RNA sequencing provides evidence for allelism of determinants of the N-, B- or NB-tropism of murine leukemia viruses.

Previous genetic and biochemical studies identified three large RNAase T1-resistant oligonucleotides, each associated with either the N-, B- or NB-tropism of murine C-type viruses of BALB/c origin. These oligonucleotides were shown to lie in the 5' third of the oligonucleotide maps of their respective viruses. We sequenced the three oligonucleotides and found that they share a 10 base sequence. Together these observations provide good evidence that the determinants of N-, B- or NB-tropism monitored by the three oligonucleotides are allelic. The oligonucleotides associated with N- and B-tropism differ in sequence at four of sixteen nucleotides, while the B- and NB-tropism-associated oligonucleotides differ in sequence by only one base out of sixteen. These results are consistent with the possibilities that B-tropic viruses may arise from N-tropic viruses by recombination, while NB-tropic viruses may arise from B-tropic virus by mutation. An unexplained finding was that a 10 base sequence present in the oligonucleotide associated with N-tropism is also found in the 3' third of the genomes of the N-, B- and NB-tropic viruses studied.     

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.     

Evidence for recombination between N- and B-tropic murine leukemia viruses: analysis of three virion proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

We have sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the virion proteins of an N- and a B-tropic C-type virus derived from the BALB/c mouse and 21 putative recombinants, designated XLP-N viruses, obtained from seven crosses between these N- and B-tropic viruses. All the XLP-N viruses are N-tropic but posses the XC plaque morphology of their B-tropic virus parent. Three virion proteins, p15, p30, and gp70, of the parental viruses each differ in electrophoretic mobility. Two recombinants were found that possess a p15 that comigrates with p15 of the B virus; 19 possess a p15 that comigrates with N virus p15. Sixteen recombinants possess a gp70 that migrates like the gp70 of the B virus: four have gp70 with an electrophoretic mobility like that of the N virus gp70. All 21 recombinants possess a p30 that comigrates with p30 of their N virus parent. Given the origin and phenotype of XLP-N viruses, these results would seem to provide good evidence that these viruses are recombinants.     

T1 oligonucleotide maps of N-, B-, and B leads to NB-tropic murine leukemia viruses derived from BALB/c.

We previously described and characterized RNase T1 RNA fingerprints of an N-, a B-, and five B leads to NB-tropic murine leukemia viruses derived from BALB/c mice (Faller and Hopkins, J. Virol. 23:188-195, 1977, and J. Virol. 24:609-617, 1977). These viruses share the majority of their large RNase T1-resistant oligonucleotides, but each possesses some "unique" oligonucleotides relative to the others. We have ordered the large T1-resistant oligonucleotides of the N-, the B-, and one NB-tropic virus relative to the 3' end of their genomes to obtain oligonucleotide maps. These maps indicate that (i) the large T1 oligonucleotides shared by the N-, B-, and NB-tropic viruses probably occupy the same relative positions on their genomes; (ii) the 14 T1 oligonucleotides that differ between the N- and B-tropic viruses are derived from regions scattered along the genomes; and (iii) an oligonucleotide that is present in five NB-tropic viruses but not in their B-tropic virus progenitors lies toward the 5' end of the NB-tropic virus oligonucleotide map.     

Chromatographic Separation and Antigenic Analysis of Proteins of the Oncornaviruses IV. Biochemical Typing of Murine Viral Proteins

Tryptic peptide maps were prepared for four purified structural proteins derived from several murine leukemia viruses (MuLV's). Analyses of these peptide maps reveal that the p30 proteins of Rauscher, Moloney, and Gross MuLV's are very similar to each other, as are the p10's obtained from these three viruses. In contrast, the peptide maps of the individual p15's and p12's from the same viruses establish that each of these polypeptides is highly strain specific. For all four polypeptides studied, unique peptides appear in the Rauscher MuLV and Moloney MuLV tryptic profiles that are not present in the corresponding Gross MuLV profile. By this method of analysis it was possible to distinguish the p30's of N-tropic and B-tropic MuLV's derived from the same BALB/c mouse.     

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.     

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.     

