Adaptation of chimeric retroviruses in vitro and in vivo: isolation of avian retroviral vectors with extended host range

We have designed and characterized two new replication-competent avian sarcoma/leukosis virus-based retroviral vectors with amphotropic and ecotropic host ranges. The amphotropic vector RCASBP-M2C(797-8), was obtained by passaging the chimeric retroviral vector RCASBP-M2C(4070A) (6) in chicken embryos. The ecotropic vector, RCASBP(Eco), was created by replacing the env-coding region in the retroviral vector RCASBP(A) with the env region from an ecotropic murine leukemia virus. It replicates efficiently in avian DFJ8 cells that express murine ecotropic receptor. For both vectors, permanent cell lines that produce viral stocks with titers of about 5 x 10(6) CFU/ml on mammalian cells can be easily established by passaging transfected avian cells. Some chimeric viruses, for example, RCASBP(Eco), replicate efficiently without modifications. For those chimeric viruses that do require modification, adaptation by passage in vitro or in vivo is a general strategy. This strategy has been used to prepare vectors with altered host range and could potentially be used to develop vectors that would be useful for targeted gene delivery.     

Inhibition of infectious murine leukemia virus production by Fv-4 env gene products exerting dominant negative effect on viral envelope glycoprotein

Fv-4 is a mouse gene that confers resistance against ecotropic murine leukemia virus (MLV) infection on mice. While receptor interference by the Fv-4 env gene product, Fv-4 Env, that can bind to the ecotropic MLV receptor has been shown to play an important role in the resistance, other mechanisms have also been suggested because it confers extremely efficient, complete resistance in vivo. Here, we have examined the effect of Fv-4 Env on infectious MLV production. Infectious MLV titers in supernatants obtained after transfection with a Friend MLV (FMLV) Env-expressing plasmid from MLV gag-pol producer cells harboring a retroviral vector were largely reduced by coexpression of Fv-4 Env. Syncytia formation mediated by R-peptide-deleted FMLV Env in NIH 3T3 cells was impaired by Fv-4 Env coexpression. Similarly, Fv-4 Env inhibited infectious amphotropic MLV production and syncytia formation mediated by R-peptide-deleted amphotropic MLV Env. Immunoprecipitation analysis revealed interaction of Fv-4 Env with amphotropic MLV Env as well as FMLV Env. These results indicate that Fv-4 Env inhibits infectious ecotropic and amphotropic MLV production by exerting dominant negative effect on MLV Env, suggesting contribution of this inhibitory effect to the resistance against ecotropic MLV infection in Fv-4-expressing mice.     

Entry of Amphotropic and 10A1 Pseudotyped Murine Retroviruses Is Restricted in Hematopoietic Stem Cell Lines

Although transduction with amphotropic murine leukemia virus (MLV) vectors has been optimized successfully for hematopoietic differentiated progenitors, gene transfer to early hematopoietic cells (stem cells) is still highly restricted. A similar restriction to gene transfer was observed in the mouse stem cell line FDC-Pmix compared with transfer in the more mature myeloid precursor cell line FDC-P1 and the human erythroleukemia cell line K562. Gene transfer was not improved when the vector was pseudotyped with gp70^SU of the 10A1 strain of MLV, which uses the receptor of the gibbon ape leukemia virus (Pit1), in addition to the amphotropic receptor (Pit2). Although 10A1 and amphotropic gp70^SU bound to FDC-P1, K562, and fibroblasts, no binding to FDC-Pmix cells was detected. This indicates that FDC-Pmix cells lack functional Pit2 and Pit1 receptors. Pseudotyping with the vesicular stomatitis virus G protein improved transduction efficiency in FDC-Pmix stem cells by 2 orders of magnitude, to fibroblast levels, confirming a block to retroviral infection at the receptor level.     

