Characterization of liver specific promoters in a foamy viral vector pMD09.

Foamy viruses (FVs) or spumaviruses are retroviruses that are explored as vectors for gene therapy. The good feature of foamy viruses is its broad tropism; however, their infections result in non-targeted gene expression. Here, we attempted to design the liver targeted viral gene delivery by employing liver specific gene promoters like albumin (ALB), transthyretin (TTR) and hepatitis B virus (HBV) promoters. We compared the relative gene expression of liver specific promoters versus the U3 promoter in liver cell line (HepG2) and non-liver cell lines: human fibrosarcoma cell line (HT1080), baby hamster kidney cell line (BHK), human embryonic kidney cell line (HEK 293T) and cervical cancer cell line (HeLa). We have found that the promoter exchange didn't affect viral assembly. The ability to drive gene expression was best with TTR promoter which was followed by HBV and ALB promoter. The use of TTR, HBV and ALB promoters are helpful in achieving liver specific gene expression. Keywords: foamy virus; gene therapy; liver; albumin; transthyretin promoter; HBV promoter.

Stimulation of the human RAD51 nucleofilament restricts HIV-1 integration in vitro and in infected cells.

Stable HIV-1 replication requires the DNA repair of the integration locus catalyzed by cellular factors. The human RAD51 (hRAD51) protein plays a major role in homologous recombination (HR) DNA repair and was previously shown to interact with HIV-1 integrase (IN) and inhibit its activity. Here we determined the molecular mechanism of inhibition of IN. Our standard in vitro integration assays performed under various conditions promoting or inhibiting hRAD51 activity demonstrated that the formation of an active hRAD51 nucleofilament is required for optimal inhibition involving an IN-DNA complex dissociation mechanism. Furthermore we show that this inhibition mechanism can be promoted in HIV-1-infected cells by chemical stimulation of the endogenous hRAD51 protein. This hRAD51 stimulation induced both an enhancement of the endogenous DNA repair process and the inhibition of the integration step. Elucidation of this molecular mechanism leading to the restriction of viral proliferation paves the way to a new concept of antiretroviral therapy based on the enhancement of endogenous hRAD51 recombination activity and highlights the functional interaction between HIV-1 IN and hRAD51.

Transduction of human neural progenitor cells with foamy virus vectors for differentiation-dependent gene expression.

Neural progenitor cells are potential vehicles for delivery of therapeutic agents into the brain. Differentiation-dependent promoters may be useful to target the therapeutic transgene expression to specific neural cell types. Here we explored the potential of vectors based on the foamy virus (FV) for genetic engineering of neural progenitor cells. We demonstrate that FV vectors can mediate stable long-term constitutive expression of the enhanced green fluorescent protein (EGFP) in neural progenitor cells. For differentiation-dependent gene expression, we constructed a FV vector with an internal expression cassette containing the human 2.2 kb promoter (Gfa2) of the astrocyte-specific glial fibrillary acidic protein (GFAP) and sequences encoding EGFP. We show FV-vector-mediated delivery of the Gfa2-egfp transgene into the human neural stem cell line HNSC.100 and differentiation-dependent expression in stably transduced cell populations. Differentiation of the FV-transduced HNSC.100 cells to astrocytes upregulated expression of both the Gfa2-egfp transgene and the native gfap gene, confirming differentiation-dependent activation of the transduced Gfa2 promoter. These results demonstrate that differentiation-dependent gene expression can be achieved by FV-vector-mediated gene transfer to neural progenitor cells. Our findings support the use of FV vectors for the genetic engineering of neural progenitor cells for therapeutic and research applications.

The therapeutic potential of siRNA in gene therapy of neurodegenerative disorders.

RNA interference using small inhibitory RNA (siRNA) has become a powerful tool to downregulate mRNA levels by cellular nucleases that become activated when a sequence homology between the siRNA and a respective mRNA molecule is detected. Therefore siRNA can be used to silence genes involved in the pathogenesis of various diseases associated with a known genetic background. As for many neurodegenerative disorders a causative therapy is unavailable, siRNA holds a promising option for the development of novel therapeutic strategies. Here we discuss different siRNA target strategies aiming for an allele-specific degradation of disease-inducing mRNA and we review the literature in the field of siRNA and its application in animal models of neurodegenerative diseases, including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD) and spinocerebellar ataxia (SCA1).

Foamy virus--adenovirus hybrid vectors.

