gag-mos Polyproteins encoded by variants of the Moloney strain of mouse sarcoma virus

Stanker, Larry H.; Horn, Jacqueline Peltier; Gallick, Gary E.; Kloetzer, William S.; Murphy, Edwin C.; Blair, Donald G.; Arlinghaus, Ralph B.

doi:10.1016/0042-6822(83)90483-x

Cited by 20 publications

(29 citation statements)

References 24 publications

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“…It is clear, however, that P85gag-ms is phosphorylated in vivo and that this phosphorylation occurs at both serine and threonine residues (19). The in vitro phosphorylated P85gag-mos also contains phosphoserine and phosphothreonine.…”

Section: Discussionmentioning

confidence: 99%

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P85 ^gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity

Kloetzer

Maxwell

Arlinghaus

1983

Proc. Natl. Acad. Sci. U.S.A.

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A protein identified as P859'9-°was shown to be phosphorylated when immunoprecipitates from tsllO Moloney murine sarcoma virus transformed nonproducer cells (clone 6m2) were incubated with [y-32P]ATP. The in vitro-labeled 85,000-dalton phosphoprotein comigrated on NaDodSO4/polyacrylamide gels with authentic phosphorylated P859'9-°. Immunoprecipitates obtained with antisera prepared against Rauscher murine leukemia virus core protein p30 were active in the immune complex kinase assay but anti-murine leukemia virus plO precipitates were not. Previous studies have shown that anti-p30 but not antiplO antisera recognize P859'9-°. The 6m2 clone has been shown to express P85gag-"w8 at 330C but not at 39TC. Anti-p30 immune complexes from 6m2 cells maintained at 390C failed to phosphorylate the 85,000-dalton protein. Furthermore, the in vitro phosphorylated 85,000-dalton protein gave the same pattern of V8 protease-generated cleavage products as in vivo 32P-labeled P85gag-'.8, We conclude from these results that P85gag`m06 is phosphorylated in anti-p30 immune complex kinase reactions. Phosphoamino acid analyses indicated that the in vitro phosphorylated P85gag-tno contained phosphoserine and phosphothreonine. Our findings indicate that incubation of anti-p30 immunoprecipitates at 39°C drastically reduced, in a specific way, the kinase activity associated with P85gag-1l8. This result and other data suggest that the kinase is virus-encoded. Because P85gag-"1w8, but not Pr65gag, is phosphorylated in anti-p30 immunoprecipitates from MuLVMuSV tsllO producer cells, the kinase enzyme is associated with P85gag-' and not gag gene products. A second major polypeptide of the size of P58gag was also phosphorylated in anti-p30 immunoprecipitates from cells maintained at 33°C but not at 39°C. Since 6m2 cells at 39°C contain P58wag, this is also consistent with the kinase activity being associated with P85gag-11W.

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Section: Discussionmentioning

confidence: 99%

“…The first is that P85gag-m's is phosphorylated in vivo at serine and threonine residues (19 (20). Cell extracts (0.5 ml) were incubated with 10 ,ul of absorbed antisera for 2 hr at 0°C.…”

mentioning

confidence: 99%

P85 ^gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity

Kloetzer

Maxwell

Arlinghaus

1983

Proc. Natl. Acad. Sci. U.S.A.

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“…Thus, the 3.8-kilobase (kb) MuSVtsllO transcript can be translated into a truncated gag gene precursor, P58gag, only (11). (In a number of previous studies [5,8,11,22,23,32], the MuSVtsllO RNAs were estimated to be 4.0 and 3.5 kb in size. Precise definition of the deletion boundaries and splice sites [23] indicates that the unspliced and spliced MuSVtsllO RNAs are actually 3.8 and 3.3 kb in size, respectively.)…”

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confidence: 99%

“…In 6m2 cultures, revertants to the transformed state arise spontaneously at 39°C (2, 3). Two revertant cell lines, designated 54-5A4 and 204-3, have been studied (3,32). We recently established that there is a 5-base deletion (-AGTGT-) at the usual 3' splice site in the MuSVtsllO viral DNA in 54-5A4 cells (5).…”

