Expression and characterization of glycophospholipid-anchored human immunodeficiency virus type 1 envelope glycoproteins.

Four chimeric human immunodeficiency virus type 1 (HIV-1) env genes were constructed which encoded the extracellular domain of either the wild-type or a cleavage-defective HIV-1 envelope glycoprotein (gp160) fused at one of two different positions in env to a C-terminal glycosyl-phosphatidylinositol (GPI) attachment signal from the mouse Thy-1.1 glycoprotein. All four of the constructs encoded glycoproteins that were efficiently expressed when Rev was supplied in trans, and the two cleavable forms were processed normally to gp120 and a chimeric "gp41." The chimeric glycoproteins, in contrast to the wild-type glycoprotein, could be cleaved from the surface of transfected cells by treatment with phosphatidylinositol-specific phospholipase C, indicating that they were anchored in the plasma membrane by a GPI moiety. These GPI-anchored glycoproteins were transported intracellularly at a rate only slightly lower than that of the full-length HIV-1 glycoprotein and were present on the cell surface in equivalent amounts. Nevertheless, all four glycoproteins were defective in mediating both cell-cell and virus-cell fusion as determined by syncytium formation in COS-1-HeLa-T4 cell mixtures and trans complementation of an env-defective HIV-1 genome.     

Mutational analysis of conserved domains within the cytoplasmic tail of gp41 from human immunodeficiency virus type 1: effects on glycoprotein incorporation and infectivity

The transmembrane (TM) glycoprotein gp41 of human immunodeficiency virus type 1 possesses an unusually long ( approximately 150 amino acids) and highly conserved cytoplasmic region. Previous studies in which this cytoplasmic tail had been deleted partially or entirely have suggested that it is important for virus infectivity and incorporation of the gp120-gp41 glycoprotein complex into virions. To determine which regions of the conserved C-terminal domains are important for glycoprotein incorporation and infectivity, several small deletions and amino acid substitutions which modify highly conserved motifs were constructed in the infectious proviral background of NL4.3. The effects of these mutations on infectivity and glycoprotein incorporation into virions produced from transfected 293-T cells and infected H9 and CEMx174 cells were determined. With the exception of a mutation deleting amino acids QGL, all of the constructs resulted in decreased infectivity of the progeny virus both in a single-round infectivity assay and in a multiple-infection assay in H9 and CEMx174 cells. For most mutations, the decreased infectivity was correlated with a decreased incorporation of glycoprotein into virions. Substitution of the arginines (residues 839 and 846) with glutamates also reduced infectivity, but without a noticeable decrease in the amount of glycoprotein incorporated into virus produced from infected T cells. These results demonstrate that minor alterations in the conserved C-terminal region of the gp41 cytoplasmic tail can result in reductions in infectivity that correlate for most but not all constructs with a decrease in glycoprotein incorporation. Observed cell-dependent differences suggest the involvement of cellular factors in regulating glycoprotein incorporation and infectivity.     

Dynamics of the diradylglycerol responses of stimulated phagocytes

The generation of diradylglycerols (sn-1,2 diacylglycerols (DAG) and 1-O-alkyl-2-acylglycerols (AAG] was investigated in human polymorphonuclear leukocytes stimulated with fMet-Leu-Phe, phorbol myristate acetate (PMA), or A23187. With each stimulus, the elevations in the mass of DAG clearly preceded that of AAG. The levels of both lipids increased over time, peaked by 15-20 min (fMet-Leu-Phe) or 45-60 min (PMA or A23187) and returned slowly toward base line thereafter. The base-line levels of DAG were some 4-fold higher than levels of AAG. On stimulation, the relative increases in AAG (approximately 4-fold, fMet-Leu-Phe; approximately 20-fold, PMA and A23187) were much greater than the corresponding relative increases in the levels of DAG (approximately 2-fold fMet-Leu-Phe; approximately 5-fold, PMA and A23187). The diradylglycerol responses were dependent upon agonist concentration. Prior treatment with cytochalasin B augmented the fMet-Leu-Phe diradylglycerol responses but did not alter unstimulated or PMA- or A23187-stimulated diradylglycerol responses. Depletion of extracellular Ca2+ blocked responses to fMet-Leu-Phe, but not to PMA. Treatment with pertussis toxin: (a) completely blocked the responses to fMet-Leu-Phe, (b) slightly suppressed the AAG but not the DAG response to PMA, and (c) did not affect the responses to A23187. Gas chromatographic/mass spectral analyses indicated that the AAG generated during cell activation consists of a mixture of species differentiated by 1-O-alkyl chains of 16:0, 18:0, 18:1 and an additional species that remains uncharacterized. Since DAG and AAG are reportedly activators and inhibitors, respectively, of protein kinase C activities, the sequential generation of these lipid messengers may provide for a system to critically control the activation of protein kinase C.     

