Continuous production of Rous sarcoma virus free of transformation defective virus in clones of established RSV-transformed quail cells

Quail embryo fibroblasts were infected with a Schmidt-Ruppin strain RSV × chf recombinant virus. Virus-transformed cells were established as a permanent line and then cloned in methyl cellulose. Out of 140 clones isolated four clones were capable of indefinite growth. These clones were examined for (i) production of sarcoma and td virus particles, (ii) number of integrated virus genome equivalents, and (iii) deletions of the src gene in the provirus. We found that the clones yield about 106 focus-forming units of the sarcoma virus per milliliter of the culture medium. No td virus could be detected by plating of the virus at the endpoint dilution and no 35 S td virus RNA but only 38 S sarcoma virus RNA was found in virions. Hybridization kinetic studies indicated that three different clones contain about 2 virus genome equivalents, and one clone contains about 4 virus genome equivalents per diploid cell. Upon transfection the proviruses of different clones generated sarcoma viruses and no td viruses. Finally digestion with EcoRI restriction endonuclease released in all four clones a 1.9 × 106-dalton fragment characteristic of the complete src gene, while no 0.8 × 10⁶-dalton fragment characteristic of a td provirus could be detected. We concluded that the clones of RSV-transformed quail cells contain only nondefective sarcoma proviruses and produce from these proviruses nondefective focus-forming virions in the absence of any segregant td virions.     

Molecular clones of endogenous murine leukemia virus-related DNA sequences from Balb/c mice: characterization of integration sites

Eight recombinant DNA clones of endogenous murine leukemia virus (MuLV)-related DNA sequences have been isolated from a lambdaphage genomic library of Balb/c mouse DNA. Each clone contains LTR (long terminal repeat) and gag-related sequences, as well as 5' cellular DNA sequences. The virus-related sequences in each clone show an organization similar to that of integrated proviruses; those clones with the greatest length of MuLV-related sequences also contain pol and env gene-related sequences. One clone appears to contain an intact endogenous provirus. Unique cellular DNA segments from three of these clones were subcloned and used as specific "integration site" hybridization probes. Restriction fragment-length polymorphisms (RFLPs) were observed for these integration sites in the DNA of a number of different inbred mouse strains. One provirus-containing fragment was observed only in Balb/c mice while two others were observed in some but not all of the inbred mouse strains tested. Further restriction enzyme mapping of these three loci in the genomic DNA of Balb/c and C3H/HeJ or C57BL/6 mice indicated that the observed RFLPs were due to the presence of proviral DNA sequences in the Balb/c strain at these three integration sites which were lacking in the other mouse strains. The strain distribution of these three provirus insertions suggests that the BE 1 and 7 proviruses were widely, although not universally, present among the progenitors of modern inbred mouse strains, while the BE 16 provirus may be a recent addition to the genome of Balb/c mice.     

New endogenous proviruses in the genome of brown Leghorn chickens

Genetic differences and differences in localization of endogenous proviruses in chicken genomes were found in studies on endogenous provirus sequences in DNA of white and brown Leghorn chickens. In brown Leghorn DNA, proviruses both similar with Raus-associated virus (endogenous virus of white Leghorns) and differing from it in the pattern of hydrolysis by EcoRI restricting endonuclease were found, the latter occurring more frequently. Different sets of variously localized proviruses were found in DNA of individual chickens; none of the proviruses was found in DNA of all brown Leghorns examined. No single provirus common for chickens of the two species was found. The genetic diversity of endogenous proviruses observed in brown Leghorns indicates the possibility of coexistence in the chicken DNA of message on several significantly different endogenous viruses.     

ERV3, a full-length human endogenous provirus: chromosomal localization and evolutionary relationships

A full-length human endogenous provirus termed ERV3 was isolated from a human fetal recombinant DNA library by low stringency hybridization with two probes: baboon endogenous virus LTR; and a pol-env subclone from the endogenous chimpanzee provirus, CH2. DNA sequencing within the clone and comparisons with other retroviruses revealed that ERV3 contains gag and pol gene sequences that are significantly related to those of mammalian type C retroviruses and previously described human endogenous proviruses. The ERV3 genome was determined to reside at a single locus on human chromosome 7 using a panel of rodent X human somatic cell hybrids.     

