Overlapping enhancer/promoter and transcriptional termination signals in the lentiviral long terminal repeat

We have studied the effects associated with two single amino acid substitution mutations in HIV-1 capsid (CA), the E98A and E187G. Both amino acids are well conserved among all major HIV-1 subtypes. HIV-1 infectivity is critically dependent on proper CA cone formation and mutations in CA are lethal when they inhibit CA assembly by destabilizing the intra and/or inter molecular CA contacts, which ultimately abrogate viral replication. Glu98, which is located on a surface of a flexible cyclophilin A binding loop is not involved in any intra-molecular contacts with other CA residues. In contrast, Glu187 has extensive intra-molecular contacts with eight other CA residues. Additionally, Glu187 has been shown to form a salt-bridge with Arg18 of another N -terminal CA monomer in a N-C dimer. However, despite proper virus release, glycoprotein incorporation and Gag processing, electron microscopy analysis revealed that, in contrast to the E187G mutant, only the E98A particles had aberrant core morphology that resulted in loss of infectivity.     

Transcriptional activation of the human immunodeficiency virus long terminal repeat sequences by cis-platin

We constructed a recombinant plasmid, pBHIV1 carrying the long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1), linked to the chloramphenicol acetyl transferase (CAT) gene plasmid. Plasmid pBHIV1 also contains the aminoglycoside phosphotransferase gene as a selectable marker. We introduced pBHIV1 in rat 208F fibroblasts and obtained stable geneticin resistant RFBHIV1-1 transfectant cells. A further control used was plasmid p202A, which carries the mutant T24 H-ras1 promoter linked to the promotorless cat gene. Plasmid p202A also carries the aph gene as a selectable marker and was transfected into 208F cells to obtain stable transfectant RF202A-1 cells. Both RFBHIV1-1 and RF202A-1 cells expressed CAT activity from the HIV LTR and T24 H-ras1 promoters. The response to cis-platin, a platin derivative and hexadecyl-phosphocholine was studied on the HIV LTR and H-ras1 regulated CAT activity in RFBHIV1-1 and RF202A-1 cells. It was found that at 5 x 10(-5) M concentrations cis-platin stimulates by 22-fold the expression of CAT from the HIV LTR, whereas only a 4-fold stimulation was observed on the T24 H-ras1 promoter. Our results suggest caution against therapy including this compound at cytotoxic concentrations in the treatment of AIDS patients.     

Characterization of enhancer elements in the long terminal repeat of Moloney murine sarcoma virus

A series of recombinant molecules were constructed which direct the expression of the easily assayed gene chloramphenicol acetyltransferase. We have used these recombinants to show that the 73/72-base-pair tandem repeat unit from the Moloney murine sarcoma virus long terminal repeat shares a number of properties with the prototypic enhancer element, the simian virus 40 72-base-pair repeat. Specifically, the Moloney murine sarcoma virus sequence significantly enhances the level of gene expression at both 5' and 3' locations and in either orientation relative to the test gene. It is able to enhance gene activity both from its own promoter and from a heterologous (simian virus 40) promoter. The 73/72-base-pair subunits of the Moloney murine sarcoma virus enhancer differ in sequence by four nucleotides and also in the strength of their enhancer function. The promoter distal A repeat is at least three times as active as the promoter proximal B repeat in enhancing chloramphenicol acetyltransferase expression. Results of these studies also show that the enhancer sequence alone is unable to induce gene activity but requires other promoter elements, including a proximal GC-rich sequence and the Goldberg-Hogness box.     

Transcriptional targeting of B cells with viral vectors

In order to optimize viral gene transfer into hematopoietic stem cells we developed retroviral and lentiviral vectors with B cell-specificity. Using fragments of the human CD19 promoter we demonstrate in mice that upon lethal irradiation and reconstitution with virus-treated bone marrow transgene expression is specific for the B cell-lineage. We compare various viral constructs with different promoter length and with or without B cell-specific enhancer regions in retro- and lentiviral backbones. Our data suggest that B cell-targeting for gene therapy approaches is feasible, leads to stable expression, and can be modulated by using different transduction and expression systems.     

At least two nuclear proteins bind specifically to the Rous sarcoma virus long terminal repeat enhancer.

