Development and characterization of promoterless helper RNAs for the production of alphavirus replicon particle

Kamrud, Kurt I.; Alterson, Kim; Custer, Max; Dudek, J.; Goodman, Christin H.; Owens, Gary; Smith, Jonathan

doi:10.1099/vir.0.020081-0

Cited by 25 publications

(28 citation statements)

References 22 publications

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“…Although the H/C helper proved to be effective in PIV genome packaging, its application could potentially result in recombination events between the PIV genome and helper RNA (33,34). These events are in all likelihood very rare, but they are still possible and could potentially lead to formation of viral genomes encoding an RNA packaging-competent capsid protein under the control of an additional subgenomic promoter.…”

Section: Mutations In Rna-binding Domain Of Veev Capsid Protein Have mentioning

confidence: 99%

Pseudoinfectious Venezuelan Equine Encephalitis Virus: a New Means of Alphavirus Attenuation

Atasheva

Kim

Akhrymuk

et al. 2013

J Virol

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bVenezuelan equine encephalitis virus (VEEV) is a reemerging virus that causes a severe and often fatal disease in equids and humans. In spite of a continuous public health threat, to date, no vaccines or antiviral drugs have been developed for human use. Experimental vaccines demonstrate either poor efficiency or severe adverse effects. In this study, we developed a new strategy of alphavirus modification aimed at making these viruses capable of replication and efficient induction of the immune response without causing a progressive infection, which might lead to disease development. To achieve this, we developed a pseudoinfectious virus (PIV) version of VEEV. VEE PIV mimics natural viral infection in that it efficiently replicates its genome, expresses all of the viral structural proteins, and releases viral particles at levels similar to those found in wild-type VEEV-infected cells. However, the mutations introduced into the capsid protein make this protein almost incapable of packaging the PIV genome, and most of the released virions lack genetic material and do not produce a spreading infection. Thus, VEE PIV mimics viral infection in terms of antigen production but is safer due to its inability to incorporate the viral genome into released virions. These genome-free virions are referred to as virus-like particles (VLPs). Importantly, the capsid-specific mutations introduced make the PIV a very strong inducer of the innate immune response and add self-adjuvant characteristics to the designed virus. This unique strategy of virus modification can be applied for vaccine development against other alphaviruses.

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Section: Mutations In Rna-binding Domain Of Veev Capsid Protein Have mentioning

confidence: 99%

Pseudoinfectious Venezuelan Equine Encephalitis Virus: a New Means of Alphavirus Attenuation

Atasheva

Kim

Akhrymuk

et al. 2013

J Virol

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“…Doing this reduces the theoretical number of possible recombination events that would still result in functional structural protein expression from the recombinant replicon, because the helper-replicon recombination must occur in a precise way that places the structural protein under the control of the subgenomic promoter that was present in the replicon, as opposed to the structural protein gene having an attached promoter that can function from a variety of recombination sites. This approach can be used successfully without a reduction in VRP titer [49], but the theoretical benefit has yet to be tested experimentally to show that the rate of RCV is actually reduced in this system. RCV attenuation.…”

Section: Safety Improvementsmentioning

confidence: 92%

“…One design innovation that may be able to reduce the frequency of repliconhelper recombination events is the removal of the subgenomic promoter from the helper RNAs [49]. Doing this reduces the theoretical number of possible recombination events that would still result in functional structural protein expression from the recombinant replicon, because the helper-replicon recombination must occur in a precise way that places the structural protein under the control of the subgenomic promoter that was present in the replicon, as opposed to the structural protein gene having an attached promoter that can function from a variety of recombination sites.…”

Section: Safety Improvementsmentioning

confidence: 99%

Alphavirus Particle-Based Vaccine Vectors

Balsitis¹,

Beard²,

Mason³

2010

Replicating Vaccines

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Most of the vaccines in use today are live attenuated, killed, or protein subunit vaccines. Although these vaccines have saved countless lives, there is still a need to develop safer and more efficacious ones. These improved new generation vaccines will enable us to protect more people from a greater number of different infectious disease threats. One such new vaccine technology is derived from the alphaviruses, which are single-strand, positive-sense RNA viruses in the family Togaviridae. By removing the genes that encode for the viral coat proteins, and replacing them with an antigen-encoding gene, these viruses become a replicon that can replicate its genome but cannot propagate new virus particles. The resources that the virus once expended to make new progeny are now diverted to making vaccine antigen within the host. As a result of this molecular alteration, the alphavirus particle-based vectors serve as an attractive technology for the development of new and improved vaccines.

