Biomedical subjects

B S Schnierle

Publications and source records attributed to B S Schnierle.

11 recordsLinked to original sources

PubMed2001

A packaging cell line generating CD4-specific retroviral vectors for efficient gene transfer into primary human T-helper lymphocytes.

Murine leukemia virus (MuLV) can be pseudotyped with a variant of the human immunodeficency virus (HIV) envelope gene encoding the surface glycoprotein gp120-SU and a carboxy-terminally truncated transmembrane (TM) protein with only seven cytoplasmic amino acids. MuLV/HIV-1 pseudotyped retroviral vectors selectively target gene transfer to human cells expressing both CD4 and CXCR4. To apply this vector system to gene therapy of human diseases, we generated a stable packaging cell line, FLY-HIV-87, expressing the MuLV gag and pol genes and the C-terminally truncated variant of the HIV-1 envelope gene, but no retroviral vector genome. Production of infectious vector particles was tested after the introduction of different vector genomes and was in the range of 5x10(5) IU/ml. The vector particles could be concentrated up to 25-fold. Specific and efficient gene transfer into CD4/CXCR4 expressing cell lines and stimulated primary human CD4+ peripheral blood lymphocytes was achieved. Thus the packaging cell line FLY-HIV-87 is highly suitable for gene therapy of disorders of human T-helper cells.

CD4 Antigens↗

PubMed2001

Membrane-anchored peptide inhibits human immunodeficiency virus entry.

Peptides derived from the heptad repeats of human immunodeficiency virus (HIV) gp41 envelope glycoprotein, such as T20, can efficiently inhibit HIV type 1 (HIV-1) entry. In this study, replication of HIV-1 was inhibited more than 100-fold in a T-helper cell line transduced with a retrovirus vector expressing membrane-anchored T20 on the cell surface. Inhibition was independent of coreceptor usage.

Amino Acid Sequence↗

PubMed1999

Truncation of the human immunodeficiency virus-type-2 envelope glycoprotein allows efficient pseudotyping of murine leukemia virus retroviral vector particles.

The incorporation of human immunodeficiency virus-type-2 (HIV-2) envelope glycoprotein into murine leukemia virus (MuLV) particles was studied in a transient transfection packaging cell system. We observed that wild-type HIV-2 envelope protein or a frameshift mutant with 187 unrelated carboxyl-terminal residues did not allow the formation of infectious retroviral particles. In view of recent findings that an HIV-1 envelope protein variant with a shortened cytoplasmic domain was incorporated into MuLV particles, we constructed carboxyl-terminal truncations of the HIV-2 envelope protein. An envelope variant with 18 cytoplasmic amino acids formed only very few viral pseudotypes. The further removal of an additional 11 amino acids allowed the efficient pseudotyping of MuLV particles. As with the HIV-1 envelope protein, an HIV-2 envelope variant with 7 cytoplasmic amino acids was incorporated into functional MuLV particles. The pseudotyped vectors obtained are able to infect human CD4/CXCR4-expressing cells. Cell lines expressing human CD4 and other coreceptors could not be infected. This retroviral vector will prove useful for the study of HIV infection events mediated by the HIV-2 envelope glycoproteins, as well as for the targeting of CD4+ cells in the context of gene therapy of AIDS.

Animals↗

PubMed1999

Stable expression of the ecotropic retrovirus receptor in amphotropic packaging cells facilitates the transfer of recombinant vectors and enhances the yield of retroviral particles.

Retroviral vectors are used widely as gene transfer vehicles. Vector particles are generated by packaging cell lines, which supply the structural proteins gag, pol and env needed to package the retroviral vector RNA. The most efficient way to introduce the vector genome into the packaging cell line is cross-infection with a retroviral vector. Since the infection of a packaging cell line by the produced virus is blocked due to the down regulation of the retrovirus receptor by the envelope glycoprotein, the vector genome should be introduced by a virus with a host tropism different from the one of the packaging cell line. The murine ecotropic retrovirus receptor was expressed in the human amphotropic packaging cell line FLYA13 to generate a cell line which can be infected by murine ecotropic retroviruses. Vector transfer can now be facilitated by cross-infection with the appropriate ecotropic retroviral vectors and provides a simple and efficient method for the generation of amphotropic packaging lines.

3T3 Cells↗

PubMed1997

Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells.