Mouse strain resistant to N-, B-, and NB-tropic murine leukemia viruses.

Mouse strain G was studied for its susceptibility to various strains of murine leukemia and sarcoma viruses. Both N- and NB-tropic Friend leukemia viruses neither induced splenomegaly nor grew efficiently in strain G mice. Using the XC test, cultured embryo cells were found to be resistant, but not absolutely, to all the tested viruses, N-tropic AKR virus, N- and NB-tropic Friend leukemia viruses, NB-tropic Rauscher leukemia virus, B-tropic WN1802B virus, NB-tropic Moloney leukemia and sarcoma viruses, and N-tropic Kirsten sarcoma virus, although the resistance to Moloney leukemia and sarcoma viruses is sometimes not as strong as that for other viruses. Thus, the strain G mice are unique among mouse strains because they show resistance that is not related to the N-B tropism of murine leukemia viruses.     

Physical mapping of the Fv-1 tropism host range determinant of BALB/c murine leukemia viruses

The murine leukemia viruses (MuLVs) have different host ranges and were originally designated N-tropic and B-tropic if they replicated preferentially in vitro on NIH and BALB/c fibroblasts, respectively. It was later found that N-tropic MuLVs were in fact restricted in BALB/c cells, that B-tropic MuLVs were restricted in NIH cells, and that both viruses were restricted in (BALB X NIH) F1 cells. A single gene, Fv-1, with two alleles, Fv-1b and Fv-1n, determines this dominant restriction. A virus-encoded protein seems to carry the viral host range determinant which is recognized by the Fv-1 gene product. To map the viral DNA sequences encoding this determinant, we constructed viral DNA recombinants in vitro between the cloned infectious viral DNA genomes from BALB/c N-tropic and B-tropic MuLVs. Infectious recombinant MuLVs were recovered by microinjecting these recombinant DNAs into murine Fv-1- SC-1 cells and were subsequently tested in vitro for their host ranges (N- or B-tropic). We found that a short 302-base pair 5'-end fragment was necessary and sufficient to confer a specific host range to a recombinant. Our sequencing data revealed that this fragment codes for amino acid sequences in gag p30. They also showed that only two consecutive amino acid differences, Gln-ArgN- and Thr-GluB-, in p30 are responsible for the N- and B-tropic host ranges of the BALB/c MuLVs, respectively. Therefore, it appears that the Fv-1b and Fv-1n gene products can discriminate between these two p30 amino acid sequences.     

Polymorphism of B-tropic leukemia viruses from BALB/c mice: association of a p30 antigen with N- versus B-tropism.

Comparison of a number of murine leukemia virus clones by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed extensive protein polymorphism among B-tropic, but not N-tropic, isolates from BALB/c mice, particularly in migration of p30 proteins. A type-specific radioimmunoassay for p30 was developed which uniformly discriminated all B-tropic viruses from N-tropic viruses of BALB/c origin. N- and B-tropic viruses of C57BL/6 and AKR Fv-1b/b origin could also be distinguished by this assay.     

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.     

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.     

T1 oligonucleotides that segregate with tropism and with properties of gp70 in recombinants between N- and B-tropic murine leukemia viruses.

We have analyzed large RNase T1-resistant oligonucleotides derived from the genomes of 16 recombinants between N- and B-tropic murine leukemia viruses of BALB/c. The parental viruses, designated SP-N and LP-B, differ in several phenotypic or biochemically defined properties: N- or B-tropism; XC plaque morphology, electrophoretic mobility of three virion proteins (p15, p30, and gp70); ability to induce GIX antigen on infected cells; presence of 6 to 8 (out of 36 to 38 analyzable) large T1 oligonucleotides. One SP-N-specific T1 oligonucleotide was inherited by all 16 N-tropic recombinants and, thus, appears to be linked to N-tropism. This oligonucleotide lies in the 5' third of the oligonucleotide map of SP-N. One LP-B-specific T1 oligonucleotide was inherited by all 11 recombinants whose gp70 has an electrophoretic mobility like that of LP-B gp70 and that, like LP-B, fail to induce GIX antigen. This oligonucleotide lies in the 3' third of the oligonucleotide map of LP-B.     

Fv-1 determinants in xenotropic murine leukemia viruses studied with biological assay systems: Isolation of xenotropic virus with N-tropic Fv-1 activity in the cryptic form.

By a biological assay system using phenotypically mixed ecotropic and xenotropic murine leukemia viruses, we investigated whether in the virions of a xenotropic virus there is N- or B-tropic Fv-1 determinant in active form. The existence of N-tropic Fv-1 determinant was demonstrated in SL-XT-1 xenotropic virus isolated from the spleen of a 3-month-old SL mouse, and the N-tropic Fv-1 tropism was confirmed by analysis of the phenotypically mixed viruses harvested from clonal SC-1 cells doubly infected with the SL-XT-1 and B-tropic ecotropic viruses. However, neither N- nor B-tropic Fv-1 determinant was demonstrated in any xenotropic viruses isolated from embryo cells of BALB/c, NZB, or DBA/2 mice, or Cas E #1-IU, and xenotropic-like virus isolated from a wild mouse.     

Analysis of Proteins of Mouse Sarcoma Pseudotype Viruses: Type-Specific Radioimmunoassays for Ecotropic Virus p30''s

Murine sarcoma virus pseudotypes were prepared by infection of nonproducer cells (A1-2), which were transformed by the Gazdar strain of mouse sarcoma virus, with Gross (N-tropic), WN1802B (B-tropic), or Moloney (NB-tropic) viruses. The respective host range pseudotype sarcoma viruses were defined by the titration characteristics on cells with the appropriate Fv-1 genotype. Proteins from virus progeny were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bands present in both the 65,000- and the 10,000- to 20,000- molecular-weight regions of the gel distinguished the pseudotype viruses from their respective helpers. Furthermore, two protein bands were noted in the p30 region of murine sarcoma virus (Gross), one corresponding to Gross virus p30, and another of slightly slower mobility. However, since the mobility of the putative sarcoma p30 is nearly indentical to that of WN1802B, its presence could not be established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Type-specific radioimmunoassays for Gross virus p30 and for WN1802B p30 were applied for analysis of pseudotype preparations, and among several ecotropic viruses tested, only the homologous virus scored in the respective assay. By use of these assays, pseudotype viruses were found to contain only 8 to 48% helper-specific p30's; the remainder is presumably derived from the sarcoma virus.     

In Vitro Selection of High-Infectious, Leukemogenic Virus from Low-Infectious, Non-Leukemogenic Type C Virus from a Malignant ST/a Mouse Cell Line

Low-infectious, nontransforming type C virus was isolated from an in vitro spontaneously transformed ST/a mouse cell line, ST-L1. The virus released by ST-L1 cells was NB-tropic and XC(-). It gave rise to very small peroxidase antibody plaques (PAP) in cultures which initially were nonproducing. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the structural proteins of the ST-L1 virus showed an envelope glycoprotein with an apparent mass of 65 kilodaltons (kdal). The mouse cells SC-1, BALB/3T3, and NIH/3T3 could be productively infected with cell-free supernatants from the ST-L1 cell line; however, virus was detected in supernatant fluids only after two to four subcultures of the infected cells. The virus thus produced was XC(+) and a large plaque former. The virus released from infected SC-1 cells was N-tropic, whereas the viruses from infected NIH/3T3 and BALB/3T3 cells were NB-tropic. The structural proteins of the N- and NB-tropic viruses could be distinguished on SDS polyacrylamide gels, the major dissimilarity being a difference in the mobility of the p30. All these viruses had an envelope glycoprotein with an apparent mass of 70 kdal. The infectivity of the viruses, measured as PAP per nanogram of p30, was 30- to 60-fold lower for the virus released from the ST-L1 cell line than that of the viruses after passage in SC-1, NIH/3T3, and BALB/3T3 cells. None of the viruses could infect rabbit or mink cells. Inoculation of the viruses into newborn mice showed that the ST-L1 virus was non-leukemogenic, whereas the NB-tropic virus selected from this after passage in BALB/3T3 or NIH/3T3 cells was highly leukemogenic. Viruses isolated from leukemic animals were indistinguishable with respect to host range and protein mobilities in SDS gels from the ones with which the mice were inoculated. Although the SC-1-selected virus was highly infectious in vitro, it was only weakly, if at all, leukemogenic.     

