scholarly journals Enhanced transformation by a simian virus 40 recombinant virus containing a Harvey murine sarcoma virus long terminal repeat.

1983 ◽  
Vol 3 (3) ◽  
pp. 325-339 ◽  
Author(s):  
M Kriegler ◽  
M Botchan
Keyword(s):  
Long Terminal Repeat ◽  
Recombinant Virus ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Viral Dna ◽  
Sv40 Virus ◽  
Transformed Cell ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Murine Sarcoma

We have constructed a recombinant simian virus 40 (SV40) DNA containing a copy of the Harvey murine sarcoma virus long terminal repeat (LTR). This recombinant viral DNA was converted into an infectious SV40 virus particle and subsequently infected into NIH 3T3 cells (either uninfected or previously infected with Moloney leukemia virus). We found that this hybrid virus, SVLTR1, transforms cells with 10 to 20 times the efficiency of SV40 wild type. Southern blot analysis of these transformed cell genomic DNAs revealed that simple integration of the viral DNA within the retrovirus LTR cannot account for the enhanced transformation of the recombinant virus. A restriction fragment derived from the SVLTR-1 virus which contains an intact LTR was readily identified in a majority of the transformed cell DNAs. These results suggest that the LTR fragment which contains the attachment sites and flanking sequences for the proviral DNA duplex may be insufficient by itself to facilitate correct retrovirus integration and that some other functional element of the LTR is responsible for the increased transformation potential of this virus. We have found that a complete copy of the Harvey murine sarcoma virus LTR linked to well-defined structural genes lacking their own promoters (SV40 early region, thymidine kinase, and G418 resistance) can be effectively used to promote marker gene expression. To determine which element of the LTR served to enhance the biological activity of the recombinant virus described above, we deleted DNA sequences essential for promoter activity within the LTR. SV40 virus stocks reconstructed with this mutated copy of the Harvey murine sarcoma virus LTR still transform mouse cells at an enhanced frequency. We speculate that when the LTR is placed more than 1.5 kilobases from the SV40 early promoter, the cis-acting enhancer element within the LTR can increase the ability of the SV40 promoter to effectively operate when integrated in a murine chromosome. These data are discussed in terms of the apparent cell specificity of viral enhancer elements.

Enhanced transformation by a simian virus 40 recombinant virus containing a Harvey murine sarcoma virus long terminal repeat

1983 ◽  
Vol 3 (3) ◽  
pp. 325-339
Author(s):  
M Kriegler ◽  
M Botchan
Keyword(s):  
Long Terminal Repeat ◽  
Recombinant Virus ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Viral Dna ◽  
Sv40 Virus ◽  
Transformed Cell ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Murine Sarcoma

We have constructed a recombinant simian virus 40 (SV40) DNA containing a copy of the Harvey murine sarcoma virus long terminal repeat (LTR). This recombinant viral DNA was converted into an infectious SV40 virus particle and subsequently infected into NIH 3T3 cells (either uninfected or previously infected with Moloney leukemia virus). We found that this hybrid virus, SVLTR1, transforms cells with 10 to 20 times the efficiency of SV40 wild type. Southern blot analysis of these transformed cell genomic DNAs revealed that simple integration of the viral DNA within the retrovirus LTR cannot account for the enhanced transformation of the recombinant virus. A restriction fragment derived from the SVLTR-1 virus which contains an intact LTR was readily identified in a majority of the transformed cell DNAs. These results suggest that the LTR fragment which contains the attachment sites and flanking sequences for the proviral DNA duplex may be insufficient by itself to facilitate correct retrovirus integration and that some other functional element of the LTR is responsible for the increased transformation potential of this virus. We have found that a complete copy of the Harvey murine sarcoma virus LTR linked to well-defined structural genes lacking their own promoters (SV40 early region, thymidine kinase, and G418 resistance) can be effectively used to promote marker gene expression. To determine which element of the LTR served to enhance the biological activity of the recombinant virus described above, we deleted DNA sequences essential for promoter activity within the LTR. SV40 virus stocks reconstructed with this mutated copy of the Harvey murine sarcoma virus LTR still transform mouse cells at an enhanced frequency. We speculate that when the LTR is placed more than 1.5 kilobases from the SV40 early promoter, the cis-acting enhancer element within the LTR can increase the ability of the SV40 promoter to effectively operate when integrated in a murine chromosome. These data are discussed in terms of the apparent cell specificity of viral enhancer elements.

scholarly journals Alternate utilization of two regulatory domains within the Moloney murine sarcoma virus long terminal repeat.

