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A lentivirus packaging system based on alternative RNA transport mechanisms to express helper and gene transfer vector RNAs and its use to study the requirement of accessory proteins for particle formation and gene delivery.

ABSTRACT: A lentivirus-based packaging system was designed to reduce the chance of recombination between helper and gene transfer vector sequences by using the constitutive transport element (CTE) derived from Mason-Pfizer monkey virus for expression of the viral proteins and the Rev-Rev response element (RRE) combination for expression of the gene transfer vector. Using this approach, we evaluated a series of human immunodeficiency virus type 1 packaging constructs that express one or more accessory proteins (Vif, Vpr, and Vpu), in addition to the Gag and Pol proteins, for particle formation and virus stock production for gene transfer. Constructs that also express Vpr or both Vpr and Vpu produced more particles, as measured by a p24 assay, than did plasmids that did not contain these sequences. Transactivation experiments showed that the packaging plasmids that encode Vpr or both Vpr and Vpu also expressed a functional single-exon Tat protein. For these constructs, high-titer virus stocks could be prepared in the absence of a cotransfected Tat-expressing plasmid. Amphotropic-envelope-pseudotyped virus stocks prepared with all of the packaging constructs, irrespective of whether any of the accessory proteins were coexpressed, were equally efficient in transducing growth-arrested HeLa cells. The combination/mixed packaging system was compared to systems that were based on either the CTE alone or Rev and RRE for expression of both the packaging plasmid as well as the gene transfer vector. The combination/mixed packaging system was comparable to the other systems for production of virus stocks, suggesting that this design may prove to be safer for the eventual deployment of lentivirus vectors for therapeutic purposes.

SUBMITTER: Srinivasakumar N

PROVIDER: S-EPMC112994 | biostudies-literature | 1999 Nov

REPOSITORIES: biostudies-literature

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A lentivirus packaging system based on alternative RNA transport mechanisms to express helper and gene transfer vector RNAs and its use to study the requirement of accessory proteins for particle formation and gene delivery.

Srinivasakumar N N Schuening F G FG

Journal of virology 19991101 11

A lentivirus-based packaging system was designed to reduce the chance of recombination between helper and gene transfer vector sequences by using the constitutive transport element (CTE) derived from Mason-Pfizer monkey virus for expression of the viral proteins and the Rev-Rev response element (RRE) combination for expression of the gene transfer vector. Using this approach, we evaluated a series of human immunodeficiency virus type 1 packaging constructs that express one or more accessory prot ...[more]

PMID: 10516068

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Project description:All lentiviruses encode the accessory protein Rev, whose main biological function is to mediate the nuclear export of unspliced and incompletely spliced viral transcripts by binding to a viral cis-acting element (termed the Rev-responsive element, RRE) within the env-encoding region. Equine infectious anemia virus (EIAV) is a member of the lentivirus genus in the Retroviridae family and is considered an important model for the study of lentivirus pathogenesis. Here, we identified a novel transcript from the EIAV genome that encoded a viral protein, named Mat, with an unknown function. The transcript mat was fully spliced and comprised parts of the coding regions of MA and TM. Interestingly, the expression of Mat depended on Rev and the chromosome region maintenance 1 (CRM1) pathway. Rev could specifically bind to Mat mRNA to promote its nuclear export. We further identified that the first exon of Mat mRNA, which was located within the Gag-encoding region, acted as an unreported RRE. Altogether, we identified a novel fully spliced transcript mat with an unusual RRE, which interacted with Rev for nuclear export through the CRM1 pathway. These findings updated the EIAV genome structure, highlighted the diversification of posttranscriptional regulation patterns in EIAV, and may help to expand the understanding of gene transcription and expression of lentivirus. IMPORTANCE In lentiviruses, the nuclear export of viral transcripts is an important step in controlling viral gene expression. Generally, the unspliced and incompletely spliced transcripts are exported via the CRM1-dependent export pathway in a process mediated by the viral Rev protein by binding to the Rev-responsive element (RRE) located within the Env-coding region. However, the completely spliced transcripts are exported via an endogenous cellular pathway, which was Rev independent. Here, we identified a novel fully spliced transcript from EIAV and demonstrated that it encoded a viral protein, termed Mat. Interestingly, we determined that the expression of Mat depended on Rev and identified that the first exon of Mat mRNA could specifically bind to Rev and be exported to the cytoplasm, which suggested that the first exon of Mat mRNA was a second RRE of EIAV. These findings provided important insights into the Rev-dependent nuclear export of completely spliced transcripts in lentiviruses.

Dataset Information

A lentivirus packaging system based on alternative RNA transport mechanisms to express helper and gene transfer vector RNAs and its use to study the requirement of accessory proteins for particle formation and gene delivery.

Publications

A lentivirus packaging system based on alternative RNA transport mechanisms to express helper and gene transfer vector RNAs and its use to study the requirement of accessory proteins for particle formation and gene delivery.

Similar Datasets

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