Genetic transmission of endogenous N- and B-tropic murine leukemia viruses in low-leukemic strain C57BL/6.

Spontaneous expression of endogenous N- and B-tropic murine leukemia viruses was stu1bb), DDD (Fuv-1nn), DDD-Fvr (fv-1nn), (DDD or DDD-Fvr times C57BL/6)F1, and 16 partially inbredlines with either the Fv-1nn or Fv-1bb genotype, which had been established from hybrids between C57BL/6 and DDD-Fvr. When tested at middle age, virus-positive mice were found in C57BL/6, F1 hybrids, and 9 out of 16 partially inbred lines. N-tropic viruses were isolated from Fv-1nn, Fv-1bb mice, whereas B-tropic viruses, except for one isolate, were from Fv-1bb mice only. C57BL/6 mice were positive for both N- and B-tropic viruses, whereas DDD-Fvr mice were negative. With respect to the Fv-1 genotype and the presence of endogenous murine leukemia viruses, the partially inbred lines were grouped into five types: (i) Fv-1bb, both N- and B-tropic virus positive, like C57BL/6; (ii) Fv-1nn, virus negative, like DDD-Fvr; (iii) Fv-1bb, virus negative; (iv) Fv-1nn, only N-tropic virus positive; and (v) less convincingly, Fv-1bb, only B-tropic virus positive. These findings indicate that the transmission of N- and B-tropic viruses in C57BL/6 is genetically controlled and that the expression of B-tropic virus, but not of N-tropic virus, is closely associated with the Fv-1 genotype.     

Biochemical analysis of murine leukemia viruses isolated from radiation-induced leukemias of strain BALB/c.

Murine leukemia viruses isolated from radiation-induced BALB/c leukemias were characterized with respect to viral proteins and RNA. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the viral structural proteins revealed that for p12, p15, p30, and gp70, three of four electrophoretic variants of each could be detected. There was no correlation found between any of these mobilities and N- or B-tropism of the viruses. Proteins of all xenotropic viral isolates were identical in their gel electrophoretic profiles. The similar phenotypes of multiple viral clones from individual leukemias and of isolates grown in different cells suggest that the polymorphism of ecotropic viruses was generated in vivo rather than during in vitro virus growth. By two-dimensional fingerprinting of RNase T1-resistant oligonucleotides from 70S viral DNA, the previously reported association of N- and B-tropism with two distinct oligonucleotides was confirmed. The presence of two other oligonucleotides was correlated with positive and negative phenotypes of the virus-coded GIX cell surface antigen. The RNAs of two B-tropic isolates with distinctive p15 and p12 phenotypes differed from the RNA of a prototype N-tropic virus by the absence of three oligonucleotides mapping in the 5' portion (gag region) of the prototype RNA. In addition, one small-plaque B-tropic virus displayed extensive changes in the RNA sequences associated with the env region of the prototype.     

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).     

Effect of the Fv-1 locus on the titration of murine leukemia viruses.

Titration of N- and B-tropic murine leukemia viruses on sensitive and resistant cell lines has been studied by direct XC plaque assay and infective center assay. The titration of cloned B-tropic virus by infective center assay on BALB/3T3 (Fv-1b/b) and NIH/3T3 (Fv-1n/n) cells gave one-hit patterns, with 100-fold less infected NIH/3T3 cells than BALB/3T3 cells. The titration of B-tropic virus on DBA/2 cells (Fv-1n/n) was also a one-hit. The titration of a one-hit curve, and there were about 100-fold less infected BALB/3T3 cells than NIH/3T3 cells. Comparable results were obtained by titrating the cloned N-tropic virus on congenic SIM (Fv-1n/n) and SIM.R (Fv-1b/b) cells or the Gross N-tropic virus on BALB/3T3 cells. Therefore, our data indicate that the multiple-hit phenomenon described previously may not be an essential part of the Fv-1 gene restriction.