Nucleotide sequence and structure of integrated bovine leukemia virus long terminal repeats

Bovine leukemia virus (BLV) proviruses, harbored by the productively infected fetal lamb kidney (FLK-BLV) cell line, were cloned in bacteriophage lambda L47. The nucleotide sequence of the proviral long terminal repeats (LTR) with flanking cell and virus DNA have been determined. The BLV LTR is 531 bp in length and is bounded by the dinucleotides 5'-TG...CA-3', which are part of a 3-bp inverted repeat. The integrated provirus is flanked by 6-bp direct repeats of cellular DNA. A tRNApro primer binding site is present starting 2 bp downstream of the 5' LTR. In addition to sequencing integrated proviral DNA clones, the nucleotide sequence of a cDNA clone, representing the 3' end of genomic viral RNA, was determined; thus revealing the RNA polyadenylation site and R:U5 boundary within the LTR. Unlike most other retroviruses, a consensus polyadenylation signal, "AATAAA," is not located proximal to the BLV polyadenylation site. The RNA initiation site, defining the U3:R boundary, was located in the BLV LTR by S1 nuclease mapping. This site is approximately 25 bp downstream of an A + T-rich region which probably encompasses a Goldberg-Hogness ("TATAA") box and about 90 bp downstream of a potential "CCAAT" box. The BLV LTR possesses a U3 region of 204 bp, an unusually long R region of 241 bp, and a U5 region of 86 bp.     

Sequence homology of the simian retrovirus genome with human T-cell leukemia virus type I

A retrovirus highly related to human T-cell leukemia virus type I (HTLV-I) was isolated from a T-cell line established from a seropositive pig-tailed monkey and the provirus genome was molecularly cloned using HTLV-I as a probe. The monkey virus (STLV) had the genomic structure of the LTR-gag-pol-env-pX-LTR. Analysis of the env-pX-LTR region revealed the 90% homology of the nucleotide sequence with that of HTLV-I in each region. This high homology of the sequence indicates that STLV is a member of the HTLV family, but apparently different from HTLV-I. This suggests that the possibility of recent interspecies transmission from monkeys to humans in the endemic area is very small. From its similarity to HTLV, STLV should be useful as an animal model in studies on natural HTLV infection and leukemogenesis of HTLV in humans.     

MuLV IN mutants responsive to HDAC inhibitors enhance transcription from unintegrated retroviral DNA

For Moloney murine leukemia virus (M-MuLV), sustained viral infections require expression from an integrated provirus. For many applications, non-integrating retroviral vectors have been utilized to avoid the unwanted effects of integration, however, the level of expression from unintegrated DNA is significantly less than that of integrated provirus. We find that unintegrated DNA expression can be increased in the presence of HDAC inhibitors, such as TSA, when applied in combination with integrase (IN) mutations. These mutants include an active site mutation as well as catalytically active INs bearing mutations of K376 in the MuLV C-terminal domain of IN. MuLV IN K376 is homologous to K266 in HIV-1 IN, a known substrate for acetylation. The MuLV IN protein is acetylated by p300 in vitro, however, the effect of HDAC inhibitors on gene expression from unintegrated DNA is not dependent on the acetylation state of MuLV IN K376.     

Nucleotide sequence of the first exon of the rat c-myc gene: proviral insertions in murine leukemia virus-induced lymphomas do not affect exon 1

We have previously reported that proviruses are integrated adjacent to the c-myc gene in rat thymomas induced by murine leukemia viruses. In order to characterize these insertions, we have isolated recombinant DNA clones from normal rat DNA containing all of the normal rat c-myc gene, and from two Moloney murine leukemia virus-induced lymphomas containing both proviral and adjacent rat c-myc sequences. We determined the DNA sequence of portions of the normal and one tumor-derived clone. The normal and tumor-derived exon 1 sequences are identical. By comparing our sequence to the sequences of mouse and human c-myc, we located the first exon of the rat c-myc gene. Analysis of the tumor-derived rat c-myc clones showed that proviral integration occurred approximately 1.4 kb upstream of exon 1 of c-myc in the case of one tumor and 0.55 kb upstream of c-myc exon 1 in the other. Thus, we conclude that the proviral insertions in these tumors did not affect the rat c-myc gene by altering the structure of the c-myc RNA. Consistent with this, the c-myc RNA present in a cell line derived from one of these tumors is identical in size to the normal c-myc RNA. Furthermore, the level of c-myc expression is not dramatically elevated in this cell line. Exon 1 of the rat c-myc gene contains no ATG start codons and contains multiple stop codons in all three reading frames, indicating that it, like the chicken and mouse exon 1 sequences, is noncoding. The extent of homology between our sequence of rat c-myc exon 1 and the published sequence of human c-myc exon 1 is similar to the extent of homology between the sequences of mouse and human c-myc exon 1. The rat and mouse c-myc exon 1 sequences differ from each other by about the amount predicted from the known divergence times of mice from rats. Exon 1 of c-myc is only slightly conserved, evolving at a rate similar to that seen for introns and pseudogenes.     

