Selective pressure of a quinoxaline nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on HIV-1 replication results in the emergence of nucleoside RT-inhibitor-specific (RT Leu-74-->Val or Ile and Val-75-->Leu or Ile) HIV-1 mutants.

The quinoxaline nonnucleoside RT inhibitor (NNRTI) (S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4- dihydroquinoxaline-2(1H)-thione (HBY 097) was used to select for drug-resistant HIV-1 variants in vitro. The viruses first developed mutations affecting the NNRTI-binding pocket, and five of six strains displayed the RT G190-->E substitution, which is characteristic for HIV-1 resistance against quinoxalines. In one variant, a new mutant (G190-->Q) most likely evolved from preexisting G190-->E mutants. The negative charge introduced by the G190-->E substitution was maintained at that site of the pocket by simultaneous selection for V179-->D together with G190-->Q. After continued exposure to the drug, mutations at positions so far known to be specific for resistance against nucleoside RT inhibitors (NRTIs) (L74-->V/I and V75-->L/I) were consistently detected in all cultures. The inhibitory activities of the cellular conversion product of 2',3'-dideoxyinosine (ddI, didanosine), 2',3'-dideoxyadenosine (ddA) and of 2',3'-didehydro-3'-deoxythymidine (d4T, stavudine) against these late-passage viruses were shown to be enhanced with the L74-->V/I RT mutant virus as compared with the wild-type (wt) HIV-1MN isolate. Clonal analysis proved linkage of the codon 74 and codon 75 mutations to the NNRTI-specific mutations in all RT gene fragments. The nonnucleoside- and nucleoside-resistance mutation sites are separated by approximately 35 A. We propose that the two sites "communicate" through the template-primer which is situated in the DNA-binding cleft between these two sites. Quinoxalines cause high selective pressure on HIV-1 replication in vitro; however, the implication of these findings for the treatment of HIV-1 infection has yet to be determined.

Crystal structure of the membrane-exposed domain from a respiratory quinol oxidase complex with an engineered dinuclear copper center.

Cytochrome oxidase is a membrane protein complex that catalyzes reduction of molecular oxygen to water and utilizes the free energy of this reaction to generate a transmembrane proton gradient during respiration. The electron entry site in subunit II is a mixed-valence dinuclear copper center in enzymes that oxidize cytochrome c. This center has been lost during the evolution of the quinoloxidizing branch of cytochrome oxidases but can be restored by engineering. Herein we describe the crystal structures of the periplasmic fragment from the wild-type subunit II (CyoA) of Escherichia coli quinol oxidase at 2.5-A resolution and of the mutant with the engineered dinuclear copper center (purple CyoA) at 2.3-A resolution. CyoA is folded as an 11-stranded mostly antiparallel beta-sandwich followed by three alpha-helices. The dinuclear copper center is located at the loops between strands beta 5-beta 6 and beta 9-beta 10. The two coppers are at a 2.5-A distance and symmetrically coordinated to the main ligands that are two bridging cysteines and two terminal histidines. The residues that are distinct in cytochrome c and quinol oxidases are around the dinuclear copper center. Structural comparison suggests a common ancestry for subunit II of cytochrome oxidase and blue copper-binding proteins.

The sigma factor sigma s affects antibiotic production and biological control activity of Pseudomonas fluorescens Pf-5.