Inhibition of infectious murine leukemia virus production by Fv-4 env gene products exerting dominant negative effect on viral envelope glycoprotein

Fv-4 is a mouse gene that confers resistance against ecotropic murine leukemia virus (MLV) infection on mice. While receptor interference by the Fv-4 env gene product, Fv-4 Env, that can bind to the ecotropic MLV receptor has been shown to play an important role in the resistance, other mechanisms have also been suggested because it confers extremely efficient, complete resistance in vivo. Here, we have examined the effect of Fv-4 Env on infectious MLV production. Infectious MLV titers in supernatants obtained after transfection with a Friend MLV (FMLV) Env-expressing plasmid from MLV gag–pol producer cells harboring a retroviral vector were largely reduced by coexpression of Fv-4 Env. Syncytia formation mediated by R-peptide-deleted FMLV Env in NIH 3T3 cells was impaired by Fv-4 Env coexpression. Similarly, Fv-4 Env inhibited infectious amphotropic MLV production and syncytia formation mediated by R-peptide-deleted amphotropic MLV Env. Immunoprecipitation analysis revealed interaction of Fv-4 Env with amphotropic MLV Env as well as FMLV Env. These results indicate that Fv-4 Env inhibits infectious ecotropic and amphotropic MLV production by exerting dominant negative effect on MLV Env, suggesting contribution of this inhibitory effect to the resistance against ecotropic MLV infection in Fv-4-expressing mice.     

Comparison of Rous sarcoma virus RNA processing in chicken and mouse fibroblasts: evidence for double-spliced RNA in nonpermissive mouse cells.

Rous sarcoma virus, an avian retrovirus, transforms but does not replicate in mammalian cells. To determine to what extent differences in RNA splicing might contribute to this lack of productive infection, cloned proviral DNA derived from the Prague A strain of Rous sarcoma virus was transfected into mouse NIH 3T3 cells, and the viral RNA was compared by RNase protection with viral RNA from transfected chicken embryo fibroblasts by using a tandem antisense riboprobe spanning the three major splice sites. The levels of viral RNA in NIH 3T3 cells compared with those in chicken embryo fibroblasts were lower, but the RNA was spliced at increased efficiency. The difference in the ratio of unspliced to spliced RNA levels was not due to the increased lability of unspliced RNA in NIH 3T3 cells. Although chicken embryo fibroblasts contained equal levels of src and env mRNAs, spliced viral mRNAs in NIH 3T3 cells were almost exclusively src. In NIH 3T3 cells the env mRNA was further processed by using a cryptic 5' splice site located within the env coding sequences and the normal src 3' splice site to form a double-spliced mRNA. This mRNA was identical to the src mRNA, except that a 159-nucleotide sequence from the 5' end of the env gene was inserted at the src splice junction. Smaller amounts of single-spliced RNA were also present in which only the region between the cryptic 5' and src 3' splice sites was spliced out. The aberrant processing of the viral env mRNA in NIH 3T3 cells may in part explain the nonpermissiveness of these cells to productive Rous sarcoma virus infection.     

Packaging system for rapid production of murine leukemia virus vectors with variable tropism.

A method for rapidly producing helper-free murine leukemia virus (MLV) without using packaging cell lines is described. Viruses bearing ecotropic or amphotropic MLV or Rous sarcoma virus envelope glycoprotein and containing various retroviral vector genomes have been prepared with titers 30 to 40-fold higher than those produced by transient transfection of standard packaging cells. This system can be used to alter the cellular tropism of MLV by incorporating other envelope glycoproteins and to prepare retroviral vector stocks without establishing stable producer cell lines. This method will be particularly useful for preparing viruses that encode toxic proteins and for the rapid analysis of panels of mutant envelope glycoproteins.     

Characterization of a polytropic murine leukemia virus proviral sequence associated with the virus resistance gene Rmcf of DBA/2 mice

The DBA/2 mouse Rmcf gene is responsible for in vivo and in vitro resistance to infection by the polytropic mink cell focus-forming (MCF) virus subgroup of murine leukemia viruses (MLVs). Previous studies suggested that Rmcf resistance is mediated by expression of an interfering MCF MLV envelope (Env) gene. To characterize this env gene, we examined resistance in crosses between Rmcf(r) DBA/2 mice and Mus castaneus, a species that lacks endogenous MCF env sequences. In backcross progeny, inheritance of Rmcf resistance correlated with inheritance of a specific endogenous MCF virus env-containing 4.6-kb EcoRI fragment. This fragment was present in the DBA/2N substrain with Rmcf-mediated resistance but not in virus-susceptible DBA/2J substrain mice. This fragment contains a provirus with a 5' long terminal repeat and the 5' half of env; the gag and pol genes have been partially deleted. The Env sequence is identical to that of a highly immunogenic viral glycoprotein expressed in the DBA/2 cell line L5178Y and closely resembles the env genes of modified polytropic proviruses. The coding sequence for the full-length Rmcf Env surface subunit was amplified from DNAs from virus-resistant backcross mice and was cloned into an expression vector. NIH 3T3 and BALB 3T3 cells stably transfected with this construct showed significant resistance to infection by MCF MLV but not by amphotropic MLV. This study identifies an Rmcf-linked MCF provirus and indicates that, like the ecotropic virus resistance gene Fv4, Rmcf may mediate resistance through an interference mechanism.     

