Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors.

We wished to construct cell lines that supply the gene products of gag, pol, and env for the growth of replication-defective reticuloendotheliosis retrovirus vectors without production of the helper virus. To do this, first we located by S1 mapping the donor and acceptor splice sites of reticuloendotheliosis virus strain A. The donor splice site is ca. 850 base pairs from the 5' end of proviral DNA. It is close to or overlaps the encapsidation sequences for viral RNA. The splice acceptor site is ca. 5.6 kilobase pairs from the 5' end of proviral DNA. Therefore, the encapsidation sequences and the donor splice site were removed from viral DNA to give expression of the gag and pol genes without virus production. The promoter in the long terminal repeat was fused to a site near the first ATG codon of the env gene, thereby deleting the encapsidation sequences and the gag and pol genes to give expression of the env gene without virus production. The permissive canine cell line D17 was transfected with the two modified viral DNAs. Two cell clones that contain both modified viral DNAs support the production of replication-defective spleen necrosis virus-thymidine kinase recombinant retrovirus vectors without the production of helper virus. To prevent recombination, the vector contains deletions that overlap with deletions in the integrated helper virus DNAs. This helper cell-vector system will be useful to derive infectious recombinant virus stocks of high titer (over 10(5) thymidine kinase transforming units per ml) which are able to infect avian, rat, and dog cells without the aid of helper virus.     

Improved retroviral vectors for gene transfer and expression

We describe a set of murine retrovirus-based vectors that include unique cloning sites for insertion of cDNAs such that the cDNA can be driven by either the retroviral long terminal repeat, the immediate early promoter of human cytomegalovirus, or the simian virus 40 early promoter. The vectors carry the neomycin phosphotransferase gene expressed from an alternate promoter as a selectable marker. These vectors have been constructed to prevent viral protein synthesis from the remaining viral sequences, to yield high-titer virus stocks after introduction into retrovirus packaging cells, and to eliminate homologous overlap with viral DNAs present in retrovirus packaging cells in order to prevent helper virus production. Methods for generating high-titer virus are described.     

A transient three-plasmid expression system for the production of high titer retroviral vectors.

We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium butyrate to further increase expression of each component, we achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components. This system can be used both for the generation of high titer retroviral stocks for transduction and for the rapid screening of a large number of MLV gag-pol or env mutants.     

Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.

Retrovirus vectors can be made in the absence of helper virus by using retrovirus packaging cell lines. Helper-free virus is critical for a variety of gene transfer studies. The most useful packaging cell lines contain helper virus DNA from which the signal required for packaging of the viral RNA genome into virions has been deleted. However, we showed that the ability to package virus is conferred at very low frequency to cells infected with virus from these packaging cell lines, presumably by low-frequency transmission of the deleted virus genome. In addition, these packaging cell lines can interact with some retroviral vectors to yield replication-competent virus. We constructed packaging cell lines containing helper virus DNA that had several alterations in addition to deletion of the packaging signal. The new packaging cells retained the useful features of previously available lines but did not yield helper virus after introduction of any of the vectors tested, and transfer of the packaging function was not detected.     

Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus.

We have constructed hybrid retrovirus packaging cell lines that express the gibbon ape leukemia virus env and the Moloney murine leukemia virus gag-pol proteins. These cells were used to produce a retrovirus vector at over 10(6) CFU/ml, with a host range that included rat, hamster, bovine, cat, dog, monkey, and human cells. The gag-pol and env expression plasmids were separately transfected to reduce the potential for helper virus production, which was not observed. The NIH 3T3 mouse cells from which the packaging lines were made are not infectable by gibbon ape leukemia virus; thus, the generation and spread of possible recombinant viruses in the packaging cells is greatly reduced. These simian virus-based packaging cells extend the host range of currently available murine and avian packaging cells and should be useful for efficient gene transfer into higher mammals.     

The spleen focus-forming virus (SFFV) envelope gene, when introduced into mice in the absence of other SFFV genes, induces acute erythroleukemia.

Previous studies in our laboratory and others have been consistent with the hypothesis that the envelope (env) gene of the spleen focus-forming virus (SFFV) is the only gene essential for the induction of acute erythroleukemia. However, no studies have been carried out with the SFFV env gene in the complete absence of other SFFV sequences. To accomplish this goal, we isolated the sequences that encode the envelope glycoprotein, gp52, of SFFVA and expressed them in a Moloney murine leukemia virus-based double-expression vector containing the neomycin resistance gene. The method used to produce retrovirus stocks in tissue culture cells affected the expression of the gp52 gene in the vector and the subsequent pathogenicity of the vector in mice. Highly pathogenic virus stocks were obtained by cotransfection of vector and helper virus DNAs into fibroblasts, followed by virus replication and spread through the cell population. Mice infected with this stock developed a rapid erythroid disease that was indistinguishable from that induced by the entire SFFV genome, and the virus stock transformed erythroid cells in vitro. Spleen cells from the diseased mice expressed the SFFV env gene product but not the SFFV gag gene product. As expected, mice given the virus containing the SFFV env gene in the reverse orientation did not express the SFFV env gene product or develop erythroleukemia. This study, therefore, demonstrated (i) that double-expression retroviral vectors can be used under specific conditions to produce viruses expressing high levels of a particular gene and (ii) that incorporation of the SFFV env gene into such a vector in the absence of other SFFV sequences results in a retrovirus which is as pathogenic as the original SFFV.     

Q vectors, bicistronic retroviral vectors for gene transfer

We have developed a retroviral vector that incorporates unique features of some previously described vectors. This vector includes: 3' long terminal repeats (LTRs) of the self-inactivating class; a 5' LTR that is a hybrid of the cytomegalovirus (CMV) enhancer and the mouse sarcoma virus promoter; an internal CMV immediate early region promoter to drive expression of the transduced gene and the neomycin phosphotransferase selectable marker; an expanded multiple cloning site and an internal ribosome entry site. An SV40 ori was introduced into the vector backbone to promote high copy number replication in packaging cell lines that express the SV40 large T antigen. We demonstrate that these retroviral constructs, designated Q vectors, can be used in applications where high viral titers and high level stable or transient gene expression are desirable.     

An array of murine leukemia virus-related elements is transmitted and expressed in a primate recipient of retroviral gene transfer.

Direct RNA-PCR analyses of T-cell lymphomas that developed in rhesus macaques during a gene transfer experiment revealed the presence of several different recombinant murine leukemia viruses (MuLV). Most prominent was the expected MuLV recombinant, designated MoLTRAmphoenv in which the amphotropic env of the helper packaging virus was joined to the long terminal repeat (LTR) of the Moloney MuLV-derived vector. This retrovirus does not exist in nature. An additional copy of the core enhancer acquired from the vector LTR may have augmented the replicative properties of MoLTRAmphoenv MuLV in several different rhesus cell types compared with the prototype amphotropic MuLV4070A. Unexpectedly, at least two types of mink cell focus-forming MuLV elements, arising from endogenous retroviral sequences expressed in the murine packaging cell line, were also transmitted and highly expressed in one of the macaques. Furthermore, murine virus-like VL-30 sequences were detected in the rhesus lymphomas, but these were not transcribed into RNA. The unanticipated presence of an array of MuLV-related structures in a primate gene transfer recipient demands ever-vigilant scrutiny for the existence of transmissible retroviral elements and replication-competent viruses possessing altered tropic or growth properties in packaging cells producing retroviral vectors.     

