Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

Bosselman, R A; Hsu, Rou-Yin; Bruszewski, Joan; Hu, Sylvia; Martin, Francis H.; Nicolson, Margery

doi:10.1128/mcb.7.5.1797

Cited by 60 publications

(24 citation statements)

References 55 publications

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“…However, RCV does not appear to be an issue with the split structural protein gene PCL. Modification of the Sindbis virus PCL into a split structural protein gene configuration somewhat paralleled the evolution of retrovirus vector PCLs, whereby the required gene products were expressed from individual cassettes (31)(32)(33). In the case of alphavirus vector PCL, expression of the capsid protein and envelope glycoproteins from distinct cassettes rather than as the native polyprotein reduced the level of contaminating RCV to below the limit of detection.…”

Section: Discussionmentioning

confidence: 99%

Stable alphavirus packaging cell lines for Sindbis virus- and Semliki Forest virus-derived vectors

Polo¹,

Belli²,

Driver³

et al. 1999

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

134

109

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Alphavirus vectors are being developed for possible human vaccine and gene therapy applications. We have sought to advance this field by devising DNA-based vectors and approaches for the production of recombinant vector particles. In this work, we generated a panel of alphavirus vector packaging cell lines (PCLs). These cell lines were stably transformed with expression cassettes that constitutively produced RNA transcripts encoding the Sindbis virus structural proteins under the regulation of their native subgenomic RNA promoter. As such, translation of the structural proteins was highly inducible and was detected only after synthesis of an authentic subgenomic mRNA by the vectorencoded replicase proteins. Efficient production of biologically active vector particles occurred after introduction of Sindbis virus vectors into the PCLs. In one configuration, the capsid and envelope glycoproteins were separated into distinct cassettes, resulting in vector packaging levels of 10 7 infectious units͞ml, but reducing the generation of contaminating replication-competent virus below the limit of detection. Vector particle seed stocks could be amplified after low multiplicity of infection of PCLs, again without generating replicationcompetent virus, suggesting utility for production of largescale vector preparations. Furthermore, both Sindbis virusbased and Semliki Forest virus-based vectors could be packaged with similar efficiency, indicating the possibility of developing a single PCL for use with multiple alphavirusderived vectors.The use of virus-derived expression vectors for gene therapy and vaccine applications increasingly is being pursued, with a number of diverse virus types and approaches. Alphaviruses are attractive candidates for such applications because of their high levels of replication and gene expression, their ability to infect a variety of diverse cell types, and the ability to manipulate cDNA clones from which infectious viral RNA may be transcribed (for review, see refs. 1 and 2). The alphavirus genome is a single-stranded, positive-sense RNA of approximately 11.7 kb and is encapsidated within an icosahedral capsid protein shell (for review, see ref.3). Nucleocapsids, in turn, are surrounded by a host-derived lipid envelope from which the viral spike glycoproteins E1 and E2 protrude. Cytoplasmic replication of the RNA genome is mediated by four viral-encoded nonstructural proteins and proceeds through a full-length negative-sense intermediate. Subsequent positive-strand RNA synthesis results in both progeny genome RNA and an abundant, internally initiated subgenomic mRNA. The virus structural proteins are translated from the subgenomic mRNA as a polyprotein that is processed into the individual components of the virion.The general strategy for construction of alphavirus-based expression vectors has been to substitute the viral structural protein genes with a heterologous gene, maintaining transcriptional control via the highly active subgenomic RNA promoter (1, 2, 4). As such, these vector r...

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Section: Discussionmentioning

confidence: 99%

Stable alphavirus packaging cell lines for Sindbis virus- and Semliki Forest virus-derived vectors

Polo¹,

Belli²,

Driver³

et al. 1999

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

134

109

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“…Since, for most gene transfer applications, the generation of pure stocks of recombinant virus free of replication-competent helper virus is desirable, there has been considerable interest in the development of cell lines that produce the necessary viral gene products for encapsidation yet do not themselves yield detectable helper virus or transfer of viral genes (1)(2)(3)(4)(5)(6). In the first generation of such "helper-free" packaging cell lines, expression of the necessary viral proteins was achieved through the stable introduction of a mutant Moloney murine leukemia virus (Mo-MuLV) proviral genome containing a 350-base-pair (bp) deletion of the 4i sequence (2), a sequence required for efficient encapsidation of the Mo-MuLV genome.…”

mentioning

confidence: 99%

“…5 and see below). In addition, in a minority of cases, the encapsidation of the 4i-genome appears to lead to the generation of wild-type virus through recombinational events involving a copackaged recombinant genome carrying the q sequence (4)(5)(6)(7)(8)(9).…”

mentioning

confidence: 99%

Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges.

Danos

Mulligan

1988

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

824

386

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We have constructed a set of packaging cell lines useful for the generation of helper-free recombinant retroviruses with amphotropic and ecotropic host ranges. Most often, the initial step in the generation of recombinant retrovirus for mammalian gene transfer studies is the introduction of a suitable proviral DNA vector into fibroblastic cells that produce the necessary viral proteins for encapsidation ofthe desired recombinant RNA.

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“…[22][23][24][25] The rate of RCR generation has been decreased by minimizing sequence homology between vector and packaging cell sequences and the creation of split packaging cell lines that segregate gag-pol and env genes onto separate plasmids. [26][27][28][29][30][31] Split packaging cell lines have successfully decreased the frequency of RCR generation from these packaging plasmids. However, a variety of novel RCRs have been generated which contain endogenous retroviral sequences.…”

Section: Testing For Replication-competent Retrovirusmentioning

confidence: 99%

Retroviral vector production in the National Gene Vector Laboratory at Indiana University

2005

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Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

Cited by 60 publications

References 55 publications

Stable alphavirus packaging cell lines for Sindbis virus- and Semliki Forest virus-derived vectors

Stable alphavirus packaging cell lines for Sindbis virus- and Semliki Forest virus-derived vectors

Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges.

Retroviral vector production in the National Gene Vector Laboratory at Indiana University

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