To confer adenovirus vectors (AdV), the feature of integration into the host cell genome hybrid vectors were characterized in vitro, which express vectors derived from the prototypic foamy virus (FV) in the backbone of a high-capacity AdV. FVs constitute a subfamily of retroviruses with a distinct replication pathway and no known pathogenicity. In the absence of envelope glycoprotein, the prototypic FV behaves like a retrotransposon, while it behaves like an exogenous retrovirus in its presence. Two principle types of vectors, which either allows the intracellular (HC-FAD-7) or, in addition, the extracellular (HC-FAD-2) pathway were constructed. In both chimeras the expression of the FV vector was controlled by the tetracycline-regulatable system. Hybrids were produced close to 10(10) infectious units/ml. By Southern blotting, the functionality of the hybrid vectors to generate host cell genomic integrants was shown. However, the efficiency of HC-FAD-7 to establish stable transgene expression was rather low, while around 70% of cells were stably transduced in secondary round following primary transduction with HC-FAD-2 at an MOI of 100. Given the benign characteristics of high-capacity adenovirus and FV vectors, hybrids based on HC-FAD-2 are probably suited for an in vivo application.

The replication strategy of foamy viruses.

The replication strategy of foamy viruses diverges in many aspects from what is commonly accepted as the rules of retroviral replication. Although many questions on the details of the replication pathway are still unanswered, it appears that foamy viruses have adopted a strategy which functionally bridges the retroviral and the hepadnaviral replication pathways. A number of experimental findings in favour of the view that foamy viruses are reverse transcribing DNA viruses which integrate into the host cell genome are discussed.

Fatal myocarditis associated with acute parvovirus B19 and human herpesvirus 6 coinfection.

We report on the case of a healthy young boy who developed a fulminant myocarditis due to acute coinfection with erythrovirus (parvovirus B19) and human herpesvirus 6 (HHV-6) in the absence of an antiviral immune response. We suggest that the HHV-6-induced immunosuppression enhanced dissemination of parvovirus B19, which led to fatal myocarditis.

A particle-associated glycoprotein signal peptide essential for virus maturation and infectivity.

Signal peptides (SP) are key determinants for targeting glycoproteins to the secretory pathway. Here we describe the involvement in particle maturation as an additional function of a viral glycoprotein SP. The SP of foamy virus (FV) envelope glycoprotein is predicted to be unusually long. Using an SP-specific antiserum, we demonstrate that its proteolytic removal occurs posttranslationally by a cellular protease and that the major N-terminal cleavage product, gp18, is found in purified viral particles. Analysis of mutants in proposed signal peptidase cleavage positions and N-glycosylation sites revealed an SP about 148 amino acids (aa) in length. FV particle release from infected cells requires the presence of cognate envelope protein and cleavage of its SP sequence. An N-terminal 15-aa SP domain with two conserved tryptophan residues was found to be essential for the egress of FV particles. While the SP N terminus was found to mediate the specificity of FV Env to interact with FV capsids, it was dispensable for Env targeting to the secretory pathway and FV envelope-mediated infectivity of murine leukemia virus pseudotypes.

Phenotypic analysis of the sensitivity of HIV-1 to inhibitors of the reverse transcriptase, protease, and integrase using a self-inactivating virus vector system.

Conventional phenotypic analysis of resistance of the human immunodeficiency virus (HIV) to antiviral therapy is time-consuming and requires culture of infectious virus. Although phenotypic analyses may be desirable, rapid generation of test results and decentralized availability of the test system will be important to achieve utility in the clinical practice. This study describes the design of an alternative phenotypic resistance test using replication incompetent viral vectors. Chimeric HIV vectors containing a marker gene were generated. The env and most of the regulatory and accessory genes of HIV were removed. In addition, the 3'U3 region was deleted to obtain a self-inactivating construct. Cotransfection of the plasmid with a plasmid that provided the vesicular stomatitis virus glycoprotein resulted in the production of replication-incompetent virus vectors. Infection of susceptible cells with the vectors led to marker gene expression. Vector production in the presence of protease (PR) inhibitors, or infection in the presence of reverse transcriptase (RT) or integrase (IN) inhibitors reduced marker gene expression in a dose-dependent manner. Marker gene activity was preserved at higher drug levels if vectors contained RT and PR genes from resistant virus isolates. Sensitivity to nucleoside and non-nucleoside RT inhibitors, protease and integrase inhibitors could be determined in 10 working days. The phenotypic drug resistance test using replication-incompetent HIV vectors significantly speeds up drug resistance measurements and allows testing at reduced biosafety levels. This will make clinical use of phenotypic assessment of antiviral resistance more feasible.