Packaging Cells Based on Inducible Gene Amplification for the Production of Adeno-Associated Virus Vectors

Although vectors based on adeno-associated virus (AAV) offer several unique advantages, their usage has been hampered by the difficulties encountered in vector production. In this report, we describe a new AAV packaging system based on inducible amplification of integrated helper and vector constructs containing the simian virus 40 (SV40) replication origin. The packaging and producer cell lines developed express SV40 T antigen under the control of the reverse tetracycline transactivator system, which allows inducible amplification of chromosomal loci linked to the SV40 origin. Culturing these cells in the presence of doxycycline followed by adenovirus infection resulted in helper and vector gene amplification as well as higher vector titers. Clonal producer cell lines generated vector titers that were 10 times higher than those obtained by standard methods, with approximately 10⁴ vector particles produced per cell. These stocks were free of detectable replication-competent virus. The lack of a transfection step combined with the reproducibility of stable producer lines makes this packaging method ideally suited for the large-scale production of vector stocks for human gene therapy.     

Comparison of promoter suppression in avian and murine retrovirus vectors.

Previously, we described "promoter suppression" in infectious retrovirus vectors with two genes and an internal promoter. Here, we examined several parameters of promoter suppression and found that the amount of suppression in an integrated retrovirus vector was dependent both on whether the vector was derived from spleen necrosis virus or murine leukemia virus and on which internal promoter was present in the vector. Murine leukemia virus vectors showed less suppression than analogous spleen necrosis virus vectors. Furthermore, the amount of suppression was not dependent on either the relative strengths of the promoters nor the distance between the promoters. Moreover, we found that in vectors in which one promoter was suppressed, there was an inverse correlation between the DNaseI sensitivity of the chromatin surrounding a promoter and the suppression of its expression.     

Packaging cell lines for simian foamy virus type 1 vectors

Foamy viruses are nonpathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. We have previously demonstrated the utility of simian foamy virus type 1 (SFV-1) as a vector system by transient expression assay (M. Wu et al., J. Virol. 72:3451-3454, 1998). In this report, we describe the first stable packaging cell lines for foamy virus vectors based on SFV-1. We developed two packaging cell lines in which the helper DNA is placed under the control of either a constitutive cytomegalovirus (CMV) immediate-early gene or inducible tetracycline promoter for expression. Although the constitutive packaging expressing cell line had a higher copy number of packaging DNA, the inducible packaging cell line produced four times more vector particles. This result suggested that the structural gene products in the constitutively expressing packaging cell line were expressed at a level that is not toxic to the cells, and thus vector production was reduced. The SFV-1 vector in the presence of vesicular stomatitis virus envelope protein G (VSV-G) produced an insignificant level of transduction, indicating that foamy viruses could not be pseudotyped with VSV-G to generate high-titer vectors. The availability of stable packaging cell lines represents a step toward the use of an SFV-1 vector delivery system that will allow scaled-up production of vector stocks for gene therapy.     

In vivo analysis of a new lacZ retrovirus vector suitable for cell lineage marking in avian and other species

To obtain a replication-defective retrovirus vector well suited for cell lineage marking in early avian embryos, we have constructed and tested a derivative of the avian spleen necrosis virus (SNV) carrying the marker gene lacZ. Consistently high titers of this virus, designated CXL, were produced from retroviral packaging cells with no evidence of contaminating helper virus even after 12 months of continuous culture. CXL expresses lacZ strongly and stably in avian cells and has a host range that extends to other avian and some mammalian species. We show that CXL has the potential to mark a wide variety of chick embryo cell types by infection in ovo. The high titers obtainable with this virus can provide a significant advantage over alternative lacZ vectors, especially in lineage marking of early stage embryos. As an example of this, we show that CXL can be used to mark cells of the precardiac mesoderm in stage 4-5 chick embryos.     

Single-step conversion of cells to retrovirus vector producers with herpes simplex virus-Epstein-Barr virus hybrid amplicons

We report here on the development and characterization of a novel herpes simplex virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV-Epstein-Barr virus (EBV) hybrid amplicons to efficiently convert cells to retrovirus vector producer cells after single-step transduction. The retrovirus genes gag-pol and env (GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV hybrid amplicon. Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. Retrovirus vector sequences encoding lacZ were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 10(6) and 10(7) transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery.     