Pseudomonas fluorescens Pf-5, a rhizosphere-inhabiting bacterium that suppresses several soilborne pathogens of plants, produces the antibiotics pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol. A gene necessary for pyrrolnitrin production by Pf-5 was identified as rpoS, which encodes the stationary-phase sigma factor sigma s. Several pleiotropic effects of an rpoS mutation in Escherichia coli also were observed in an RpoS- mutant of Pf-5. These included sensitivities of stationary-phase cells to stresses imposed by hydrogen peroxide or high salt concentration. A plasmid containing the cloned wild-type rpoS gene restored pyrrolnitrin production and stress tolerance to the RpoS- mutant of Pf-5. The RpoS- mutant overproduced pyoluteorin and 2,4-diacetyl-phloroglucinol, two antibiotics that inhibit growth of the phytopathogenic fungus Pythium ultimum, and was superior to the wild type in suppression of seedling damping-off of cucumber caused by Pythium ultimum. When inoculated onto cucumber seed at high cell densities, the RpoS- mutant did not survive as well as the wild-type strain on surfaces of developing seedlings. Other stationary-phase-specific phenotypes of Pf-5, such as the production of cyanide and extracellular protease(s) were expressed by the RpoS- mutant, suggesting that sigma s is only one of the sigma factors required for the transcription of genes in stationary-phase cells of P. fluorescens. These results indicate that a sigma factor encoded by rpoS influences antibiotic production, biological control activity, and survival of P. fluorescens on plant surfaces.

Temporally and spectrally resolved subpicosecond energy transfer within the peripheral antenna complex (LH2) and from LH2 to the core antenna complex in photosynthetic purple bacteria.

We report studies of energy transfer from the 800-nm absorbing pigment (B800) to the 850-nm absorbing pigment (B850) of the LH2 peripheral antenna complex and from LH2 to the core antenna complex (LH1) in Rhodobacter (Rb.) sphaeroides. The B800 to B850 process was studied in membranes from a LH2-reaction center (no LH1) mutant of Rb. sphaeroides and the LH2 to LH1 transfer was studied in both the wild-type species and in LH2 mutants with blue-shifted B850. The measurements were performed by using approximately 100-fs pulses to probe the formation of acceptor excitations in a two-color pump-probe measurement. Our experiments reveal a B800 to B850 transfer time of approximately 0.7 ps at 296 K and energy transfer from LH2 to LH1 is characterized by a time constant of approximately 3 ps at 296 K and approximately 5 ps at 77 K. In the blue-shifted B850 mutants, the transfer time from B850 to LH1 becomes gradually longer with increasing blue-shift of the B850 band as a result of the decreasing spectral overlap between the antennae. The results have been used to produce a model for the association between the ring-like structures that are characteristic of both the LH2 and LH1 antennae.

Inhibition of human immunodeficiency virus type 1 transcription and replication by DNA sequence-selective plant lignans.

A plant lignan, 3'-O-methyl nordihydroguaiaretic acid (3'-O-methyl NDGA, denoted Malachi 4:5-6 or Mal.4; molecular weigth 316), was isolated from Larrea tridentata and found to be able to inhibit human immunodeficiency virus (HIV) Tat-regulated transactivation in vivo, induce protection of lymphoblastoid CEM-SS cells from HIV (strain IIIB) killing, and suppress the replication of five HIV-1 strains (WM, MN, VS, JR-CSF, and IIIB) in mitogen-stimulated peripheral blood mononuclear cells, all in a dose-dependent manner. Mal.4 inhibits both basal transcription and Tat-regulated transactivation in vitro. The target of Mal.4 has been localized to nucleotides -87 to -40 of the HIV long terminal repeat. Mal.4 directly and specifically interferes with the binding of Sp1 to Sp1 sites in the HIV long terminal repeat. By inhibiting proviral expression, Mal.4 may be able to interrupt the life cycles of both wild-type and reverse transcriptase or protease mutant viruses in HIV-infected patients.

Trans-dominant inhibitory human immunodeficiency virus type 1 protease monomers prevent protease activation and virion maturation.

Production of infectious human immunodeficiency virus (HIV) requires proper polyprotein processing by the dimeric viral protease. The trans-dominant inhibitory activity of a defective protease monomer with the active site Asp-25 changed to Asn was measured by transient transfection. A proviral plasmid that included the drug-selectable Escherichia coli gpt gene was used to deliver the wild-type (wt) or mutant proteases to cultured cells. Coexpression of the wt proviral DNA (HIV-gpt) with increasing amounts of the mutant proviral DNA (HIV-gpt D25N) results in a concomitant decrease in proteolytic activity monitored by in vivo viral polyprotein processing. The viral particles resulting from inactivation of the protease were mostly immature, consisting predominantly of unprocessed p55gag and p160gag-pol polyproteins. In the presence of HIV-1 gp160 env, the number of secreted noninfectious particles correlated with the presence of increasing amounts of the defective protease. Greater than 97% reduction in infectivity was observed at a 1:6 ratio of wt to defective protease DNA. This provides an estimate of the level of inhibition required for effectively preventing virion processing. Stable expression of the defective protease in monkey cells reduced the yield of infectious particles from these cells by 90% upon transfection with the wt proviral DNA. These results show that defective subunits of the viral protease exert a trans-dominant inhibitory effect resulting from the formation of catalytically compromised heterodimers in vivo, ultimately yielding noninfectious viral particles.

Expression of endogenous peptide-major histocompatibility complex class II complexes derived from invariant chain-antigen fusion proteins.

CD4+ T cells recognize major histocompatibility complex (MHC) class II-bound peptides that are primarily obtained from extracellular sources. Endogenously synthesized proteins that readily enter the MHC class I presentation pathway are generally excluded from the MHC class II presentation pathway. We show here that endogenously synthesized ovalbumin or hen egg lysozyme can be efficiently presented as peptide-MHC class II complexes when they are expressed as fusion proteins with the invariant chain (Ii). Similar to the wild-type Ii, the Ii-antigen fusion proteins were associated intracellularly with MHC molecules. Most efficient expression of endogenous peptide-MHC complex was obtained with fusion proteins that contained the endosomal targeting signal within the N-terminal cytoplasmic Ii residues but did not require the luminal residues of Ii that are known to bind MHC molecules. These results suggest that signals within the Ii can allow endogenously synthesized proteins to efficiently enter the MHC class II presentation pathway. They also suggest a strategy for identifying unknown antigens presented by MHC class II molecules.

Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene.

The scl gene encodes a basic-helix-loop-helix transcription factor which was identified through its involvement in chromosomal translocations in T-cell leukemia. To elucidate its physiological role, scl was targeted in embryonic stem cells. Mice heterozygous for the scl null mutation were intercrossed and their offspring were genotyped. Homozygous mutant (scl-/-) pups were not detected in newborn litters, and analysis at earlier time points demonstrated that scl-/- embryos were dying around embryonic day 9.5. The scl-/- embryos were pale, edematous, and markedly growth retarded after embryonic day 8.75. Histological studies showed complete absence of recognizable hematopoiesis in the yolk sac of these embryos. Early organogenesis appeared to be otherwise normal. Culture of yolk sac cells of wild-type, heterozygous, and homozygous littermates confirmed the absence of hematopoietic cells in scl-/- yolk sacs. Reverse transcription PCR was used to examine the transcripts of several genes implicated in early hematopoiesis. Transcripts of GATA-1 and PU.1 transcription factors were absent from RNA from scl-/- yolk sacs and embryos. These results implicate scl as a crucial regulator of early hematopoiesis.

Insertional mutagenesis and marker rescue in a protozoan parasite: cloning of the uracil phosphoribosyltransferase locus from Toxoplasma gondii.

Nonhomologous integration vectors have been used to demonstrate the feasibility of insertional mutagenesis in haploid tachyzoites of the protozoan parasite Toxoplasma gondii. Mutant clones resistant to 5-fluorouracil were identified at a frequency of approximately 10(-6) (approximately 2 x 10(-5) of the stable transformants). Four independent mutants were isolated, all of which were shown to lack uracil phosphoribosyl-transferase (UPRT) activity and harbor transgenes integrated at closely linked loci, suggesting inactivation of the UPRT-encoding gene. Genomic DNA flanking the insertion point (along with the integrated vector) was readily recovered by bacterial transformation with restriction-digested, self-ligated total genomic DNA. Screening of genomic libraries with the recovered fragment identified sequences exhibiting high homology to known UPRT-encoding genes from other species, and cDNA clones were isolated that contain a single open reading frame predicted to encode the 244-amino acid enzyme. Homologous recombination vectors were exploited to create genetic knock-outs at the UPRT locus, which are deficient in enzyme activity but can be complemented by transient transformation with wild-type sequences--formally confirming identification of the functional UPRT gene. Mapping of transgene insertion points indicates that multiple independent mutants arose from integration at distinct sites within the UPRT gene, suggesting that nonhomologous integration is sufficiently random to permit tagging of the entire parasite genome in a single transformation.

Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries.

A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementarity-determining regions (CDRs) in a single-chain Fv specific for the O polysaccharide of Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10% while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv.

Exit from G0 and entry into the cell cycle of cells expressing p21Sdi1 antisense RNA.

p21Sdi1 (also known as Cip1 and Waf1), an inhibitor of DNA synthesis cloned from senescent human fibroblasts, is an inhibitor of G1 cyclin-dependent kinases (Cdks) in vitro and is transcriptionally regulated by wild-type p53. In addition, p21Sdi1 has been found to inhibit DNA replication by direct interaction with proliferating cell nuclear antigen. In this study we analyzed normal human fibroblast cells arrested in G0 and determined that an excess of p21Sdi1 was present after immunodepletion of various cyclins and Cdks, in contrast to mitogen-stimulated cells in early S phase. Expression of antisense p21Sdi1 RNA in G0-arrested cells resulted in induction of DNA synthesis as well as entry into mitosis. These results suggest that p21Sdi1 functions in G0 and early G1 and that decreased expression of the gene is necessary for cell cycle progression.

Recombinant vesicular stomatitis viruses from DNA.

We assembled a DNA clone containing the 11,161-nt sequence of the prototype rhabdovirus, vesicular stomatitis virus (VSV), such that it could be transcribed by the bacteriophage T7 RNA polymerase to yield a full-length positive-strand RNA complementary to the VSV genome. Expression of this RNA in cells also expressing the VSV nucleocapsid protein and the two VSV polymerase subunits resulted in production of VSV with the growth characteristics of wild-type VSV. Recovery of virus from DNA was verified by (i) the presence of two genetic tags generating restriction sites in DNA derived from the genome, (ii) direct sequencing of the genomic RNA of the recovered virus, and (iii) production of a VSV recombinant in which the glycoprotein was derived from a second serotype. The ability to generate VSV from DNA opens numerous possibilities for the genetic analysis of VSV replication. In addition, because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.

In vivo assembly of rhodopsin from expressed polypeptide fragments.

Rhodopsin folding and assembly were investigated by expression of five bovine opsin gene fragments separated at points corresponding to proteolytic cleavage sites in the second or third cytoplasmic regions. The CH(1-146) and CH(147-348) gene fragments encode amino acids 1-146 and 147-348 of opsin, while the TH(1-240) and TH(241-348) gene fragments encode amino acids 1-240 and 241-348, respectively. Another gene fragment, CT(147-240), encodes amino acids 147-240. All five opsin polypeptide fragments were stably produced upon expression of the corresponding gene fragments in COS-1 cells. The singly expressed polypeptide fragments failed to form a chromophore with 11-cis-retinal, whereas coexpression of two or three complementary fragments [CH(1-146) + CH(147-348), TH(1-240) + TH(241-348), or CH(1-146) + CT(147-240) + TH(241-348)] formed pigments with spectral properties similar to wild-type rhodopsin. The NH2-terminal polypeptide in these rhodopsins showed a glycosylation pattern characteristic of wild-type COS-1 cell rhodopsin and was noncovalently associated with its complementary fragment(s). Further, the CH(1-146) + CH(147-348) rhodopsin showed substantial light-dependent activation of transducin. We conclude that the functional assembly of rhodopsin is mediated by the association of at least three protein-folding domains.