Detection and characterization of simian retroviruses homologous to human T-cell leukemia virus type I

Lymphoid cell lines were established from five different species of monkeys which were positive in antibodies cross-reactive with human T-cell leukemia virus type I (HTLV-I) and were shown to contain provirus sequences homologous to HTLV-I. Gene-specific probes of HTLV-I, gag, pol, env, pX, and LTR, hybridized efficiently with monkey DNAs from these cell lines under stringent conditions, indicating that the proviruses are very similar to HTLV-I along with whole viral genomes. However, the preliminary restriction mapping turned out the difference between simian retroviruses and HTLV-I and also among simian retroviruses. These findings suggest a common ancestor of simian and human retroviruses, but exclude the recent interspecies transmission between monkeys and humans.     

Transformation studies with a human T-cell leukemia virus type 1 molecular clone

In in vitro studies human T-cell leukemia virus type 1 (HTLV-1) may be produced by stable or transient transfection of target cells with an infectious molecular clone. Studies using primary human T cells, the natural targets of HTLV-1 infection, are hampered by difficulty in achieving significant infection with cell-free virus and a poor efficiency of transfection of primary cells. A method is described for the generation of stable cell lines expressing HTLV-1 from an infectious proviral clone. The stably transfected cells can be irradiated and cocultured with human peripheral blood mononuclear cells (PBMC) resulting in infected primary cells. These cells become immortalized, IL-2 dependent lines, which contain integrated copies of provirus and express a full spectrum of viral proteins. Analysis of cellular markers indicates that immortalized cell lines consist of CD3+/CD4+ T cells, matching the most common adult T-cell leukemia (ATL) cell phenotype. The method described has great utility in the study of the replication and transformation capacity of HTLV and HTLV mutant viruses in their natural targets, primary human T lymphocytes.     

Truncated forms of human and simian immunodeficiency virus in infected individuals and rhesus macaques are unique or rare quasispecies

Truncated proviruses of variable sizes are present in peripheral blood mononuclear cells (PBMC) of human immunodeficiency virus type 1 (HIV-1)-infected persons and simian immunodeficiency virus (SIV)-infected rhesus macaques. Here, we investigated whether the highly deleted HIV and SIV proviruses are present in infected organisms as multiple copies or whether each truncated provirus is unique. Using end-point dilution, multiple long-distance (LD) DNA PCR assays were run in parallel using DNA extracted from PBMC of seropositive, treatment-naive persons and from lymph nodes of a rhesus monkey inoculated with cloned, full-length SIVmac239 DNA. The PCR products were titrated and mapped. Most truncated proviruses were present in the DNA samples tested as single, nonintegrated molecules that differed from one another in size and/or nucleotide sequence. These results indicate that truncated primate lentiviral sequences found in infected tissues are unique or rare quasispecies that do not replicate significantly.     

Feline syncytium-forming virus proviral DNA Time of synthesis and relationship to the host cell genome

An infectious DNA assay has been used to study the time of synthesis of feline syncytium-forming virus (FSFV) proviral DNA, and also its relationship to the host cell genome. When extracted late in infection, infectious FSFV proviral DNA was associated with high molecular weight DNA with the same buoyant density as host cell DNA. Furthermore, the apparent size of this DNA could be reduced by shearing, suggesting that the fragments containing FSFV provirus also contained sequences not essential for viral replication. These data suggest that the DNA provirus of FSFV was integrated into chromosomal DNA late in infection. Under single-cycle growth conditions, infectious FSFV DNA was detected after 1 hr in the Hirt supernatant and after 2 hr in the Hirt pellet DNA. Progeny virus was not detected until at least 5 hr postinfection. It was concluded that the synthesis of free proviral DNA and its subsequent integration into the host cell genome may be essential events in the replication of foamy viruses.     

