Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors.

Retroviral vectors with self-inactivating (SIN) long-terminal repeats not only increase the autonomy of the internal promoter but may also reduce the risk of insertional upregulation of neighboring alleles. However, gammaretroviral as opposed to lentiviral packaging systems produce suboptimal SIN vector titers, a major limitation for their clinical use. Northern blot data revealed that low SIN titers were associated with abundant transcription of internal rather than full-length transcripts in transfected packaging cells. When using the promoter of Rous sarcoma virus or a tetracycline-inducible promoter to generate full-length transcripts, we obtained a strong enhancement in titer (up to 4 x 10(7) transducing units per ml of unconcentrated supernatant). Dual fluorescence vectors and Northern blots revealed that promoter competition is a rate-limiting step of SIN vector production. SIN vector stocks pseudotyped with RD114 envelope protein had high transduction efficiency in human and non-human primate cells. This study introduces a new generation of efficient gammaretroviral SIN vectors as a platform for further optimizations of retroviral vector performance.

Towards hematopoietic stem cell-mediated protection against infection with human immunodeficiency virus.

The failure of pharmacological approaches to cure infection with the human immunodeficiency virus (HIV) has renewed the interest in gene-based therapies. Among the various strategies that are currently explored, the blockade of HIV entry into susceptible T cells and macrophages promises to be the most powerful intervention. For long-term protection of both of these lineages, genetic modification of hematopoietic stem cells (HSCs) would be required. Here, we tested whether HSCs and their progeny can be modified to express therapeutic levels of M87o, a gammaretroviral vector encoding an artificial transmembrane molecule that blocks fusion-mediated uptake of HIV. In serial murine bone marrow transplantations, efficient and multilineage expression of M87o was observed for more than 1 year (range 37-75% of mononuclear cells), without signs of toxicity related to the transmembrane molecule. To allow enrichment of M87o-modified HSCs after transplant, we constructed vectors coexpressing the P140K mutant of O(6)-methylguanine-DNA-methyltransferase (MGMT-P140K). This clinically relevant selection marker mediates a survival advantage in HSCs if exposed to combinations of methylguanine-methyltransferase (MGMT) inhibitors and alkylating agents. A bicistronic vector mediated sufficient expression of both M87o and MGMT to confer a selective survival advantage in the presence of HIV and alkylating agents, respectively. These data encourage further investigations in large animal models and clinical trials.

Woodchuck hepatitis virus post-transcriptional regulatory element deleted from X protein and promoter sequences enhances retroviral vector titer and expression.

Introduction of the post-transcriptional regulatory element (PRE) of woodchuck hepatitis virus (WHV) into the 3' untranslated region of retroviral and lentiviral gene transfer vectors enhances both titer and transgene expression. Optimal use of the PRE is often necessary to obtain vectors with sufficient performance for therapeutic applications. The enhancing activity of the PRE depends on the precise configuration of its sequence and the context of the vector and cell into which it is introduced. However, data obtained in the context of WHV-associated hepatocellular carcinomas suggests that the PRE might potentially contribute to tumorigenesis, especially if encoding a truncated version of the WHV X protein. Oncogenic side effects of lentiviral vectors containing the PRE have reinforced these safety concerns, although a causal role of the PRE remained unproven. Here, we demonstrate that PRE mutants can be generated that are devoid of X protein open reading frames (ORFs) as well as other ORFs exceeding 25 amino acids, without significant loss of RNA enhancement activity. Furthermore, the X protein promoter could be deleted without compromising the enhancement of vector titers and transgene expression. Such a modified PRE sequence appears useful for future vector design.

Self-inactivating retroviral vectors with improved RNA processing.

