In vitro methylation of specific regions of the cloned Moloney sarcoma virus genome inhibits its transforming activity

The transforming activity of cloned Moloney sarcoma virus (MSV) proviral DNA was inhibited by in vitro methylation of the DNA at cytosine residues, using HpaII and HhaI methylases before transfection into NIH 3T3 cells. The inhibition of transforming activity due to HpaII methylation was reversed by treatment of the transfected cells with 5-azacytidine, a specific inhibitor of methylation. Analysis of the genomic DNA from the transformed cells which resulted from the transfection of methylated MSV DNA revealed that the integrated MSV proviral DNA was sensitive to HpaII digestion in all cell lines examined, suggesting that loss of methyl groups was necessary for transformation. When cells were infected with Moloney murine leukemia virus at various times after transfection with methylated MSV DNA, the amount of transforming virus produced indicated that the loss of methyl groups occurred within 24 h. Methylation of MSV DNA at HhaI sites was as inhibitory to transforming activity as methylation at HpaII sites. In addition, methylation at both HpaII and HhaI sites did not further reduce the transforming activity of the DNA. These results suggested that; whereas methylation of specific sites on the provirus may not be essential for inhibiting the transforming activity of MSV DNA, methylation of specific regions may be necessary. Thus, by cotransfection of plasmids containing only specific regions of the MSV provirus, it was determined that methylation of the v-mos gene was more inhibitory to transformation than methylation of the viral long terminal repeat.

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Generation of a recombinant Moloney murine leukemia virus carrying the v-src gene of avian sarcoma virus: transformation in vitro and pathogenesis in vivo.

Journal of Virology ◽

10.1128/jvi.54.3.804-816.1985 ◽

1985 ◽

Vol 54 (3) ◽

pp. 804-816 ◽

Cited By ~ 4

Author(s):

M H Feuerman ◽

B R Davis ◽

P K Pattengale ◽

H Fan

Keyword(s):

Murine Leukemia Virus ◽

Leukemia Virus ◽

Moloney Murine Leukemia Virus ◽

Src Gene ◽

Sarcoma Virus ◽

Virus Transformation ◽

Avian Sarcoma ◽

Murine Leukemia

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Highly inducible cell lines derived from mice genetically transmitting the Moloney murine leukemia virus genome.

Journal of Virology ◽

10.1128/jvi.29.3.899-906.1979 ◽

1979 ◽

Vol 29 (3) ◽

pp. 899-906 ◽

Cited By ~ 5

Author(s):

L Bacheler ◽

R Jaenisch ◽

H Fan

Keyword(s):

Cell Lines ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Virus Genome ◽

Moloney Murine Leukemia Virus ◽

Murine Leukemia

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Effect of Small Polyanions on In Vitro Assembly of Selected Members of Alpha-, Beta- and Gammaretroviruses

Viruses ◽

10.3390/v13010129 ◽

2021 ◽

Vol 13 (1) ◽

pp. 129

Author(s):

Alžběta Dostálková ◽

Barbora Vokatá ◽

Filip Kaufman ◽

Pavel Ulbrich ◽

Tomáš Ruml ◽

...

Keyword(s):

Equine Infectious Anemia Virus ◽

Electron Tomography ◽

Leukemia Virus ◽

Virus Like Particles ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Anemia Virus ◽

Immature Virus ◽

Hiv 1

The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.

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Sequential activation of the three protomers in the Moloney murine leukemia virus Env

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1617264114 ◽

2017 ◽

Vol 114 (10) ◽

pp. 2723-2728 ◽

Cited By ~ 3

Author(s):

Mathilda Sjöberg ◽

Robin Löving ◽

Birgitta Lindqvist ◽

Henrik Garoff

Keyword(s):

Murine Leukemia Virus ◽

Leukemia Virus ◽

Moloney Murine Leukemia Virus ◽

Virus Envelope ◽

Sequential Activation ◽

Virus Envelope Protein ◽

Membrane Fusion Proteins ◽

Viral Membrane Fusion ◽

Murine Leukemia

Viral membrane fusion proteins of class I are trimers in which the protomeric unit is a complex of a surface subunit (SU) and a fusion active transmembrane subunit (TM). Here we have studied how the protomeric units of Moloney murine leukemia virus envelope protein (Env) are activated in relation to each other, sequentially or simultaneously. We followed the isomerization of the SU-TM disulfide and subsequent SU release from Env with biochemical methods and found that this early activation step occurred sequentially in the three protomers, generating two asymmetric oligomer intermediates according to the scheme (SU-TM)3→ (SU-TM)2TM → (SU-TM)TM2→ TM3. This was the case both when activation was triggered in vitro by depleting stabilizing Ca2+from solubilized Env and when viral Env was receptor triggered on rat XC cells. In the latter case, the activation reaction was too fast for direct observation of the intermediates, but they could be caught by alkylation of the isomerization active thiol.