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confidence: 99%

“…We recently established that there is a 5-base deletion (-AGTGT-) at the usual 3' splice site in the MuSVtsllO viral DNA in 54-5A4 cells (5). This deletion prevents splicing at this site but aligns the gag and mos genes into a continuous open reading frame, allowing the translation of transforming PlO0gag-mos from the unspliced RNA at any growth temperature (5,32 investigate the effect that loss of the AG dinucleotide might have on splicing reactions in vivo other than accurate exon ligation at the usual 3' splice site, we examined 5'-splice-site cleavage of viral RNA in 54-5A4 cells grown at various temperatures. We found that 5'-splice-site cleavage could be observed, but only at growth temperatures that normally would be permissive for MuSVtsllO splicing (i.e., 33°C and lower) (Fig.…”

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Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo.

Cizdziel¹,

Mars²,

Murphy³

1988

Mol. Cell. Biol.

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The spliced form of MuSVtsllO viral RNA is approximately 20-fold more abundant at growth temperatures of 33°C or lower than at 37 to 41°C. This difference is due to changes in the efficiency of MuSVtsllO RNA splicing rather than selective thermolability of the spliced species at 37 to 41°C or general thermosensitivity of RNA splicing in MuSVtsllO-infected cells. Moreover, RNA transcribed from MuSVtsllO DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVtsllOinfected cells (6m2 cells) at 39°C. However, after a short (about 30-min) lag following a shift to 33°C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrentiy. Splicing of viral RNA in the MuSVtsllO revertant 54-5A4, which lacks the sequence -AG/TGT-at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA. Increased splicing at this site in 54-5A4 viral RNA is probably driven by the unavailabilit of the usual 3' splice site for exon ligation.

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Temperature‐dependent transmembrane potential changes in cells infected with a temperature‐sensitive moloney sarcoma virus

Lai¹,

Gallick²,

Arlinghaus³

et al. 1984

Journal Cellular Physiology

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Normal rat kidney cells (NRK) infected with the temperature-sensitive (ts) transformation mutant of Moloney murine sarcoma virus yielded a clone of cells, 6m2, that exhibited a transformed morphology at 33 degrees C and a normal morphology at 39 degrees C. Transmembrane potential (Em) was measured fluorometrically using a cyanine dye diS-C3-(5). Fluorescence was inversely correlated with Em. Cells at 33 degrees C had lower Em. Em changes were recorded within 15 minutes of temperature shift from 33 degrees C to 39 degrees C in both directions, increasing in the 33 degrees C to 39 degrees C direction and decreasing in the 39 degrees C to 33 degrees C direction. Uninfected NRK cells when shifted under the same condition exhibited small fluorescence changes in the 33 degrees C to 39 degrees C direction. Shifting from 39 degrees C to 33 degrees C resulted in Em changes similar to those in 6m2 cells. Also studied was a cell line infected with a spontaneous revertant of the ts mutant, designated 54-5A4; it was transformed at both temperatures. Shifting from 33 degrees C to 39 degrees C in both directions yielded small changes. Transmembrane potential changes in 6m2 cells precede other transformation-specific changes that occur after a temperature shift.

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gag-mos Polyproteins encoded by variants of the Moloney strain of mouse sarcoma virus

Cited by 20 publications

References 24 publications

P85 ^gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity

P85 ^gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity

Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo.

Temperature‐dependent transmembrane potential changes in cells infected with a temperature‐sensitive moloney sarcoma virus

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gag-mos Polyproteins encoded by variants of the Moloney strain of mouse sarcoma virus

Cited by 20 publications

References 24 publications

P85 gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity

P85 gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity

Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo.

Temperature‐dependent transmembrane potential changes in cells infected with a temperature‐sensitive moloney sarcoma virus

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P85 ^gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity

P85 ^gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity