MuLV Genome Research Articles

Profound Amplification of Pathogenic Murine Polytropic Retrovirus Release from Coinfected Cells

Previous studies indicate that mice infected with mixtures of mouse retroviruses (murine leukemia viruses [MuLVs]) exhibit dramatically altered pathology compared to mice infected with individual viruses of the mixture. Coinoculation of the ecotropic virus Friend MuLV (F-MuLV) with Fr98, a polytropic MuLV, induced a rapidly fatal neurological disease that was not observed in infections with either virus alone. The polytropic virus load in coinoculated mice was markedly enhanced, while the ecotropic F-MuLV load was unchanged. Furthermore, pseudotyping of the polytropic MuLV genome within ecotropic virions was nearly complete in coinoculated mice. In an effort to better understand these phenomena, we examined mixed retrovirus infections by utilizing in vitro cell lines. Similar to in vivo mixed infections, the polytropic MuLV genome was extensively pseudotyped within ecotropic virions; polytropic virus release was profoundly elevated in coinfected cells, and the ecotropic virus release was unchanged. A reduced level of polytropic SU protein on the surfaces of coinfected cells was observed and correlated with a reduced level of nonpseudotyped polytropic virion release. Marked amplification and pseudotyping of the polytropic MuLV were also observed in mixed Fr98-F-MuLV infections of cell lines derived from the central nervous system (CNS), the target for Fr98 pathogenesis. Additional experiments indicated that pseudotyping contributed to the elevated polytropic virus titer by increasing the efficiency of packaging and release of the polytropic genomes within ecotropic virions. Mixed infections are the rule rather than the exception in retroviral infection, and the ability to examine them in vitro should facilitate a more thorough understanding of retroviral interactions in general.

In Vivo Interactions of Ecotropic and Polytropic Murine Leukemia Viruses in Mixed Retrovirus Infections

Mixed retrovirus infections are the rule rather than the exception in mice and other species, including humans. Interactions of retroviruses in mixed infections and their effects on disease induction are poorly understood. Upon infection of mice, ecotropic retroviruses recombine with endogenous proviruses to generate polytropic viruses that utilize different cellular receptors. Interactions among the retroviruses of this mixed infection facilitate disease induction. Using mice infected with defined mixtures of the ecotropic Friend murine leukemia virus (F-MuLV) and different polytropic viruses, we demonstrate several dramatic effects of mixed infections. Remarkably, inoculation of F-MuLV with polytropic MuLVs completely suppressed the generation of new recombinant viruses and dramatically altered disease induction. Co-inoculation of F-MuLV with one polytropic virus significantly lengthened survival times, while inoculation with another polytropic MuLV induced a rapid and severe neurological disease. In both instances, the level of the polytropic MuLV was increased 100- to 1,000-fold, whereas the ecotropic MuLV level remained unchanged. Surprisingly, nearly all of the polytropic MuLV genomes were packaged within F-MuLV virions (pseudotyped) very soon after infection. At this time, only a fractional percentage of cells in the mouse were infected by either virus, indicating that the co-inoculated viruses had infected the same small subpopulation of susceptible cells. The profound amplification of polytropic MuLVs in coinfected mice may be facilitated by pseudotyping or, alternatively, by transactivation of the polytropic virus in the coinfected cells. This study illustrates the complexity of the interactions between components of mixed retrovirus infections and the dramatic effects of these interactions on disease processes.

Analysis of Variability among Endogenous Ecotropic MuLV Loci in Laboratory Mice

We have isolated a molecular clone of an ecotropic murine leukemia virus from the ovaries of an SWR/J × RF/J hybrid female. The molecularly cloned virus, named pSR3, was demonstrated to induce virus production upon transfection into SWR/J immortalized fibroblasts and to promote germ line integration of proviruses in a fraction of the offspring germline when inoculated to neonate SWR/J females. Sequence analysis reveals that pSR3 is closely related to p623, a plasmid derived from Emv-11 (also referred to as AKV-1). Alignment of the pSR3 sequence with the partial nucleotide sequence of Emv-11 (an endogenous virus carried by BALB/c and C3H/He mice) together with p623 then allows a comparison between three vital sequences. Analysis of these data gives (a) an estimation of the natural divergence rate of MuLV genomes in the course of viral replication (1-5 × 10-5 mutations per cycle and per nucleotide) and (b) molecular evidence for a recent origin through germ line infection of endogenous loci. From additional clues, Emv -11 appears to be the probable ancestor of at least some of these loci.

