In vitro synthesis of a 9 kbp terminally redundant DNA carrying the infectivity of moloney murine leukemia virus

Gilboa, Eli; Goff, Stephen P.; Shields, Anthony F.; Yoshimura, Fayth K.; Mitra, Sudha W.; Baltimore, David

doi:10.1016/0092-8674(79)90101-6

Cited by 185 publications

(143 citation statements)

References 20 publications

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“…To overcome these problems we have developed an alternative strategy for cloning retrovirus-transmitted VL30 genes. This exploits the ability of detergentdisrupted retrovirus particles to synthesize abundant quantities of retroviral DNA [12,13]. In this report we show that VL30 DNA can also be synthesized in such endogenous reactions.…”

Section: Introductionmentioning

confidence: 90%

A novel approach to cloning transcriptionally active retrovirus-like genetic elements from mouse cells

Carter¹,

Norton²,

Avery

1983

Nucl Acids Res

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A family of dispersed, moderately repeated mouse genetic elements is expressed as retrovirus-like 30S RNA species (VL30 RNA) which can be transmitted to other cells when packaged as a pseudovirion complex by murine leukemia viruses (MuLV). Using the endogenous reverse transcriptase reaction of VL30 RNA-containing MuLV particles, full-length VL30 DNA was synthesized and cloned in pAT153. Analysis of a number of clones identified long terminal repeat structures (LTRs) characteristic of retrovirus proviruses and transposable genetic elements. Whilst the unique region of all clones was identical, the LTRs displayed some heterogeneity. Comparison of the unique region of cloned VL30 DNA with mouse genomic VL30 sequences showed the retrovirus-derived clones to be encoded by only a few members of the divergent VL30 gene family. These findings thus demonstrate a method for cloning a defined sub-class of retrovirus-like cellular genes which are both transcriptionally active and transmissible by a retrovirus.

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Section: Introductionmentioning

confidence: 90%

A novel approach to cloning transcriptionally active retrovirus-like genetic elements from mouse cells

Carter¹,

Norton²,

Avery

1983

Nucl Acids Res

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“…This in vitro reaction, however, does not yield closed circular viral DNA with two LTR copies. Most importantly, the infectivity of viral DNA made in vitro is several orders of magnitude lower than that of viral DNA made early during infection (10,23,28). This suggests that cell factors are required during the reverse transcription process.…”

mentioning

confidence: 88%

“…Several of these steps seem to be carried out by reverse transcriptase alone or possibly in combination with other virion proteins. Indeed, infectious double-stranded linear viral DNA, as well as circular viral DNA with one LTR copy can be synthesized in vitro by purified virions (1,2,5,6,10). This in vitro reaction, however, does not yield closed circular viral DNA with two LTR copies.…”

mentioning

confidence: 99%

An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

Richter¹,

Ozer²,

DesGroseillers³

et al. 1984

Mol. Cell. Biol.

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To identify specific cellular factors which could be required during the synthesis of retroviral DNA, we have studied the replication of murine leukemia virus in mouse cells temperature sensitive for cell DNA synthesis (M. L. Slater and H. L. Ozer, Cell 7:289-295, 1976) and in several of their revertants. This mutation has previously been mapped on the X chromosome. We found that a short incubation of mutant cells at a nonpermissive temperature (39°C) during the early part of the virus cycle (between 0-to 20-h postinfection) greatly inhibited virus production. This The replication of the RNA genome of retroviruses by reverse transcriptase is a complex process. Linear doublestranded DNA molecules with segmented (+) strand are first synthesized with a redundant sequence at each end, the long terminal repeats (LTR) (for review, see reference 36). LTR are composed of a sequence derived from the 3' end and another one from the 5' end of the RNA genome. The linear DNA molecules mature as nonsegmented, fully doublestranded molecules and appear to be converted in the nucleus into two species of covalently closed circular viral DNAs (20, 32), one shorter species harboring one LTR copy and the larger species harboring two LTR copies (31, 42). Several of these steps seem to be carried out by reverse transcriptase alone or possibly in combination with other virion proteins. Indeed, infectious double-stranded linear viral DNA, as well as circular viral DNA with one LTR copy can be synthesized in vitro by purified virions (1,2,5,6,10). This in vitro reaction, however, does not yield closed circular viral DNA with two LTR copies. Most importantly, the infectivity of viral DNA made in vitro is several orders of magnitude lower than that of viral DNA made early during infection (10,23,28). This suggests that cell factors are required during the reverse transcription process. Other experiments also indicate that the cell provides essential * Corresponding author.factors during some steps of reverse transcription. Serum starvation has been shown to markedly affect the production of unintegrated viral DNA in newly infected avian (7,8,37) or murine (14) cells. Cycloheximide added early after infection prevented the formation of both species of supercoiled viral DNA, suggesting that newly synthesized proteins are required to complete this step (41). The treatment of newly infected murine (4, 13) or avian (16) cells with aphidicolin, an inhibitor of cellular DNA polymerase a (17) and 5 (24), also prevented the formation of supercoiled viral DNA, suggesting that cellular DNA polymerases may be involved at this step of the virus cycle.Although very suggestive, these experiments do not permit one to distinguish between a number of factors possibly affected by the inhibitors. Also, it is not clear whether the effect of these treatments is directly on retroviral DNA synthesis or only indirectly influencing it. It would therefore be helpful to identify the cellular factors required in the reverse transcription process by using cell mutan...

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“…Gilboa et al 4 worked out many of the mechanisms of reverse transcription several years ago. Some details, however, are just emerging.…”

Section: Using Retroviruses To Study Hiv Signalingmentioning

confidence: 99%

Viruses as gene delivery vectors: Application to gene function, target validation, and assay development

Lotze

Kost

2002

Cancer Gene Ther

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as part of an ongoing series designed to highlight and review areas important to modern drug development ( Figure 1 ). Briefly introduced by Tom Kost ( GlaxoSmithKline ) and Michael Lotze ( University of Pittsburgh ), the focus was on the intersection of genomics, proteomics, and now ''viromics.'' The latter term refers to the use of viruses and viral gene transfer to explore the complexity arising from the vast array of new targets available from the human and murine genomes. Indeed, access to large numbers of genes using viral vectors is a key tool for drug discovery and drug delivery. With 38,000 genes identified within the human genome, only 5000 are considered readily druggable. Generating tools such as these to validate targets represents a major part of the armamentarium of the postgenomic scientist. During the last 12 years alone, there have been over 26,000 publications on virus vectors. Many of them have been found useful in target validation, assay development, and evaluation in in vivo models and gene therapy. Thus, there is now an extensive knowledge base for several viral vectors, with unique attributes within each of them providing versatility, efficiency, and ease of use. The individual scientists presenting at the meeting illustrated many of the unique and useful characteristics of such vector systems including retrovirus, adenovirus, herpes virus, simbis virus, and baculovirus.

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In vitro synthesis of a 9 kbp terminally redundant DNA carrying the infectivity of moloney murine leukemia virus

Cited by 185 publications

References 20 publications

A novel approach to cloning transcriptionally active retrovirus-like genetic elements from mouse cells

A novel approach to cloning transcriptionally active retrovirus-like genetic elements from mouse cells

An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

Viruses as gene delivery vectors: Application to gene function, target validation, and assay development

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