Six-NB-tropic murine leukemia viruses derived from a B-tropic virus of BALB/c have altered p30.

We have examined the electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels of three virion proteins of B-tropic murine leukemia virus from BALB/c and six of its NB-tropic derivatives. The gp70 protein and a 13,000-molecular-weight virion protein tentatively identified as p15 of the NB-tropic viruses migrated with the corresponding B virus proteins. However, the major internal structural protein of type C virions, p30, of all the NB-tropic viruses migrated more rapidly than the p30 of their B virus progenitor. Although this change in p30 raises the possibility that p30 may be involved in determining the N-, B-, or NB-tropism of MuLV's, it is also possible that the change accompanies but does not directly determine the change in tropsim.     

Donation of N- or B-tropic phenotype to NB-tropic murine leukemia virus during mixed infections.

The IC isolate of Moloney murine leukemia virus (MuLV), which is NB-tropic, was grown in cells producing conditionally defective or defective virus particles derived from N- or B-tropic MuLV. The infectious MuLV that was then released was found to be sensitive to Fv-1 restriction but produced NB-tropic progeny upon passage. These results indicate that this NB-tropic MuLV can acquire sensitivity to Fv-1 restriction by phenotypic mixing with N- or B-tropic MuLV. It is thus suggested that NB-tropic MuLV is insensitive to Fv-1 restriction simply because it lacks the determinants of tropism.     

Evidence for recombination between N- and B-tropic murine leukemia viruses.

After co-infection of Sc-1 cells with N- and B-tropic murine leukemia viruses that differ in their XC plaque morphology, Hopkins et al. (1976) obtained viruses, designated XLP-N, that appeared to be recombinants, since they possess the N-tropism of one parent and the XC plaque morphology of the other (the B-tropic virus) parent. Here we present evidence, based on antigenicity and electrophoretic mobility, that some clonal isolates of XLP-N have inherited gp70 gene of their B-tropic virus parent. In addition to providing evidence that XLP-N viruses are recombinants, the fact that an N-tropic virus may apparently possess a gp70 derived from a B-tropic virus provides evidence, which is in agreement with the findings of others (Huang et al., 1973; Krontiris et al., 1973) that the N- or B-tropism of murine leukemia virus does not reside in gp70.     

N-Tropic variants obtained after co-infection with N- and B-tropic murine leukemia viruses.

Sc-1 cells co-infected with small XC plaque-forming N-tropic and large XC plaque-forming B-tropic murine leukemia viruses produced, in addition to parental types, progeny with the phenotype, large XC plaque morphology, and N-tropism. This phenotype remained stable through end point titration and plaque purification on NIH/3T3 cells and growth on BALB/3T3 cells. These N-tropic viruses (XLP-N virus) grow to unusually high titer and make very large XC plaques.     

Production of B-tropic murine leukemia virus by somatic cell hybrids between mouse peritoneal macrophages and simian virus 40-transformed human cells.

Simian virus 40 (SV40)-transformed human cells (LN-SV) were fused with BALB/c peritoneal macrophages (BALB/c X LN-SV) and with C57BL peritoneal macrophages (C57BL X LN-SV) and hybrid clones, all of which had segregated human chromosomes and contained the entire complement of mouse chromosomes, were isolated. All 15 BALB/c X LN-SV hybrid clones were producing varying titers (10 to 10(6) plaque-forming units/ml) of B-tropic murine leukemia virus, whereas none of the nine C57BL X LN-SV hybrid clones was producing detectable ecotropic murine leukemia virus.     

Structure of products of the Moloney murine leukemia virus endogenous DNA polymerase reaction.

