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Biomedical subjects

G Lemay

Publications and source records attributed to G Lemay.

At least 19 recordsLinked to original sources
PubMed2001

Human Jurkat lymphocytes clones differ in their capacity to support productive human immunodeficiency virus type 1 multiplication.

CD4-positive human lymphocytic cell lines are essential tools for the study of human immunodeficiency virus (HIV) replication. Jurkat cells are among cells more frequently used for this purpose. In the current study, various cell clones or cells stocks derived from this cell line were shown to vary substantially in their response to viral infection and in their ability to support productive virus multiplication. The formation of syncytia, the effect of Vpu on viral export, and especially the specific infectivity of the viruses released, can vary significantly among independent cell stocks. This suggests that the choice of an adequate cell clone or cell line could be critical while evaluating specific properties of the virus and further stresses the limitations of tissue culture models. However, these observations also raise the possibility of exploiting these differences among cells to study specific aspects of host-cell interactions contributing to viral multiplication.

Clone Cells↗
PubMed2000

Functional studies of a chimeric protein containing portions of the Na(+)/glucose and Na(+)/myo-inositol cotransporters.

We obtained cDNA chimeras between Na/glucose cotransporter (SGLT1) and the homologous Na(+)/myo-inositol cotransporter (SMIT) by creating random chimeras in plasmids. Of 12 chimeras, two were functional when expressed in Xenopus laevis oocytes but, upon sequencing, only one of them (C1) produced an actual chimeric protein. In C1, the first 69 amino acids of SGLT1 were replaced by the corresponding 50 amino acids of SMIT. C1 transports the same sugars as does SGLT1. C1's affinity for all sugar substrates was systematically increased by a factor of 3.3+/-0.4 but the V(max) was diminished by a factor of 15-40. In contrast, the cotransport affinity for Na(+) was unchanged. The surface expression of C1 was one seventh that of SGLT1, which explains part of the reduced V(max) and implies a significant reduction in turnover rate. N-terminal truncated constructs of SGLT1 cDNA showed that deleting amino acids 2-14 does not affect cotransporter activity, but that the pentapeptide T(14)RPVET(19) is important for normal levels of SGLT1 current. The main result of a kinetic analysis of the systematic increase in apparent affinity for sugars, together with the intact Na apparent affinity, suggests enhanced access to the sugar binding site in C1.

3-O-Methylglucose↗
PubMed1999

Expression of the human immunodeficiency virus frameshift signal in a bacterial cell-free system: influence of an interaction between the ribosome and a stem-loop structure downstream from the slippery site.

A-1 frameshift event is required for expression of the pol gene when ribosomes translate the mRNA of human immunodeficiency virus type-1 (HIV-1). In this study, we inserted the frameshift region of HIV-1 (a slippery heptanucleotide motif followed by a stem-loop) in a reporter gene coding for firefly luciferase. The ability of the corresponding mRNA, generated by in vitro transcription, to be translated in an Escherichia coli cell-free extract is the first demonstration that the HIV-1 frameshift can be reproduced in a bacterial cell-free extract, providing a powerful approach for analysis of the frameshift mechanism. The responses of the frameshift signal to chloramphenicol, an inhibitor of peptide bond formation, and spectinomycin, an inhibitor of translocation, suggest that the frameshift complies with the same rules found in eukaryotic translation systems. Furthermore, when translation was performed in the presence of streptomycin and neamine, two error-inducing antibiotics, or with hyperaccurate ribosomes mutated in S12, the frameshift efficiency was increased or decreased, respectively, but only in the presence of the stem-loop, suggesting that the stem-loop can influence the frameshift through a functional interaction with the ribosomes.

Animals↗
PubMed1999

A glycosyl hydrolase activity of mammalian reovirus sigma1 protein can contribute to viral infection through a mucus layer.

The mammalian reovirus sigma1 protein is responsible for viral attachment to host cells and hemagglutination properties of the virus. In the present study, sequence similarity between sigma1 and chicken-type lysozymes prompted us to investigate additional functions of the sigma1 protein. Expression in Pichia pastoris yeast cells showed that sigma1 can actually cleave lysozyme substrates, including complex sugars found in bacterial cell walls. Replacement by site-directed mutagenesis of acidic amino acid residues in sigma1 by their respective isosteric, uncharged, amino acid residues has allowed us to identify Glu36 and Asp54 as the catalytic pair involved in sigma1-mediated glycosidase activity. The enzyme appears inactive in virions but its activity is unmasked upon generation of infectious subviral particles (ISVPs) by partial proteolytic removal of the outer capsid proteins. Purified sigma1 protein and ISVPs can also hydrolyze mucins, heavily glycosylated glycoproteins that are a major component of the mucus layer overlaying the intestinal epithelium. Furthermore, reovirus infection of epithelial Madin Darby canine kidney cells was inhibited tenfold in cells expressing mucin at their apical surface, while this inhibition was overcome by ISVPs. Unmasking of sigma1 mucinolytic activity in the intestine, consecutive to proteolytic cleavage of virions to ISVPs, thus likely contributes to the known increase in infectivity of reovirus ISVPs compared to complete virions. This work presents the first evidence that some mammalian viruses have evolved mechanisms to facilitate their penetration through the protective barrier of the mucus layer in the intestinal tract.