1985 ◽  
Vol 5 (8) ◽  
pp. 1959-1968 ◽  
Author(s):  
B J Graves ◽  
S P Eisenberg ◽  
D M Coen ◽  
S L McKnight
Keyword(s):  
Long Terminal Repeat ◽  
Transcriptional Activation ◽  
Substantial Effect ◽  
Positive Control ◽  
Terminal Repeat ◽  
Temporal Shift ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Moloney Murine Sarcoma Virus ◽  
Murine Sarcoma

The Moloney murine sarcoma virus long terminal repeat (LTR) harbors two distinct positive activators of transcription, namely, a distal signal and an enhancer. In this report we demonstrate that infection by herpes simplex virus (HSV) can markedly affect the utilization of these two Moloney murine sarcoma virus transcription signals. We investigated the HSV-mediated trans-acting effects with two goals in mind: first, to gain insight into LTR function, and second, to probe the mechanisms used by HSV to establish its own transcription cascade. In mock-infected cells, LTR-mediated expression was heavily dependent on the Moloney murine sarcoma virus enhancer but was effectively distal signal independent. HSV infection mobilized the use of the LTR distal signal and concomitantly alleviated enhancer dependence. Indeed, enhancer function may actually be inhibited by HSV trans-acting factors. These results suggest that the two positive control signals of the Moloney murine sarcoma virus LTR facilitate transcriptional activation by two different pathways. We further observed that the identity of the structural gene driven by the LRT, as well as the state of integration of a transfected template, can exert a substantial effect on the response of a template to HSV infection. According to these findings, we propose a tentative model to account for the initial temporal shift of the HSV transcriptional cascade.

Alternate utilization of two regulatory domains within the Moloney murine sarcoma virus long terminal repeat

1985 ◽  
Vol 5 (8) ◽  
pp. 1959-1968
Author(s):  
B J Graves ◽  
S P Eisenberg ◽  
D M Coen ◽  
S L McKnight
Keyword(s):  
Long Terminal Repeat ◽  
Transcriptional Activation ◽  
Substantial Effect ◽  
Positive Control ◽  
Terminal Repeat ◽  
Temporal Shift ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Moloney Murine Sarcoma Virus ◽  
Murine Sarcoma

The Moloney murine sarcoma virus long terminal repeat (LTR) harbors two distinct positive activators of transcription, namely, a distal signal and an enhancer. In this report we demonstrate that infection by herpes simplex virus (HSV) can markedly affect the utilization of these two Moloney murine sarcoma virus transcription signals. We investigated the HSV-mediated trans-acting effects with two goals in mind: first, to gain insight into LTR function, and second, to probe the mechanisms used by HSV to establish its own transcription cascade. In mock-infected cells, LTR-mediated expression was heavily dependent on the Moloney murine sarcoma virus enhancer but was effectively distal signal independent. HSV infection mobilized the use of the LTR distal signal and concomitantly alleviated enhancer dependence. Indeed, enhancer function may actually be inhibited by HSV trans-acting factors. These results suggest that the two positive control signals of the Moloney murine sarcoma virus LTR facilitate transcriptional activation by two different pathways. We further observed that the identity of the structural gene driven by the LRT, as well as the state of integration of a transfected template, can exert a substantial effect on the response of a template to HSV infection. According to these findings, we propose a tentative model to account for the initial temporal shift of the HSV transcriptional cascade.

The long terminal repeat of an endogenous intracisternal A-particle gene functions as a promoter when introduced into eucaryotic cells by transfection

1984 ◽  
Vol 4 (10) ◽  
pp. 2128-2135
Author(s):  
K K Lueders ◽  
J W Fewell ◽  
E L Kuff ◽  
T Koch
Keyword(s):  
Long Terminal Repeat ◽  
Promoter Activity ◽  
Terminal Repeat ◽  
Flanking Sequence ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Mouse Cells ◽  
Moloney Murine Sarcoma Virus ◽  
Intracisternal A Particle ◽  
Murine Sarcoma

We describe experiments designed to determine whether an endogenous intracisternal A-particle (IAP) gene randomly selected from a mouse embryo library has the potential to be transcriptionally active. Assays for IAP gene transcription were done with permanently transformed rat cells and transiently transfected monkey and mouse cells. The rat cells, which had integrated IAP gene copies, contained IAP RNA. A start site within the IAP 5' long terminal repeat (LTR) was localized by S1 mapping. The promoter activity of the IAP LTR was also measured in cells 48 h after the introduction of recombinant plasmids in which bacterial chloramphenicol acetyl transferase (CAT) encoding sequences were under the control of the LTR. The IAP LTR promoted CAT activity in mouse and monkey cells. In mouse L-cells, the levels of CAT activity were 10 to 25% of those promoted by an analogous recombinant containing the Moloney murine sarcoma virus LTR as the promoter. In contrast to the Moloney murine sarcoma virus LTR, the IAP LTR was five- to eightfold more active in monkey cells than in mouse cells. The 5' and 3' LTRs were equally active, and promoter activity was dependent on having the orientation of the LTRs with respect to the CAT gene the same as their orientation with respect to the IAP gene. A 5'-flanking sequence containing a member of the highly repetitive R-sequence family increased CAT activity in COS cells 11-fold when present along with the LTR. Our results indicate that the LTR of an endogenous mouse IAP gene can function as an efficient promoter in heterologous as well as homologous cells.

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