Molecular and Physicochemical Factors Governing Solubility of the HIV gp41 Ectodomain.

The HIV gp41 ectodomain (e-gp41) is an attractive target for the development of vaccines and drugs against HIV because of its crucial role in viral fusion to the host cell. However, because of the high insolubility of e-gp41, most biophysical and structural analyses have relied on the production of truncated versions removing the loop region of gp41 or the utilization of nonphysiological solubilizing conditions. The loop region of gp41 is also known as principal immunodominant domain (PID) because of its high immunogenicity, and it is essential for gp41-mediated HIV fusion. In this study we identify the aggregation-prone regions of the amino acid sequence of the PID and engineer a highly soluble mutant that preserves the trimeric structure of the wild-type e-gp41 under physiological pH. Furthermore, using a reverse mutagenesis approach, we analyze the role of mutated amino acids upon the physicochemical factors that govern solubility of e-gp41. On this basis, we propose a molecular model for e-gp41 self-association, which can guide the production of soluble e-gp41 mutants for future biophysical analyses and biotechnological applications.

Reprogramming a GFP reporter gene subjects it to complex lentiviral gene regulation.

Late human immunodeficiency virus (HIV)-derived RNAs encoding relevant therapeutic targets or promising vaccine compounds, such as the HIV-1 group-specific antigen (Gag), are translocated from the nucleus into the cytoplasm via sophisticated export machinery. Relevant steps include the concerted action of several cis-acting RNA elements with the viral Rev-shuttle protein and several cellular components (Ran1/Exportin; Crm1). Based on detailed understanding of the molecular mechanisms guiding this complex process, we used rational codon usage modification to design and reprogram a GFP encoding reporter RNA now exactly mimicking the complex transcriptional and posttranscriptional regulation of late lentiviral mRNAs.

Broad antibody mediated cross-neutralization and preclinical immunogenicity of new codon-optimized HIV-1 clade CRF02_AG and G primary isolates.

Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary "street strain" isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA) to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G). Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G) and consensus tat (CRF02_AG and G) antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G.

Industrial scale gene synthesis.

The most recent developments in the area of deep DNA sequencing and downstream quantitative and functional analysis are rapidly adding a new dimension to understanding biochemical pathways and metabolic interdependencies. These increasing insights pave the way to designing new strategies that address public needs, including environmental applications and therapeutic inventions, or novel cell factories for sustainable and reconcilable energy or chemicals sources. Adding yet another level is building upon nonnaturally occurring networks and pathways. Recent developments in synthetic biology have created economic and reliable options for designing and synthesizing genes, operons, and eventually complete genomes. Meanwhile, high-throughput design and synthesis of extremely comprehensive DNA sequences have evolved into an enabling technology already indispensable in various life science sectors today. Here, we describe the industrial perspective of modern gene synthesis and its relationship with synthetic biology. Gene synthesis contributed significantly to the emergence of synthetic biology by not only providing the genetic material in high quality and quantity but also enabling its assembly, according to engineering design principles, in a standardized format. Synthetic biology on the other hand, added the need for assembling complex circuits and large complexes, thus fostering the development of appropriate methods and expanding the scope of applications. Synthetic biology has also stimulated interdisciplinary collaboration as well as integration of the broader public by addressing socioeconomic, philosophical, ethical, political, and legal opportunities and concerns. The demand-driven technological achievements of gene synthesis and the implemented processes are exemplified by an industrial setting of large-scale gene synthesis, describing production from order to delivery.

The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization.

One of the main advantages of de novo gene synthesis is the fact that it frees the researcher from any limitations imposed by the use of natural templates. To make the most out of this opportunity, efficient algorithms are needed to calculate a coding sequence, combining different requirements, such as adapted codon usage or avoidance of restriction sites, in the best possible way. We present an algorithm where a "variation window" covering several amino acid positions slides along the coding sequence. Candidate sequences are built comprising the already optimized part of the complete sequence and all possible combinations of synonymous codons representing the amino acids within the window. The candidate sequences are assessed with a quality function, and the first codon of the best candidates' variation window is fixed. Subsequently the window is shifted by one codon position. As an example of a freely accessible software implementing the algorithm, we present the Mr. Gene web-application. Additionally two experimental applications of the algorithm are shown.