Successful preemptive cidofovir treatment for CMV antigenemia after dose-reduced conditioning and allogeneic blood stem cell transplantation.

BACKGROUND: Cidofovir (CDV) is a nucleotide analogue with proven in vitro effects against cytomegalovirus (CMV) and adenovirus and has been successfully used in the treatment of CMV retinitis in AIDS patients. METHODS: We performed a prospective study to evaluate the efficacy of CDV in 17 patients with hematological malignancies after allogeneic blood stem cell transplantation from related (n=3) and unrelated (n=14) donors. Dose-reduced conditioning (DRC) regimen consisted of busulfan (Bu)/fludarabine (Flu) (n=9) and idarubicin/cytosine arabinoside/Flu (n=1). Myeloablative conditioning (MC) was performed with Bu/cyclophosphamide (Cy)/etoposide (Eto) (n=4), Bu/Cy (n=2), and total body irradiation (TBI)/Cy/Eto (n=1). Antithymocyte globulin (ATG) was used in seven patients with DRC and in six patients with MC. In all patients, either the donor, host, or both were CMV IgG positive pretransplant. Indication for therapy was preemptive treatment of primary CMV antigenemia defined as two consecutive positive tests of pp65 antigenemia assay after transplant. In case of response with a decreasing number of pp65-positive leukocytes, CDV was scheduled in a dosage of 5 mg/kg body weight once a week for 2 weeks followed by maintenance therapy every 2 weeks in an outpatient setting. All patients received probenecid and prehydration as recommended. Patients were monitored using an immunostaining assay for pp65 antigen and a qualitative and quantitative CMV polymerase chain reaction (PCR). Success of treatment was defined as negativity for the pp65 antigen. RESULTS: After DRC, nine of ten patients (90%) showed a response with seven of nine revealing a complete clearance of the virus (pp65 negative, qualitative PCR negative). In the remaining two responders, treatment was changed to ganciclovir because of either renal impairment or slow clearance of antigenemia. Only one of seven patients in the MC group experienced a temporary clearance of pp65 antigen. After MC, two patients experienced CMV disease. Treatment-related toxicity rate was moderate with four patients developing reversible renal impairment (creatinine 133-180 micromol/L); one patient with proteinuria and three patients with complaints of nausea and vomiting. CONCLUSION: Our data suggest the feasibility of CDV administration in patients after allogeneic transplantation. In the recommended dose, it might be used successfully for low-risk patients, e.g., after DRC or organ transplantation, in an outpatient setting.

Efficient intracellular retrotransposition of an exogenous primate retrovirus genome.

The foamy virus (FV) subgroup of Retroviridae reverse transcribe their RNA (pre-)genome late in the replication cycle before leaving an infected cell. We studied whether a marker gene-transducing FV vector is able to shuttle to the nucleus and integrate into host cell genomic DNA. While a potential intracellular retrotransposition of vectors derived from other retroviruses was below the detection limit of our assay, we found that up to 5% of cells transfected with the FV vector were stably transduced, harboring 1 to approximately 10 vector integrants. Generation of the integrants depended on expression of functional capsid, reverse transcriptase and integrase proteins, and did not involve an extracellular step. PCR analysis of the U3 region of the 5' long terminal repeat and determination of proviral integration sites showed that a reverse transcription step had taken place to generate the integrants. Co-expression of a mutated envelope allowing particle egress and avoiding extracellular infection resulted in a significantly increased rescue of cells harboring integrants, suggesting that accumulation of proviruses via intracellular retrotransposition represents an integral part of the FV replication strategy.

An evolutionarily conserved positively charged amino acid in the putative membrane-spanning domain of the foamy virus envelope protein controls fusion activity.