CD4-expressing T cells in lymphoid organs are infected by the primary strains of HIV and represent one of the main sources of virus replication. Gene therapy strategies are being developed that allow the transfer of exogenous genes into CD4(+) T lymphocytes whose expression might prevent viral infection or replication. Insights into the mechanisms that govern virus entry into the target cells can be exploited for this purpose. Major determinants of the tropism of infection are the CD4 molecules on the surface of the target cells and the viral envelope glycoproteins at the viral surface. The best characterized and most widely used gene transfer vectors are derived from Moloney murine leukemia virus (MuLV). To generate MuLV-based retroviral gene transfer vector particles with specificity of infection for CD4-expressing cells, we attempted to produce viral pseudotypes, consisting of MuLV capsid particles and the surface (SU) and transmembrane (TM) envelope glycoproteins gp120-SU and gp41-TM of HIV type 1 (HIV-1). Full-length HIV-1 envelope glycoproteins were expressed in the MuLV env-negative packaging cell line TELCeB6. Formation of infectious pseudotype particles was not observed. However, using a truncated variant of the transmembrane protein, lacking sequences of the carboxyl-terminal cytoplasmic domain, pseudotyped retroviruses were generated. Removal of the carboxyl-terminal domain of the transmembrane envelope protein of HIV-1 was therefore absolutely required for the generation of the viral pseudotypes. The virus was shown to infect CD4-expressing cell lines, and infection was prevented by antisera specific for gp120-SU. This retroviral vector should prove useful for the study of HIV infection events mediated by HIV-1 envelope glycoproteins, and for the targeting of CD4(+) cells during gene therapy of AIDS.

CD4 Antigens↗

PubMed1996

Expression of chimeric envelope proteins in helper cell lines and integration into Moloney murine leukemia virus particles.

New retroviral constructs with a grafted specificity of infection could become useful gene delivery vehicles with applications in systemic gene therapy. We have constructed retroviral vectors to target gene transfer to human tumor cells. Chimeric envelope proteins have been expressed to obtain viral particles with a defined specificity of infection. Two tumor cell-specific recognition domains were cloned and fused with the viral envelope gene. A recognition domain specific for ErbB-2 expressing tumor cells was derived from a monoclonal antibody directed against the ErbB-2 receptor in the form of a single chain antibody domain (scFv-erbB-2). The receptor binding domain was derived from the heregulin gene (HRG70). This domain provides recognition specificity for ErbB-3 and ErbB-4 receptor expressing tumor cells. The recognition domains were inserted at the amino terminal end into the MoMLV envelope gene. Helper cell lines were established which express the recombinant envelope protein genes, the gag and pol genes and packageable retroviral RNA. The analysis of the helper cell line revealed that the recombinant ErbB-2 scFv-envelope protein was expressed, but not incorporated into viral particles. The scFv-erbB-2 envelope protein was not inserted into the cell membrane and the assembly of retroviral particles was not completed. In contrast, the HRG70-envelope protein was expressed on the surface of the helper cells and incorporated into retroviral particles. The HRG70-envelope protein, however, did not alter the host range of infection. Only cells expressing the ecotropic viral receptor could be infected.

3T3 Cells↗

PubMed1996

Retroviral targeted delivery.

The systemic delivery of genes will open new applications for gene therapy. The deployment of retroviral vectors for this purpose is being considered and requires the development of retroviral vectors with a defined target cell specificity of infection. Several reports have recently described attempts to engineer the envelope protein of murine retroviruses in order to expand the host range and enable them to infect specific human cells. The strategies are based on the introduction of binding sites specific for receptors on the surface of target cells. Although the attempts to manipulate the specificity of viral target cell recognition are ambitious and promising, they are not without pitfalls. We summarize the results obtained and the difficulties encountered.

Gene Products, env↗

PubMed1996

Cytolysis of tumor cells expressing the Neu/erbB-2, erbB-3, and erbB-4 receptors by genetically targeted naive T lymphocytes.

