Genetically Stable Picornavirus Expression Vectors with Recombinant Internal Ribosomal Entry Sites

Dufresne, Andrew T.; Dobrikova, Elena Y.; Schmidt, S.; Gromeier, Matthias

doi:10.1128/jvi.76.17.8966-8972.2002

Cited by 11 publications

(9 citation statements)

References 27 publications

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“…Deletion of SLD VI from type 1 IRESes has been shown to yield viable viruses with reduced neurovirulence (14,26,31,43). In our constructs a highly conserved cryptic AUG codon upstream of the authentic initiating codon was placed into optimal Kozak context to allow translation in the absence of SLD VI (10,12) (Fig. 3A).…”

Section: Rps Is More Attenuated Than Pv1/s(ires) In Vitromentioning

confidence: 99%

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Genetic Determinants of Cell Type-Specific Poliovirus Propagation in HEK 293 Cells

Campbell

Lin

Dobrikova

et al. 2005

J Virol

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The ability of poliovirus to propagate in neuronal cells can be reduced by introducing appropriate nucleotide substitutions into the viral genome. Specific mutations scattered throughout the poliovirus genome yielded the live attenuated vaccine strains of poliovirus. Neuron-specific propagation deficits of the Sabin strains are partially encrypted within a confined region of the internal ribosomal entry site (IRES), which carries attenuating point mutations in all three serotypes. Recently, high levels of neurovirulence attenuation were achieved with genetically engineered polioviruses containing heterologous IRES elements. This is exemplified with poliovirus recombinants replicating under control of a human rhinovirus type 2 (HRV2) IRES element. We have carried out experiments delineating the genetic basis for neuronal IRES function. Neuronal dysfunction of the HRV2 IRES is determined mainly by IRES stem-loop domain V, the locus for attenuating point mutations within the Sabin strains. Neuronal incompetence associated with HRV2 IRES domain V is substantially more pronounced than that observed with the attenuating IRES point mutation of the Sabin serotype 1 vaccine strain. Mix-and-match recombination of polio and HRV2 IRES domain V suggests that the attenuation phenotype correlates with overall structural features rather than primary sequence. Our experiments have identified HEK 293 cells as a novel system for the study of neuron-specific replication phenotypes of poliovirus. This cell line, originally derived from embryonic human kidney, has recently been described to display neuronal characteristics. We report propagation properties in HEK 293 cells for poliovirus recombinants with attenuated neurovirulence in experimental animals that corroborate this observation.Poliovirus (PV), a positive-strand RNA virus of approximately 7,500 nucleotides (nt), is the prototypic member of the genus Enterovirus in the family Picornaviridae. PV is the causative agent of poliomyelitis, a paralytic condition complicating ϳ1% of all PV infections. The incidence of poliomyelitis has been drastically reduced since the introduction of two effective vaccines-the live attenuated (Sabin) strains and the formalininactivated (Salk) vaccine. Serial passage of wild-type (wt) PV in various culture systems yielded the serotype 1 and 3 Sabin strains, while serotype 2 is a naturally occurring variant with inherently reduced neurovirulence (48).It is generally assumed that the excellent safety record of the Sabin strains (the rate of vaccine-associated paralytic poliomyelitis [VAPP] is estimated at 0.14 per 1,000,000 doses) (60) is a direct reflection of tissue type-specific deficiencies that affect their propagation in the human central nervous system (CNS). Nevertheless, despite over 40 years of successful application, the molecular basis for neuroattenuation of the Sabin strains is not completely understood (16).Elucidation of the genomic sequences of PV type 1 (Sabin) [PV1(S)] (38) and PV3(S) (53, 58) and comparison to their wt progenitor...