Amino Acid Sequence↗
PubMed1999

Molecular characterization of an inwardly rectifying K+ channel from HeLa cells.

Previous patch-clamp studies have shown that the potassium permeability of the plasma membrane in HeLa cells, a cell line derived from an epidermoid carcinoma of the cervix, is controlled by various K+-selective pores including an IRK1 type inwardly rectifying K+ channel. We used the sequence previously reported for the human heart Kir2.1 channel to design a RT-PCR strategy for cloning the IRK1 channel in HeLa cells. A full-length clone of 1.3 kb was obtained that was identical to the human cardiac Kir2.1 inward rectifier. The nature of the cloned channel was also confirmed in a Northern blot analysis where a signal of 5.3 kb corresponding to the molecular weight expected for a Kir2.1 channel transcript was identified not only in HeLa cells, but also in WI-38, ECV304 and bovine aortic endothelial cells. The HeLa IRK1 channel cDNA was subcloned in an expression vector (pMT21) and injected into Xenopus oocytes. Cell-attached and inside-out single channel recordings obtained from injected oocytes provided evidence for a voltage-independent K+-selective channel with current/voltage characteristics typical of a strong inward rectifier. The single channel conductance for inward currents measured in 200 mm K2SO4 conditions was estimated at 40 +/- 1 pS (n = 3), for applied voltages ranging from -100 to -160 mV, in agreement with the unitary conductance for the IRK1 channel identified in HeLa cells. In addition, the single channel conductance for inward currents, Gamma, was found to vary as a function of alphaK, the external K+ ion activity, according to Gamma = Gamma0 [alphaK]delta with Gamma0 = 3.3 pS and delta = 0.5. Single channel recordings from injected oocytes also provided evidence of a voltage-dependent block by external Cs+ and Ba2+. The presence of 500 micron Cs+ caused a voltage-dependent flickering, typical of a fast channel blocking process which resulted in a reduction of the channel open probability at increasingly negative membrane potential values. The fractional electrical distance computed for the Cs+ blocking site was greater than 1 indicating a multiple ion channel occupation. In contrast, external Ba2+ at concentrations ranging from 25 to 100 micron caused a slow channel block, consistent with the binding of a single Ba2+ ion at a site located at half the membrane span. It is concluded on the basis of these observations that HeLa cells expressed a Kir2.1 type inwardly rectifying channel likely to be involved in maintaining and regulating the cell resting potential.

Animals↗
PubMed1999

Computational sequence analysis of mammalian reovirus proteins.

In the present study, computer-assisted searches for sequence similarities were performed with amino acid sequences from mammalian reovirus proteins. These analysis revealed that many proteins of reovirus are partially similar to known viral or cellular proteins. Consensus sequences have been identified that are in accordance with already suspected functions of reovirus proteins. The analysis has also revealed unexpected similarities of some reovirus proteins with specific classes of proteins which sequences are present in the databases. This could suggest yet unidentified activities for some of the reovirus proteins.

Amino Acid Sequence↗
PubMed1999

Polarized human immunodeficiency virus budding in lymphocytes involves a tyrosine-based signal and favors cell-to-cell viral transmission.

Maturation and release of human immunodeficiency virus type 1 (HIV-1) is targeted at the pseudopod of infected mononuclear cells. However, the intracellular mechanism or targeting signals leading to this polarized viral maturation are yet to be identified. We have recently demonstrated the presence of a functional YXXL motif for specific targeting of HIV-1 virions to the basolateral membrane surface in polarized epithelial Madin-Darby canine kidney cells (MDCK). Site-directed mutagenesis was used to demonstrate that the membrane-proximal tyrosine in the intracytoplasmic tail of the HIV-1 transmembrane glycoprotein (gp41) is an essential component of this signal. In the present study, immunolocalization of viral budding allowed us to establish that this tyrosine-based signal is involved in determining the exact site of viral release at the surface of infected mononuclear cells. Substitution of the critical tyrosine residue was also shown to increase the amount of envelope glycoprotein at the cell surface, supporting previous suggestions that the tyrosine-based motif can promote endocytosis. Although alteration of the dual polarization-endocytosis motif did not affect the infectivity of cell-free virus, it could play a key role in cell-to-cell viral transmission. Accordingly, chronically infected lymphocytes showed a reduced ability to transmit the mutant virus to a cocultivated cell line. Overall, our data indicate that the YXXL targeting motif of HIV is active in various cell types and could play an important role in viral propagation; this may constitute an alternative target for HIV therapeutics and vaccine development.