Amplification and novel locations of endogenous mouse mammary tumor virus genomes in mouse T-cell lymphomas

Endogenous mouse mammary tumor virus genomes are amplified and located in novel cell DNA sequences in many mouse T-cell lymphomas. Transplanted tumors recovered from the same mouse strain and shown to be of independent origin by chromosomal analysis, by the presence of JH immunoglobulin gene rearrangements, or by the integration patterns of exogenous Moloney MuLV genomes frequently showed similar patterns of novel mouse mammary tumor virus-containing cell DNA fragments. This process of amplification and relocation can occur within a limited number of cell generations and in C57BL/6 mice does not lead to the synthesis of mature virus-encoded proteins. In some instances, amplified mouse mammary tumor virus genomes contained novel restriction cleavage sites in the gag-pol region. The restricted time course of occurrence, lack of synthesis of mature virion proteins, and apparent site specificity indicate that this process of retrovirus amplification differs significantly from virus replication after exogenous infection.

Endogenous mouse leukemia viruses.

The earlier demonstration that genes of the mouse greatly influenced the spontaneous incidence of lymphoma was among the more persistent barriers to general acceptance of a viral etiology of this disease. We now can be fairly certain that some of those mouse genes are the DNA life phase of a class of retrovirus known as murine leukemia virus. These MuLV, although related in sequence to each other, are a collection of viruses that show diverse patterns of host range and tissue tropisms. The host-range properties of MuLV serve as means of classifying them into related families known as ecotropic, xenotropic, and amphotropic, and are probably dictated by determinants on gp70. The preferential abilities to replicate in different tissues, on the other hand, may be dictated by the controlling sequences located at the 3' end of the genome, known as U3. MuLV genomes are located at many different sites in the mouse genome. The viral genomes found at those sites can be induced to be expressed with different efficiencies spontaneously in vivo, by chemicals in vitro, or by DNA transfection. Certain MuLV genomes can also interact to increase expression perhaps by recombination or trans complementation. Although the molecular mechanisms that explain these phenomena are not yet clear, the phenomenon of differential expression has important pathological consequences, particularly in the development of lymphoma. The complex process by which endogenous MuLV induce leukemia appears to involve the expression and interaction of multiple MuLV genomes. It seems apparent that expression of an MCF-like gp70 is an invariant aspect of this process, and that observation suggests that this molecule, like the SFFV gp52, may indeed serve to stimulate cell proliferation. The most common means of expressing such a molecule at elevated levels appears to involve recombining it into an ecotropic genome that replicates with high efficiency. Thus, the viral requirements for leukemogenesis may depend on both efficient and perhaps tissue tropic replication as well as on the expression of a particular gp70.(ABSTRACT TRUNCATED AT 400 WORDS)

Variation of Long-Terminal-Repeat Size in Molecular Clones of the BALB/c Endogenous Ecotropic Murine Leukemia Virus

Publisher Summary This chapter discusses the analyses of molecular clones of an endogenous ecotropic virus of BALB/c mice, WN1802N, to identify possible changes that occur in the long terminal repeat (LTR) region. In these analyses, covalently-closed circular DNA intermediates were isolated from cell cultures 48 hours post-infection and cloned by constructing Hin dIII recombinants with Charon 9 bacteriophage. The DNA of 15 independent isolates was first analyzed by Hin dIII restriction and endonuclease digestion, agarose gel electrophoresis, and Southern blot hybridization. Seven of the clones (λWN7, λWN12, λWN26, λWN30, λWN36, and λWN41) have an insert size that corresponds approximately to the complete MuLV genome containing two LTRs. There are four different size LTRs in this population of clones. The most abundant size class is 520 bp and it is probable that this is the size of the endogenous provirus LTR. Seven of these are infectious. There are three isolates with a 600-bp LTR, one of which is infectious, one with 570 bp, and one with 670 bp. Neither of these last two are infectious, but it is yet to be determined whether this is related to the LTR.

Transfer of murine leukaemia and murine sarcoma virus genetic information by transfection with isolated metaphase chromosomes.

Chromosome-mediated transfer of murine leukaemia (MuLV) and murine sarcoma (MuSV) virus genetic information to uninfected recipient cells was investigated. Metaphase chromosomes from AKR MuLV-infected SC-1 mouse cells were incubated with NIH/3T3 cells. After several passages (1 to 3 weeks), infectious virions exhibiting reverse transcriptase activity and the characteristic host range of ecotropic, N-tropic AKR virus appeared in the supernatant fluids of the treated cells. Restriction endonuclease analysis of genomic DNA from transfected cells indicated that AKR proviral DNA was associated with the high molecular weight DNA of the host. These results demonstrate that the AKR MuLV genome can be stably transferred to uninfected recipient cells via isolated metaphase chromosomes. Although AKR virions are not able to infect heterologous cells, chromosome-mediated transfection resulted in the establishment of productive AKR MuLV infection in mink cells. Thus, the use of chromosomes to transfer virus genes can circumvent the natural host restriction barrier. In other experiments, it was shown that normal NIH/3T3 cells were transformed after exposure to metaphase chromosomes isolated from an MuSV-infected, non-producer line. Foci were detected 14 to 21 days after chromosome treatment and were shown to contain true viral transformants since transforming virus was produced after superinfection with MuLV.