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“…The 281-bp fragment was digested with SphI and NotI restriction enzymes and then ligated into ⌬26S capsid (dHcap6-m1) and ⌬26S GP (dHgp6-m1) helper plasmids linearized with the same two enzymes. The dHcap6-m1 and dHgp6-m1 helpers have been described previously (37). The resulting helper plasmids were designated dHcap6-m1 3Ј RC1-6-plus and dHgp6-m1 3Ј RC1-6-plus (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Early "split helper" RNA designs contain the 5Ј and 3Ј sequences required for replication as well as an alphavirus 26S subgenomic promoter that normally controls production of the alphavirus structural protein mRNA. Recently, second-generation split helpers have been designed, where the 26S promoter has been removed from the helper RNAs (⌬26S helpers) (37). Removal of the 26S promoter from the helper RNAs further reduces the probability of functional recombination events between helper RNAs and the replicon RNA, as multiple, precise, nonhomologous recombinations are required (37).…”

mentioning

confidence: 99%

In Vitro and In Vivo Characterization of MicroRNA-Targeted Alphavirus Replicon and Helper RNAs

Kamrud

Coffield

Owens

et al. 2010

J Virol

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Alphavirus-based replicon vector systems (family Togaviridae) have been developed as expression vectors with demonstrated potential in vaccine development against both infectious diseases and cancer. The singlecycle nature of virus-like replicon particles (VRP), generated by supplying the structural proteins from separate replicable helper RNAs, is an attractive safety component of these systems. MicroRNAs (miRNAs) have emerged as important cellular RNA regulation elements. Recently, miRNAs have been employed as a mechanism to attenuate or restrict cellular tropism of replication-competent viruses, such as oncolytic adenoviruses, vesicular stomatitis virus, and picornaviruses as well as nonreplicating lentiviral and adenoviral vectors. Here, we describe the incorporation of miRNA-specific target sequences into replicable alphavirus helper RNAs that are used in trans to provide the structural proteins required for VRP production. VRP were found to be efficiently produced using miRNA-targeted helper RNAs if miRNA-specific inhibitors were introduced into cells during VRP production. In the absence of such inhibitors, cellular miRNAs were capable of downregulating helper RNA replication in vitro. When miRNA targets were incorporated into a replicon RNA, cellular miRNAs were capable of downregulating replicon RNA replication upon delivery of VRP into animals, demonstrating activity in vivo. These data provide the first example of miRNA-specific repression of alphavirus replicon and helper RNA replication and demonstrate the feasibility of miRNA targeting of expression vector helper functions that are provided in trans.MicroRNAs (miRNAs) are small RNAs of approximately 21 nucleotides (nt) in length that have been identified in cells and are associated with regulation of mRNA translation and stability (46, 49, 50). The first miRNA described (lin-4) was shown to control the translation of a cellular mRNA (lin-14) involved in the timing of Caenorhabditis elegans larval development (51, 76). Subsequently, numerous miRNAs that control cellular activities, such as proliferation, cell death, fat metabolism, neuronal patterning in nematodes, hematopoietic differentiation, and plant development, have been identified (4,11,16,17,21,36,60). These small regulatory RNAs have been identified in a wide range of animals, including mammals, fish, worms, and flies (1,2,18,33,42,47,48,56,57,59). The miRNA sequences are transcribed from specific miRNA genes, as independent transcriptional units, throughout the genome (47, 49, 53) or may be produced in coordination with intron processing of specific mRNAs (66, 78).The cellular production of miRNAs begins with transcription of large precursor primary miRNAs which are processed by a nuclear RNase III-like enzyme, Drosha (52,73). The large precursor RNAs are termed pri-miRNAs, and the smaller Drosha-processed species, termed pre-miRNAs, are exported from the nucleus by Exportin 5 (77). The pre-miRNA, exported from the nucleus, is then processed in the cytoplasm by another RNase III-like enzy...

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Development and characterization of promoterless helper RNAs for the production of alphavirus replicon particle

Cited by 25 publications

References 22 publications

Pseudoinfectious Venezuelan Equine Encephalitis Virus: a New Means of Alphavirus Attenuation

Pseudoinfectious Venezuelan Equine Encephalitis Virus: a New Means of Alphavirus Attenuation

Alphavirus Particle-Based Vaccine Vectors

In Vitro and In Vivo Characterization of MicroRNA-Targeted Alphavirus Replicon and Helper RNAs

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