We used the sensitive gel electrophoresis DNA-binding assay and DNase I footprinting to detect at least two protein factors (EFI and EFII) that bound specifically to the Rous sarcoma virus (RSV) enhancer in vitro. These factors were differentially extracted from quail cell nuclei, recognized different nucleotide sequences in the U3 region of the RSV long terminal repeat, and appeared to bind preferentially to opposite DNA strands as monitored by the DNase I protection assay. The EFI- and EFII-protected regions within U3 corresponded closely to sequences previously demonstrated by deletion mutagenesis to be required for enhancer activity, strongly suggesting a functional significance for these proteins. Only weak homologies between other enhancer consensus sequence motifs and the EFI and EFII recognition sites were observed, and other viral enhancers from simian virus 40 and Moloney murine sarcoma virus did not compete effectively with the RSV enhancer for binding either factor.     

Bystander macrophages silence transgene expression driven by the retroviral long terminal repeat

The Moloney murine leukemia virus (MLV)-based retroviral vector has been widely used for transfer of exogenous genes to various organs and tissues. Although the long terminal repeat (LTR) of MLV allows for transgene expression in a wide range of cell type, its activity is often silenced in vivo. In reporter macrophages transduced with a MLV-based retroviral vector, activity of the LTR was transiently and reversibly suppressed following stimulation by lipopolysaccharide (LPS). When unstimulated reporter macrophages were co-cultured with LPS-stimulated, untransduced macrophages, the LTR activity was similarly depressed. Activity of the LTR in retrovirus-transduced, mesangial cells was also down-regulated when co-cultured with activated macrophages. This suppressive effect was reproduced by cross-feeding with culture media conditioned by activated macrophages. LPS-stimulated macrophages abundantly expressed cytokines including IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1). When externally added, TNF-alpha and/or TGF-beta1, but not IL-1beta, depressed activity of the LTR in reporter macrophages and reporter mesangial cells. These results raise a possibility that expression of transgenes driven by the MLV-LTR may be silenced in vivo when the retrovirally-transduced cells are co-localized with activated macrophages.     

Surface-engineering of lentiviral vectors

Vectors derived from retroviridae offer particularly flexible properties in gene transfer applications given the numerous possible associations of various viral surface glycoproteins (determining cell tropism) with different types of retroviral cores (determining genome replication and integration). Lentiviral vectors should be preferred gene delivery vehicles over vectors derived from onco-retroviruses such as murine leukemia viruses (MLVs) that cannot transduce non-proliferating target cells. Generating lentiviral vectors pseudotyped with different viral glycoproteins (GPs) may modulate the physicochemical properties of the vectors, their interaction with the host immune system and their host range. There are however important gene transfer restrictions to some non-proliferative tissues or cell types and recent studies have shown that progenitor hematopoietic stem cells in G(0), non-activated primary blood lymphocytes or monocytes were not transducible by lentiviral vectors. Moreover, lentiviral vectors that have the capacity to deliver transgenes into specific tissues are expected to be of great value for various gene transfer applications in vivo. Several innovative approaches have been explored to overcome such problems that have given rise to novel concepts in the field and have provided promising results in preliminary evaluations in vivo. Here we review the different approaches explored to upgrade lentiviral vectors, aiming at developing vectors suitable for in vivo gene delivery.     

Mapping of functional regions of murine retrovirus long terminal repeat enhancers: enhancer domains interact and are not independent in their contributions to enhancer activity.

We have used deletion and recombinant long terminal repeat (LTR) mutants to examine enhancer activity differences between LTRs of the nonpathogenic Akv and the thymus lymphomagenic MCF13 murine retroviruses. Deletion mutant analysis revealed that major control regions for MCF13 and Akv LTR enhancer activity were similar but not identical. For both LTRs, major control regions were distinctly different in a murine T-cell and a fibroblast cell line. Recombinant enhancer analysis showed that LTRs could be divided into three regions capable of altering the level of enhancer activity through cooperative or antagonistic interaction. The contribution of each region to enhancer activity was dependent on its context with respect to the other regions. LTR enhancer function in different cell types appears to be the result of the interaction of enhancer modular elements.