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.     

Inhibition of RNA splicing at the Rous sarcoma virus src 3' splice site is mediated by an interaction between a negative cis element and a chicken embryo fibroblast nuclear factor.

In permissive Rous sarcoma virus-infected chicken embryo fibroblasts (CEF), approximately equimolar amounts of env and src mRNAs are present. In nonpermissive mammalian cells, the src mRNA level is elevated and env mRNA level is reduced. A cis element in the region between the env gene and the src 3' splice site, which we have termed the suppressor of src splicing (SSS), acts specifically in CEF but not in human cells to reduce src mRNA levels. The splicing inhibition in CEF is not caused by a base-paired structure which is predicted to form between the SSS and the src 3' splice site. To further investigate the mechanism of the inhibition, we have used human HeLa cell nuclear extracts to compare in vitro the rates of splicing of RNA substrates containing the Rous sarcoma virus major 5' splice site and either the env or src 3' splice sites. We show that the src 3' splice site is used approximately fivefold more efficiently than the env 3' splice site. The efficiency of in vitro splicing at the src 3' splice site is specifically reduced by addition of CEF nuclear extract. The inhibition is dependent on the presence of the SSS element and can be abrogated by addition of competitor RNA. We propose that the SSS region represents a binding site for a negative-acting CEF splicing factor(s).     

A Proline-Rich Motif Downstream of the Receptor Binding Domain Modulates Conformation and Fusogenicity of Murine Retroviral Envelopes

The entry of retroviruses into cells depends on receptor recognition by the viral envelope surface subunit SU followed by membrane fusion, which is thought to be mediated by a fusion peptide located at the amino terminus of the envelope transmembrane subunit TM. Several fusion determinants have been previously identified in murine leukemia virus (MLV) envelopes, but their functional interrelationships as well as the processes involved in fusion activation upon retroviral receptor recognition remain unelucidated. Despite both structural and functional similarities of their envelope glycoproteins, ecotropic and amphotropic MLVs display two different postbinding properties: (i) while amphotropic MLVs fuse the cells at neutral pH, penetration of ecotropic MLVs is relatively acid pH dependent and (ii) ecotropic envelopes are more efficient than amphotropic envelopes in inducing cell-to-cell fusion and syncytium formation. By exploiting the latter characteristic in the analysis of chimeras of ecotropic and amphotropic MLV envelopes, we show here that substitution of the ecotropic MLV proline-rich region (PRR), located in the SU between the amino-terminal receptor binding domain and the TM-interacting SU carboxy-terminal domains, is sufficient to revert the amphotropic low-fusogenic phenotype into a high-fusogenic one. Furthermore, we have identified potential β-turns in the PRR that control the stability of SU-TM associations as well as the thresholds required to trigger either cell-to-cell or virus-to-cell fusion. These data, demonstrating that the PRR functions as a signal which induces envelope conformational changes leading to fusion, have enabled us to derive envelopes which can infect cells harboring low levels of available amphotropic receptors.     

Transforming potential of the v-myb oncogene from avian myeloblastosis virus: alterations in the oncogene product may reveal a new target specificity

Transfection of brown leghorn chicken embryo fibroblasts by DNA containing v-myb sequences cloned either in a complete AMV proviral DNA or in a retroviral derived vector has led to the isolation of two kinds of transformed cells. A characterization of the proviral sequences retained and expressed in these transformed cells revealed that they contained either new or altered v-myb-related RNA species. The experiments presented in this paper also show that both types of transformants expressed truncated myb-related polypeptides, suggesting that alterations of the v-myb product may result in a new target specificity, leading to the transformation of chicken embryo fibroblasts.     