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Mutational analysis of a ras catalytic domain

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2646-2654.1986 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2646-2654

Author(s):

B M Willumsen ◽

A G Papageorge ◽

H F Kung ◽

E Bekesi ◽

T Robins ◽

...

Keyword(s):

Catalytic Domain ◽

Mutational Analysis ◽

Nucleotide Binding ◽

Membrane Localization ◽

Wide Range ◽

Murine Sarcoma Virus ◽

Sarcoma Virus ◽

Transforming Activity ◽

Type V

We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target.

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Design of a Retrovirus-Derived Vector for Expression and Transduction of Exogenous Genes in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.3.6.1123-1132.1983 ◽

1983 ◽

Vol 3 (6) ◽

pp. 1123-1132

Author(s):

Archibald S. Perkins ◽

Paul T. Kirschmeier ◽

Sebastiano Gattoni-Celli ◽

I. Bernard Weinstein

Keyword(s):

Mammalian Cells ◽

High Efficiency ◽

Leukemia Virus ◽

Restriction Enzymes ◽

Virus Genome ◽

Opposite Polarity ◽

High Yield ◽

Animal Cells ◽

Murine Sarcoma Virus ◽

Sarcoma Virus

We have developed a transfection vector for animal cells that contains long terminal repeat (LTR) sequences to promote expression. Plasmid p101/101, a derivative of plasmid pBR322 containing the complete Moloney murine sarcoma virus genome, was cut with restriction enzymes and religated so that both the 5′ and 3′ LTRs were retained and all but about 700 base pairs of the intervening viral sequences were removed. To test this vector, the Escherichia coli gene gpt was cloned into a unique Pst I site, between the two LTRs, with guanine and cytosine tailing, a method that can be generalized for insertion of any DNA segment into this vector. When DNA from recombinant plasmids in which the gpt gene was inserted in the same transcriptional polarity as the LTR sequences was transfected onto murine or rat fibroblast cultures, we obtained a high yield of Gpt + colonies. However, plasmid constructs with the gpt gene in the opposite polarity were virtually devoid of activity. With gpt in the proper orientation, restriction enzyme cuts within the LTRs or between the 5′ LTR and the gpt gene reduced transfection by more than 98%, whereas a cut between the gpt gene and the 3′ LTR gave an 80% reduction in activity. Thus, both 5′ and 3′ LTR sequences are essential for optimal gpt expression, although the 5′ LTR appears to play a more important role. When the LTR- gpt plasmid was transfected onto murine leukemia virus-infected mouse fibroblasts, we obtained evidence that RNA copies became pseudotyped into viral particles which could transfer the Gpt + phenotype into rodent cells with extremely high efficiency. This vector should prove useful for high-efficiency transduction of a variety of genes in mammalian cells.

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Retrovirus-mediated gene transfer of human adenosine deaminase: expression of functional enzyme in murine hematopoietic stem cells in vivo

Molecular and Cellular Biology ◽

10.1128/mcb.7.10.3459-3465.1987 ◽

1987 ◽

Vol 7 (10) ◽

pp. 3459-3465

Author(s):

B Lim ◽

D A Williams ◽

S H Orkin

Keyword(s):

Adenosine Deaminase ◽

Leukemia Virus ◽

Hematopoietic Cells ◽

Phosphoglycerate Kinase ◽

Moloney Murine Leukemia Virus ◽

Systematic Evaluation ◽

Hematopoietic Stem ◽

Specific Sequences

Simplified Moloney murine leukemia virus-based recombinant retrovirus vectors have been constructed which transduce human adenosine deaminase (ADA) cDNA. ADA transcription is under the control of the constitutive promoter for the human X chromosome phosphoglycerate kinase (pgk) gene. In these simplified vectors, dominant selectable markers are not included and selection is dependent on overproduction of functional ADA enzyme. Primary murine hematopoietic cells were infected with helper-free recombinant ADA virus generated from Psi-2 packaging cells. Protein analysis revealed that human ADA enzyme was expressed in progenitor-derived hematopoietic colonies in vitro and CFU-S-derived spleen colonies in vivo. Enzyme expression was dependent on transcription from the pgk promoter. ADA expression in primary murine hematopoietic cells directed by the internal promoter was not adversely affected by the presence of the Moloney virus long terminal repeat enhancer sequence. Use of these vectors allows systematic evaluation of the effects of specific sequences in recombinant retrovirus vectors on expression in primary murine hematopoietic cells in vivo.

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