Ras farnesyltransferase inhibitors suppress the phenotype resulting from an activated ras mutation in Caenorhabditis elegans.

Attachment of Ras protein to the membrane, which requires farnesylation at its C terminus, is essential for its biological activity. A promising pharmacological approach of antagonizing oncogenic Ras activity is to develop inhibitors of farnesyltransferase. We use Caenorhabditis elegans vulval differentiation, which is controlled by a Ras-mediated signal transduction pathway, as a model system to test previously identified farnesyltransferase inhibitors. We show here that two farnesyltransferase inhibitors, manumycin and gliotoxin, suppress the Multivulva phenotype resulting from an activated let-60 ras mutation, but not the Multivulva phenotype resulting from mutations in the lin-1 gene or the lin-15 gene, which act downstream and upstream of let-60 ras, respectively, in the signaling pathway. These results are consistent with the idea that the suppression of the Multivulva phenotype of let-60 ras by the two inhibitors is specific for Ras protein and that the mutant Ras protein might be more sensitive than wild-type Ras to the farnesyltransferase inhibitors. This work suggests that C. elegans vulval development could be a simple and effective in vivo system for evaluation of farnesyltransferase inhibitors against Ras-activated tumors.

Novel microbicidal agents derived from Naja atra cardiotoxin 1 (CTX-1)

During my PhD course, I focused my research on antimicrobial peptides (AMPs), in particular on the aspects of their computational design and development. This work led to the development of a new family of AMPs that I designed, starting from the amino acid sequence of a snake venom toxin, the cardiotoxin 1 (CTX-1) of Naja atra. Naja atra atra cardiotoxin 1, produced by Chinese cobra snakes belonging to Elapidae family, is included in the three-finger toxin family and exerts high cytotoxicity and antimicrobial activity too. This toxin family is characterized by specific folding of three beta-sheet loops (“fingers”) extending from the central core and by four conserved disulfide bridges. Using as template the first loop of this toxin, different sequences of 20 amino acids linear cationic peptides have been designed in order to avoid toxic effects but to maintain and strengthen the antimicrobial activity. As a result, the sequence NCP-0 (Naja Cardiotoxin Peptide-0) was designed as ancestor and subsequently other 4 variant sequences of NCP0 were developed. These variant sequences have shown microbicidal activity towards a panel of reference strains of Gram-positive and Gram-negative bacteria, fungi and an enveloped virus. In particular, the sequence designed as NCP-3 (Naja Cardiotoxin Peptide-3) and its variants NCP-3a and NCP-3b have shown the best antimicrobial activity together with low cytotoxicity against eukaryotic cells and low hemolytic activity. Bactericidal activity has been demonstrated by minimum bactericidal concentration (MBC) assay at values below 10 μg/ml for Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ( clinical isolates), Moraxella catharralis ATCC 25238, MRSA ATCC 43400, while towards Staphylococcus aureus ATCC 25923, Enterococcus hirae ATCC 10541 and Streptococcus agalactiae ATCC 13813 the bactericidal activity was demonstrated even below 1.6 μg/ml concentration. This potent antimicrobial activity was confirmed even for unicellular fungi Candida albicans, Candida glabrata and Malassezia pachydermatis (MBC 32.26-6.4 μg/ml), and also against the fast-growing mycobacteria Mycobacterium smegmatis DSMZ 43756 and Mycobacterium fortuitum DSMZ 46621 (MBC 100 μg/ml). Moreover, NCP-3 has shown a virucidal activity on the enveloped virus Bovine Herpesvirus 1 (BoHV1) belonging to herpesviridae family. The bactericidal activity is maintained in a high salt concentration (125 and 250 mM NaCl) medium and PB +20% Mueller Hinton Medium for E. coli, MRSA and Pseudomonas aeruginosa reference strains. Considering these in vitro obtained data, we propose NCP-3 and its variants NCP-3a and NCP-3b as promising antimicrobial candidates. For this reason, the whole novel AMPs family has been protected by a national patent (n°102015000015951).