Establishment of novel cell lines latently infected with human immunodeficiency virus 1

Many human immunodeficiency virus 1 (HIV-1) researchers focus on the developing new anti-reservoir therapy to eradicate HIV-1 provirus from the HIV-1-infected patients. HIV-1 provirus is the major obstacle for effective HIV-1 treatment because it integrates into the host genome and can produce a virus progeny after stopping highly active antiretroviral therapy (HAART). We established two novel cell lines latently infected with HIV-1 by limiting dilution cloning of A3.01 cells infected with HIV-1. Analysis of the flanking sequence of HIV-1 proviral DNA integrated into chromosomal cellular DNA revealed that proviral DNA was inserted into different sites of different chromosomes in the two examined cell lines. In these lines, virus reactivation could be induced by a phorbol 12-myristate 13-acetate (PMA) treatment that resulted in a marked increase of the production HIV-1 p24 antigen and appearance of the infectious virus. The novel cell lines latently infected with HIV-1 represent further tool for the study of molecular mechanisms of viral latency and development of anti-reservoir therapy of HIV-1 infection.     

The characterization of Mason-Pfizer monkey virus-induced cell fusion.

The characteristics and requirements of multinucleate cell (syncytium) induction by Mason-Pfizer monkey virus (M-PMV) on human and non-human primate cells have been investigated. Multinucleate cell induction by this “D”-type retrovirus shows single-hit kinetics on human foreskin and rhesus monkey fetal lung cells. The peak of syncytium-forming activity in an isopycnic sucrose gradient coincides with the peak of M-PMV virions as assessed by electron microscopy and analysis of viral polypeptides. Unlike the paramyxoviruses, M-PMV does not induce “early” cell fusion when added in high concentrations to the size of the syncytia remains constant after this time. Ultraviolet irradiation of M-PMV reduces its ability to form syncytia and to replicate with single-hit kinetics, suggesting that a functional viral genome is required for syncytium formation. Proviral DNA synthesis and assembly of virions are not necessary for cell fusion since the addition of cytosine arabinoside at concentrations which block virus replication has little effect on multinucleate cell formation. Moreover both multinucleate cells lacking detectable intracellular virus polypeptides, and groups of individual, nonfused but brightly staining cells can be observed in immunofluorescence assays at times when multinucleate cell formation is maximal. Cell fusion is inhibited by the addition of cycloheximide during the first 12 hr of infection, suggesting that de novo protein synthesis is required for multinucleate cell formation. The possibility that translation of genomic RNA yields a fusion-inducing product is discussed.     

Factors affecting long-term stability of Moloney murine leukemia virus-based vectors

We have examined the long-term functional and structural stability of retroviral vectors in infected murine cells. We have used Moloney murine leukemia virus-based vectors expressing human HPRT, firefly luciferase (luc), and Escherichia coli beta-galactosidase (lacZ) as reporter genes, and the human HPRT and the transposon Tn5 neomycin resistance (neo) gene as selectable markers. All vectors, whether single or double gene, yielded both stable and unstable clones. Stability of the proviruses was dependent on a number of factors, including the nature of the infected cell, the reporter gene, the integration site of the provirus, the relative positions of the component genes in multigene vectors, and the presence or absence of selection pressure. Selection pressure was helpful, but not universally effective, in maintaining provirus structural and functional integrity. Reporter gene expression from an internal promoter was likely to be unstable with or without selection for an upstream, LTR-driven neo gene. In some clones, loss of proviral gene expression was accompanied by deletions, while other inactive clones retained an apparently intact provirus. In the latter clones, treatment with 5-azacytidine failed to reactivate the reporter genes, but superinfection with helper virus resulted in the reappearance of transmissible vector, indicating a reversible epigenetic mechanism for proviral shutdown. The design of effective retroviral vectors and their possible use in vivo will require further characterization of these determinants of provirus stability.