Jan Mous

Transcript imaging of the development of human T helper cells using oligonucleotide arrays.

Many pathological processes, including those causing allergies and autoimmune diseases, are associated with the presence of specialized subsets of T helper cells at the site of inflammation. Understanding the genetic program that controls the functional properties of T helper type 1 (Th1) versus T helper type 2 (Th2) cells may provide insight into the pathophysiology of inflammatory diseases. We compared the gene-expression profiles of human Th1 and Th2 cells using high-density oligonucleotide arrays with the capacity to display transcript levels of 6,000 human genes. Here we analyse the data sets derived from five independent experiments using statistical algorithms. This approach resulted in the identification of 215 differentially expressed genes, encoding proteins involved in transcriptional regulation, apoptosis, proteolysis, and cell adhesion and migration. A subset of these genes was further upregulated by exposure of differentiated Th1 cells to interleukin-12 (IL-12), as confirmed by kinetic PCR analysis, indicating that IL-12 modulates the effector functions of Th1 cells in the absence of antigenic stimulation. Functional assays and in vivo expression of selected genes have validated the biological relevance of our study. Our results provide new insight into the transcriptional program controlling the functional diversity of subsets of T helper cells.

Characterization of human immunodeficiency virus type 1 mutantswith decreased sensitivity to proteinase inhibitor Ro 31-8959

A human immunodeficiency virus type 1 (HIV-1) variant with highly reduced susceptibility to Ro 31-8959, an inhibitor of the viral proteinase, has been selected by repeated passage of wild-type virus in CEM cells in the presence of increasing concentrations of the inhibitor. Peptide sequences of the proteinase of selected virus were obtained from proviral DNA. Sequence comparison to wild-type (wt) proteinase demonstrated two amino acid substitutions in the resistant virus, a Gly to Val exchange at position 48 and a Leu to Met exchange at position 90. Furthermore, sequences of intermediate passage virus suggest contributions from positions 12, 36, 57, and 63 in early steps of resistance development. The selected virus showed a ca. 40-fold increase in 50% inhibitory concentration of Ro 31-8959. Growth kinetics of resistant virus were comparable to wild-type virus and the resistant genotype proved to be stable in the absence of inhibitor. Directed mutagenesis of the HIV-1 HXB2 proteinase at positions 48 and 90 suggested that each mutation alone led to a moderate decrease in sensitivity of the recombinant virus to proteinase inhibitor. However, a recombinant virus carrying both mutations in the proteinase gene showed a significant reduction in its sensitivity to Ro 31-8959 thus proving the importance of these exchanges for the resistance phenotype.

Identification of amino acid residues critical for endonuclease and integration activities of HIV-1 in protein in vitro

HIV-IN protein, tagged with a hexahistidine tail was expressed in Escherichia coli and purified by a one-step nickel chelate affinity chromatography procedure. The purified IN protein was characterized in terms of its endonuclease and integrase properties in vitro. Specific cleavage and integration of HIV U5 LTR ends were observed in the presence of 2-5 mM Mg2+ or Ca2+. In the presence of 2 mM Mn2+, cleavage and integration occurred at additional sites indicating a decreased specificity. The properties of mutant IN proteins were examined in vitro. Deletion of 39 amino acids from the N-terminus and a minimum of 25 residues from the C-terminus impaired IN-mediated cleavage and integration activities. The results of site-directed mutagenesis experiments showed that residues critical for IN function are highly conserved. Mutations of conserved residues Asp64, Pro109, Asp116, and Glu152 adversely affected IN function in vitro. Mutations of nonconserved residues Gly189 and Thr112 had no effect. Mutation of a conserved Thr115 to Ala caused a near complete loss of Mg(2+)-dependent integration activity, but only partially effected endonucleolytic cleavage activity of IN. These results suggest that not all conserved residues are involved in both endonucleolytic cleavage and integration activities of HIV-IN.

