Wulin Teo

Restriction of Feline Immunodeficiency Virus by Ref1, Lv1, and Primate TRIM5α Proteins

ABSTRACT The Ref1 and Lv1 postentry restrictions in human and monkey cells have been analyzed for lentiviruses in the primate and ungulate groups, but no data exist for the third (feline) group. We compared feline immunodeficiency virus (FIV) to other restricted (human immunodeficiency virus type 1 [HIV-1], equine infectious anemia virus [EIAV]) and unrestricted (NB-tropic murine leukemia virus [NB-MLV]) retroviruses across wide ranges of viral inputs in cells from multiple primate and nonprimate species. We also characterized restrictions conferred to permissive feline and canine cells engineered to express rhesus and human TRIM5α proteins and performed RNA interference (RNAi) against endogenous TRIM5α. We find that expression of rhesus or human TRIM5α proteins in feline cells restricts FIV, impairing pseudotyped vector transduction and viral replication, but rhesus TRIM5α is more restricting than human TRIM5α. Notably, however, canine cells did not support restriction by human TRIM5α and supported minimal restriction by rhesus TRIM5α, suggesting that these proteins may not function autonomously or that a canine factor interferes. Stable RNAi knockdown of endogenous rhesus TRIM5α resulted in marked increases in FIV and HIV-1 infectivities while having no effect on NB-MLV. A panel of nonprimate cell lines varied widely in susceptibility to lentiviral vector transduction, but normalized FIV and HIV-1 vectors varied concordantly. In contrast, in human and monkey cells, relative restriction of FIV compared to HIV-1 varied from none to substantial, with the greatest relative infectivity deficit for FIV vectors observed in human T-cell lines. Endogenous and introduced TRIM5α restrictions of FIV could be titrated by coinfections with FIV, HIV-1, or EIAV virus-like particles. Arsenic trioxide had complex and TRIM5α-independent enhancing effects on lentiviral but not NB-MLV infection. Implications for human gene therapy are discussed.

Feline Immunodeficiency Virus-Based Lentiviral Vectors

Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This article describes the production and use of advanced generation FIV vectors. Key properties are discussed in comparison to other lentiviral vectors. Additional topics include the practical implications of species-specific retroviral restriction factors and the production of nonintegrating FIV vectors.

Titration of Feline Immunodeficiency Virus-Based Lentiviral Vector Preparations

Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This protocol describes methods for measuring and calculating vector titers in transducing units (TU)/mL. Alternate methods are provided for green fluorescent protein (GFP) vectors and for β-galactosidase vectors.

Production and harvest of feline immunodeficiency virus-based lentiviral vector from cells grown in T75 tissue-culture flasks.

Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This protocol describes the production and harvesting of vector from cells grown in T75 tissue-culture flasks. The methods are for production for basic science laboratory use and in vivo experimentation. They do not meet standards for clinical-grade (good manufacturing practice [GMP]) production.