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Section: Rps Is More Attenuated Than Pv1/s(ires) In Vitromentioning

confidence: 99%

“…3A). Deletion of IRES SLD VI yields a viable virus with slightly reduced viral replication and gene expression in HeLa cells (10,12). We constructed PV1/M(␦6), PV1/S(␦6), and RPS(␦6) based on the parents PV1/M(IRES), PV1/ S(IRES), and RPS (Fig.…”

Section: Rps Is More Attenuated Than Pv1/s(ires) In Vitromentioning

confidence: 99%

Genetic Determinants of Cell Type-Specific Poliovirus Propagation in HEK 293 Cells

Campbell

Lin

Dobrikova

et al. 2005

J Virol

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“…The potential for genetic instability for many recombinant viral vectors is well described and a range of largely vector-specific strategies have been adopted to limit the problem [46,[49][50][51][52][53]. In the Kunjin replicon system deletion of inserted genes appears to occur in packaging cells during VLP manufacture.…”

Section: Discussionmentioning

confidence: 99%

Kunjin replicon-based simian immunodeficiency virus gag vaccines

Anraku

Mokhonov

Rattanasena

et al. 2008

Vaccine

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An RNA-based, non-cytopathic replicon vector system, based on the flavivirus Kunjin, has shown considerable promise as a new vaccine delivery system. Here we describe the testing in mice of four different SIVmac239 gag vaccines delivered by Kunjin replicon virus-like-particles. The four vaccines encoded the wild type gag gene, an RNA-optimised gag gene, a codon-optimised gag gene and a modified gag-pol gene construct. The vaccines behaved quite differently for induction of effector memory and central memory responses, for mediation of protection, and with respect to insert stability, with the SIV gag-pol vaccine providing the optimal performance. These results illustrate that for an RNA-based vector the RNA sequence of the antigen can have profound and unforeseen consequences on vaccine behaviour.

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“…Although the genome structure and replication mechanism of a particular virus is a major determinant of the strategy to be used to modify its genome, viral vectors are commonly engineered in two ways: as infectious forms and non-infectious replicon forms. In the first approach, a foreign gene of interest is inserted into the viral genome in such a way that all the viral genes that are essential for productive viral infection are functionally intact (Dufresne et al, 2002;Jia et al, 2002;Yun et al, 2003). In contrast, the second approach aims to generate a subgenomic replicon that contains only a limited number of the viral genes required for RNA replication and lacks part or all of the struc-tural protein-coding and accessory protein-coding regions of the viral genome (Khromykh, 2000;Schlesinger, 2000;Enjuanes et al, 2005).…”

Section: (A) (B)mentioning

confidence: 99%

Packaging of porcine reproductive and respiratory syndrome virus replicon RNA by a stable cell line expressing its nucleocapsid protein

Song

Kim

et al. 2011

J Microbiol.

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Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae family, is one of the most common and economically important swine pathogens. Although both live-attenuated and killed-inactivated vaccines against the virus have been available for a decade, PRRSV is still a major problem in the swine industry worldwide. To explore the possibility of producing single-round infectious PRRSV replicon particles as a potential vaccine strategy, we have now generated two necessary components: 1) a stable cell line (BHK/Sinrepl9/PRRSV-N) that constitutively expresses the viral nucleocapsid (N) protein localized to the cytoplasm and the nucleolus and 2) a PRRSV replicon vector (pBAC/PRRSV/Replicon-AN) with a 177-nucleotide deletion, removing the 3'-half portion of ORF7 in the viral genome, from which the self-replicating propagation-defective replicon RNAs were synthesized in vitro by SP6 polymerase run-off transcription. Transfection of this replicon RNA into N protein-expressing BHK-21 cells led to the secretion of infectious particles that packaged the replicon RNA, albeit with a low production efficiency of 0.4 × 10(2) to 1.1 × 10(2) infectious units/ml; the produced particles had only single-round infectivity with no cell-to-cell spread. This trans-complementation system for PRRSV provides a useful platform for studies to define the packaging signals and motifs present within the viral genome and N protein, respectively, and to develop viral replicon-based antiviral vaccines that will stop the infection and spread of this pathogen.

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Genetically Stable Picornavirus Expression Vectors with Recombinant Internal Ribosomal Entry Sites

Cited by 11 publications

References 27 publications

Genetic Determinants of Cell Type-Specific Poliovirus Propagation in HEK 293 Cells

Genetic Determinants of Cell Type-Specific Poliovirus Propagation in HEK 293 Cells

Kunjin replicon-based simian immunodeficiency virus gag vaccines

Packaging of porcine reproductive and respiratory syndrome virus replicon RNA by a stable cell line expressing its nucleocapsid protein

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