Three RNA features have been identified that elevate retroviral transgene expression: an intron in the 5' untranslated region (5'UTR), the absence of aberrant translational start codons and the presence of the post-transcriptional regulatory element (PRE) of the woodchuck hepatitis virus in the 3'UTR. To include such elements into self-inactivating (SIN) vectors with potentially improved safety, we excised the strong retroviral promoter from the U3 region of the 3' long terminal repeat (LTR) and inserted it either downstream or upstream of the retroviral RNA packaging signal (Psi). The latter concept is new and allows the use of an intron in the 5'UTR, taking advantage of retroviral splice sites surrounding Psi. Three LTR and four SIN vectors were compared to address the impact of RNA elements on titer, splice regulation and transgene expression. Although titers of SIN vectors were about 20-fold lower than those of their LTR counterparts, inclusion of the PRE allowed production of more than 10(6) infectious units per ml without further vector optimizations. In comparison with state-of-the-art LTR vectors, the intron-containing SIN vectors showed greatly improved splicing. With regard to transgene expression, the intron-containing SIN vectors largely matched or even exceeded the LTR counterparts in all cell types investigated (embryonic carcinoma cells, fibroblasts, primary T cells and hematopoietic progenitor cells).

In vitro transcription of PrfA-dependent and -independent genes of Listeria monocytogenes.

In vitro transcription starting from the promoters of the Listeria monocytogenes genes hly, plcA, actA, mpl, prfA and iap has been studied. Whereas transcription from Phly, PplcA and PactA is strictly PrfA-dependent, that from Piap, PprfA1/2 and, unexpectedly, also from Pmpl is independent. Initiation of in vitro transcription at all tested promoters except PprfA requires high concentrations of ATP but not GTP. The nucleotides required in higher concentrations for efficient in vitro transcription are always included in the first three nucleotides of the corresponding transcript. RNA polymerase prepared from L. monocytogenes cultured either in rich culture medium (RNAP(BHI)), exposed to heat shock conditions (RNAP48) or conditioned in minimal essential medium (RNAP(MEM)) shows significant differences in the transcription efficiencies when transcription is initiated at these promoters. Transcription starting from the PrfA-dependent promoters PactA and Phly is enhanced with RNAP48 and RNAP(MEM) (in relation to Piap-mediated transcription), while transcription from the other promoters is reduced when compared with RNAP(BHI). These data suggest that in vivo transcription of the genes actA and hly may not function optimally with RNA polymerase loaded with the vegetative sigma factor 43, but may require a modified RNA polymerase, possibly loaded with an alternative sigma factor.

A new RNA element located in the coding region of a murine endogenous retrovirus can functionally replace the Rev/Rev-responsive element system in human immunodeficiency virus type 1 Gag expression.

Nuclear export of incompletely spliced RNAs is a prerequisite for retroviral replication. Complex retroviruses like human immunodeficiency virus (HIV) encode a viral transport factor (Rev), which binds to its target sequence on the RNA genome and directs it into the Crm-1-mediated export pathway. Other retroviruses, like Mason-Pfizer monkey virus, contain cis-acting constitutive RNA transport elements (CTE) which achieve nuclear export of intron-containing RNA via cellular transport factors. Here, we describe the identification and characterization of a novel cis-acting orientation-dependent RNA expression element in the coding region of the murine intracisternal A-type particle (IAP) MIA14. This IAP expression element (IAPE) can functionally replace the Rev system in the expression of HIV-1 Gag proteins but functions independently of Crm-1. The presence of this element is needed for the expression of the IAP Gag proteins, indicating its biological significance. The IAPE can be functionally replaced by placing a CTE on the MIA14 RNA, further supporting its role in mRNA export. Northern blot analysis revealed that total RNA, as well as cytoplasmic RNA, was increased when the element was present. The element was mapped to a predicted stem-loop structure in the 3' part of the pol open reading frame. There was no overall homology between the IAPE and the CTE, but there was complete sequence identity between short putative single-stranded loops. Deletion of these loops from the IAPE severely reduced Rev-independent Gag expression.

Context dependence of different modules for posttranscriptional enhancement of gene expression from retroviral vectors.

We present a systematic comparison of three modules that enhance expression from retroviral gene transfer vectors at a posttranscriptional level: (i) splice signals (SS) that create an intron in the 5' untranslated region; (ii) constitutive RNA transport elements (CTE), originally discovered in D-type retroviruses; and (iii) the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE). Here we show that enhancement of expression depends not only on the specific element, but also on the gene of interest, implying context-dependent activity of the RNA elements. Interestingly, different results were obtained for genes that normally require or do not require such control elements. Expression of the HIV-1 gag-protease gene, which normally depends on the viral export factor Rev, was strongly enhanced by an oligomeric CTE, while WPRE had only a marginal effect. On the other hand, both CTE and WPRE compensated for the lack of an intron in the expression of human beta-globin. In this case, the strongest stimulation of RNA production was observed when functional SS were combined with the WPRE. Both CTE and, in particular, WPRE also enhanced expression of cDNAs that do not normally require any such element (green fluorescent protein, human multidrug resistance-1). In this study, functional SS and WPRE acted in an additive manner, resulting in a 10-fold higher level of expression. Our results indicate that the described modules act on different levels of RNA processing, transport, and translation and that the correct choice of a posttranscriptional enhancer configuration depends on the type of cDNA to be expressed.

PrfA mediates specific binding of RNA polymerase of Listeria monocytogenes to PrfA-dependent virulence gene promoters resulting in a transcriptionally active complex.

There is accumulating evidence that the coordinate transcription of the virulence genes in Listeria monocytogenes constitutes a very complex regulation mechanism which might require other factors in addition to PrfA. We previously described an unknown proteinaceous component from crude bacterial cell extracts, which, together with PrfA, formed a specific complex (CI) in electrophoretic mobility shift assays (EMSA) with an hly promoter probe. Here we identify the RNA polymerase (RNAP) of L. monocytogenes as an essential component of the CI complex. Addition of purified RNAP plus PrfA to the hly promoter probe allowed reconstitution of a complex migrating at the same height as CI. By using EMSA and DNaseI footprint experiments it could be shown that PrfA leads to an enhanced and specific binding of RNAP. Transcriptional activity of RNAP in vitro, using the actA promoter, was strictly dependent on PrfA.

Stability of the Agrobacterium tumefaciens VirB10 protein is modulated by growth temperature and periplasmic osmoadaption.

Export of oncogenic T-DNA from the phytopathogen Agrobacterium tumefaciens is mediated by the products of the virB operon. It has recently been reported (K. J. Fullner and E. W. Nester, J. Bacteriol. 178:1498-1504, 1996) that DNA transfer does not occur at elevated temperatures; these observations correlate well with much earlier studies on the temperature sensitivity of crown gall tumor development on plants. In testing the hypothesis that this loss of DNA movement reflects a defect in assembly or maintenance of a stable DNA transfer machinery at high temperature, we have found that steady-state levels of VirB10 are sensitive to growth temperature while levels of several other VirB proteins are considerably less affected. This temperature-dependent failure to accumulate VirB10 is exacerbated in an attachment-deficient mutant strain (chvB) which exhibits pleiotropic defects in periplasmic osmoadaption, and virulence of a chvB mutant can be partially restored by lowering the temperature at which the bacteria and the plant tissue are cocultivated. Furthermore, the stability of VirB10 is diminished in cells lacking functional VirB9, but only under conditions of low osmolarity. We propose that newly synthesized VirB10 is inherently labile in the presence of a large osmotic gradient across the inner membrane and is rapidly degraded unless it is stabilized by VirB9-dependent assembly into oligomeric complexes. The possibility that VirB10-containing complexes are not assembled properly at elevated temperatures suggests an explanation for the decades-old observation that tumor formation is exquisitely sensitive to ambient temperature.

The Ti plasmid increases the efficiency of Agrobacterium tumefaciens as a recipient in virB-mediated conjugal transfer of an IncQ plasmid.