Animals↗
PubMed1998

Characterization of the thermosensitive ts453 reovirus mutant: increased dsRNA binding of sigma 3 protein correlates with interferon resistance.

The mutation harbored by the reovirus ts453 thermosensitive mutant has been assigned to the S4 gene encoding the major outer capsid protein sigma 3. Previous gene sequencing has identified a nonconservative amino acid substitution located near the zinc finger of sigma 3 protein in the mutant. Coexpression in COS cells of the sigma 3 protein presenting this amino acid substitution (N16K), together with the other major capsid protein mu 1, has also revealed an altered interaction between the two proteins; this altered interaction prevents the sigma 3-dependent cleavage of mu 1 to mu 1C. This could explain the lack of outer capsid assembly observed during ts453 virus infection at nonpermissive temperature. In the present study, we pursued the characterization of this mutant sigma 3 protein. Although the N16K mutation is located close to the zinc finger region, it did not affect the ability of the protein to bind zinc. In contrast, this mutation, as well as mutations within the zinc finger motif itself, can increase the binding of the protein to double-stranded RNA (dsRNA). It also appears that the N16K mutant protein is more efficiently transported to the nucleus than the wild-type protein, an observation consistent with the postulated role of dsRNA binding in sigma 3 nuclear presence. The lack of association with mu 1, and/or the increased dsRNA-binding activity of sigma 3, could be responsible for a partial resistance of the ts453 virus to interferon treatment and this could have important consequences in the context of protein synthesis regulation during natural reovirus infection.

Animals↗
PubMed1998

MuLV-based vectors pseudotyped with truncated HIV glycoproteins mediate specific gene transfer in CD4+ peripheral blood lymphocytes.

Human immunodeficiency virus (HIV) infection ultimately leads to the destruction of the CD4+ lymphocyte subset and the onset of AIDS. In recent years, several gene therapy procedures making use of retroviral vectors that selectively target HIV susceptible cells have been proposed in order to interfere with HIV productive infection. However, the HIV glycoproteins' inability to be incorporated in other heterologous retroviruses considerably limits true HIV cell tropism of such vectors. We now report the use of murine leukemia virus (MuLV) viral particles harboring a truncated form of the HIV glycoprotein for specific gene delivery. Reporter lacZ gene transfer was determined to be appropriately specific to CD4+ cells when HeLaCD4 cells or peripheral blood lymphocytes (PBLs) were infected with these pseudotyped MuLV virus vectors. In contrast, MuLV viruses harboring amphotropic MuLV envelope glycoproteins displayed a broad and nonspecific infection of PBL subpopulations. This new approach, taking advantage of the ability of truncated HIV envelope glycoproteins to be incorporated into heterologous retroviral particles, may foreseeably be used in future interventions based on the coordinated delivery of therapeutic gene products specifically to cell types susceptible to HIV infection.

Blotting, Southern↗
PubMed1997

Characterization of the reovirus lambda1 protein RNA 5'-triphosphatase activity.

Characterization of the phosphohydrolytic activities of recombinant reovirus lambda1 protein demonstrates that, in addition to the previously reported nucleoside triphosphate phosphohydrolase and helicase activities, the protein also possesses RNA 5'-triphosphatase activity. This activity was absolutely dependent on the presence of a divalent cation, Mg2+ or Mn2+, and specifically removes the 5'-gamma-phosphate at the end of triphosphate-terminated RNAs. Kinetic competition analysis showed that nucleoside triphosphate phosphohydrolase and RNA 5'-triphosphatase reactions are carried out at a common active site. These results strongly support the idea that, in addition to its role as an RNA helicase during transcription of the viral genome, lambda1 also participates during formation of the cap structure at the 5' end of newly synthesized reovirus mRNAs. The lambda1 protein represents only the third RNA triphosphatase whose primary structure is known and the first described in a double-stranded RNA virus.

Acid Anhydride Hydrolases↗
PubMed1997

Characterization of the nucleoside triphosphate phosphohydrolase and helicase activities of the reovirus lambda1 protein.

Previous studies have shown that the reovirus lambda1 core protein harbors a putative nucleotide-binding motif and exhibits an affinity for nucleic acids. In addition, a nucleoside triphosphate phosphohydrolase activity present in reovirus cores has been recently assigned to lambda1 using gene reassortment analysis. In this study, it was demonstrated that the recombinant lambda1 protein, expressed in the yeast Pichia pastoris, is able to hydrolyze nucleoside 5'-triphosphates or deoxynucleoside 5'-triphosphates. This activity was absolutely dependent on the presence of a divalent cation, Mg2+ or Mn2+. The protein can also unwind double-stranded nucleic acid molecules in the presence of a nucleoside 5'-triphosphate or deoxynucleoside 5'-triphosphate. These results provide the first biochemical evidence that the reovirus lambda1 protein is a nucleoside triphosphate phosphohydrolase/helicase and strongly support the idea that lambda1 participates in transcription of the viral genome.

Acid Anhydride Hydrolases↗