Identification of farnesoid X receptor beta as a novel mammalian nuclear receptor sensing lanosterol

ABSTRACT Nuclear receptors are ligand-modulated transcription factors. On the basis of the completed human genome sequence, this family was thought to contain 48 functional members. However, by mining human and mouse genomic sequences, we identified FXRβ as a novel family member. It is a functional receptor in mice, rats, rabbits, and dogs but constitutes a pseudogene in humans and primates. Murine FXRβ is widely coexpressed with FXR in embryonic and adult tissues. It heterodimerizes with RXRα and stimulates transcription through specific DNA response elements upon addition of 9-cis-retinoic acid. Finally, we identified lanosterol as a candidate endogenous ligand that induces coactivator recruitment and transcriptional activation by mFXRβ. Lanosterol is an intermediate of cholesterol biosynthesis, which suggests a direct role in the control of cholesterol biosynthesis in nonprimates. The identification of FXRβ as a novel functional receptor in nonprimate animals sheds new light on the species differences in cholesterol metabolism and has strong implications for the interpretation of genetic and pharmacological studies of FXR-directed physiologies and drug discovery programs.

HIV-1 RT-associated ribonuclease H displays both endonuclease and 3'----5' exonuclease activity.

We have analysed the mechanism of ribonuclease H (RNaseH) induced cleavage of a defined RNA‐DNA hybrid by human immuno‐deficiency virus (HIV‐1) reverse transcriptase (RT). An in vitro transcribed RNA labelled at the 3′ end was hybridized to a pentadecameric DNA oligonucleotide complementary to an internal region of the RNA. Upon incubation of this RNA‐DNA hybrid with recombinant p66 or p66/p51 HIV‐1 reverse transcriptase, RT‐RNaseH mediated cleavage is observed at most nucleotides within the short hybridized stretch, resulting in a spectrum of RNA fragments extending from the 3′ label to this region and differing in length by one nucleotide. The same RNA, this time labelled at the 5′ end, yields only one or two major cleavage products corresponding to RNA species extending from the 5′ label to the middle of the hybridized region. Such a result can be explained by the action of both endonuclease and 3′‐‐‐‐5′ exonuclease activities inherent to the C‐terminal domain of p66 RT. To investigate how RNaseH cleavage is coupled to reverse transcription, a combination of deoxynucleoside triphosphates was used which allowed controlled extension of the primer DNA. Concomitantly with the elongation of the oligonucleotide primer, RNaseH cleavage proceeds towards the 5′ end of the RNA with identical increments, suggesting a simultaneous action of both activities.

Active site mutagenesis of the AIDS virus protease and its alleviation by trans complementation.

Replacement of the putative active site Asp residue of cloned HIV‐1 protease with Ala yields a molecule incapable of autocatalytic processing. Similarly, protease/reverse transcriptase and protease/reverse transcriptase/endonuclease polyproteins containing the same mutation accumulate as enzymatically inert polyproteins. Introduction of a second, wild‐type, copy of protease in trans alleviates this defect, leading in the case of individually cloned protease to cleavage of the mutant protein, and with the polyprotein mutants to release of the reverse transcriptase and endonuclease polypeptides, the former of which recover enzymatic activity. In related experiments, a similar inhibition and trans‐complementation of a genetically engineered gag–protease fusion protein was observed.

Expression of biologically active human T-cell lymphotropic virus type III reverse transcriptase in Bacillus subtilis

A 2.4-kb DNA fragment from the pol region of the human T-cell lymphotropic virus, encoding the protease, reverse transcriptase and a portion of the endonuclease N-terminus was stably introduced into a high-level Bacillus subtilis expression system under inducible control of the Escherichia coli lac regulatory elements. The major expression plasmid, pRTL11, contains a bacteriophage T5 promoter/lac operator element, which is controlled by lac repressor, supplied by the secondary plasmid, pBL1. Upon IPTG induction, a 90-kDa polyprotein is synthesised and subsequently proteolytically cleaved to reveal 64-kDa and 52-kDa polypeptides. Partial purification reveals that reverse transcriptase activity co-migrates with these two polypeptides.