Foamy viruses (FVs) are highly fusogenic, and their replication induces massive syncytium formation in infected cell cultures which is believed to be mediated by expression of the envelope (Env) protein. The FV Env is essential for virus particle egress. The unusually long putative membrane-spanning domain (MSD) of the transmembrane subunit carries dispersed charged amino acids and has an important function for particle envelopment. To better understand the capsid-envelope interaction and Env-mediated cell fusion, we generated a variety of FV MSD mutations. C-terminal deletions revealed the cytoplasmic domain to be dispensable but the full-length MSD to be required for fusogenic activity. The N-terminal 15 amino acids of the MSD were found to be sufficient for membrane anchorage and promotion of FV particle release. Expression of wild-type Env protein rarely induced syncytia due to intracellular retention. Coexpression with FV Gag-Pol resulted in particle export and a dramatic increase in fusion activity. A nonconservative mutation of K(959) in the middle of the putative MSD resulted in increased fusogenic activity of Env in the absence of Gag-Pol due to enhanced cell surface expression as well as structural changes in the mutant proteins. Coexpression with Gag-Pol resulted in a further increase in the fusion activity of mutant FV Env proteins. Our results suggest that an interaction between the viral capsid and Env is required for FV-induced giant-cell formation and that the positive charge in the MSD is an important determinant controlling intracellular transport and fusogenic activity of the FV Env protein.

Complex effects of deletions in the 5' untranslated region of primate foamy virus on viral gene expression and RNA packaging.

Due to various advantageous features there is current interest in retroviral vectors derived from primate foamy viruses (PFVs). Two PFV cis-acting sequences have been mapped in the 5' region of the RNA (pre-)genome and in the 3' pol genomic region. In order to genetically separate PFV packaging constructs from vector constructs, we investigated the effect of deletions in the 5' untranslated region (UTR) of PFV packaging constructs and vectors on gene expression and RNA incorporation into viral particles. Our results indicate that the 5' UTR serves different previously unknown functions. First, the R region of the long terminal repeat was found to be required for PFV gag gene expression. This regulation of gene expression appeared to be mainly posttranscriptional. Second, constructs with sequence deletions between the R region and the gag gene start codon packaged as much viral mRNA into particles as the undeleted construct, and RNA from such a 5'-UTR-deleted packaging construct was copackaged into vector-virus particles, together with vector RNA which was preferentially packaged. Finally, in the U5 region a sequence was identified that was required to allow cleavage of the Gag precursor protein by the pol gene-encoded protease, suggesting a role of RNA in PFV particle formation. Taken together, the results indicate that complex interactions of the viral RNA, capsid, and polymerase proteins take place during PFV particle formation and that a clear separation of PFV vector and packaging construct sequences may be difficult to achieve.

Replication of a foamy virus mutant with a constitutively active U3 promoter and deleted accessory genes.

Foamy viruses (FVs) are complex retroviruses which require for their replication the activity of a transcriptional trans-activator (Tas) as well as Tas-responsive elements in the viral promoters. A mutant of the chimpanzee FV strain, CFV/hu (previously called human FV), genome in which most of the U3 promoter of the CFV long terminal repeat was substituted by the constitutively active human cytomegalovirus immediate early gene enhancer/promoter was constructed. This plasmid (pTS12) and a derivative (pTS13), which has a deletion in the tas gene, gave rise to replication-competent virus. Compared with parental CFV, both mutants replicated only very poorly, with retarded growth kinetics and maximal cell-free virus titres reduced by approximately three orders of magnitude. Mutation of the DD35E motif of the CFV integrase to DA35E rendered the recombinant TS virus replication-deficient. This indicated that provirus integration is probably still required for this FV derivative, which had been converted from a complex regulated retrovirus into a simple one by incorporation of a constitutively active promoter from another virus which regularly does not integrate into the host cell genome.

An active foamy virus integrase is required for virus replication.

Foamy viruses (FVs) make use of a replication strategy which is unique among retroviruses and shows analogies to hepadnaviruses. The presence of an integrase (IN) and obligate provirus integration distinguish retroviruses from hepadnaviruses. To clarify whether a functional IN is required for FV replication, a mutant in the highly conserved DD35E motif of the active centre was analysed. This mutant was found to be able to express Gag and Pol protein precursors and cleavage products and to generate and deliver cDNA. However, this mutant was replication-deficient. The junctions of individual foamy proviruses with cellular DNA were sequenced. The findings suggest that FV integration is asymmetrical, because the proviruses started with what is believed to be the U3 end of the free linear DNA to generate the conventional TG dinucleotide, while apparently two nucleotides from the U5 end were cleaved to create the complementary CA dinucleotide. Alignment of known FV genome sequences indicated that this mechanism of integration is not restricted to the two FV isolates from which integrates were studied, but appears to be a common feature of this retrovirus subfamily. In conclusion, with respect to the necessity of a functionally active IN for virus replication FVs behave like other retroviruses; their mechanism of integration, however, is probably unique.

