Hexamethylene bisacetamide leads to reduced helper virus-free HSV-1 amplicon expression titers via suppression of ICP0

The herpes simplex virus (HSV)-derived amplicon vector has evolved into a promising gene transfer platform for widespread DNA delivery in gene replacement strategies and vaccine development given its ease of molecular manipulation, large transgene capacity, and transduction efficiencies of numerous cell types in vivo. The recent development of helper virus-free packaging methodologies bodes well for this vector system in its eventual implementation as a clinically viable therapeutic modality. For realization of clinical application, efforts have been made to enhance yields and quality of helper-free amplicon stocks. Hexamethylene bisacetamide (HMBA), a hybrid polar compound that exhibits stimulatory activity of HSV-1 immediate-early gene expression, has been employed as a standard reagent in helper virus-free packaging given its purported mode of action on virus gene expression kinetics. Unexpectedly, we have found that HMBA exhibits no titer-enhancing activity; in contrast, the compound enhances the proportion of amplicon virions that are non-expressive. Omission of HMBA during vector packaging led to a marked reduction in the ratios of vector genome-transducing to transgene-expressing virions. This effect was neither packaging-cell-specific nor amplicon-promoter-dependent. Analysis of resultant vector stocks indicated amplicon genome replication/concatenation was unaffected, but the level of particle-associated ICP0 was reduced in stocks packaged in the presence of HMBA. Inclusion of a co-transfected, ICP0-expressing plasmid into the packaging process led to significant rescue of amplicon expression titers, indicating that regulation of ICP0 concentrations is critical for maintenance of the amplicon genome expressive state.     

ICP34.5 protein of herpes simplex virus facilitates the initiation of protein translation by bridging eukaryotic initiation factor 2alpha (eIF2alpha) and protein phosphatase 1

The ICP34.5 protein of herpes simplex virus type 1 is a neurovirulence factor that plays critical roles in viral replication and anti-host responses. One of its functions is to recruit protein phosphatase 1 (PP1) that leads to the dephosphorylation of the α subunit of translation initiation factor eIF2 (eIF2α), which is inactivated by infection-induced phosphorylation. As PP1 is a protein phosphatase with a wide range of substrates, the question remains to be answered how ICP34.5 directs PP1 to specifically dephosphorylate eIF2α. Here we report that ICP34.5 not only binds PP1 but also associates with eIF2α by in vitro and in vivo assays. The binding site of eIF2α is identified at amino acids 233-248 of ICP34.5, which falls in the highly homologous region with human gene growth arrest and DNA damage 34. The interaction between ICP34.5 and eIF2α is independent of the phosphorylation status of eIF2α at serine 51. Deletion mutation of this region results in the failure of dephosphorylation of eIF2α by PP1 and, consequently, interrupts viral protein synthesis and replication. Our data illustrated that the binding between viral protein ICP34.5 and the host eIF2α is crucial for the specific dephosphorylation of eIF2α by PP1. We propose that herpes simplex virus protein ICP34.5 bridges PP1 and eIF2α via their binding motifs and thereby facilitates the protein synthesis and viral replication.     

Ligand induced stabilization of the melting temperature of the HSV-1 single-strand DNA binding protein using the thermal shift assay

We have adapted the thermal shift assay to measure the ligand binding properties of the herpes simplex virus-1 single-strand DNA binding protein, ICP8. By measuring SYPRO Orange fluorescence in microtiter plates using a fluorescence-enabled thermal cycler, we have quantified the effects of oligonucleotide ligands on the melting temperature of ICP8. We found that single-stranded oligomers raise the melting temperature of ICP8 in a length- and concentration-dependent manner, ranging from 1 °C for (dT)₅ to a maximum of 9 °C with oligomers ?10 nucleotides, with an apparent K_d of <1 μM for (dT)₂₀. Specifically, the results indicate that ICP8 is capable of interacting with oligomers as short as 5 nucleotides. Moreover, the observed increases in melting temperature of up to 9 °C, indicates that single-strand DNA binding significantly stabilizes the structure of ICP8. This assay may be applied to investigate the ligand binding proteins of other single-strand DNA binding proteins and used as a high-throughput screen to identify compounds with therapeutic potential that inhibit single-strand DNA binding. As proof of concept, the single-strand DNA binding agent ciprofloxacin reduces the ligand induced stabilization of the melting temperature of ICP8 in a dose-dependent manner.     

Environmental drivers of ectomycorrhizal communities in Europe's temperate oak forests

Ectomycorrhizal fungi are major ecological players in temperate forests, but they are rarely used in measures of forest condition because large‐scale, high‐resolution, standardized and replicated belowground data are scarce. We carried out an analysis of ectomycorrhizas at 22 intensively monitored long‐term oak plots, across nine European countries, covering complex natural and anthropogenic environmental gradients. We found that at large scales, mycorrhizal richness and evenness declined with decreasing soil pH and root density, and with increasing atmospheric nitrogen deposition. Shifts in mycorrhizas with different functional traits were detected; mycorrhizas with structures specialized for long‐distance transport related differently to most environmental variables than those without. The dominant oak‐specialist Lactarius quietus, with limited soil exploration abilities, responds positively to increasing nitrogen inputs and decreasing pH. In contrast, Tricholoma, Cortinarius and Piloderma species, with medium‐distance soil exploration abilities, show a consistently negative response. We also determined nitrogen critical loads for moderate (9.5–13.5 kg N/ha/year) and drastic (17 kg N/ha/year) changes in belowground mycorrhizal root communities in temperate oak forests. Overall, we generated the first baseline data for ectomycorrhizal fungi in the oak forests sampled, identified nitrogen pollution as one of their major drivers at large scales and revealed fungi that individually and/or in combination with others can be used as belowground indicators of environmental characteristics.     

Expression of HSV-1 ICP0 antigen peptide in prokaryotic cells and preparation of specific antibody

As an immediate-early protein of herpes simplex virus, infected-cell polypeptide 0 (ICP0) exhibits complicated interactions with host cells, and its regulatory function on gene expression is of great importance. Since the ICP0 encoding sequence contains many rare codons which are absent in E.coli, and ICP0 is highly unstable in prokaryotic cells, expression of entire ICP0 in prokaryotic cells has never been reported. In order to further investigate the function of ICP0, a recombinant plasmid was constructed by subcloning a cDNA fragment encoding an amino-terminal of 105 residues of the ICP0 protein into pGEX-5x-1 vector. The resulting GST-105 fusion antigen peptide was expressed with high efficiency in E.coli. Antibodies prepared after the immunization of mice with purified fusion protein can recognize not only the denatured ICP0 protein, but also the native ICP0 protein with normal biological conformation. Foundation items: National natural science foundation items (30570081 and 30670094)