Structure-activity relationship studies of flavopiridol analogues

Cyclin dependent kinases (CDKs) along with the complementary cyclins form key regulatory checkpoint controls on the cell cycle. Flavopiridol is a synthetic flavone that shows potent and selective cyclin-dependent kinase inhibitory activity. In this paper, we report modifications of the 3-hydroxy-1-methylpiperidinyl (D ring) of flavopiridol and their effect on CDK inhibitory activity.     

Truncations of the simian immunodeficiency virus transmembrane protein confer expanded virus host range by removing a block to virus entry into cells.

We have investigated how truncation of the cytoplasmic domain of the transmembrane (TM) glycoprotein of simian immunodeficiency virus (SIV) modulates the host range of this virus. Termination codons were introduced into the env gene of SIVmac239 which resulted in the truncation of the transmembrane protein from a wild-type 354 amino acids (TM354) to 207 (TM207) and 193 (TM193) amino acids. Expression of the wild-type and mutant env genes from a simian virus 40-based vector resulted in normal biosynthesis and processing of the glycoproteins to gp130 and gp41 or the truncated TM proteins (gp28 and gp27). When expressed on the surface of COS-1 cells, all three glycoproteins mediated fusion of both CEMX174 and HUT78 cells. Virions containing the wild-type and mutant glycoproteins were capable of efficient replication in macaque peripheral blood lymphocytes and CEMX174 cells; in contrast, only virions that contained TM207 were capable of rapid infection of HUT78 cells. Both truncated glycoproteins were capable of efficiently mediating infection of both CEMX174 and HUT78 cells by an env-deficient human immunodeficiency virus. The wild-type SIV glycoprotein, however, was unable to mediate human immunodeficiency virus infection of HUT78 cells when assayed with this system. An analysis of the protein composition of SIV released from infected CEMX174 cells showed that the mutant virions contained significantly higher levels of glycoprotein compared with the wild type. These results demonstrate that truncation of the SIV cytoplasmic domain removes a block at the level of glycoprotein-mediated virus entry into HUT78 cells and points to a role for glycoprotein density in determining virus tropism.     

Mutations in the leucine zipper of the human immunodeficiency virus type 1 transmembrane glycoprotein affect fusion and infectivity.

Many retroviruses, including the human and simian immunodeficiency viruses, contain a leucine zipper-like repeat in a highly conserved region of the external domain of the transmembrane (TM) glycoprotein. This region has been postulated to play a role in stabilizing the oligomeric form of these molecules. To determine what role this region might play in envelope structure and function, several mutations were engineered into the middle isoleucine of the leucine zipper-like repeat of the human immunodeficiency virus type 1 (HIV-1) TM protein. A phenotypic analysis of these mutants demonstrated that conservative mutations (Ile to Val or Leu) did not block the ability of the viral glycoprotein to mediate cell-cell fusion or affect virus infectivity. In contrast, each of the other mutations, except for the Ile-to-Ala change, completely inhibited the ability of the glycoprotein to fuse HeLa-T4 cells and of mutant virions to infect H9 cells. The alanine mutation produced an intermediate phenotype in which both cell fusion and infectivity were significantly reduced. Thus, the biological activity of the glycoprotein titrates with the hydrophobicity of the residue in this position. None of the mutations affected the synthesis, oligomer formation, transport, or processing of the HIV glycoprotein complex. Although these results do not rule out a role for the leucine zipper region in glycoprotein oligomerization, they clearly point to a critical role for it in a post-CD4 binding step in HIV membrane fusion and virus entry.     