A novel truncated env gene isolated from a feline leukemia virus-induced thymic lymphosarcoma

We PCR amplified the exogenous feline leukemia virus (FeLV)-related env gene species from lymphosarcomas induced by intradermally administered plasmid DNA of either the prototype FeLV, subgroup A molecular clone, F6A, or a new molecular clone, FeLV-A, Rickard strain (FRA). Of the nine tumors examined, six showed the presence of deleted env species of variable sizes in the tumor DNA. One env mutant, which was detected in a FRA-induced thymic lymphosarcoma, had a large internal deletion beginning from almost the N-terminal surface glycoprotein (SU) up to the middle region of the transmembrane (TM) protein of the env gene. The deduced polypeptide of this truncated env (tenv) retained the complete signal peptide and seven amino acids of the N-terminal mature SU of FRA env gene, followed by eight amino acids from the frameshift in the TM region. To study the biological function of tenv, we used a murine retrovirus vector to produce amphotropic virions. Infection of feline fibroblasts (H927), human fibrosarcoma cells (HT1080), or human B-lymphoma cells (Raji) led to pronounced cytotoxicity, while the tenv virus did not induce significant cytotoxicity to feline T-lymphoma cells (3201B) or human T-lymphoma cells (CEM). Together, these results convincingly demonstrated that the genetic events that led to truncation in the env gene occurred de novo in FeLV lymphomagenesis and that such a product, tenv could induce cytotoxicity to fibroblastic and B-lymphoid cells but not to T-lymphoid tumor cells. This type of selective toxicity might be potentially important in the development of the neoplastic disease.     

Isolation of microcell hybrid clones containing retroviral vector insertions into specific human chromosomes.

We sought an efficient means to introduce specific human chromosomes into stable interspecific hybrid cells for applications in gene mapping and studies of gene regulation. A defective amphotropic retrovirus was used to insert the gene conferring G418 resistance (neo), a dominant selectable marker, into the chromosomes of diploid human fibroblasts, and the marked chromosomes were transferred to mouse recipient cells by microcell fusion. We recovered five microcell hybrid clones containing one or two intact human chromosomes which were identified by karyotype and marker analysis. Integration of the neo gene into a specific human chromosome in four hybrid clones was confirmed by segregation analysis or by in situ hybridization. We recovered four different human chromosomes into which the G418 resistance gene had integrated: human chromosomes 11, 14, 20, and 21. The high efficiency of retroviral vector transformation makes it possible to insert selectable markers into any mammalian chromosomes of interest.     

Retrovirus-induced spongiform neurodegeneration is mediated by unique central nervous system viral targeting and expression of env alone

Certain murine leukemia viruses (MLVs) can induce progressive noninflammatory spongiform neurodegeneration similar to that caused by prions. The primary MLV determinants responsible have been mapped to within the env gene; however, it has remained unclear how env mediates disease, whether non-Env viral components are required, and what central nervous system (CNS) cells constitute the critical CNS targets. To address these questions, we examined the effect of transplanting engraftable C17.2 neural stem cells engineered to pseudotype, disseminate, and trans-complement neurovirulent (CasBrE, CasE, and CasES) or non-neurovirulent (Friend and SFF-FE) env sequences (SU or SU/TM) within the CNS using either the "non-neurovirulent" amphotropic helper virus, 4070A, or pgag-polgpt (a nonpackaged vector encoding Gag-Pol). These studies revealed that acute MLV-induced spongiosis results from two separable activities of Env. First, Env causes neuropathology through unique viral targeting within the CNS, which was efficiently mediated by ecotropic Envs (CasBrE and Friend), but not 4070A amphotropic Env. Second, Env induces spongiosis through a toxin activity that is MLV-receptor independent and does not require the coexpression of other viral structural proteins. CasBrE and 4070A Envs possess the toxin activity, whereas Friend Env does not. Although the identity of the critical viral target cell(s) remains unresolved, our results appear to exclude microglia and oligodendrocyte lineage cells, while implicating viral entry into susceptible neurons. Thus, MLV-induced disease parallels prionopathies in that a single protein, Env, mediates both the CNS targeting and the toxicity of the infectious agent that manifests itself as progressive vacuolar neurodegeneration.     

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.