Guy Lemay

The membrane‐proximal intracytoplasmic tyrosine residue of HIV‐1 envelope glycoprotein is critical for basolateral targeting of viral budding in MDCK cells

Budding of retroviruses from polarized epithelial Madin–Darby canine kidney cells (MDCK) takes place specifically at the basolateral membrane surface. This sorting event is suspected to require a specific signal harbored by the viral envelope glycoprotein and it was previously shown that, as for most basolateral proteins, the intracytoplasmic domain plays a crucial role in this targeting phenomenon. It is well known that tyrosine‐based motifs are a central element in basolateral targeting signals. In the present study, site‐directed mutagenesis was used to generate conservative or non‐conservative substitutions of each four intracytoplasmic tyrosines of the human immunodeficiency virus (HIV‐1) envelope glycoprotein. This approach revealed that the membrane‐proximal tyrosine is essential to ensure both the basolateral localization of envelope glycoprotein and the basolateral targeting of HIV‐1 virions. Substitutions of the membrane‐proximal tyrosine did not appear to affect incorporation of envelope glycoprotein into the virions, as assayed by virion infectivity and protein content, nor its capability to ensure its role in viral infection, as determined by viral multiplication kinetics. Altogether, these results indicate that the intracytoplasmic domain of the HIV‐1 envelope glycoprotein harbors a unique, tyrosine‐based, basolateral targeting signal. Such a tyrosine‐based targeting signal may play a fundamental role in HIV transmission and pathogenesis.

Characterization of the Nucleoside Triphosphate Phosphohydrolase and Helicase Activities of the Reovirus λ1 Protein

Previous studies have shown that the reovirus λ1 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 λ1 using gene reassortment analysis. In this study, it was demonstrated that the recombinant λ1 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 λ1 protein is a nucleoside triphosphate phosphohydrolase/helicase and strongly support the idea that λ1 participates in transcription of the viral genome.

A novel group I intron in Candida dubliniensis is homologous to a Candida albicans intron

In the present study, we determined the sequence of group I self-splicing introns found in the large ribosomal RNA subunit of Candida albicans, Candida stellatoidea and the recently-described species Candida dubliniensis. It was found that both the intron and ribosomal RNA nucleotide sequences are almost perfectly identical between different C. albicans strains as well as between C. albicans and C. stellatoidea strains. Comparisons of ribosomal RNA sequences suggest that local isolates of atypical C. albicans from individuals infected with human immunodeficiency virus can be assigned to the C. dubliniensis species. C. dubliniensis strains also harbor a group I intron in their ribosomal RNA, as observed in about 40% of C. albicans strains and all C. stellatoidea strains. This novel C. dubliniensis group I intron is identical to the C. albicans and C. stellatoidea intron, except for two widely divergent stem-loop regions. Despite these differences, the C. dubliniensis intron possesses self-splicing ability in an in vitro assay. Taken together, these data support the idea that C. albicans and C. stellatoidea should be joined together as variants of the same species while C. dubliniensis is a distinct but closely related microorganism. To our knowledge, the C. albicans and C. dubliniensis introns are the first example of a pair of homologous group I introns differing only by the presence of apparently facultative sequences in some stem-loops suspected to be involved in stabilization of tertiary structure.

Electrogenic amino acid exchange via the rBAT transporter

A cDNA clone was isolated from rabbit renal cortex using DNA‐mediated expression cloning, which caused alanine‐dependent outward currents when expressed in Xenopus oocytes. The cDNA encodes rBAT, a Na‐independent amino acid transporter previously cloned elsewhere. Exposure of cDNA‐injected oocytes to neutral amino acids led to voltage‐dependent outward currents, but inward currents were seen upon exposure to basic amino acids. Assuming one charge/alanine, the outward current represented 38% of the rate of uptake of radiolabelled alanine, and was significantly reduced by prolonged preincubation of oocytes in 5 mM alanine. The currents were shown to be due to countertransport of basic amino acids for external amino acids using the cut‐open oocyte system. This transport represents a major mode of action of this protein, and may help in defining a physiological role for rBAT in the apical membrane of renal and intestinal cells.