Resurrecting the ancestral enzymatic role of a modulatory subunit

In the post-genomic era, functional prediction of genes is largely based on sequence similarity searches, but sometimes the homologues bear different roles because of evolutionary adaptations. For instance, the existence of enzyme and non-enzyme homologues poses a difficult case for function prediction and the extent of this phenomenon is just starting to be surveyed. Different evolutionary paths are theoretically possible for the loss or acquisition of enzyme function. Here we studied the ancestral role of a model non-catalytic modulatory subunit. With a rational approach, we "resurrected" enzymatic activity from that subunit to experimentally prove that it derived from a catalytic ancestor. We show that this protein (L subunit ADP-glucose pyrophosphorylase) evolved to have a regulatory role, losing catalytic residues more than 130 million years ago, but preserving, possibly as a by-product, the substrate site architecture. Inactivation of catalytic subunits could be the consequence of a general evolutionary strategy to explore new regulatory roles in hetero-oligomers.     

Siderophore-redox active ionophore host-guest assemblies: A prototype for selective metal ion compartmentalization

The reduction potentials for two Wurster’s crowns, aza crown ethers which incorporate the redox active N,N,N′,N′-tetraalkyl-1,4-phenylenediamine into the structure of 18-crown-6, were studied in the presence of the siderophore ferrioxamine B, FeHDFB⁺. Addition of FeHDFB⁺ resulted in a positive shift in the host reduction potential for both aza crown ethers studied. This shift is explained in terms of host-guest supramolecular assembly formation, which was independently verified by FAB-MS. An enhanced affinity for host-guest formation of the reduced aza crown ether was calculated for each aza crown ether-siderophore assembly using a thermochemical cycle. These differences in host binding affinity as a function of redox state can be harnessed for use in specific metal ion compartmentalization with application, for example, to environmental remediation.     

Serologic survey for pathogens potentially affecting pronghorn (Antilocapra americana) fawn recruitment in Arizona, USA

During the 1990s, pronghorn (Antilocapra americana) populations declined in Arizona, USA. To investigate potential causes of decline, we collected blood samples from hunter-harvested male pronghorn from 2001 to 2003 on four Arizona sites. Sera were tested for antibody to parainfluenza virus type 3 (PI3), bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, epizootic hemorrhagic disease virus (EHDV), bluetongue virus (BTV), and Chlamydia psittaci. Antibody against PI3 was found in 33% of the samples, whereas antibody against BTV/EHDV was found in 77%. Antibodies to other pathogens were found at low prevalence rates. Although pronghorn decline in Arizona is probably not directly related to disease, potential reproductive effects of BTV/EHDV and PI3 infection on pronghorn in Arizona merit further study.     

Eulemur, me lemur: the evolution of scent-signal complexity in a primate clade

Signal complexity has been linked to social complexity in vocal, but not chemical, communication. To address this gap, we examined the chemical complexity of male and female glandular secretions in eight species of Eulemur. In this diverse clade of macrosmatic primates, species differ by social or mating system and dominance structure. We applied principal component and linear discriminate analyses to data obtained by gas chromatography/mass spectrometry. Beyond the significant effects on chemical signals of gland type, sex, season and species, we found effects of social variables and phylogeny. Notably, female odours were more chemically complex in multimale-multifemale species than pair-bonded species, whereas male odours were more chemically complex in codominant species than female-dominant species. Also, the traditional sexual dimorphism, whereby male signal complexity exceeds that of females, was present in codominant species, but reversed in female-dominant species. Lastly, a positive relationship between the species' pairwise chemical distances and their pairwise phylogenetic distances supported a gradual, but relatively fast mode of signal evolution. We suggest that the comparative method can be a powerful tool in olfactory research, revealing species differences relevant to the understanding of current signal utility and evolutionary processes. In particular, social complexity in lemurs may have selected for olfactory complexity.