Minimum Requirements for Efficient Transduction of Dividing and Nondividing Cells by Feline Immunodeficiency Virus Vectors

Abstract

The development of gene delivery vectors based on feline immunodeficiency virus (FIV) is an attractive alternative to vectors based on primate sources for the delivery of genes into humans. To investigate the requirements for efficient transduction of dividing and nondividing cells by vector particles based on FIV, a series of packaging and vector constructs was generated for which viral gene expression was minimized and from which unnecessarycis-acting sequences were deleted. Pseudotyped vector particles produced in 293T cells were used to transduce various target cells, including contact-inhibited human skin fibroblasts and growth-arrested HT1080 cells. FIV vectors in which the U3 promoter was replaced with the cytomegalovirus promoter gave rise to over 50-fold-higher titers than FIV vectors containing the complete FIV 5′ long terminal repeat (LTR). Comparison of the transduction efficiencies of vectors containing different portions of the FIV Gag coding region indicates that at least a functional part of the FIV packaging signal (Ψ) is located within an area which includes the 5′ LTR and the first 350 bp of gag. Transduction efficiencies of vectors prepared without FIV vif and orf2 accessory gene expression did not differ substantially from those of vectors prepared with accessory gene expression in either dividing or nondividing cells. The requirement for FIV rev-RRE was, however, demonstrated by the inefficient production of vector particles in the absence of rev expression. Together, these results demonstrate the efficient transduction of nondividing cells in vitro by a multiply attenuated FIV vector and contribute to an understanding of the minimum requirements for efficient vector production and infectivity. In addition, we describe the ability of an FIV vector to deliver genes in vivo into hamster muscle tissue.

Human, Pig, and Mouse Interferon-Induced Transmembrane Proteins Partially Restrict Pseudotyped Lentiviral Vectors

“…Lentiviral vectors were produced and titered by the University of Iowa Viral Vector Core (Iowa City, IA; www.medicine.uiowa.edu/vectorcore). The FIV vector system used in this study 27,28 expressed firefly luciferase or green fluorescent protein (GFP) from the FIV3.3RSV backbone. 29 VSV-G, Machupo virus (MACV), and baculovirus (GP64)-pseudotyped FIV vector particles were generated by transient transfection, concentrated 250-fold by centrifugation, and titered using real-time PCR as previously described.…”

Lentiviral Vector Gene Transfer to Porcine Airways

“…Vector production. The FIV vector system utilized in this study 22,50 expressed either secreted (Gaussia) luciferase, mCherry, or firefly luciferase. Pseudotyped FIV vector particles were generated by transient transfection, concentrated 250-fold by centrifugation, and titered using real-time PCR as previously described.…”

FIV as a Model for HIV: An Overview

“…33 Subsequent studies focused on FIV vector development revealed that the FIV U3 domain was the determinant responsible for restricted virus replication in human cells and showed that replacement of U3 with the cytomegalovirus immediate early promoter produced FIV vectors capable of viral gene expression in human cells. 225,260 Studies reported by one research group showing that FIV is capable of productive infection of primate cells both in vitro and in vivo 261,262 have not been confirmed by other independent researchers but suggest FIV infection of nonfeline cells may warrant further examination.…”

“…218,222 Multiple reports have now confirmed that cell cultureadapted FIV isolates are capable of using CXCR4 exclusively for virus entry and infection. 33,36,[218][219][220][221]223,224 Results of subsequent reports suggesting FIV usage of chemokine receptors CCR5 and CCR3 223,225 have conflicted with observations of other investigators and may have resulted from enhanced expression of CXCR4 associated with ectopic expression of CCR5. 224 Use of other chemokine receptors by particular FIV isolates is not clear at this time and is under investigation.…”

“…260 Subsequent studies reported by other research groups described biology and cell tropism for similar but modified FIV vector systems also based on FIV 34TF10. 225,609,610 All FIV vector systems described to date use three plasmids: a transfer vector for encoding the gene of interest, a packaging vector for expression of structural and enzymatic genes, and a plasmid for expression of the vesicular stomatitis virus (VSV)-G envelope protein for pseudotyping of vector particles. 606 A chimeric FIV LTR, composed of a human cytomegalovirus (hCMV) immediate early gene promoter replacing the FIV U3 element and fused to the R/U5 LTR domains, is found in all FIV transfer vectors and is necessary for FIV vector production from human cells.…”