Packaging of intron-containing genes into retrovirus vectors by alphavirus vectors

Abstract

Efficient and controllable expression of a transgene usually requires the presence of intron sequences and much efforts have been made to produce retrovirus vectors that can transduce and integrate genes with introns. However, this has proven difficult because the viral RNA is spliced when it is synthesized in the nucleus of a producer cell. We describe a novel approach to avoid this problem. In our system the retroviral RNA is synthesized in the cytoplasm of the cell, not in the nucleus, in a reaction driven by the Semliki Forest virus (SFV) expression system. The approach was tested with a recombinant Moloney murine leukemia virus genome containing the chloramphenicol acetyltransferase (CAT) gene in association with an intron. This was inserted into a SFV transcription plasmid and the corresponding SFV vector RNA was transcribed in vitro. BHK-21 cells were then transfected with this vector RNA together with two additional SFV vectors that encode the Moloney murine leukemia virus packaging proteins. Retrovirus vectors containing intron-CAT sequences were produced at titers up to 1.3 x 10(6) infectious particles per ml during a 5-hr incubation period. The vectors faithfully transduced the intron-containing CAT gene into NIH 3T3 cells, where the intron-CAT RNA was subjected to efficient splicing and used for high level enzyme expression. Thus, the results show that intron containing genes can be efficiently packaged into retrovirus vectors by the SFV expression system.

Figures

Figure 1

(A) Schematic representation of pSFV1-I-CAT and pSFV1-CAT constructs. Only the SFV recombinant regions are shown. The SP6 promoter is indicated by an open arrow. The subgenomic SFV promoter and the SV40 promoter are indicated with solid arrows. SFVnsp1–4 represents the coding region of the SFV nonstructural proteins 1 to 4. The recombinant retroviral genome (R-U5-ψ+-NEO-SV40-IN-CAT-pA-U3-R) is inserted downstream of the subgenomic SFV promoter. The SV40 early promoter is used to initiate the transcription of the CAT gene. A chimeric intron (IN) is inserted between the SV40 promoter and the CAT gene in pSFV1-I-CAT. In pSFV1-CAT, this intron has been removed. An internal, SV40-derived poly(A) signal (pA) is present downstream of the CAT gene in both constructs. (B) Titers of retrovirus vector preparations. RNA was prepared from plasmid constructs and used for vector production in BHK-21 cells. Media were harvested and titrated for neoR-transduction competent retrovirus vectors on NIH 3T3 cells. The titers shown are the number of neomycin-resistant colonies obtained from 1 ml of medium. The maps are not drawn to scale.

Figure 2

CAT expressions in cells infected with ecotropic retroviral vectors carrying an I-CAT gene or an intron-free CAT gene (CAT). (A) Transient expression of the CAT gene. NIH 3T3 cells were plated into 60-mm culture dishes at 5 × 105 cells per dish 24 hr before infection. The cells in one dish were incubated with 1 × 105 infectious retroviral vectors carrying the I-CAT or the intron-free CAT gene at 37°C for 24 hr in the presence of 4 μg/ml polybrene. Then the medium was replaced with fresh medium and the cells were further incubated at 37°C for 28 hr. The CAT expression was measured as described in Materials and Methods. Noninfected NIH 3T3 cells were used as a control. (B) CAT gene expression in G418 selected cells. NIH 3T3 cells were infected with the I-CAT or CAT retroviral vectors and then incubated for 6 days in the presence of 600 μg/ml G418. The cells were trypsinized, replated (1 × 106 cells per 60-mM dish), cultured at 37°C for 24 hr, and tested for CAT activity. (C) CAT gene expression in cell clones. The infected cells were incubated for 11 days in the presence of 600 μg/ml G418. Nine resistant colonies were picked from each infection (CAT 1–9 and I-CAT 1–9) and expanded by incubation for 11–18 days. The CAT activity of the cells was measured as described in B. Bars = SD.