Identification of a DNA fragment from a molecularly cloned mink cell focus-inducing murine leukemia virus specific for xenotropic virus-related sequences.

Molecular clones of closed circular DNA molecules of a mink cell focus-inducing murine leukemia virus (MCF-13 MuLV) were generated. Closed circular DNA molecules isolated from a Hirt extraction of recently infected NIH/3T3 cells were inserted at their unique EcoRI site into lambda gtWES.lambda B. Restriction endonuclease analysis of inserts of two clones indicated that they represented intact MCF-13 MuLV genomes. One viral insert contained two large terminal repeat sequences, and the other contained only one. A 300-base-pair DNA fragment located in the envelope region of the MCF-13 MuLV genome was determined to be related to xenotropic MuLV sequences.

Moloney murine leukemia virus-induced tumors: recombinant proviruses in active chromatin regions

The DNase I sensitivity of chromosomal DNA regions carrying integrated proviral genomes of Moloney (M-MuLV) and AKR Murine Leukemia Virus (AKR-MuLV), and the cellular homologue of the mos-gene (c-mos) of Moloney Sarcoma Virus (MSV) were studied in tumor tissues of leukemic mice. The genetically transmitted sequences of M-MuLV, AKR-MuLV, and the c-mos gene are all in DNase I resistant chromatin conformations in M-MuLV-induced tumors. Each M-MuLV-induced tumor contained at least one somatically acquired integrated recombinant MuLV genome that displayed two main characteristic features of active chromatin: a) a configuration hypersensitive to DNase I, and b) extensive hypomethylation. DNase I hypersensitive sites were mapped at the junction of cellular sequences and the 5'-viral large terminal repeat (LTR). Expression of a recombinant MuLV seems therefore to be a necessary feature to maintain the transformed state.

Dilute (d) coat colour mutation of DBA/2J mice is associated with the site of integration of an ecotropic MuLV genome.

The single endogenous DBA/2J ectropic provirus segregated concordantly with the dilute (d) coat colour mutation on chromosome 9 in 53/53 DBA/2J-derived recombinant inbred mouse strains and all seven inbred and mutant strains tested that carry the d allele. Analysis of DNA from a spontaneous DBA/2J d revertant (d+2J) showed that these mice lack ecotropic-specific MuLV DNA sequences and suggested that the dilute mutation resulted from integration of an ecotropic provirus into the mouse genome.

Heterogeneity of “virus-like” genes encoding retrovirus-associated 30S RNA and their organization within the mouse genome

The mouse genome harbors a set of genes reiterated 20–50 times that normally expresses 30S RNA transcripts. These transcripts can be rescued specifically into virions of helper-independent C-type viruses as pseudotypes and are thought to be defective endogenous retroviruses. We cloned DNA sequences coding “virus-like” 30S RNA from a gene library of the inbred mouse strain BALB/c in bacteriophage lambda. We identified the sequences by their hybridization with an RNA abundant in preparation of MuLV virions produced by mouse cells but sharing no sequence homology with the MuLV genome, and by the ability of the cloned DNAs to hybridize specifically with the poly(A)-containing 30S RNA present in the cytoplasm of uninfected mouse cells. The 30S RNAs are encoded by uninterrupted 5 kb DNA sequences in the genome. Individual copies of virus-like genes were distributed at different loci and were flanked by cellular DNA sequences that shared no apparent sequence homology. Each of the virus-like genes is related but not identical to all other copies. Two levels of genetic heterogeneity were detected: the first results from relatively large, continuous sequences of nonhomology up to 300 bp long; the second results from a pattern of scattered base substitutions suggesting that some DNA sequences might have been conserved more than others.

Physical map of biologically active Harvey sarcoma virus unintegrated linear DNA

BALB/c JLS V9 cells recently infected with Harvey sarcoma virus-murine leukemia virus (HSV-MuLV) complex contained unintegrated HSV linear DNA of 6.0-kilobase pair mass. The cells also contained two HSV closed circular DNA species along with MuLV-encoded linear and closed circular DNA species. HSV 6.0-kilobase pair linear DNA induced focal transformation upon transfection of NIH 3T3 mouse fibroblasts, and the biological activity of HSV DNA did not require helper MuLV functions. A physical map of restriction endonuclease cleavage sites along HSV 6.0-kilobase pair linear DNA was derived. Comparison of this map with one for Moloney MuLV DNA showed that the HSV and Moloney MuLV genomes are identical near their viral RNA 3' ends.