Specific binding of recombinant foamy virus envelope protein to host cells correlates with susceptibility to infection.

The interaction of simian foamy viruses (FVs) with their putative cellular receptor(s) was studied with two types of recombinant envelope protein (Env). Transient expression of full-length Env in BHK-21 cells induced syncytia formation. However, selected stable transfectants fused with naive cells but not with each other. A soluble fusion protein of the Env surface domain with the Fc fragment of a human IgG1 heavy chain (EnvSU-Ig) was produced in the baculovirus expression system, purified to homogeneity, and used for binding and competition analyses. EnvSU-Ig but not unrelated Ig fusion proteins bound to cells specifically. Neutralizing serum blocked binding of EnvSU-Ig and, vice versa, serum-mediated neutralization was abrogated by the chimeric protein. Concomitant reduction of EnvSU-Ig binding and FV susceptibility was seen in Env-expressing target cells. Although EnvSU-Ig did not inhibit FV infection, very likely due to its displacement by multivalent virus-cell interactions, this divalent ligand should help to characterize functionally and to identify the ubiquitous FV receptor.

Foamy virus capsids require the cognate envelope protein for particle export.

Unlike other subclasses of the Retroviridae the Spumavirinae, its prototype member being the so-called human foamy virus (HFV), require the expression of the envelope (Env) glycoprotein for viral particle egress. Both the murine leukemia virus (MuLV) Env and the vesicular stomatitis virus G protein, which efficiently pseudotype other retrovirus capsids, were not able to support export of HFV particles. Analysis of deletion and point mutants of the HFV Env protein revealed that the HFV Env cytoplasmic domain (CyD) is dispensable for HFV particle envelopment, release, and infectivity, whereas deletion of the membrane-spanning-domain (MSD) led to an accumulation of naked capsids in the cytoplasm. Neither alternative membrane association of HFV Env deletion mutants lacking the MSD and CyD via phosphoglycolipid anchor nor domain swapping mutants, with the MSD or CyD of MuLV Env and VSV-G exchanged against the corresponding HFV domains, could restore particle envelopment and the release defect of pseudotypes. However, replacement of the HFV MSD with that of MuLV led to budding of HFV capsids at the intracellular membranes. These virions were of apparently wild-type morphology but were not naturally released into the supernatant and they were noninfectious.

Cells expressing the human foamy virus (HFV) accessory Bet protein are resistant to productive HFV superinfection.

Bet is a foamy virus (FV) accessory protein not required for virus replication. The function of Bet is not understood. We report on the generation of cell lines stably expressing the HFV Bet protein. In Bet+ cells, HFV replication was reduced by approximately 3-4 orders of magnitude compared with control cells. The HFV Bet-expressing cells only partially resisted infection by the distantly related feline FV (FFV). Pseudotyping experiments, using murine retroviral vectors with an HFV envelope, revealed that the resistance was not due to downregulation of the unknown HFV receptor. In transfection experiments, using proviral reporter gene constructs and infectious proviruses, no significant differences were detected between Bet+ and control cells. In infection experiments, HFV vectors expressing an indicator gene under control of the HFV promoters showed no activity in Bet+ cells. The results are best compatible with the hypothesis that the main block to productive superinfection of Bet+ cells occurs at an early stage of replication between virus entry and provirus establishment. We suggest that inhibition of provirus integration by Bet protein may serve a distinct function in the unique foamy virus replication cycle.

Characterization of a cis-acting sequence in the Pol region required to transfer human foamy virus vectors.

To identify cis-acting elements in the foamy virus (FV) RNA pregenome, we developed a transient-vector-production system based on cotransfection of indicator gene-bearing vector and gag-pol and env expression plasmids. Two elements which were critical for vector transfer were found and mapped approximately. The first element was located in the RU5 leader and the 5' gag region (approximately up to position 650 of the viral RNA). The second element was located in an approximately 2-kb sequence in the 3' pol region. Although small 5' and 3' deletions, as well as internal deletions of the latter element, were tolerated, both elements were found to be absolutely required for vector transfer. The functional characterization of the pol region-located cis-acting element revealed that it is essential for efficient incorporation or the stability of particle-associated virion RNA. Furthermore, virions derived from a vector lacking this sequence were found to be deficient in the cleavage of the Gag protein by the Pol precursor protease. Our results suggest that during the formation of infectious virions, complex interactions between FV Gag and Pol and the viral RNA take place.