Figure 3

PCR analysis of proviruses and RT-PCR analysis of CAT gene transcripts in neoR transduced cell clones. Schematic, not to scale, gene maps of the proviruses are shown above each gel. The transcription initiation site in the 5′LTR is indicated by an open arrow and that of the SV40 promoter by a solid arrow. Transcripts are shown as partially gray and dashed lines. The dashed regions are only present on transcripts where the retroviral promoter and/or the poly(A) site has been used. Primers used for PCR and RT-PCR are indicated with small arrows and labeled 1–4. Solid lines below the provirus in A and B indicate expected PCR fragments. Solid lines below the transcripts in C–F indicate fragments that are expected to be generated by RT-PCR from spliced and unspliced RNA, respectively. (A and B) PCR analyses of DNA isolated from I-CAT clones 1–9 and CAT clones 1–9 with primers 1 and 2. (C and D) RT-PCR analyses of RNA isolated from I-CAT clones 1–9 and CAT clones 1–9 with the primers 1 and 2. (E) RT-PCR analyses of RNA isolated from I-CAT clones 1–9 with primers 3 and 2. (F) RT-PCR analyses of RNA isolated from CAT clones 1–9 with primers 4 and 2. (G) Comparisons of PCR and RT-PCR analyses shown in A–F. Lanes 2 and 3 show the PCR products with DNA from I-CAT-1 and CAT-1, respectively. Lanes 4 and 5 show the RT-PCR products with RNA from I-CAT-1 and CAT-1 cells, respectively, by primers 1 and 2. Lane 6 shows the RT-PCR product with RNA from I-CAT-1 cells by primers 3 and 2. Lane 7 shows the RT-PCR product with RNA from CAT-1 cells by primers 4 and 2. λ/R+H, Lambda DNA/EcoRI + HindIII markers. The 947-, 1,375-, and 1,587-bp markers (from bottom to top) are indicated by dots to the left of the gel. The reaction products corresponding to the expected fragments are indicated by an arrow to the right of the gel.

Figure 3

PCR analysis of proviruses and RT-PCR analysis of CAT gene transcripts in neoR transduced cell clones. Schematic, not to scale, gene maps of the proviruses are shown above each gel. The transcription initiation site in the 5′LTR is indicated by an open arrow and that of the SV40 promoter by a solid arrow. Transcripts are shown as partially gray and dashed lines. The dashed regions are only present on transcripts where the retroviral promoter and/or the poly(A) site has been used. Primers used for PCR and RT-PCR are indicated with small arrows and labeled 1–4. Solid lines below the provirus in A and B indicate expected PCR fragments. Solid lines below the transcripts in C–F indicate fragments that are expected to be generated by RT-PCR from spliced and unspliced RNA, respectively. (A and B) PCR analyses of DNA isolated from I-CAT clones 1–9 and CAT clones 1–9 with primers 1 and 2. (C and D) RT-PCR analyses of RNA isolated from I-CAT clones 1–9 and CAT clones 1–9 with the primers 1 and 2. (E) RT-PCR analyses of RNA isolated from I-CAT clones 1–9 with primers 3 and 2. (F) RT-PCR analyses of RNA isolated from CAT clones 1–9 with primers 4 and 2. (G) Comparisons of PCR and RT-PCR analyses shown in A–F. Lanes 2 and 3 show the PCR products with DNA from I-CAT-1 and CAT-1, respectively. Lanes 4 and 5 show the RT-PCR products with RNA from I-CAT-1 and CAT-1 cells, respectively, by primers 1 and 2. Lane 6 shows the RT-PCR product with RNA from I-CAT-1 cells by primers 3 and 2. Lane 7 shows the RT-PCR product with RNA from CAT-1 cells by primers 4 and 2. λ/R+H, Lambda DNA/EcoRI + HindIII markers. The 947-, 1,375-, and 1,587-bp markers (from bottom to top) are indicated by dots to the left of the gel. The reaction products corresponding to the expected fragments are indicated by an arrow to the right of the gel.