Overall, a new copy appears about every 12 generations of inbreeding. Some of the unique proviral DNA copies contain internal alterations, as reflected by restriction enzyme maps that differ from those of prototype ecotropic proviruses.     

Nucleotide sequences of a feline leukemia virus subgroup A envelope gene and long terminal repeat and evidence for the recombinational origin of subgroup B viruses.

Molecular clones of the subgroup A feline leukemia virus FeLV-A/Glasgow-1 have been obtained. Nucleotide sequence analysis of the 3' end of the proviral genome and comparison with the published sequence of FeLV-B/Gardner-Arnstein showed that the most extensive differences are located within the 5' domain of the env gene. Within this domain, several divergent regions of env are separated by more conserved segments. The 3' end of env is highly conserved, with only a single amino acid coding difference in p15env. The proviral long terminal repeats are also highly conserved, differing by only eight base substitutions and one base insertion. Specific probes constructed from the FeLV-A or FeLV-B env genes were used to compare the env genes of various exogenous FeLV isolates and the endogenous FeLV-related proviruses of normal cat DNA. An FeLV-A-derived env probe showed no hybridization to normal cat DNA but detected all FeLV-A and FeLV-C isolates tested. In contrast, an FeLV-B env probe detected independent FeLV-B isolates and a family of endogenous FeLV-related proviruses. Our observations provide strong evidence to support the hypothesis that FeLV-B viruses have arisen by recombination between FeLV-A and endogenous proviral elements in cat DNA.     

Haplotype Analysis of the Human Endogenous Retrovirus Locus HERV-K(HML-2.HOM) and Its Evolutionary Implications

We and others recently identified an almost-intact human endogenous retrovirus (HERV), termed HERV-K(HML-2.HOM), that is usually organized as a tandem provirus. Studies on HERV proviral loci commonly rely on the analysis of single alleles being taken as representative for a locus. We investigated the frequency of HERV-K(HML-2.HOM) single and tandem alleles in various human populations. Our analysis revealed that another HERV-K(HML-2) locus, the so-called HERV-K(II) provirus, is also present as a tandem provirus allele in the human population. Proviral tandem formations were identified in various nonhuman primate species. We furthermore examined single nucleotide polymorphisms (SNPs) within the HERV-K(HML-2.HOM) proviral gag, prt, and pol genes, which all result in nonsense mutations. We identified four proviral haplotypes displaying different combinations of gag, prt, and pol SNPs. Haplotypes harboring completely intact proviral genes were not found. For the left provirus of the tandem arrangement a haplotype displaying intact gag and prt genes and a mutated pol was found in about two-thirds of individuals from different ethnogeographic origins. The same haplotype was always found in the right provirus. The various haplotypes point toward multiple recombination events between HERV-K(HML-2.HOM) proviruses. Based on these findings we derive a model for the evolution of the proviral locus since germ line integration. [Reviewing Editor : Dr. Martin Kreitman]     

Drosophila germline invasion by the endogenous retrovirus gypsy: involvement of the viral env gene

The endogenous retrovirus gypsy is expressed at high levels in mutant flamenco female flies. Gypsy viral particles extracted from such flies can infect naive flamenco individuals raised in the presence of these extracts mixed into their food. This results in the integration of new proviruses into the germline genome. These proviruses can then increase their copy number by (1) expression in the flamenco female somatic cells, (2) transfer into the oocyte and (3) integration into the genome of the progeny. Surprisingly, unlike the infection observed in the feeding experiments, this strategy of endogenous proviral multiplication does not seem to involve the expression of the viral env gene.     

Mechanism of c-erbB transduction: newly released transducing viruses retain poly(A) tracts of erbB transcripts and encode C-terminally intact erbB proteins

We have previously shown that avian leukosis virus (ALV) induces erythroblastosis by insertional activation of the c-erbB gene. In 25% of the ALV-induced leukemic samples we have analyzed, acute retroviruses that have captured the activated erbB oncogene were released. The unusually high frequency at which erbB transduction occurs makes this an ideal system for studying the mechanism of oncogene transduction. In addition, these leukemic samples provide a rich source for the isolation of novel erbB-transducing viruses. We report here our characterization of several new erbB-transducing proviruses. The 5' recombination points of all these viruses mapped to the same intron in which proviral insertions cluster, supporting the hypothesis that transduction begins with proviral insertion near the oncogene. The 3' recombination points usually occurred within the 3' untranslated region downstream from the termination codon of the c-erbB gene. Three of the erbB-containing proviruses were molecularly cloned and analyzed in detail. Two of them were capable of releasing acute viruses, and interestingly, both retained poly(A) tracts of erbB messages in their genomes. A stretch of six adenosine residues in the ALV env gene appeared to mediate the 3' recombination events required for the generation of these viruses. These data provide further insight into the mechanism by which oncogenes are transduced into retroviral genomes.     

The Putative Alpha Helix 2 of Human Immunodeficiency Virus Type 1 Vpr Contains a Determinant Which Is Responsible for the Nuclear Translocation of Proviral DNA in Growth-Arrested Cells

Several viral determinants were shown to play a role in the ability of human immunodeficiency virus type 1 (HIV-1) to infect nondividing cells. In particular, Vpr and Gag matrix (MA) were recognized to be involved in the nuclear transport of the viral preintegration complex. The goal of the present study was to evaluate the ability of isogenic HIV-1 viruses harboring different vpr and gag genes to infect nondividing cells. Surprisingly, our results reveal that the introduction of mutations in the MA nuclear localization signal marginally affected the ability of proviruses to establish infection in growth-arrested HeLa or MT4 cells. In contrast, we show that in our experimental system, the absence of Vpr expression leads to a reduction in viral infectivity and production which correlates with a decrease in the synthesis and nuclear transport of proviral DNA as determined by PCR analysis. Moreover, our data demonstrate that this reduction of viral replication is also observed with proviruses containing different mutated Vpr alleles. In particular, the Vpr Q65E mutant, which contains a substitution in the second predicted amphipathic alpha-helical structure located in the central region of the protein, is associated with an impairment of the protein nuclear localization and a concomitant reduction of the nuclear transport of proviral DNA. The results of this study provide evidence that a putative amphipathic alpha-helical structure in the central region of Vpr contains a determinant involved in the nuclear translocation of the preintegration complex in nondividing cells.     

Molecular cloning of MuLV proviruses integrated into the genome of mouse erythroleukemia cells. I. Characteristics of endogenous proviruses

The library of genes was obtained from erythroleukemic AKR cells (C-1), that were maintained as suspension culture. Thirty four clones that had homology with 60-70S RNA of Rauscher Leukemia virus (RLV) were separated from this library. The restriction mapping was carried out with 14 clones, that contained most extensive proviral sequences. One clone (107) contains proviral sequences that are derived from one of the components of the RLV complex. The other 13 clones contain sequences of endogenous xenotropic viruses. The endogenous retroviral sequences obtained differ in restrictive maps from proviruses of ecotropic and xenotropic infectious endogenous MuLV and, apparently, might be attributed as non-inducible infectious xenotropic MuLV of class III. Some of the cloned retroviral sequences had symmetrical structure, that is typical for integrated proviruses, i. e. these sequences were separated from flanking cellular ones by long terminal repeats. All investigated retroviral sequences are deletion mutants of MuLV proviruses. It was shown that the inner regions of proviruses diverged more than the long terminal repeats. The expression of the main inner MuLV polypeptide (p30) was detected in NIH 3T3 cells, transfected with DNA of some clones.     

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.     

Proviruses of avian sarcoma virus are terminally redundant, co-extensive with unintegrated linear DNA and integrated at many sites.

We have analyzed the DNA from 15 clones of avian sarcoma virus (ASV)-transformed rat cells with restriction endonucleases and molecular hybridization techniques to determine the location and structure of proviral DNA. All twenty units of proviral DNA identified in these 15 clones appear to be inserted at different sites in host DNA. In each of the ten cases that could be sufficiently well mapped, entirely different regions of cellular DNA were involved. Thus ASV DNA can be accommodated at many positions in cellular DNA, but the existence of preferred sites has not been excluded. Six of the 15 clones carry only one normal provirus, two contain two normal proviruses, and seven harbor either one or two proviruses that appear anomalous in physical mapping tests. Both ends of at least 18 proviruses, however, were found to contain sequences specific to both the 3' and 5' termini of viral RNA. The organization of these terminally redundant sequences appeared identical to that of the 300 base pair (bp) repeats found at the ends of unintegrated linear DNA (Shank et al., 1978). Proviral DNA is therefore co-extensive, or nearly co-extensive, with unintegrated linear DNA and has a structure we denote as CELL DNA-3'5'----------3'5'-CELL DNA. Three of the four anomalous proviruses which were fully analyzed were deletion mutants lacking 25--65% of the genetic content of ASV; the fourth provirus had a novel site for cleavage by Eco RI but was otherwise normal. Tests for the biological competence of proviral DNA, based upon rescue of transforming virus after fusion with chicken cells, were generally consistent with the physical mapping studies.     

Expression of Middle-Component RNA of Cowpea Mosaic Virus: In Vitro Generation of a Precursor to Both Capsid Proteins by a Bottom-Component RNA-Encoded Protease from Infected Cells

The internal organization of endogenous xenotropic murine leukemia virus proviruses was determined in a series of blot hybridization experiments in which DNA from several different inbred mouse strains, digested with restriction enzymes known to cleave xenotropic proviral DNAs at least twice, was annealed to generalized murine leukemia virus or xenotropic env-specific DNA probes. Comigrating bands of variable intensity which hybridized to the xenotropic env probe were identified in all inbred mouse DNA preparations. At least seven classes of endogenous xenotropic proviral DNA with respect to SacI cleavage maps were detected in mouse DNA. Two of the seven classes were indistinguishable from proviruses associated with known infectious xenotropic murine leukemia viruses. These results are consistent with the existence of related but organizationally distinct families of endogenous xenotropic proviral DNA that are present in different relative abundances in mouse genomic DNA.     

Identification of the avian myeloblastosis virus genome. II. Restriction endonuclease analysis of DNA from lambda proviral recombinants and leukemic myeoblast clones.

Two lambda proviral DNA recombinants were characterized with a number of restriction endonucleases. One recombinant contained a complete presumptive avian myeloblastosis virus (AMV) provirus flanked by cellular sequences on either side, and the second recombinant contained 85% of a myeloblastosis-associated virus type 1 (MAV-1)-like provirus with cellular sequences adjacent to the 5' end of the provirus. Comparing the restriction maps for the proviral DNAs contained in each lambda hybrid showed that the putative AMV and MAV-1-like genomes shared identical enzyme sites for 3.6 megadaltons beginning at the 5' termini of the proviruses with respect to viral RNA. Two enzyme sites near the 3'-end of the MAV-1-like provirus were not present in the putative AMV genome. We also examined a number of leukemic myeloblast clones for proviral content and cell-provirus integration sites. The presumptive AMV provirus was present in all the leukemic myeloblast clones regardless of the endogenous proviral content of the target cells or the AMV pseudotype used for conversion. Multiple cellular sites were suitable for integration of the putative AMV genome and the helper genomes. The proviral genomes were all integrated colinearly with respect to linear viral DNA.