The innate immune response, clinical outcomes, and ex vivo HCV antiviral efficacy of a TLR7 agonist (PF-4878691).

Hepatitis C virus (HCV) infection is an issue of global concern, and studies are ongoing to identify new therapies that are both effective and safe. PF-4878691 is a Toll-like receptor 7 (TLR7) agonist modeled so as to dissociate its antiviral activities from its inflammatory activities. In a proof-of-mechanism study in healthy volunteers who received doses of 3, 6, and 9 mg of PF-4878691 twice a week for 2 weeks, PF-4878691 induced biomarkers of the immune and interferon (IFN) responses in a dose-dependent and dose-frequency-related manner. A novel finding was induction of TLR7 expression in vivo in response to PF-4878691, leading to an amplified biomarker response. A nonresponder at the 9-mg dose had a polymorphism in the IFN-α receptor 1 subunit (Val168Leu). Two subjects who had received 9-mg doses experienced serious adverse events (SAEs), characterized by flu-like symptoms, hypotension, and lymphopenia, leading to early termination of the study. TLR7 stimulation results in a pharmacologic response at levels commensurate with predicted antiviral efficacy, but these doses are associated with SAEs, raising concerns about the therapeutic window of this class of compounds for the treatment of HCV infection.

Lack of alternative coreceptor use by pediatric HIV-1 R5 isolates for infection of primary cord or adult peripheral blood mononuclear cells.

HIV-1 infection of neonates results in an extended acute period of virus replication, frequent neurological problems and reduced survival compared to adults. In adults, R5 viruses mainly infect CCR5(+) CD4(+) memory T-cells. In neonates, CCR5(+) memory T-cells form a substantially smaller fraction of total lymphocytes. We therefore tested whether alternative coreceptors confer infection of lymphocytes by pediatric isolates. Pediatric HIV-1 R5 isolates failed to replicate in Delta32/Delta32 CCR5 PBMCs or in cord PBMCs treated with a CCR5 inhibitor. These results do not indicate a role for alternative coreceptors and provide support for CCR5 inhibitors in the therapy of HIV-1(+) neonates.

Transactivation of a cellular promoter by the NS1 protein of the parvovirus minute virus of mice through a putative hormone-responsive element.

The promoter of the thyroid hormone receptor alpha gene (c-erbA-1) is activated by the nonstructural protein 1 (NS1) of parvovirus minute virus of mice (prototype strain [MVMp]) in ras-transformed FREJ4 cells that are permissive for lytic MVMp replication. This stimulation may be related to the sensitivity of host cells to MVMp, as it does not take place in parental FR3T3 cells, which are resistant to the parvovirus killing effect. The analysis of a series of deletion and point mutants of the c-erbA-1 promoter led to the identification of an upstream region that is necessary for NS1-driven transactivation. This sequence harbors a putative hormone-responsive element and is sufficient to render a minimal promoter NS1 inducible in FREJ4 but not in FR3T3 cells, and it is involved in distinct interactions with proteins from the respective cell lines. The NS1-responsive element of the c-erbA-1 promoter bears no homology with sequences that were previously reported to be necessary for NS1 DNA binding and transactivation. Altogether, our data point to a novel, cell-specific mechanism of promoter activation by NS1.

Upstream CREs participate in the basal activity of minute virus of mice promoter P4 and in its stimulation in ras-transformed cells.

The activity of the P4 promoter of the parvovirus minute virus of mice (prototype strain MVMp) is stimulated in ras-transformed FREJ4 cells compared with the parental FR3T3 line. This activation may participate in the oncolytic effect of parvoviruses, given that P4 drives a transcriptional unit encoding cytotoxic nonstructural proteins. Our results suggest that the higher transcriptional activity of promoter P4 in FREJ4 cells is mediated at least in part by upstream CRE elements. Accordingly, mutations in the CRE motifs impair P4 function more strongly in the FREJ4 derivative than in its FR3T3 parent. Further evidence that these elements contribute to hyperactivity of the P4 promoter in the ras transformant is the fact that they form distinct complexes with proteins from FREJ4 and FR3T3 cell extracts. This difference can be abolished by treating the FREJ4 cell extracts with cyclic AMP-dependent protein kinase (PKA) or treating original cultures with a PKA activator. These findings can be linked with two previously reported features of ras-transformed cells: the activation of a PKA-inhibited protein kinase cascade and the reduction of PKA-induced protein phosphorylation. In keeping with these facts, P4-directed gene expression can be up- or downmodulated in vivo by exposing cells to known inhibitors or activators of PKA, respectively.

NF-Y controls transcription of the minute virus of mice P4 promoter through interaction with an unusual binding site.

Electrophoretic mobility shift assays performed with nuclear extracts from human fibroblasts revealed the formation of two major protein complexes with an oligonucleotide (nucleotides 78 to 107) from the palindromic region located upstream from the minute virus of mice (MVM) P4 promoter. It was shown that this oligonucleotide bound USF at the enhancer E box CACATG. The second complex contained the transcription factor NF-Y, whose association was surprising because its target sequence lacks the canonical CCAAT motif present in all mammalian NF-Y binding sites identified so far. The MVM NF-Y recognition element instead contains the CCAAC sequence. USF and NF-Y had distinct but overlapping sequence requirements for binding, suggesting that their associations with MVM DNA were mutually exclusive. Because of the palindromic nature of MVM DNA terminal sequences, NF-Y associated with the three nucleotide configurations corresponding to the hairpin structure and to the external and internal arms of the extended duplex replication form, respectively. However, owing to the imperfection of the palindrome, the binding of USF was restricted to the internal arm. Point mutations that suppressed the in vitro binding of NF-Y to the internal palindromic arm reduced the activity of the resident P4 promoter, while those preventing complex formation with USF did not, as determined by transient expression assays using the luciferase reporter gene. The data led to the identification of a novel P4 upstream regulatory region capable of interacting with two transcription factors, from which one (NF-Y) appeared to upmodulate the activity of the promoter.

Cruciform structure of a DNA motif of parvovirus minute virus of mice (prototype strain) involved in the attenuation of gene expression.

It has previously been reported that the region between nucleotides 259 and 383 immediately downstream from the P4 early promoter of parvovirus minute virus of mice, prototype strain (MVMp) is responsible for transcriptional attenuation. Attenuation results from the premature pausing of RNA polymerase II within this sequence (designated to as att) and seems to depend on potential RNA secondary structure. To assess the attenuation capacity of att under near physiological conditions, the early transcription unit of MVMp was replaced by the chloramphenicol acetyltransferase reporter gene under control of the early P4 promoter, in the presence or absence of att. The resulting recombinant vectors were encapsidated in parvovirus particles and replicated in cells after co-infection with the wild-type virus. The att fragment reduced the rate of expression of the reporter gene by approximately threefold, confirming previously reported data from transfection experiments performed in the same cellular system. This attenuation factor is unexpectedly high, considering that the 'readthrough' fold of the nascent viral transcript is thermodynamically more stable than the 'attenuation' configuration. In an attempt to elucidate this point, we sought for the presence of secondary structures in the template DNA molecule. In vitro nuclease probing of viral dsDNA revealed that the att fragment had a cruciform configuration with both complementary strands folding into the computer-predicted stem-loop 'attenuation' structure. These observations lead us to propose that the secondary structure of the DNA template may prompt the formation of the 'attenuation' stem-loop in nascent mRNAs by bringing corresponding self-complementary sequences into close proximity.

Mapping of upstream regulatory elements in the P4 promoter of parvovirus minute virus of mice.

The early promoter (P4) of the autonomous parvovirus minute virus of mice (prototype strain) directs the expression of the transcription unit coding for the nonstructural proteins NS1 and NS2. Although proximal promoter elements (GC and TATA boxes) are essential for P4 activity in vivo, additional upstream sequences appear to be required for optimal transcription. Therefore, associations of proteins with the upstream regulatory region of promoter P4 were studied in the rat fibroblast cell line FREJ4 by gel retardation and in vitro as well as in vivo footprinting assays. This led to the identification of at least four distinct upstream elements that interacted with cellular proteins. The functionality of these elements was supported by the reduced level of gene expression driven by corresponding linker-substitutive mutants of promoter P4.

Nucleolin forms a specific complex with a fragment of the viral (minus) strand of minute virus of mice DNA.

Nucleolin, a major nucleolar protein, forms a specific complex with the genome (a single-stranded DNA molecule of minus polarity) of parvovirus MVMp in vitro. By means of South-western blotting experiments, we mapped the binding site to a 222-nucleotide motif within the non-structural transcription unit, referred to as NUBE (nucleolin-binding element). The specificity of the interaction was confirmed by competitive gel retardation assays. DNaseI and nuclease S1 probing showed that NUBE folds into a secondary structure, in agreement with a computer-assisted conformational prediction. The whole NUBE may be necessary for the interaction with nucleolin, as suggested by the failure of NUBE subfragments to bind the protein and by the nuclease footprinting experiments. The present work extends the previously reported ability of nucleolin to form a specific complex with ribosomal RNA, to a defined DNA substrate. Considering the tropism of MVMp DNA replication for host cell nucleoli, these data raise the possibility that nucleolin may contribute to the regulation of the parvoviral life-cycle.

Programmed killing of human cells by means of an inducible clone of parvoviral genes encoding non-structural proteins.

Although its dependence on the target cell type is well established, the cytopathogenicity of parvoviruses has remained elusive to date as far as its mechanism is concerned. However, indirect evidence suggested that parvoviral non-structural (NS) proteins may be the cytotoxic effectors. In order to test this hypothesis, a molecular clone of parvovirus MVMp was modified, by replacing the P4 promoter of the NS transcription unit by the glucocorticoid-inducible promoter of the mouse mammary tumour virus. Clones of neoplastic human cells that had incorporated this construct and that were induced to produce NS proteins by dexamethasone, showed a cytopathic effect and eventually died. Our data strongly suggest that the intracellular accumulation of parvoviral NS products jeopardizes the survival of the cells, which cannot be detected unless a threshold protein concentration is reached. Interestingly, a cell variant could be isolated which resisted dexamethasone-induced killing, although it was fully inducible for the production of NS proteins. This variant was also unusually resistant to infection with MVMp virions, thus confirming the essential role played by the NS proteins in the parvoviral cytotoxicity and indicating that the cytocidal activity of the parvoviral NS products is modulated by cellular factors that may vary from one cell to another.