Transfer of Primer Binding Site-Mutated Simian Immunodeficiency Virus Vectors by Genetically Engineered Artificial and Hybrid tRNA-Like Primers

Abstract

Simian immunodeficiency viruses (SIV) harbor primer binding sites (PBS) matching tRNA 3Lys or tRNA 5 Lys . To study determinants of primer usage in SIV, a SIVmac239-based vector was impaired by mutating the PBS to a sequence (PBS-X2) with no match to any tRNA. By cotransfection of a synthetic gene encoding a tRNA Pro -like RNA with a match to PBS-X2, the activity of this vector could be restored to a transduction efficiency slightly lower than that of the wild-type vector. A vector with a PBS matching tRNA Pro was functional at a level slightly below that of the wild-type vector, but higher transduction efficiency could be obtained by cotransfection of a gene for an engineered tRNA Pro -tRNA 3 Lys hybrid with a match to PBS-Pro. The importance of tRNA backbone identity was further analyzed by complementing the PBS-X2 vector with a gene for a matching x2 primer with a tRNA 3 Lys backbone, which led to three-to fourfold-higher titers than those observed for the x2 primer with the tRNA Pro backbone. In summary, our results demonstrate flexibility in PBS and primer usage for SIVmac239, with PBS-primer complementarity being the major determinant, in analogy with previous findings for murine leukemia viruses and human immunodeficiency virus type 1.Retroviruses replicate through reverse transcription of the viral RNA genome into a double-stranded DNA form that is integrated into the genome of the host. The virus-encoded reverse transcriptase (RT) enzyme mediates this process through the use of a host-encoded tRNA. The 3Ј-terminal 18-nucleotide (nt) segment of the tRNA primer molecule anneals to the RNA genome at a complementary sequence known as the primer binding site (PBS). In order to synthesize a double-stranded DNA copy, two template shifts are involved. In plus-strand synthesis, the 3Ј 18-nt segment of the tRNA primer is reverse transcribed into a DNA copy, which mediates the second template shift by annealing to the minus-strand

Atraumatic Oral Spray Immunization with Replication-Deficient Viral Vector Vaccines

“…PBMC, peripheral blood mononuclear cell. Although we cannot formally exclude replication of SCIV in vivo, we consider this to be highly unlikely since (i) the primer complementation approach used to generate the SCIVs reduces infectivity in single-round replication assays at least 10 4 -fold (17,18); (ii) the SCIVs have an additional deletion of vif, which severely impairs replication in primary cells and rhesus macaques (9,32), and (iii) we were never able to recover replication-competent virus from SCIV-injected animals despite repeated attempts (23).…”

Target-Cell-Derived tRNA-like Primers for Reverse Transcription Support Retroviral Infection at Low Efficiency

“…We have previously shown that replication of Akv-MLVderived vectors with a mutationally impaired PBS can be restored by engineered complementary tRNA primers ( Fig. 1A), functional in both the initiation of first-strand synthesis and second-strand transfer (Lund et al, 1997;Modin et al, 2000;Hansen et al, 2001). In this setup (producer-cell complementation) vector construct and tRNA primer expressing constructs are cotransfected into the BOSC 23 packaging cell line ( Fig.…”

“…tRNA X2pro and tRNA X2Lys3 , both able to complement the Akv-MLV-derived vector pPBS-x2m (Hansen et al, 2001) in the producer-cell complementation system, were transfected (with a transfection efficiency of approximately 30%) into NIH 3T3 cells to verify expression of the synthetic tRNAs.…”

“…While genetic analysis based on mutagenesis studies of viral RNA and proteins has already contributed valuable information, investigation of the role of tRNA in vivo via mutational analysis is hampered due to its multifunctional nature. However, combinations of PBS-modified vectors and complementary tRNAs have been found to be functional in initiation of retroviral replication in MLV (Lund et al, 1997), HIV Morrow, 2000, 2001), and SIVmac239 (Hansen et al, 2001) in vivo. In such a "producer-cell" complementation system ( Fig.…”

Reduced Mobilization of Rev-Responsive Element-Deficient Lentiviral Vectors

“…Additional mechanisms that might contribute to mobilization of SIN vectors are promoter trapping by integration of the lentiviral vector downstream of an active cellular promoter and recombination events leading to the reconstitution of a transcriptional active 3Ј U3 region of the lentiviral vector during vector production. Mutation of the primer binding site (PBS) of an SIV vector and complementation of the PBS mutations during vector production by a matched artificial tRNA provided an independent approach to reduce the risk of SIV vector mobilization by HIV-1 (8,9). However, mobilization of the SIV vector by homologous SIV was reduced to a lesser extent.…”