Inclusion of jaagsiekte sheep retrovirus proviral elements markedly increases lentivirus vector pseudotyping efficiency  Patrick L. Sinn, Erin R. Burnight, Hong Shen, Hung Fan, Paul B. McCray  Molecular Therapy  Volume 11, Issue 3, Pages 460-469 (March 2005) DOI: 10.1016/j.ymthe.2004.10.022 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Widely divergent FIV titer conferred by different configurations of the JSRV envelope GP expression cassette. FIV vector expressing nuclear targeted β-galactosidase was prepared by three-plasmid transfection and titered by limiting dilution on HT1080 cells as described under Materials and Methods. Five different configurations of the JSRV env GP are shown schematically and the resulting titers are presented as the average transducing units per milliliter ± standard error. The n for each data set is shown (*P < 0.05 versus J; **P < 0.01 versus J). Molecular Therapy 2005 11, 460-469DOI: (10.1016/j.ymthe.2004.10.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Steady-state levels of JSRV envelope RNA from J, 5′J, J3′, and 5′J3′ following transient transfection into 293T cells. (A) Total JSRV RNA levels were determined by RNA dot-blot assay and quantified by phosphorimaging (n = 6; **P < 0.01 versus J). (B) JSRV RNA was separated into nuclear and cytoplasmic fractions and subjected to dot-blot assay and quantified by phosphorimaging (n = 6; *P < 0.05 versus J; **P < 0.01 versus J). Molecular Therapy 2005 11, 460-469DOI: (10.1016/j.ymthe.2004.10.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Confirmation and contribution of a splice event. A splice event was confirmed and the splice junctions were determined by 5′ RACE of RNA derived from cells transiently transfected with pCMV3JS21ΔGP. (A) The sequence chromatograph of the spliced product is shown. (B) The location of the splice event in the pCMV3JS21ΔGP is shown schematically. The splice junction (donor and acceptor) and the JSRV env start codon are indicated. (C) Vector titer was determined from FIV prepared using either pCMV3JS21ΔGP (n = 7) or the identical construct with the exception of a mutation in the splice donor (pCMV3JS21ΔGP ΔSD; n = 3) (**P < 0.01). (D) JSRV RNA from cells transiently transfected with either pCMV3JS21ΔGP or pCMV3JS21ΔGP ΔSD was separated into nuclear and cytoplasmic fractions and subjected to dot-blot assay and phosphorimaging (n = 6; *P < 0.05). Molecular Therapy 2005 11, 460-469DOI: (10.1016/j.ymthe.2004.10.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Generation of stable cell lines expressing each of the different JSRV envelope constructs. Flp recombinase was used to generate single copy/single loci as described under Materials and Methods. (A) A single-copy integration event for each cell line was confirmed by determining JSRV env gene copies using real-time PCR amplification of genomic DNA and normalized to GAPDH gene copies (n = 6). (B) Doxycycline responsiveness was confirmed by real-time PCR amplification of JSRV RNA in the presence or absence of 10 μg/ml doxycycline (n = 6; *P < 0.05). Molecular Therapy 2005 11, 460-469DOI: (10.1016/j.ymthe.2004.10.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 RNA half-lives of JSRV Env RNA expressed from J, 5′J, J3′, and 5′J'3′. Half-lives were determined in the established stable cell lines. Cells were stimulated with doxycycline for 24 h. Following doxycycline treatment, cellular transcription was inhibited by actinomycin D treatment (10 μg/ml). Cells were collected at 6-h intervals from 0 to 24 h, total RNA was purified, and JSRV Env RNA was quantified by real-time PCR. Molecular Therapy 2005 11, 460-469DOI: (10.1016/j.ymthe.2004.10.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Steady-state levels of JSRV envelope protein from HA-tagged J and pCMV3JS21ΔGP. HA-tagged J and pCMV3JS21ΔGP constructs (A) were transiently transfected into 293T cells along with a mock-transfection negative control. 24 h later lysates were collected and subjected to Western blot analysis. Identical blots were probed with either (B) anti-HA or (C) anti-human actin antibodies. The arrow indicates the 37-kDa band of HA-tagged JSRV Env protein corresponding to TM protein. Molecular Therapy 2005 11, 460-469DOI: (10.1016/j.ymthe.2004.10.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 7 RCR assays. JSRV-FIV was generated via three-plasmid transfection into 293T packaging cells (PC) followed by supernatant collection and transduction of target cells. β-Galactosidase protein expression was confirmed by X-gal staining in (A) packaging cells and (B) target HT1080 cells designated “T1”. (C) Total RNA isolated from PC and T1 cells was used for RT-PCR with JSRV Env- or β-galactosidase-specific primers. (D) To establish the sensitivity of the assay, PCR using JSRV Env-specific primers was performed on a dilution series of known concentrations of pCMV3JS21ΔGP plasmid template. (E) PERT assay for the detection of RCR was performed as described under Materials and Methods. Concentrated vector was used to transduce 293T cells designated T1. Supernatant was collected from T1 cells, centrifuge concentrated, and used to transduce fresh cells designated T2. The indicated cellular lysates were used to supply the reverse transcriptase necessary to generate MS2 cDNA detected by real-time PCR. Mock-transfected 293T cell lysates were included. The standard curve used to determine the cDNA copies of MS2 is shown (inset). Asterisks denote that the signal was below the limit of detection. Molecular Therapy 2005 11, 460-469DOI: (10.1016/j.ymthe.2004.10.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 8 FIV vector titers resulting from Env GP expressed from pIXSL or pcDNA31. An expression plasmid was constructed including the JSRV 5′ and 3′ regions (pIXSL) and used to express four other envelope glycoproteins to generate FIV. Envelope GP was expressed from pcDNA3.1 (black bars) or from pIXSL (open bars). The n for each data set is shown (*P < 0.01). Molecular Therapy 2005 11, 460-469DOI: (10.1016/j.ymthe.2004.10.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions