scholarly journals The packaging signal of simian immunodeficiency virus is upstream of the major splice donor at a distance from the RNA cap site similar to that of human immunodeficiency virus types 1 and 2

2003 ◽  
Vol 84 (9) ◽  
pp. 2423-2430 ◽  
Author(s):  
P. M. Strappe ◽  
J. Greatorex ◽  
J. Thomas ◽  
P. Biswas ◽  
E. McCann ◽  
...  
2001 ◽  
Vol 82 (2) ◽  
pp. 425-434 ◽  
Author(s):  
Jenice D’Costa ◽  
Heidi M. Brown ◽  
Priya Kundra ◽  
Alberta Davis-Warren ◽  
Suresh K. Arya

Retroviral vectors provide the means for gene transfer with long-term expression. The lentivirus subgroup of retroviruses, such as human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), possesses a number of regulatory and accessory genes and other special elements. These features can be exploited to design vectors for transducing non-dividing as well as dividing cells with the potential for regulated transgene expression. Encapsidation of the transgene RNA in lentiviral vectors is determined by the leader sequence-based multipartite packaging signal. Embedded in the packaging signal is a major splice donor site that, this study shows, is not by itself essential for transgene expression or encapsidation. We designed HIV-2 vectors that contained all the sequence elements thought to be necessary and sufficient for vector RNA encapsidation. Unexpectedly, despite abundant expression, only a small fraction of the transgene RNA was encapsidated and the titre of the vector was low. Redesign of the vector with a mutant splice donor resulted in increased vector RNA encapsidation and yielded vectors with high titre. Inefficient encapsidation by the conventionally designed vector was not due to suboptimal Rev responsive element (RRE)–Rev function. Varying the length of RRE in the vector did not change vector RNA encapsidation, nor did the introduction of a synthetic intron into the mutant vector. The vector RNA with the intact splice donor may have been excessively spliced, decreasing the amount of packageable RNA. A titre of 105 transducing units (TU)/ml was readily obtained for vectors with the neo or GFP transgene, and the vector could be concentrated to a titre of 1–5×107  TU/ml.


2009 ◽  
Vol 90 (10) ◽  
pp. 2513-2518 ◽  
Author(s):  
Christine S. Siegismund ◽  
Oliver Hohn ◽  
Reinhard Kurth ◽  
Stephen Norley

As a prelude to primate studies, the immunogenicity of wild-type and codon-optimized versions of simian immunodeficiency virus (SIV)agm Gag DNA, with and without co-administered granulocyte–macrophage colony-stimulating factor (GM-CSF) DNA, was directly compared in two strains of mice. Gag-specific T cells in the splenocytes of BALB/c and C57BL/6 mice immunized by gene gun were quantified by ELISpot using panels of overlapping synthetic peptides (15mers) spanning the entire capsid proteins of SIVagm, SIVmac and human immunodeficiency virus type 1. Specific antibodies were measured by ELISA. Codon optimization was shown to significantly increase the immune response to the DNA immunogens, reducing the amount of DNA necessary to induce cellular and antibody responses by one and two orders of magnitude, respectively. Co-administration of murine GM-CSF DNA was necessary for the induction of high level T- and B-cell responses. Finally, it was possible to identify both known and novel T-cell epitopes in the Gag proteins of the three viruses.


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