The impact of intragenic CpG content on gene expression.

The development of vaccine components or recombinant therapeutics critically depends on sustained expression of the corresponding transgene. This study aimed to determine the contribution of intragenic CpG content to expression efficiency in transiently and stably transfected mammalian cells. Based upon a humanized version of green fluorescent protein (GFP) containing 60 CpGs within its coding sequence, a CpG-depleted variant of the GFP reporter was established by carefully modulating the codon usage. Interestingly, GFP reporter activity and detectable protein amounts in stably transfected CHO and 293 cells were significantly decreased upon CpG depletion and independent from promoter usage (CMV, EF1 alpha). The reduction in protein expression associated with CpG depletion was likewise observed for other unrelated reporter genes and was clearly reflected by a decline in mRNA copy numbers rather than translational efficiency. Moreover, decreased mRNA levels were neither due to nuclear export restrictions nor alternative splicing or mRNA instability. Rather, the intragenic CpG content influenced de novo transcriptional activity thus implying a common transcription-based mechanism of gene regulation via CpGs. Increased high CpG transcription correlated with changed nucleosomal positions in vitro albeit histone density at the two genes did not change in vivo as monitored by ChIP.

Fusion of Epstein-Barr virus nuclear antigen-1-derived glycine-alanine repeat to trans-dominant HIV-1 Gag increases inhibitory activities and survival of transduced cells in vivo.

Trans-dominant human immunodeficiency virus type 1 (HIV-1) Gag derivatives have been shown to efficiently inhibit late steps of HIV-1 replication in vitro by interfering with Gag precursor assembly, thus ranking among the interesting candidates for gene therapy approaches. However, efficient antiviral activities of corresponding transgenes are likely to be counteracted in particular by cell-mediated host immune responses toward the transgene-expressing cells. To decrease this potential immunogenicity, a 24-amino acid Gly-Ala (GA) stretch derived from Epstein-Barr virus nuclear antigen-1 (EBNA1) and known to overcome proteasomal degradation was fused to a trans-dominant Gag variant (sgD1). To determine the capacity of this fusion polypeptide to repress viral replication, PM-1 cells were transduced with sgD1 and GAsgD1 transgenes, using retroviral gene transfer. Challenge of stably transfected permissive cell lines with various viral strains indicated that N-terminal GA fusion even enhanced the inhibitory properties of sgD1. Further studies revealed that the GA stretch increased protein stability by blocking proteasomal degradation of Gag proteins. Immunization of BALB/c mice with a DNA vaccine vector expressing sgD1 induced substantial Gag-specific immune responses that were, however, clearly diminished in the presence of GA. Furthermore, recognition of cells expressing the GA-fused transgene by CD8(+) T cells was drastically reduced, both in vitro and in vivo, resulting in prolonged survival of the transduced cells in recipient mice.

Concerted inhibitory activities of Phyllanthus amarus on HIV replication in vitro and ex vivo.

Phyllanthus amarus derived preparations were previously shown to inhibit RT inhibitor-resistant HIV variants as efficiently as wild-type strains. The drugs target different steps of the HIV life cycle, thereby presenting multiple antiviral activities. Here we show that a water/alcohol extract blocks HIV-1 attachment and the HIV-1 enzymes integrase, reverse transcriptase and protease to different degrees. A gallotannin containing fraction and the isolated ellagitannins geraniin and corilagin were shown to be the most potent mediators of these antiviral activities. The P. amarus derived preparations blocked the interaction of HIV-1 gp120 with its primary cellular receptor CD4 at 50% inhibitory concentrations of 2.65 (water/alcohol extract) to 0.48 microg/ml (geraniin). Inhibition was also evident for the HIV-1 enzymes integrase (0.48-0.16 microg/ml), reverse transcriptase (8.17-2.53 microg/ml) and protease (21.80-6.28 microg/ml). In order to prove the in vivo relevance of these biological activities, plant material was administered orally to volunteers and a potent anti-HIV activity in blood could be demonstrated. Sera at a final concentration of 5% reduced HIV replication by more than 30%. These results support the conclusion that P. amarus has inhibitory effects on HIV not only in vitro but also in vivo.