Expression of human endogenous retrovirus type K envelope glycoprotein in insect and mammalian cells.

The human endogenous retrovirus type K (HERV-K) family codes for the human teratocarcinoma-derived retrovirus (HTDV) particles. The existence of the envelope protein (ENV) of HERV-K encoded by the subgenomic env mRNA has not yet been demonstrated. To study the genetic requirements for successful expression of ENV, we have constructed a series of recombinant HERV-K env expression vectors for infection and transfection experiments in insect cells and mammalian cells, respectively. Six baculovirus constructs bearing full-length or truncated HERV-K env with or without homologous or heterologous signal peptides were used for infections of insect cells. All recombinant baculoviruses yielded ENV proteins with the expected molecular masses. The full-length 80- to 90-kDa HERV-K ENV protein including the cORF leader sequence was glycosylated in insect cells. In addition, the 14-kDa cORF protein was expressed due to splicing of the full-length env mRNA. The ENV precursor protein is not cleaved to the surface (SU) and transmembrane (TM) glycoproteins; it does not appear on the surface of infected insect cells and is not secreted into the medium. For ENV expression in COS cells, plasmid vectors harboring the cytomegalovirus immediate-early promoter/intron A element and the tissue plasminogen activator (t-PA) signal peptide or the homologous HERV-K signal peptide upstream of the env gene were employed. Glycosylated and uncleaved ENV was expressed as in GH teratocarcinoma cells but at higher levels. The heterologous t-PA signal sequence was instrumental for expression of HERV-K ENV on the cell surface. Hence, we have shown for the first time that the HERV-K env gene has the potential to be expressed as a full-length envelope protein with appropriate glycosylation. In addition, our data provide explanations for the lack of infectivity of HERV-K/HTDV particles.     

Fowlpox virus recombinants expressing the envelope glycoprotein of an avian reticuloendotheliosis retrovirus induce neutralizing antibodies and reduce viremia in chickens.

Eight stable fowlpox virus (FPV) recombinants which express the envelope glycoprotein of the spleen necrosis virus (SNV) strain of reticuloendotheliosis virus (REV), an avian retrovirus, were constructed. These recombinants differ in the genomic location of the inserted genes, in the orientation of the insert relative to flanking viral sequences, and in the promoter used to drive expression of the env gene. Of these variables, promoter strength seems to be the most crucial. The P7.5 promoter of vaccinia virus, which is commonly used in the construction of both vaccinia virus and FPV recombinants, resulted in lower levels of expression of the envelope antigen in infected chicken cells compared with a strong synthetic promoter, as determined by immunofluorescence and enzyme-linked immunosorbent assay. Two peptides encoded by the env gene, the 21-kDa transmembrane peptide and a 62-kDa precursor, were detected by immunoprecipitation of labeled proteins from cells infected with recombinant FPVs, using monoclonal antibodies against REV. These peptides comigrated with those precipitated from REV-infected cells. One of the recombinants (f29R-SNenv) was used for vaccination of 1-day-old chickens. Vaccinated chicks developed neutralizing antibodies to SNV more rapidly than did unvaccinated controls following SNV challenge and were protected against both viremia and the SNV-induced runting syndrome.     

Expression from an internal AUG codon of herpes simplex thymidine kinase gene inserted in a retrovirus vector.

We identified structural features that affect the expression of an exogenous gene inserted into a retrovirus vector constructed by using spleen necrosis virus, an avian retrovirus. The thymidine kinase gene from herpes simplex virus type 1 containing deletions in the promoter and terminal sequences of the mRNA was inserted into spleen necrosis virus. We found that synthesis of thymidine kinase by the recovered virus was apparently initiated from internal AUG residues. At least in some cases, however, the level of expression depended on the number of AUGs and the nucleotide sequence around the AUGs that preceded the initiator codon of the thymidine kinase gene.