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Denys Brand

Publications and source records attributed to Denys Brand.

12 recordsLinked to original sources

Impact of natural polymorphism within the gp41 cytoplasmic tail of human immunodeficiency virus type 1 on the intracellular distribution of envelope glycoproteins and viral assembly.

The motifs involved in the various functions of the human immunodeficiency virus type 1 (HIV-1) gp41 cytoplasmic tail (CT), particularly those related to the intracellular trafficking and assembly of envelope glycoproteins (Env) onto core particles, have generally been assessed with a restricted panel of T-cell laboratory-adapted virus strains. Here, we investigated gp41 CT sequences derived from individuals infected with HIV-1 viruses of various subtypes. We identified four patients harboring HIV variants with a natural polymorphism in the membrane-proximal tyrosine-based signal Y(712)SPL or the Y(802)W(803) diaromatic motif, which are two major determinants of Env intracellular trafficking. Confocal microscopy showed that the intracellular distribution of Env with a mutation in the tyrosine or diaromatic motif differed from that of Env with no mutation in these motifs. Surprisingly, the gp41 CTs of the primary viruses also had differential effects on the intracellular distribution of Env, independently of mutations in the tyrosine or diaromatic motifs, suggesting the involvement of additional determinants. Furthermore, analyses of virus replication kinetics indicated that the effects of mutations in the tyrosine or diaromatic motifs on viral replication depended on the gp41 CT context. These effects were at least partly due to differences in the efficiency of Env incorporation into virions. Thus, polymorphisms in primary HIV-1 gp41 CTs at the quasispecies or subtype level can influence the intracellular distribution of Env, its incorporation into virions, and viral replication capacity.

Amino Acid Motifs↗

Human immunodeficiency virus serotyping on dried serum spots as a screening tool for the surveillance of the AIDS epidemic.

Many studies have demonstrated the utility of the dried blood spot (DBS) or dried plasma/serum spot (DSS) method for serological and molecular diagnosis of HIV infection. Here, we report on the description of a serotyping assay performed on DSS, and its application to a national surveillance program of HIV variants. We combined serotyping assays that we developed previously to discriminate between HIV-1 and HIV-2, between HIV-1 group O and HIV-1 group M, and between B and non-B subtypes of HIV-1 group M. The assays are based on antibody binding to either the immunodominant epitope of gp41 or the V3 domain of gp120 of these various types, groups and subtypes. Therefore, a unique enzyme-linked immunosorbent assay (ELISA) format applied to serum eluted from DSS allowed the simultaneous discrimination between infections caused by HIV-1 B, HIV-1 non-B, HIV-1 group O, and HIV-2. Together, this serotyping assay and an immunoassay for recent infection were used for a virological surveillance linked to the anonymous mandatory notification of HIV infection in France. The preliminary results of this virological surveillance allowed us to obtain estimates of the prevalence of the rare variants HIV-2 and HIV-1 group O. It also allowed identification of the two first cases of M/O dual infections reported outside the endemic group O region of the western part of equatorial Africa, and showed that non-B subtypes circulate widely in France, almost 50% of new HIV diagnoses in 2003 being due to these variants.

Acquired Immunodeficiency Syndrome↗

Core protein cleavage by signal peptide peptidase is required for hepatitis C virus-like particle assembly.

Hepatitis C virus (HCV) core protein, expressed with a Semliki Forest virus replicon, self-assembles into HCV-like particles (HCV-LP) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV assembly and morphogenesis by electron microscopy. This model was used to investigate whether the processing of the HCV core protein by the signal peptide peptidase (SPP) is required for the HCV-LP assembly. Several mutants were designed as there are conflicting reports concerning the cleavage of mutant proteins by SPP. Production of the only core mutant protein that escaped SPP processing led to the formation of multiple layers of electron-dense ER membrane, with no evidence of HCV-LP assembly. These data shed light on the HCV core residues involved in SPP cleavage and suggest that this cleavage is essential for HCV assembly.

Animals↗

Interclade neutralization and enhancement of human immunodeficiency virus type 1 identified by an assay using HeLa cells expressing both CD4 receptor and CXCR4/CCR5 coreceptors.

We report on the development and evaluation of a human immunodeficiency virus (HIV) neutralization assay that uses P4P cells, which are CD4+CXCR4+CCR5+ HeLa cells that carry the lacZ gene under the control of the HIV-1 long terminal repeat. The results of the present study suggest that the P4P assay can be used for the extensive study of both neutralizing and enhancing activity in serum samples from HIV-1-infected patients and from vaccinated individuals.

CD4 Antigens↗

Hepatitis C virus ultrastructure and morphogenesis.

Details of the ultrastructure of hepatitis C virus (HVC) virion remain unclear because it has proved extremely difficult to visualise virus particles from infected serum and tissues directly. In addition, although much is known about the viral genome, first cloned in 1989, little is known about HCV morphogenesis, due to the lack of an efficient in vitro culture system for HCV propagation. