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M L Linial

Publications and source records attributed to M L Linial.

45 records · Page 3Linked to original sources
PubMed1993

A model system for nonhomologous recombination between retroviral and cellular RNA.

A current model for the generation of transforming retroviruses proposes that read-through RNAs, containing both viral and cellular sequences, are copackaged with viral genomic RNA. It is, however, possible that a cellular mRNA is occasionally encapsidated into a retroviral particle, even though viral packaging sequences are absent. We have generated recombinant proviruses following copackaging of an avian leukosis viral genomic RNA and a neo-containing RNA completely devoid of retroviral sequences. In these studies, we used the packaging cell line SE21Q1b, which has the unique ability to randomly package cellular mRNA into retroviral particles. We describe 10 recombinants obtained following copackaging of nonhomologous RNAs. Our data show that recombination is not occurring at the DNA level in the parental SE21Q1b cells but is occurring at the RNA level, during reverse transcription. These data further suggest that reverse transcriptase can preferentially jump between templates at short stretches of homology in otherwise unrelated RNAs. We conclude that retroviral sequences are not required for packaged mRNA to be reverse transcribed and to be included in integrated proviruses.

Animals↗
PubMed1993

Overproduction of v-Myc in the nucleus and its excess over Max are not required for avian fibroblast transformation.

The cellular proto-oncogene c-myc can acquire transforming potential by a number of different means, including retroviral transduction. The transduced allele generally contains point mutations relative to c-myc and is overexpressed in infected cells, usually as a v-Gag-Myc fusion protein. Upon synthesis, v-Gag-Myc enters the nucleus, forms complexes with its heterodimeric partner Max, and in this complex binds to DNA in a sequence-specific manner. To delineate the role for each of these events in fibroblast transformation, we introduced several mutations into the myc gene of the avian retrovirus MC29. We observed that Gag-Myc with a mutated nuclear localization signal is confined predominantly in the cytoplasm and only about 5% of the protein could be detected in the nucleus (less than the amount of endogenous c-Myc). Consequently, only a small fraction of Max is associated with Myc. However, cells infected with this mutant exhibit a completely transformed phenotype in vitro, suggesting that production of enough v-Gag-Myc to tie up all cellular Max is not needed for transformation. While the nuclear localization signal is dispensable for transformation, minimal changes in the v-Gag-Myc DNA-binding domain completely abolish its transforming potential, consistent with a role of Myc as a transcriptional regulator. One of its potential targets might be the endogenous c-myc, which is repressed in wild-type MC29-infected cells. Our experiments with MC29 mutants demonstrate that c-myc down-regulation depends on the integrity of the v-Myc DNA-binding domain and occurs at the RNA level. Hence, it is conceivable that v-Gag-Myc, either directly or circuitously, regulates c-myc transcription.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and characterization of the RNA packaging-defective retrovirus SE21Q1b.

The nonconditional RNA packaging mutant SE21Q1b contains cis- and trans-acting defects which cause cellular mRNA, rather than viral genomic RNA, to be nonspecifically packaged into SE21Q1b viral particles. Using genomic libraries of the c-SE21Q1b quail cell line, we have been able to construct a molecular clone of the SE21Q1b provirus. Upon transfection into primary quail embryo fibroblasts, the SE21Q1b molecular clone is able to recapitulate the nonspecific RNA packaging phenotype of the c-SE21Q1b cell line. The RNA packaging phenotypes displayed by several SE21Q1b/avian sarcoma-leukemia virus hybrid provirus constructs have further indicated that sequences responsible for the altered RNA packaging phenotype of SE21Q1b are localized in the left third of the SE21Q1b proviral genome. DNA sequence analysis of this region has revealed that the 5' SE21Q1b deletion has removed 179 bp from the SE21Q1b left long terminal repeat and leader regions. Several differences were detected at the carboxyl terminus of the deduced SE21Q1b nucleocapsid protein sequence in comparison with that of Rous sarcoma virus PR-C. Results of site-directed oligonucleotide mutagenesis experiments indicate, however, that the presence of these residues in the nucleocapsid protein alone is not responsible for the decreased RNA packaging specificity of SE21Q1b.

Amino Acid Sequence↗
PubMed1992

gag as well as myc sequences contribute to the transforming phenotype of the avian retrovirus FH3.

The avian retrovirus FH3, like MC29 and CMII, encodes a Gag-Myc fusion protein. However, the FH3-encoded protein is larger, about 145 kDa, and contains almost the entire retroviral gag gene. In contrast to the other gag-myc avian retroviruses, FH3 fails to transform fibroblasts in vitro, although macrophages are transformed both in vitro and in vivo (C. Chen, B. J. Biegalke, R. N. Eisenman, and M. L. Linial, J. Virol. 63:5092-5100, 1989). We have used the polymerase chain reaction technique to obtain a molecular clone of FH3. Sequence analysis of the FH3 myc oncogene revealed a single proline----histidine change (position 223) relative to c-myc. However, substitution of the FH3 myc sequence with the chicken c-myc sequence did not alter the transformation potential of the virus. Hence, overexpression of the proto-oncogene as a Gag-Myc retroviral protein is sufficient for macrophage, but not fibroblast, transformation. After passage of FH3 in fibroblast cultures, a virus (FH3L) that is capable of rapidly transforming fibroblasts appears. The Gag-Myc protein encoded by FH3L is smaller (ca. 130 kDa) than that encoded by the original viral stock (FH3E). Sequencing of an FH3L molecular clone revealed a 212-amino-acid deletion within the Gag portion. Using FH3E/FH3L recombinants, we have demonstrated that the ability of encoded viruses to transform fibroblasts directly correlates with the presence of this deletion. Moreover, the addition of the Gag sequence deleted from FH3L to the MC29 oncoprotein significantly reduces its transforming activity as measured by focus assay. These data suggest that the C-terminal segment of Gag attenuates the oncogenic potential of Gag-Myc fusion proteins.

Amino Acid Sequence↗
PubMed1990

Unusual features of integrated cDNAs generated by infection with genome-free retroviruses.

We previously demonstrated that when nonretroviral RNAs are encapsidated in retroviral particles they can be reverse transcribed into cDNAs, which are then integrated into the cellular genome. This transfer of genetic information via retroviral infection has been designated retrofection. Further analyses of three genes transferred in this manner (retrogenes) revealed that each was present in a single copy at a different site in the recipient quail cell genome and included a transcriptional promoter encoded by the encapsidated neo RNA. A unique feature of the retrogenes was a common 16-nucleotide sequence at or near a recombination border, which was not present in either recombination partner. The existence of this sequence suggests a common mechanism of retrogene formation and/or integration mediated by retrofection.

Animals↗
PubMed1989

FH3, a v-myc avian retrovirus with limited transforming ability.

We have isolated a new acute avian transforming virus which contains the oncogene myc. This virus, designated FH3, was isolated after injection of a 10-day-old chick embryo with avian leukosis virus. While FH3 shares many properties with other v-myc-containing avian retroviruses, it also has several unique properties. The primary target for transformation in vitro is chicken macrophages; infection of chicken fibroblasts does not lead to complete morphological transformation. FH3 also exhibits a limited host range, in that Japanese quail macrophages and fibroblasts are infected but are not completely transformed. FH3 induces in vivo a limited tumor type if injected into 10-day-old chick embryos; only a cranial myelocytoma, which does not appear to be metastatic, can be detected. The v-myc gene of FH3 is expressed predominantly as a P145 Gag-Myc protein which is encoded by a ca. 8-kilobase genomic RNA. This FH3-encoded polyprotein is localized in the nucleus of all infected cells, whether or not they are transformed.

Animals↗
PubMed1989

Tissue-specific lability and expression of avian leukosis virus long terminal repeat enhancer-binding proteins.

Avian leukosis virus (ALV) induces bursal lymphomas in chickens, after proviral integration next to the cellular myc proto-oncogene, and subsequent c-myc hyperexpression. Our previous work suggested that labile or short-lived cellular proteins interact with the viral long terminal repeat (LTR) enhancer, and binding of these proteins appeared to be essential for high rates of LTR-enhanced transcription (A. Ruddell, M. Linial, W. Schubach, and M. Groudine, J. Virol. 62:2728-2735, 1988). This lability is specific for B-lymphoid cell types, since T cells and fibroblasts show stable high rates of LTR-enhanced transcription and stable LTR-binding activity. Moreover, the lability of these proteins may be important in determining susceptibility to bursal lymphoma. In this study, we separated and characterized the labile and stable LTR-binding proteins and examined their lability and expression in different cell types. Gel shift and DNase I footprinting analyses indicated that at least five proteins interact with the 140-base-pair LTR enhancer region. These proteins were distinct by several criteria, including lability or stability after inhibition of protein synthesis, resistance to heat denaturation, chromatographic behavior, and expression in different cell types. Two binding proteins were present in many cell types and were specifically labile in B cells. A third binding protein showed hematopoietic-cell-type-specific expression and was also labile in B cells. These findings indicate that there is tissue-specific modulation of the lability and expression of ALV LTR-binding proteins, which may be important for regulation of LTR transcription enhancement and ALV bursal lymphomagenesis.

Animals↗