Requirements for efficient production and transduction of human immunodeficiency virus type 1-based vectors

Abstract

A number of human immunodeficiency type 1 (HIV-1)-based vectors have recently been shown to transduce nondividing cells in vivo as well as in vitro. However, if these vectors are to be considered for eventual clinical use, a major consideration is to reduce the probability of unintended generation of replication-competent virus. This can be achieved by eliminating viral genetic elements involved in the generation of replication-competent virus without impairing vector production. We have designed a system to transiently produce HIV-1-based vectors by using expression plasmids encoding Gag, Pol, and Tat of HIV-1 under the control of the cytomegalovirus immediate-early promoter. Our data show that the best vector yield is achieved in the presence of the Rev/Rev-responsive element (RRE) system. However, the constitutive transport element of Mason-Pfizer monkey virus can substitute for RRE and Rev at least to some extent, whereas the posttranscriptional regulatory element of human hepatitis B virus appeared to be inefficient. In addition, we show that high-titer virus preparations can be obtained in the presence of sodium butyrate, which activates the expression of both the packaging construct and the vector genome. Finally, our results suggest that efficient infectivity of vectors defective in the accessory proteins Vif, Vpr, Vpu, and Nef depends on the nature of the target cells.

FIG. 1

Structures of expression plasmids of the HIV-1-based vector production system. (A) Packaging system; (B) minimal gag-pol constructs. Expression of packaging and VSV G protein (VSV-G) envelope constructs is driven by the CMV immediate-early promoter (CMV). The polyadenylation signal (PA) is derived from the rabbit β-globin gene. pCMV-HIV-1 contains all the HIV-1 genes except a 19-bp deletion in the packaging signal (ΔΨ) and a 600-bp deletion in the env open reading frame (Δenv). Accessory-protein open reading frames are shown as solid boxes. The RNA transport elements are presented as shaded boxes. The vector genome pv653CMV-β-gal consists of the HIV-1 LTRs flanking the RRE and the reporter gene (β-gal) driven by the CMV promoter.

FIG. 2

Stimulation of vector production by sodium butyrate. Vectors derived from either pCMV-HIV-1 or pCHGP-2 were generated in 293T cells in the presence of various concentrations of sodium butyrate as indicated. Titers were determined in HT1080 cells as described in Materials and Methods. Values are the ratios of titers with sodium butyrate to titers without sodium butyrate for each vector. This experiment was repeated once, and similar results were obtained (data not shown).

FIG. 3

Transduction efficiency in HeLa cells. Three hundred microliters of MLV-β-gal with a titer of 2.8 × 10⁶ IU/ml (■), v653CMVβ-gal(−) with a titer of 1.4 × 10⁶ IU/ml (▨), or v653CMVβ-gal(+) with a titer of 3.7 × 10⁶ IU/ml (□) was used to transduce actively dividing or growth-arrested HeLa cells in 12-well plates. The cells were harvested 2 days after transduction, and the total β-gal activity was determined by blue-cell count after X-Gal staining. The results are presented as the percentage of the vector titer observed in HT1080 cells for each viral preparation ([titer in dividing or quiescent HeLa cells/titer in HT1080 cells] × 100). The experiment was repeated with 100 and 30 μl of the same vector stocks, and similar results were observed. Transduction of the HeLa cells with different vector stocks was repeated at least twice, and similar results were obtained (data not shown).

FIG. 4

Transduction efficiency in human skin fibroblasts. Ten microliters of MLV-β-gal (■), v653CMVβ-gal(−) (▨), or v653CMVβ-gal(+) (□) was used to infect dividing and quiescent fibroblasts in a 12-well plate. Two days after transduction, the titer was determined by blue-cell counting after X-Gal staining. Data for transduction of quiescent and dividing fibroblasts are presented as the percentage of the titer observed in growing HT1080 cells for each viral preparation ([titer in growing or quiescent fibroblasts/titer in HT1080 cells] × 100). The values are averages from four experiments, with standard deviations.