Detection of AKR MuLV-specific RNA in AKR mouse cells by in situ hybridization.

Conditions for the detection of complex RNA sequences by in situ hybridization have been investigated by using a single-stranded 3H-cDNA probe complementary to the AKR MuLV genome and in vitro cultured AKR mouse cells which spontaneously produce AKR MuLV. It is shown that fixation with glutaraldehyde at low concentration allows cellular RNA to be sufficiently well retained during the annealing process and that stringent conditions in situ can be maintained by means of formamide. Some conditions which promote atypical and non-specific binding of the probe have been identified.

The integrated genome of murine leukemia virus

The Southern gel filter transfer technique has been used to characterize the integrated genome of Moloney murine leukemia virus (M-MuLV) and the genomes of the endogenous viruses of the mouse. Study of 10 clones of rat cell independently infected by M-MuLV indicates a minimum of 15 integration sites into which the M-MuLV provirus can be inserted. No common integration site is observed among these clones. Clones productively infected by M-MuLV acquire multiple proviruses, whereas infected cells unable to produce virus contain only one M-MuLV provirus. Once established, the integrated genomes are stable for at least two years after initial infection. The use of M-MuLV probe allows detection of a spectrum of Eco RI-cleaved mouse DNA fragments containing endogenous MuLV genomes. DNAs of different inbred laboratory mouse strains yield similar patterns of provirus with each strain showing minor characteristic differences. In some instances, mouse cells infected by M-MuLV reveal additional proviruses beyond those seen in the uninfected cell. DNAs from three different M-MuLV-induced thymomas indicate, as in rat cells, multiple possible integration sites.

The G-IX system. A cell surface allo-antigen associated with murine leukemia virus; implications regarding chromosomal integration of the viral genome.

This report concerns a cell surface antigen (G(IX); G = Gross) which exhibits mendelian inheritance but which also appears de novo in cells that become productively infected with MuLV (Gross), the wild-type leukemia virus of the mouse. In normal mice, G(IX) is a cell surface allo-antigen confined to lymphoid cells and found in highest amount on thymocytes. Four categories of inbred mouse strains can be distinguished according to how much G(IX) antigen is expressed on their thymocytes. G(IX) (-) strains have none; in the three G(IX) (+) categories, G(IX) (3), G(IX) (2), and G(IX) (1), the amounts of G(IX) antigen present (per thymocyte) are approximately in the ratios 3:2:1. A study of segregating populations derived mainly from strain 129 (the prototype G(IX) (3) strain) and C57BL/6 (the prototype G(IX) (-) strain) revealed that two unlinked chromosomal genes are required for expression of G(IX) on normal lymphoid cells. The phenotype G(IX) (+) is expressed only when both genes are present, as in 129 mice. C57BL/6 carries neither of them. At one locus, expression of G(IX) is fully dominant over nonexpression (G(IX) fully expressed in heterozygotes). At the second locus, which is linked with H-2 (at a distance of 36.4 +/- 2.7 units) in group IX (locus symbol G(IX)), expression is semidominant (50% expression of G(IX) in heterozygotes); gene order T:H-2:Tla:G(IX). As a rule, when cells of G(IX) (-) mice or rats become overtly infected with MuLV (Gross), an event which occurs spontaneously in older mice of certain strains and which also commonly accompanies malignant transformation, their phenotype is converted to G(IX) (+). This invites comparison with the emergence of TL(+) leukemia cells in TL(-) mouse strains which has been observed in previous studies and which implies that TL(-) --> TL(+) conversion has accompanied leukemic transformation of such cells. So far the only example of G(IX) (-) --> G(IX) (+) conversion taking place without overt MuLV infection is represented by the occurrence of GCSA(-):G(IX) (+) myelomas in BALB/c (GCSA:G(IX) (-)) mice. Unlike the other Gross cell surface antigen described earlier, GCSA, which is invariably associated with MuLV (Gross) infection and never occurs in its absence, G(IX) antigen sometimes occurs independently of productive MuLV infection; for example, thymocytes and some leukemias of 129 mice are GCSA(-):G(IX) (+), and MuLV-producing sarcomas may be GCSA(+):G(IX) (-). The frequent emergence of cells of G(IX) (+) phenotype in all mouse strains implies that the structural gene coding for G(IX) antigen is common to all mice. There is precedent for this in the TL system, in which two of the Tla genes in linkage group IX appear to be ubiquitous among mice, but are normally expressed only in strains of mice carrying a second (expression) gene. It is not yet certain whether either of the two segregating genes belongs to the MuLV genome rather than to the cellular genome. This leaves the question whether MuLV may have a chromosomal integration site still debatable. But there is a good prospect that further genetic analysis will provide the answer and so elucidate the special relationship of leukemia viruses to the cells of their natural hosts.