Multiple RNA splicing and the presence of cryptic RNA splice donor and acceptor sites may contribute to low expression levels and poor immunogenicity of potential DNA vaccines containing the env gene of equine infectious anemia virus (EIAV).
Abstract: The env gene is an excellent candidate for inclusion in any DNA-based vaccine approach against equine infectious anemia virus (EIAV). Unfortunately, this gene is subjected to mutational pressure in E. coli resulting in the introduction of stop codons at the 5' terminus unless it is molecularly cloned using very-low-copy-number plasmid vectors. To overcome this problem, a mammalian expression vector was constructed based on the low-copy-number pLG338-30 plasmid. This permitted the production of full-length EIAV env gene clones (plcnCMVenv) from which low-level expression of the viral surface unit glycoprotein (gp90) was detected following transfection into COS-1 cells. Although this suggested the nuclear export of complete env mRNA moieties at least two additional polypeptides of 29 and 20kDa (probably Rev) were produced by alternative splicing events as demonstrated by the fact that their synthesis was prevented by mutational inactivation of EIAV env splice donor 3 (SD3) site. The plcnCMVenv did not stimulate immune responses in mice or in horses, whereas an env construct containing an inactivated SD3 site (plcnCMVDeltaSD3) did induce weak humoral responses against gp90 in mice. This poor immunogenicty in vivo was probably not related to the inherent antigenicity of the proteins encoded by these constructs but to some fundamental properties of EIAV env gene expression. Attempts to modify one of these properties by mutational inactivation of known viral RNA splice sites resulted in activation of previously unidentified cryptic SD and slice acceptor sites.
Publication Date: 2002-07-24 PubMed ID: 12135633DOI: 10.1016/s0378-1135(02)00099-8Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- P.H.S.
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The research involves the study of the influence and challenges of RNA splicing and cryptic RNA splice donor and acceptor sites on the effectiveness of DNA vaccines targeting the env gene of the equine infectious anemia virus (EIAV).
Objective of the Research
- The study primarily aimed to explore why the env gene of EIAV, despite being an excellent candidate for inclusion in DNA-based vaccines, exhibits low expression levels and poor immunogenicity.
Experimental Process and Findings
- In order to circumvent the mutational pressure found in E. coli which introduces stop codons, the researchers constructed a mammalian expression vector using a low-copy-number pLG338-30 plasmid. This allowed for the production of full-length EIAV env gene clones.
- A low level of expression of viral surface unit glycoprotein (gp90) was observed, suggesting the nuclear export of complete env mRNA moieties. Alternately spliced polypeptides were also detected.
- The production of the env gene clones did not stimulate immune responses in mice or horses, indicating poor immunogenicity. A clone with an inactivated splice donor site, however, induced a weak humoral response against the virus in mice.
Conclusions and Implications of the Study
- The absence of robust immune responses was believed to be related to intrinsic properties of EIAV env gene expression rather than the antigenicity of the proteins encoded.
- Modifying these properties by inactivation of viral RNA splice sites led to the activation of hitherto unidentified cryptic splice sites, showing that an interaction exists between RNA splicing and the potential effectiveness of EIAV DNA vaccines.
- This study highlights the need for further exploration of EIAV RNA splicing to develop an effective DNA vaccine against EIAV.
Cite This Article
APA
Zhou W, Cook RF, Cook SJ, Hammond SA, Rushlow K, Ghabrial NN, Berger SL, Montelaro RC, Issel CJ.
(2002).
Multiple RNA splicing and the presence of cryptic RNA splice donor and acceptor sites may contribute to low expression levels and poor immunogenicity of potential DNA vaccines containing the env gene of equine infectious anemia virus (EIAV).
Vet Microbiol, 88(2), 127-151.
https://doi.org/10.1016/s0378-1135(02)00099-8 Publication
Researcher Affiliations
- Department of Veterinary Science, University of Kentucky, Maxwell H. Gluck Equine Research Center, Lexington, KY 40546-0099, USA.
MeSH Terms
- Amino Acid Sequence
- Animals
- Antibodies, Viral / blood
- Base Sequence
- Cloning, Molecular / methods
- Codon, Terminator
- Equine Infectious Anemia / immunology
- Equine Infectious Anemia / prevention & control
- Gene Expression Regulation, Viral
- Gene Products, env / genetics
- Genes, env
- Horses
- Infectious Anemia Virus, Equine / genetics
- Infectious Anemia Virus, Equine / immunology
- Mice
- Molecular Sequence Data
- Mutagenesis, Site-Directed
- Mutation
- RNA Splicing / genetics
- RNA, Viral / chemistry
- Transfection / veterinary
- Vaccines, DNA / genetics
- Vaccines, DNA / immunology
- Viral Vaccines / genetics
- Viral Vaccines / immunology
Grant Funding
- R01-AI25850 / NIAID NIH HHS
Citations
This article has been cited 5 times.- Chapman R, Rybicki EP. Use of a Novel Enhanced DNA Vaccine Vector for Preclinical Virus Vaccine Investigation. Vaccines (Basel) 2019 Jun 13;7(2).
- Todorova B, Adam L, Culina S, Boisgard R, Martinon F, Cosma A, Ustav M, Kortulewski T, Le Grand R, Chapon C. Electroporation as a vaccine delivery system and a natural adjuvant to intradermal administration of plasmid DNA in macaques. Sci Rep 2017 Jun 23;7(1):4122.
- Johansson C, Schwartz S. Regulation of human papillomavirus gene expression by splicing and polyadenylation. Nat Rev Microbiol 2013 Apr;11(4):239-51.
- Kutzler MA, Weiner DB. DNA vaccines: ready for prime time?. Nat Rev Genet 2008 Oct;9(10):776-88.
- Zhao X, Schwartz S. Inhibition of HPV-16 L1 expression from L1 cDNAs correlates with the presence of hnRNP A1 binding sites in the L1 coding region. Virus Genes 2008 Feb;36(1):45-53.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