The T-DNA transfer apparatus of Agrobacterium tumefaciens mediates the delivery of the T-DNA into plant cells, the transfer of the IncQ plasmid RSF1010 into plant cells, and the conjugal transfer of RSF1010 between Agrobacteria. We show in this report that the Agrobacterium-to-Agrobacterium conjugal transfer efficiencies of RSF1010 increase dramatically if the recipient strain, as well as the donor strain, carries a wild-type Ti plasmid and is capable of vir gene expression. Investigation of possible mechanisms that could account for this increased efficiency revealed that the VirB proteins encoded by the Ti plasmid were required. Although, with the exception of VirB1, all of the proteins that form the putative T-DNA transfer apparatus (VirB1-11, VirD4) are required for an Agrobacterium strain to serve as an RSF1010 donor, expression of only a subset of these proteins is required for the increase in conjugal transfer mediated by the recipient. Specifically, VirB5, 6, 11, and VirD4 are essential donor components but are dispensable for the increased recipient capacity. Defined point mutations in virB9 affected donor and recipient capacities to the same relative extent, suggesting that similar functions of VirB9 are important in both of these contexts.

Interactions between VirB9 and VirB10 membrane proteins involved in movement of DNA from Agrobacterium tumefaciens into plant cells.

The 11 VirB proteins from Agrobacterium tumefaciens are predicted to form a membrane-bound complex that mediates the movement of DNA from the bacterium into plant cells. The studies reported here on the possible VirB protein interactions in such a complex demonstrate that VirB9 and VirB10 can each form high-molecular-weight complexes after treatment with a chemical cross-linker. Analysis of nonpolar virB mutants showed that the formation of the VirB10 complexes does not occur in a virB9 mutant and that VirB9 and VirB10 are not components of the same cross-linked complex. VirB9, when stabilized by the concurrent expression of VirB7, was shown to be sufficient to permit VirB10 to cross-link into its usual high-molecular-weight forms in the absence of other Vir proteins. Randomly introduced single point mutations in virB9 resulted in Agrobacterium strains with severely attenuated virulence. Although some of the mutants contained wild-type levels of VirB9 and displayed an unaltered VirB9 cross-linking pattern, VirB10 cross-linking was drastically reduced. We conclude that specific amino acid residues in VirB9 are necessary for interaction with VirB10 resulting in the capacity of VirB10 to participate in high-molecular-weight complexes that can be visualized by chemical cross-linking.

Differences in virulence and in expression of PrfA and PrfA-regulated virulence genes of Listeria monocytogenes strains belonging to serogroup 4.

Listeria monocytogenes isolates belonging to serogroup 4 (subtypes 4a, 4ab, 4b, 4c, 4d, and 4e) exhibit different levels of virulence in mice. Molecular studies indicate that in comparison with the control strain EGD (serotype 1/2a), these strains differ in the expression of the PrfA-regulated virulence genes, including prfA itself. Strains of serotypes 4c, 4d, 4e, and especially 4a show a low level of invasiveness in Caco-2 cells, which correlates in part with the low level of expression of the inlA gene. All serotypes reach the cytoplasm, at the latest, 2 h postinfection and become surrounded by polymerized actin within the next hour, but actin tail formation by serotype 4a, 4c, 4d, and 4e strains is drastically reduced. The actA genes in these serogroup 4 strains are expressed in minimum essential medium and within the phagocytic cell line J774. However, the amounts and (in part) the sizes of the ActA proteins in these strains differ under these conditions. The reduced actin tail formation by serotype 4a, 4c, 4d, and 4e strains may be due to the low level of in vivo expression of ActA. In addition, the loss of one repeat unit in the ActA proteins of serotype 4a and 4e strains may also contribute to the less efficient actin tail formation observed with these strains.

Differential regulation of the virulence genes of Listeria monocytogenes by the transcriptional activator PrfA.

