Tumours induced by Moloney murine sarcoma virus are clonal in rats,not clonal in mice

Summary Moloney murine sarcoma virus (M-MSV) induces rapidly growing tumours in adult mice of most conventional strains. Rats are less susceptible to M-MSV oncogenesis, but the few rhabdomyosarcomas that do develop after viral inoculation of newborn animals closely resemble conventional malignancies: they develop after a long latency, grow progressively, and metastasize to regional lymph nodes and lungs. Southern blot analysis with a v-mos-specific probe of M-MSV-induced tumours in both species demonstrated an oligo-, monoclonal pattern of exogenous v-mos integration only in the rat system, while mouse tumours were not clonal in origin. Furthermore, the same type of analysis of lymph node and lung metastases showed that cell clones already present in the primary rat lesion colonized secondary sites during tumour progression. Apparently, Moloney murine leukemia virus (M-MuLV) was not involved in rhabdomyosarcoma pathogenesis since M-MuLV-specific DNA sequences could not be demonstrated in three of the six rat tumours. Finally, in all mouse tumours, unintegrated linear M-MSV proviruses could be readily detected.Abbreviations M-MSV Moloney murine sarcoma virus - M-MuLV Moloney murine leukemia virus     

Cell transformation and tumor induction by Abelson murine leukemia virus in the absence of helper virus.

We investigated the role of the Moloney helper virus, Moloney murine leukemia virus (Mo-MuLV), in cell transformation and tumor induction by the defective Abelson murine leukemia virus (Ab-MuLV). A molecular clone of Ab-MuLV (P160 strain) was transfected into the psi 2 packaging cell line, and helper virus-free Ab-MuLV (psi 2) was harvested from the supernatant medium. Ab-MuLV (psi 2) was as efficient as helper virus-containing Ab-MuLV (Mo-MuLV) in the transformation of primary bone marrow cells in vitro. Inoculation of weanling BALB/c mice with Ab-MuLV (psi 2) induced nonthymic pre-B-cell lymphomas with high efficiency and short latency (28 days). Adult BALB/c mice were less sensitive to tumor induction by a factor of 100. Ab-MuLV (psi 2) did not induce tumors in weanling C57BL/6 mice, unlike Ab-MuLV (Mo-MuLV). Examination of the proviral integration pattern in Ab-MuLV (psi 2)-induced tumor cell DNA revealed that each of the tumors contained a single integrated provirus. Immunoprecipitation of viral-encoded proteins in helper virus-free tumor cell lines detected the P160 Ab-MuLV-transforming protein; however, no trace of the gag, pol, and env helper virus-encoded proteins was found. Our results indicate that integration and expression of a single Ab-MuLV genome is sufficient for efficient transformation of primary bone marrow cells by Ab-MuLV in vitro and tumor induction in susceptible mice. However, the helper virus may contribute to tumor induction in weanling resistant mice.     

Inhibition of bacterially expressed HIV protease activity determined by an in vitro cleavage assay with MuLV Pr65gag

HIV protease is a virally coded enzyme that cleaves gag as well as gag-pol precursor polyproteins into functional products needed for virus assembly. A pUC plasmid containing an HIV insert starting at the 5' end of the pol gene and ending just inside the intergrase coding sequence was expressed in E. coli. It provided an 11 kD gene product (protease) that specifically cleaved the Gazdar MuLV Pr65gag precursor into Pr40gag (p30 + p10) and Pr27gag (p15 + p12) intermediates, as well as lower molecular weight gag-encoded products. These were detected by immunoblotting with either MuLV anti-p30 or p12 sera. Using cleavage of MuLV Pr65gag as an assay system, pepstatin A, fusidic acid, and cerulenin were observed to inhibit HIV protease cleavage by greater than 50% at concentrations of 0.1, 0.2-0.5, and 0.5 mM, respectively.     

Myristyl amino-terminal acylation of murine retrovirus proteins: An unusual post-translational protein modification

The primary structure of the NH(2)-terminal region of the gag gene encoded internal membrane-associated protein p15 has been determined for both Rauscher and Moloney murine leukemia viruses. Peptides generated by endopeptidases and purified by HPLC were subjected to semi-automated Edman degradation. Dipeptides obtained with dipeptidyl carboxypeptidase were identified by gas chromatography-mass spectrometry. The amino acid sequence of the first 16-residue segment of Rauscher p15 is identical to the sequence of Moloney p15 except for a single amino acid substitution (Gly-->Asp) at position 13. Both proteins were found to have an acylated NH(2) terminus. By mass spectroscopy, myristic acid [CH(3)(CH(2))(12)COOH] was found to be bound through an amide linkage to the NH(2)-terminal glycyl residue in both p15s. The results of liquid chromatography show that the NH(2)-terminal myristyl group greatly contributes to the strong binding of these modified proteins and peptides to hydrophobic surfaces. Because p15 is known to be derived from the NH(2)-terminal region of a precursor polyprotein Pr65(gag) by proteolytic cleavage in the assembled virus, it is suggested that myristylation in vivo takes place during the biosynthesis of Pr65(gag). Preliminary data indicate that such modification of gag precursor polyproteins may be common to mammalian retroviruses. The role of NH(2)-terminal myristyl acylation of Pr65(gag) in virus assembly and the possibility of similar NH(2)-terminal modifications of gag-related fusion proteins of transforming viruses are discussed.     

Structure and biological activity of v-raf, a unique oncogene transduced by a retrovirus.

We have molecularly cloned a unique acutely transforming replication-defective mouse type C virus (3611-MSV) and characterized its acquired oncogene. The viral genome closely resembles Moloney (M) murine leukemia virus (MuLV), except for a substitution in M-MuLV in the middle of p30 and the middle of the polymerase gene (pol). Heteroduplex analysis revealed that 2.4 kilobases of M-MuLV DNA were replaced by 1.2 kilobases of cellular DNA. The junctions between viral and cellular sequences were determined by DNA sequence analysis to be 517 nucleotides into the p30 sequence and 1,920 nucleotides into the polymerase sequence. Comparison of the transforming gene from 3611-MSV, designated v-raf, with previously isolated retrovirus oncogenes either by direct hybridization or by comparison of restriction fragments of their cellular homologs shows it to be unique. Transfection of NIH 3T3 cells with cloned 3611-MSV proviral DNA leads to highly efficient transformation and the recovered virus elicits tumors in mice typical of the 3611-MSV virus. Transfected NIH 3T3 cells express two 3611-MSV-specific polyproteins (P75 and P90), both of which contain NH2-terminal gag gene-encoded components linked to the acquired sequence (v-raf) translational product. The cellular homolog, c-raf, is present in one or two copies per haploid genome in mouse and human DNA.     

Characterization and partial nucleotide sequence of endogenous type C retrovirus segments in human chromosomal DNA.

Twenty-six different murine leukemia virus (MuLV)-related clones have been isolated from a human DNA library and characterized by restriction enzyme mapping and reciprocal nucleic acid hybridization reactions. The sequence of approximately 2,600 nucleotides, spanning more than 4.0 kilobases, of one of the MuLV-related cloned human DNAs was also determined. The deduced amino acid sequence permitted the alignment of this prototype cloned human DNA segment with the p12 gag, p30 gag, p10 gag, and pol regions of Moloney MuLV. A majority of the endogenous type C retrovirus-related segments present in human DNA are approximately 6.0 kilobases in size and appear to contain a deletion of env sequences.     

Characterization of human immunodeficiency virus type 1 Pr55gag membrane association in a cell-free system: requirement for a C-terminal domain.

Association of the human immunodeficiency virus type 1 (HIV-1) gag polyprotein precursor with cellular membranes is necessary for assembly of virions. We used in vitro synthesized HIV-1 gag to study its association with isolated cellular membranes. Rabbit reticulocyte lysates programmed with HIV-1 gag mRNA incorporated [35S]methionine and [3H]myristate into two predominant species of 55 kDa and 40 kDa. Radioimmunoprecipitation with HIV-1-specific antibodies suggested that the 55-kDa protein represented the polyprotein precursor (Pr55gag), while the 40-kDa protein was a mixture of N- or C-terminal truncations of the gag precursor. The Pr55gag protein bound to cellular membranes, while the 40-kDa mixed protein species did not. Membrane binding studies with C terminus-truncated and point mutants revealed that the seven-amino acid sequence located between the two Cys-His arrays in the nucleocapsid region was necessary for stable association to occur. Therefore, we propose that signals in addition to myristate are required for the membrane association of HIV-1 gag proteins and that these signals include a domain in the nucleocapsid protein.     

Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus.

It was previously reported that the gag proteins of mammalian type C retroviruses are modified by the addition of myristate to the N-terminal glycine residue. We have performed oligonucleotide-directed mutagenesis to change this glycine codon in the Moloney murine leukemia virus genome to an alanine codon and also to specifically delete the glycine codon. Upon transfection into mammalian cells, these mutant genomes direct the synthesis of gag proteins, but these proteins are not myristylated. The mutants do not form virus particles or any recognizable virus-specific structures visible in thin sections with the electron microscope. Further, the mutant gag proteins appear to remain in the cytosol, whereas the wild type is found principally in particulate fractions of the cell. The results are consistent with the theory that myristate is required for the association of the gag protein with the plasma membrane and that this association is necessary for virus assembly.     

Proteasome inhibition interferes with gag polyprotein processing, release, and maturation of HIV-1 and HIV-2

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6(gag), a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6(gag). Consistent with this, viruses with mutations in PR or p6(gag) were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.     

Quantitative imaging of the T cell antitumor response by positron-emission tomography

We describe a noninvasive, quantitative, and tomographic method to visualize lymphocytes within the whole animal. We used positron-emission tomography (PET) to follow the localization of adoptively transferred immune T lymphocytes. Splenic T cells from animals that had rejected a Moloney murine sarcoma virus/Moloney murine leukemia virus (M-MSV/M-MuLV)-induced tumor were marked with a PET reporter gene, injected into tumor-bearing mice, and imaged in a microPET by using a substrate specific for the reporter. Specific localization of immune T cells to the antigen-positive tumor was detected over time, by sequential imaging of the same animals. Naive T cells did not localize to the tumor site, indicating that preimmunization was required. Autoradiography and immunohistochemistry analysis corroborated the microPET data. The method we have developed can be used to assess the effects of immunomodulatory agents intended to potentiate the immune response to cancer, and can also be useful for the study of other cell-mediated immune responses, including autoimmunity.     

Retroviral insertional mutagenesis of a target allele in a heterozygous murine cell line.

A clonal murine cell line that is heterozygous at the beta 2-microglobulin locus (B2m) was obtained by Abelson murine leukemia virus (Ab-MuLV) transformation of liver cells from (C57BL/6 X BALB/c) F1 fetuses. To obtain proviral insertional mutants, we superinfected a subclone of these cells, which does not express the env surface protein of the Moloney leukemia virus (Mo-MuLV, the helper virus that was used to transmit the defective Ab-MuLV genome during transformation), with Mo-MuLV. Mutant clones that fail to express the C57BL/6 allele of B2m (B2mb) were then immunoselected by using a monoclonal antibody that specifically recognizes the B2mb gene product and not that of the B2ma allele. Of 22 independent clones obtained, one contains a proviral insertion that is near or in the first exon of the B2mb gene. Surprisingly, the insertion is of the Ab-MuLV genome and not of replication-competent Mo-MuLV. This indicates that superinfection with Mo-MuLV "rescued" the defective Ab-MuLV genome, which then inserted into the B2mb gene. We conclude that when an allele-specific selection procedure exists, proviral insertion is a potential method for obtaining mutations in heterozygous mammalian cells. This approach may thereby provide a method for molecular cloning of such selectable genes, using a retroviral hybridization probe.     

Sequence-specific antibodies show that maturation of Moloney leukemia virus envelope polyprotein involves removal of a COOH-terminal peptide.

We followed maturation of the glycosylated envelope polyprotein Pr80env of a murine retrovirus by using antisera specific to subregions of the protein, including an antiserum directed against a synthetic peptide corresponding to the COOH-terminus of Pr80env. Shortly after synthesis and glycosylation, Pr80env is cleaved into two species, gp70 and Pr15E, that are found associated, perhaps through disulfide bonds, in infected cells. Pr15E is further cleaved at the time of virus maturation to form virus protein p15E. NH2-Terminal protein sequence analysis showed that Pr15E had an NH2 terminus in common with p15E. Pr15E, but not p15E, is precipitated by antibody against the COOH-terminal peptide; hence, p15E is missing a peptide at the COOH-terminus. Our data indicate that Pr15E is the predominant species in cells and p15E is the major species in virus.     

High prevalence of circulating antibodies to MuLV p30 antigen in human sera. An autoimmune response?

To study the possible involvement of a murine leukemia virus (MuLV) related agent in human cancer, an extensive immunoblotting analysis of human sera (cancer, autoimmune as well as control normal ones) for the presence of antibodies to MuLV structural proteins was performed. Out of 350 sera, 89 reacted with gag precursor Pr65, 72 reacted with major viral core protein p30 and five with the matrix protein p15. Antibody reactivity to the env-encoded glycoprotein gp70 was detected in 7 cases out of 16 sera tested. There were no significant differences between pathological and normal sera concerning the patterns and the frequency of the reactivity. Sera from patients with various malignancies (mainly with breast cancer) generally displayed more intensive signals to MuLV p30 than normal sera. Epitope mapping revealed that MuLV p30-reactive antibodies recognize an antigenic determinant(s) located at the carboxyterminus of the protein.     

Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor.

The mature gag and pol proteins of human immunodeficiency virus (HIV) and all retroviruses derive from large gag and gag-pol polyprotein precursors by posttranslational cleavage. A highly specific, virally encoded protease is required for this essential proteolytic processing. In this study, the HIV protease gene product was expressed in Escherichia coli and shown to autocatalyze its maturation from a larger precursor. In addition, this bacterially produced HIV protease specifically processed an HIV p55 gag polyprotein precursor when coexpressed in E. coli. This system will allow detailed structure-function analysis of the HIV protease and provides a simple assay for the development of potential therapeutic agents directed against this critical viral enzyme.     

Avian sarcoma virus gag and env gene structural protein precursors contain a common amino-terminal sequence.

The initiation site for translation of the avian sarcoma virus glycoprotein precursor, Pr63env, has been determined by analyzing the amino-terminal peptides of Pr63env and the polyprotein precursor Pr76gag encoded by the viral gag gene. The acceptor splice junction used to form the env gene mRNA has also been identified. Hybrid-selected virus-specific mRNAs were translated in vitro in the presence of either L-[35S]methionine to label at every methionine residue or L-[35S]methionine-tRNAMeti to label specifically at the amino-terminal methionine residues. Tryptic peptide maps of Pr63env labeled at every methionine residue contain all of the peptides, plus one additional peptide, present in the map of Pr57env, a nonglycosylated env-encoded polypeptide of molecular weight 57,000 immunoprecipitated from tunicamycin-treated cells. Specific amino-terminal labeling of the in vitro-synthesized polypeptides showed that the peptide missing from Pr57env corresponds to the amino-terminal tryptic peptide of Pr63env, which is removed in vivo as part of the amino-terminal signal peptide. Comparison of the amino-terminal tryptic peptides of Pr63env and Pr76gag showed that they are identical. In contrast, the chymotryptic amino-terminal peptides of Pr76gag and Pr63env are not identical. The location of the acceptor-splice junction in the env mRNA of the Prague A strain of avian sarcoma virus was determined by mung bean nuclease mapping to be at nucleotide 5,078. Fusion of the gag and env gene sequences during splicing results in use of the same AUG codon to initiate synthesis of Pr76gag and Pr63env. This sequence is contained within the 397-nucleotide 5' terminal leader that is spliced to the body of the env mRNA. The possible significance of these results for the regulation of avian sarcoma virus synthesis and translation is discussed.     

Chemical determination of the m1 Moloney sarcoma virus pP60gag gene order: evidence for unique peptides in the carboxy terminus of the polyprotein.

The gene order of the ml Moloney sarcoma virus (mlMSV) specific pP60gag (P60) was determined by direct chemical analysis of the polyprotein. P60 was cleaved with cyanogen bromide (CNBr) into eight partial and complete fragments ranging in mass from 10,000 daltons to 58,000 daltons. Peptide maps of these fragments were compared to maps of p15, p12, and three CNBr fragments of p30. The polarity of p15 and p12 in a CNBr fragment of P60 was determined by carboxypeptidase A digestion; likewise the CNBr fragments of p30 were ordered by aminopeptidase digestion. The linear arrangement of P60 CNBr fragments gave the gene order of NH2-p15-p12-p30-COOH. The m3 isolate of MSV expresses a P70 gag polyprotein. Peptide maps of 48,000-dalton CNBr fragments of m3 P70 and ml P60 were similar and suggested that both polyproteins were similar through the NH2-terminal two-thirds of p30. However, the presence of peptides unique to the 10,500-dalton COOH-terminal fragment of m1MSV p30 and not present in the p30 of either m3MSV or Moloney leukemia virus suggested that the gag gene deletion in the m1 isolate begins in the p30 reading frame.