We are developing strategies to use naive T lymphocytes in cancer therapy. For this purpose, we are deriving T cells with specificity of recognition for defined tumor cells. To direct effector lymphocytes toward tumor cells, we have manipulated the recognition specificity of naive rat and mouse T lymphocytes and a mouse T-cell line. The cells were stably transduced with a chimeric T-cell receptor (TCR) component. The zeta chain of the TCR consists of a single transmembrane protein with a short extracellular domain and an intracellular domain for TCR signaling. We provided an extracellular tumor cell recognition domain to the zeta chain. Human heregulin beta1 (ligand to the erbB-3 and erbB-4 receptors) and three different single-chain antibodies specific for the human and rat Neu/erbB-2 receptors were used. One single-chain antibody (C11) is directed against the rat Neu protein, and one single-chain antibody (FRP5) is directed against the human erbB-2 receptor. The single-chain antibody (R-AK) directed against the Mr 14,000 fusion protein of orthopox viruses served as a control. An efficient procedure was devised to introduce the chimeric genes into primary rat and mouse T lymphocytes. Retrovirus-producing packaging cell lines were cocultured with the T cells activated by phytohemagglutinin and interleukin 2. T-cell lines were transduced by exposure to retrovirus-containing supernatants from helper cell lines. Expression of the fusion genes was determined by fluorescence-activated cell sorting analysis. More than 80% of the naive rat and mouse T cells and 85-100% of the cells from the established T-cell lines expressed the fusion genes within 48 h after infection. The expression of the fusion genes was maintained for at least 10 days after infection. Target cells expressing Neu/erbB-2, erbB-3, or erbB-4 were lysed in vitro with high specificity by T cells expressing the corresponding recognition proteins. No selection of a marker gene is necessary to confer a predetermined recognition specificity. The described experiments are important for a gene therapy approach to cancer treatment with autologous T cells.

3T3 Cells↗

PubMed1994

Mutational analysis of a multifunctional protein, with mRNA 5' cap-specific (nucleoside-2'-O-)-methyltransferase and 3'-adenylyltransferase stimulatory activities, encoded by vaccinia virus.

The vaccinia virus-encoded protein VP39 is a poly(A) polymerase subunit that stimulates the formation of long poly(A) tails as well as a cap-specific mRNA (nucleoside-2'-O-)-methyltransferase. We have carried out mutagenesis studies aimed at locating regions of VP39 which are important for these activities. The open reading frame encoding VP39 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. The affinity-purified protein had both mRNA modification activities, before and after removal of the glutathione S-transferase domain. Truncation, charge cluster-->Ala scanning, and Cys-->Ser substitution mutations of VP39 were made, and the proteins were synthesized, purified, and analyzed. Deletion of the RNA binding domain, experimentally localized within the carboxyl-terminal 112 amino acids, resulted in the loss of both mRNA modification activities. Eleven of the 21 charge cluster-->Ala mutated proteins had low to nondetectable methyltransferase activity. Four of those 11 also lacked adenylyl-transferase stimulatory function, whereas the remainder had amino acid substitutions that selectively affected methyltransferase activity. However, no mutated proteins lacking adenylyltransferase stimulatory function but possessing methyltransferase activity were isolated by the procedures used. Neither of the 2 cysteine residues in VP39 was necessary for either mRNA modification activity.

Amino Acid Sequence↗

PubMed1992

Cap-specific mRNA (nucleoside-O2'-)-methyltransferase and poly(A) polymerase stimulatory activities of vaccinia virus are mediated by a single protein.

The vaccinia virus gene for S-adenosyl-L-methionine:mRNA (nucleoside-O2'-)-methyltransferase, an enzyme required for the formation of the 5' cap structure of mRNA, was identified. Protein sequence analysis revealed that this cap-specific methyltransferase is derived from the same open reading frame as that previously shown to encode VP39, a Mr 39,000 dissociable subunit of poly(A) polymerase that stimulates the formation of long poly(A) tails. Consistent with this finding, methyltransferase activity was associated with the heterodimeric poly(A) polymerase, which is composed of VP55 and VP39 subunits, as well as with monomeric VP39 protein isolated from vaccinia virions. In addition, cap-specific nucleoside-O2'-methyltransferase activity is associated with recombinant VP39, which was purified to near homogeneity from mammalian cells. From these data, we concluded that the same protein functions as a methyltransferase and a poly(A) polymerase stimulatory factor to modify the 5' and 3' ends of mRNA, respectively.

Amino Acid Sequence↗

PubMed1992

Vaccinia virus-mediated inhibition of host protein synthesis involves neither degradation nor underphosphorylation of components of the cap-binding eukaryotic translation initiation factor complex eIF-4F.

Recent reports indicated that vaccinia virus late mRNAs contain a unique 5' poly(A) leader sequence and that the in vitro translation of these mRNAs may be relatively cap-independent. These observations led us to examine the possibility that the mechanism of inhibition of host protein synthesis by vaccinia virus, like that of certain other viruses, involves specific modifications of the cap-binding translation initiation factor complex eIF-4F. The eIF-4F complex was affinity-purified from human cells infected with vaccinia virus and analyzed by one- and two-dimensional electrophoresis and immunoblotting. No evidence of vaccinia virus-induced degradation of p220, as occurs during poliovirus infection, or alteration of phosphorylation of eIF-4E (p24), as occurs during adenovirus infection, was detected at the time of severe inhibition of host protein synthesis.

Eukaryotic Initiation Factor-4F↗