Evaluation of hepatitis C virus envelope proteins expressed in E. coli and insect cells for use as tools for antibody screening

BACKGROUND/METHODS The two envelope proteins of hepatitis C virus, E1 and E2, were expressed in E. coli and, as secretory proteins, in Sf9 insect cells using recombinant baculoviruses. Co-infection of insect cells with E1 and E2-recombinant baculoviruses was performed, which has been shown to result in formation of E1-E2 dimers. All envelope proteins were purified by Ni2+-NTA chromatography and used for screening of serum samples in a HCV EIA assay. Serum samples of normal blood donors, chronically HCV-infected patients, a mixed titer panel and several seroconversion panels were screened and compared to test results with Cobas Core Anti-HCV EIA. RESULTS Screening of the sera of chronically HCV-infected patients (100% positive in Cobas Core Anti-HCV EIA) revealed 10-40% anti-E1 positive sera using different Sf9-expressed, glycosylated proteins and 93% using E. coli-expressed, non-glycosylated E1 protein. When the same sera were tested with different E2 proteins expressed in Sf9 cells and in E. coli, about 70-73% showed anti-E2 reactivity. When the proteins from Sf9 cells co-infected with E1- and E2-recombinant baculoviruses were tested, 70-80% of the same sera showed anti-envelope reactivity. CONCLUSIONS Testing of these patient antisera, and those from the well-characterized mixed titer panel BBI-PHV203, showed that recombinant E1 expressed in E. coli and co-expressed E1 and E2 proteins from Sf9 cells could be used as additional tools for anti-HCV antibody screening.

Mutation of conserved N-glycosylation sites around the CD4-binding site of human immunodeficiency virus type 1 GP120 affects viral infectivity.

Infection by the human immunodeficiency virus type 1 (HIV-1) is initiated through interaction of its exterior envelope glycoprotein gp120 with the CD4 receptor on target cells. To address the possible role of N-glycosylation of HIV-1 gp120 in binding CD4, we mutated different conserved N-glycosylation site Asn-residues in the vicinity of the putative CD4 binding site, as single mutations or in combinations. Authentic and mutant gp120 proteins were produced using the baculovirus expression system. All mutant proteins were produced and secreted at similar levels and could be immunoprecipitated with an HIV(+)-serum. Furthermore, all glycosylation mutants retained the full capacity to bind CD4 except for a triple mutant which showed reduced binding. Different gp120 mutant genes were then introduced in an infectious proviral DNA clone. Upon transfection of MT-2 cells, the authentic HIV-1 clone induced maximal virus production after 6 days. In the case of the triple glycosylation mutant, comparable virus production was first reached after a delay of about 12 days. Moreover, in contrast to native HIV, the mutant virus induced no typical multinucleated giant cells. These results suggest that the attached carbohydrates around the CD4-binding site of gp120, may contribute to the generation of this protein domain required for high affinity receptor interaction.

Purification and in vitro-phospholabeling of secretory envelope proteins E1 and E2 of hepatitis C virus expressed in insect cells

The putative envelope glycoproteins of hepatitis C virus (HCV), E1 and E2, were expressed as recombinant, secretory proteins in Sf9 insect cells through infection with recombinant baculoviruses. The influenza virus hemagglutinin signal sequence (HASS) was inserted upstream of the HCV-cDNAs in order to effect secretion. Furthermore, a hexa-histidine tag for purification on a Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) column and a protein kinase A (PKA) recognition sequence for in vitro-phospholabeling were fused upstream of the HCV-cDNA. E1- and E2 proteins lacking their carboxy-terminal, hydrophobic sequence were produced by baculovirus-infected insect cells in bioreactors of 23 1. The medium was concentrated and proteins were purified under native conditions on Ni(2+)-NTA columns. Purified proteins could be phospholabeled in vitro using the catalytic subunit of protein kinase. A isolated from bovine heart and gamma-[32P]ATP. Labeled E1 and E2 proteins expressed in insect cells could be immunoprecipitated with sera from HCV-infected patients. Co-expression of these E1 and E2 proteins led to the formation of E1-E2 complexes within the insect cell and to secretion of these complexes into the medium.