Characterization of the Reovirus λ1 Protein RNA 5′-Triphosphatase Activity

Characterization of the phosphohydrolytic activities of recombinant reovirus λ1 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′-γ-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, λ1 also participates during formation of the cap structure at the 5′ end of newly synthesized reovirus mRNAs. The λ1 protein represents only the third RNA triphosphatase whose primary structure is known and the first described in a double-stranded RNA virus.

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.

Establishment of persistent reovirus infection in SC1 cells: absence of protein synthesis inhibition and increased level of double-stranded RNA-activated protein kinase

In the present study we report the establishment and characterization of an SC1 cell line persistently infected by reovirus. We observed that a significant percentage of SC1 cells was resistant to cell lysis upon infection with non-defective reovirus stocks. The apparent resistance of SC1 cells to the virus-induced inhibition of protein synthesis is probably an important factor favoring the establishment of such a persistence. The remaining cells, obtained following reovirus infection at a high multiplicity of infection, were kept as a continuous cell line and shown to have normal growth rate. They also released a high titer of virus that did not appear to differ from the original stock in neither infectivity nor genomic pattern. Electron microscopic examination further confirmed the presence of well-developed viral inclusions in the persistently infected cells. These cells were resistant to viral superinfection and exhibited a high constitutive level of the double-stranded RNA-activated protein kinase that might be involved in this resistance. We suggest that this cell line might be an interesting, and possibly more natural system than most previously used cell lines, for the continuing study of virus-host cell interactions during establishment of viral persistence using the much-studied model of reovirus infection.

Further characterization of the ts453 mutant of mammalian orthoreovirus serotype 3 and nucleotide sequence of the mutated S4 gene.

The sigma 3 protein of mammalian orthoreoviruses has multiple proven and postulated roles during viral multiplication. In this manuscript we took advantage of the availability of the ts453 thermosensitive mutant, already assigned to the S4 gene encoding sigma 3, to begin the elucidation of the relationship between the two main domains and the different roles of the sigma 3 viral protein. The alteration in the mutant appeared to affect the structural role of the protein. Nucleotide sequence determination indicated an especially significant change close to the zinc finger of the protein. These data suggest that the zinc-binding region might be especially important during the assembly of sigma 3 into the viral capsid.

Addition of exogenous polypeptides on the mammalian reovirus outer capsid using reverse genetics

Addition of exogenous peptide sequences on viral capsids is a powerful approach to study the process of viral infection or to retarget viruses toward defined cell types. Until recently, it was not possible to manipulate the genome of mammalian reovirus and this was an obstacle to the addition of exogenous sequence tags onto the capsid of a replicating virus. This obstacle has now been overcome by the availability of the plasmid-based reverse genetics system. In the present study, reverse genetics was used to introduce different exogenous peptides, up to 40 amino acids long, at the carboxyl-terminal end of the σ1 outer capsid protein. The tagged viruses obtained were infectious, produce plaques of similar size, and could be easily propagated at high titers. However, attempts to introduce a 750 nucleotides-long sequence failed, even when it was added after the stop codon, suggesting a possible size limitation at the nucleic acid level.

Site-directed mutagenesis of the double-stranded RNA binding domain of bacterially-expressed σ3 reovirus protein

The affinity of the reovirus sigma 3 protein for double-stranded RNA (dsRNA) is well established, and efforts have been made to identify the amino acids involved in this property. In the present study, we further examined the importance of two basic amino acids motifs, located in the carboxy-terminal third of the protein. Mutants, previously characterized in COS cells, were expressed in bacterial cells using the pET expression system. The capability of the different mutants to interact with dsRNA was then determined by the binding of radiolabeled dsRNA to proteins resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. It appears that the most carboxy-terminal motif is absolutely required for the binding but the second motif also contributes to this property. However, only the carboxy-terminal motif is required for normal binding upon removal of the amino-terminal domain of the protein by proteolytic cleavage, a procedure previously shown to increase dsRNA-binding. The basic charges in both motifs are important, while breaking of their potential to adopt an alpha helical configuration does not affect binding efficiency. Furthermore, alanine substitution of a single basic amino acid in the carboxy-terminal motif can be sufficient to strongly reduce the binding of dsRNA to the protein. Altogether, these data suggest that basic amino acids of the sigma 3 carboxy-terminal motif are directly involved in dsRNA binding, while the other basic motif may contribute by preventing an inhibitory effect of the amino-terminal portion of the protein.