The two Listeria monocytogenes strains EGD and NCTC 7973 display a different regulation pattern of their PrfA-dependent genes. All PrfA-dependent genes from L. monocytogenes NCTC 7973 are much more efficiently transcribed in brain-heart infusion medium than those from strain EGD. Transcription of these genes in EGD is, however, induced after shift into Minimal Essential Medium (MEM) to a level that is comparable to that of strain NCTC 7973. Expression of the internalin gene (inlA) is also influenced by PrfA, but only one (P2) out of three mapped promoters is strictly dependent on PrfA. In contrast to the other PrfA-regulated genes, transcription of inlA even from the P2 promoter is reduced in both strains after shift to MEM. The prfA deletion mutant SLCC 53 complemented with multiple copies of prfA synthesizes large amounts of monocistronic prfA transcript, but there is no concomitant increase in the transcripts of the PrfA-dependent genes. However, upon a shift into MEM, transcription of the PrfA-dependent genes (with the exception of the inlA gene) is highly induced even in the absence of de novo protein synthesis. The PrfA proteins of the two studied L. monocytogenes strains differ in their ability to activate PrfA-dependent genes. This difference is probably the result of amino acid exchange(s) in the C-terminal part of these proteins. Strain EGD supplemented with multiple copies of prfA-7973 shows a similar regulation of the PrfA-dependent genes as strain NCTC 7973, whereas multiple copies of prfA-EGD introduced into strain EGD hardly change the rate of transcription of the PrfA-dependent genes. Our data thus suggest that PrfA-mediated gene expression depends not only on the amount of the PrfA protein and the 'quality' of the putative PrfA-binding sites, but also on the 'quality' of the PrfA protein which seems to be influenced by its amino acid composition and by environmental parameters.

Transcriptional regulation of prfA and PrfA-regulated virulence genes in Listeria monocytogenes.

The ActA protein, the lecithinase PlcB and listeriolysin are the major PrfA-dependent proteins synthesized when brain-heart infusion (BHI)-cultured Listeria monocytogenes is shifted to minimum essential medium (MEM) in the presence of the transcriptional inhibitor rifampicin. Enhanced synthesis of all three proteins under these conditions depends, however, on a short incubation (about 5 min) of the bacteria in MEM without rifampicin, suggesting that induction of these proteins in MEM requires de novo transcription. The enhanced synthesis of these three proteins is observed in the L. monocytogenes wild-type strains EGD and NCTC 7973, both of which belong to the serotype 1/2 a. A significant induction of the bicistronic mRNA for ActA and PlcB is observed in both strains shortly after shifting the bacteria from BHI to MEM. This mRNA as well as the monocistronic listeriolysin (hly)-specific mRNA is highly stable in L. monocytogenes NCTC 7973 shifted to MEM. In contrast to the actA-plcB mRNA, no enhanced transcription in MEM is observed for the regulatory prfA gene or for the PrfA-controlled virulence genes hlyA and plcA in strain NCTC 7973. However, transcription of these genes is induced in strain EGD. Transcriptional induction of the mpl gene is observed in neither strain NCTC 7973 nor in strain EGD. The life-time of the prfA, plcA, and mpl transcripts is short. ActA was also found to be the most abundant newly synthesized surface protein when the two wild-type strains of L. monocytogenes replicated within the phagocytic cell line J774. ActA synthesis seemed to be induced in the cytoplasm since the non-haemolytic mutant M3 did not induce ActA when taken up by J774 cells.

Aerosol resuspension from fabric: implications for personal monitoring in the beryllium industry.

The fabric used for work clothing at an industrial site can significantly influence personal monitor (PM) exposure estimates because dust resuspension from clothing can increase the concentration at the sampler inlet. The magnitude of the effect depends on removal forces and on the interaction of the contaminant particles with work garments. Aerosol deposition and resuspension on cotton and Nomex aramid fabrics was evaluated at a beryllium refinery. Electrostatically charged cotton backdrops collected more beryllium than neutral controls, but electronegative Nomex backdrops did not. Moving fabrics collected more beryllium than did stationary controls. When contaminated fabrics were agitated, PMs mounted 2.5 cm in front of the fabric collected more beryllium than monitors above the fabric, positioned to simulate the nose or mouth. The difference between the air concentrations measured by these PMs increased with Be loading and tended to level off for highly contaminated fabric. Cotton resuspended a larger fraction of its contaminant load than Nomex. These results are consistent with current knowledge of the behavior of particles on fabric fibers. Aerosol resuspension from garments is an important consideration in assessing inhalation exposure to toxic dusts. A garment may attract and retain toxic particles. This contamination is then available for later resuspension.