We have investigated the process by which the single-stranded RNA genome of Moloney murine leukemia virus is copied into DNA in vitro. DNA synthesis if initiated near the 5' end of the genome, and the elongation of the growing chain occurs by a jumping mechanism whereby the DNA synthesized at the 5' end of the genome is elongated along the 3' end. Unique DNA fragments synthesized beyond the 5' end of the genome in vitro have, at their 5' and 3' ends, copies of unique sequences from the 5' and 3' ends of the genome. These flank a copy of the 49- to 60-nucleotide terminally redundant sequence. These results indicate that the terminal redundancy serves as a "bridge" to allow a DNA molecule synthesized at the 5' end of the genome to serve as a primer for synthesis from the 3' end.     

RNase T1-resistant oligonucleotides of B-tropic murine leukemia virus from BALB/c and five of its NB-tropic derivatives.

We used two-dimensional gel electrophoresis to obtain fingerprints of RNase T1-resistant oligonucleotides of a B-tropic murine leukemia virus from BALB/c and five NB-tropic viruses independently derived from this B virus by passage through NIH Swiss mouse embryo cells in vitro. The fingerprints of the B- and NB-tropic viruses were very similar: approximately 33 of 35 large T1-resistant oligonucleotides appeared to be shared by these viruses. However, the five NB-tropic viruses possessed an apparently common alteration relative to their B virus progenitor. This change involved the acquisition of one oligonucleotide and, tentatively, the loss of one oligonucleotide. We do not know whether these changes represent an alteration responsible for the change from B- to NB-tropism. Fingerprints of B- and NB-tropic viruses were not affected when the viruses were grown in cells of different Fv-1 type.     

Abelson murine leukemia virus mutants with alterations in the virus-specific P120 molecule.

Abelson murine leukemia virus (A-MuLV) is a replication-defective virus that transforms both fibroblasts and hematopoietic cells in vitro. The virus encodes a 120,000-molecular-weight protein (P120) that is composed of Moloney murine leukemia virus-derived gag gene sequences and A-MuLV--specific sequences. This protein is the only A-MuLV--encoded protein that has been detected, and thus P120 is a candidate for the transforming protein of A-MuLV. We now report isolation and characterization of three new A-MuLV isolates that do not synthesize P120 but do produce analogous proteins of larger (160,000 molecular weight) and smaller (100,000 and 90,000 molecular weight) size. All of these A-MuLV isolates transform fibroblasts and lymphoid cells in vitro. Because the different A-MuLV proteins vary in the A-MuLV--specific region of the molecule, these variants may set a maximum limit on the size of the A-MuLV transforming protein.     

RNase T1-resistant oligonucleotides of an N- and a B-tropic murine leukemia virus of BALB/c: evidence for recombination between these viruses.

We used two-dimensional gel electrophoresis to obtain fingerprints of 32P-labeled RNase T1-resistant oligonucleotides derived from the genomes of an N- and a B-tropic murine leukemia virus of BALB/c. These viruses share approximately 30 large T1-resistant oligonucleotides. In addition, there are eight large oligonucleotides unique to the N-tropic virus, and there are six B-trophic virus-specific oligonucleotides. Viruses, designated XLP-N, which appear by biological criteria and analysis of virion proteins to be recombinants between these N- and B-tropic viruses, possess some but not all of the N or B virus-specific oligonucleotides.     

Characterization of hortulanus endogenous murine leukemia virus, an endogenous provirus that encodes an infectious murine leukemia virus of a novel subgroup