Increase in MICs of ciprofloxacin in vivo in two closely related clinical isolates of Enterobacter cloacae.

The mechanisms of fluoroquinolone resistance in two isolates of Enterobacter cloacae, Ecl#1 and Ecl#2, from the same patient and with identical pulsed-field gel electrophoresis patterns, have been analysed. MICs of ciprofloxacin were 0.25 and 1 mg/L for Ecl#1 and Ecl#2, respectively. Ecl#2 was also more resistant to chloramphenicol and organic solvents. The quinolone resistance determining regions of gyrA/B and parC/E, and the marORA and acrB genes, were sequenced. Expression of marR, acrB, soxS, robA, ramA and fis was analysed by northern blotting. The activity of a 90 bp E. cloacae mar promoter fragment was examined with the reporter plasmid pIGJ-1mar. Sequencing the gyrAB and parCE genes revealed a single amino acid substitution in GyrA (corresponding to position 83 in GyrA of Escherichia coli) in Ecl#1 and Ecl#2 (Phe83) compared with reference strain E. cloacae DSMZ 3264 (Thr83). Ecl#2 accumulated significantly less norfloxacin and displayed higher levels of expression of marR and acrB than Exl#1, indicative of greater fluoroquinolone efflux activity. Sequencing gyrB, parC/E and marORA, and northern blotting of robA, ramA and fis, did not reveal any further differences between the two strains. No homologue of soxRS was detected in E. cloacae. Expression of GFP from pIGJ1-mar in Ecl#2 was higher than in Ecl#1. In these two closely related clinical isolates of E. cloacae, a target mutation in GyrA (Ecl#1 and Ecl#2) and increased fluoroquinolone efflux by AcrAB (Ecl#2) contribute to the resistance phenotypes, corroborating findings in vitro and in vivo about the sequential development of fluoroquinolone resistance.

A C-terminal 18 amino acid deletion in MarR in a clinical isolate of Escherichia coli reduces MarR binding properties and increases the MIC of ciprofloxacin.

As described recently, the different degree of fluoroquinolone resistance in a pair of sequential clinical isolates of Escherichia coli was due to the increased expression of the regulatory gene marA as a consequence of an 18 amino acid C-terminal deletion in the repressor MarR (MarR Delta). To further investigate the molecular mechanism of the loss of repressor function, we purified recombinant wild-type and mutated MarR, and tested their respective ability to form dimers and their specific DNA binding properties to the operator region marO. The dimerization capacity was analysed by non-reducing SDS-PAGE and by disuccinimidyl suberate-mediated cross-linking of the recombinant proteins. The binding of MarR was studied using the recombinant proteins and DNA probes containing the two identified binding sites in marO in the presence or absence of specific and non-specific DNA fragments. Dimerization of MarR Delta was reduced compared with MarR: the dimer portion was 33.8% (MarR) and 12.4% (MarR Delta) at a protein concentration of 10 IM. In mobility-shift assays MarR Delta showed a highly reduced complex formation. Footprinting analysis confirmed reduced binding of MarR Delta to its target sites, compared with MarR. The biochemical data are in full agreement with the crystal structure of MarR, which shows that the N- and C-terminal regions of MarR contribute to dimer formation. The data also indicate a major role of the MarR dimer as opposed to the monomer in DNA binding.