Simple retroviruses present a unique opportunity for examining the host-virus relationship. Following exogenous infection and integration into the germ line, copies of these viruses can become fixed within the genome. The resulting endogenous proviral "fossils" represent a record of past retroviral infections and forms. Previous work in our laboratory has been directed at dissecting the extensive nonecotropic murine leukemia virus content of the mouse genome. One such provirus, hortulanus endogenous murine leukemia virus (HEMV), found in a single copy in the genome of Mus spicilegus, was remarkable for characteristics that suggested that it was ancient and related to the hypothetical common ancestor of murine leukemia viruses (MLVs) and other gammaretroviral species. In the present study, we have analyzed its functional properties. Transfection of a molecular clone of the HEMV provirus into mouse-derived cell lines revealed that it is replication competent. Furthermore, host range and interference studies revealed a strictly ecotropic host range and the use of a receptor distinct from those used by other classical MLVs. The identity of nucleotide sequence of the long terminal repeats (LTRs) further suggested that HEMV is a relatively recent insertion into the M. spicilegus genome at the distal end of chromosome 7. Although unique to M. spicilegus, its presence in a homozygous state in three individuals obtained from different regions implies that it has been present long enough to become fixed in this species. Exhaustive phylogenetic analysis of all regions of the HEMV genome supported the previously assigned ancestral position of HEMV relative to other MLV-related viruses. Thus, HEMV is a relatively recent introduction into the Mus germ line but is representative of a relatively ancestral MLV group.     

Polyamines stimulate natural RNA-directed DNA synthesis by Rauscher murine leukemia virus DNA polymerase

In the presence of optimal concentrations of Mg²⁺, spermine and spermidine were found to stimulate rabbit globin mRNA-directed cDNA synthesis by Rauscher murine leukemia virus (R-MuLV) DNA polymerase. Stimulation of DNA synthesis did not occur with the polyamines putrescine or cadaverine, nor could exogenously provided salt or ammonium ions duplicate the stimulation. Analysis of the mechanism of stimulation showed that inclusion of spermine in reaction mixtures a) increased Vmax and decreased apparent Km with respect to the globin mRNA-oligo(dT) tem?late-primer complex, and b) decreased the quantity of oligo (dT) required for optimal rates of cDNA synthesis on a fixed quantity of mRNA template. Genomic 70S RNA-directed cDNA synthesis was also stimulated by spermine addition to reaction mixtures, but only at supra-optimal RNA concentrations. Our results suggest that stimulation of R-MuLV DNA polymerase activity by polyamines is primarily due to stabilization of the enzyme-templateprimer initiation complex resulting in increased efficiency of initiation of cDNA synthesis.     

Reversal of Fv-1 host range by in vitro restriction endonuclease fragment exchange between molecular clones of N-tropic and B-tropic murine leukemia virus genomes.

We molecularly cloned unintegrated viral DNA of the BALB/c endogenous N-tropic and B-tropic murine leukemia retroviruses and in vitro passaged N-tropic Gross (passage A) murine leukemia retroviruses. Recombinant genomes were constructed in vitro by exchanging homologous restriction enzyme fragments from N- or B-tropic parents and subsequent recloning. Infectious virus was recovered after transfection of these recombinant genomes into NIH-3T3 cells and cocultivation with the Fv-1 nonrestrictive SC-1 cells. XC plaque assays of recombinant virus progeny on Fv-ln and Fv-lb cells indicated that the Fv-l host range was determined by sequences located between the BamHI site in the p30 region of the gag gene (1.6 kilobase pairs from the left end of the map) and the HindIII site located in the pol gene (2.9 kilobase pairs from the left end of the map).     

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.     

Characterization of recombinant murine leukemia virus integrase.

Retroviral integration involves two DNA substrates that play different roles. The viral DNA substrate is recognized by virtue of specific nucleotide sequences near the end of a double-stranded DNA molecule. The target DNA substrate is recognized at internal sites with little sequence preference; nucleosomal DNA appears to be preferred for this role. Despite this apparent asymmetry in the sequence, structure, and roles of the DNA substrates in the integration reaction, the existence of distinct binding sites for viral and target DNA substrates has been controversial. In this report, we describe the expression in Escherichia coli and purification of Moloney murine leukemia virus integrase as a fusion protein with glutathione S-transferase, characterization of its activity by using several model DNA substrates, and the initial kinetic characterization of its interactions with a model viral DNA substrate. We provide evidence for functionally and kinetically distinct binding sites for viral and target DNA substrates and describe a cross-linking assay for DNA binding at a site whose specificity is consistent with the target DNA binding site.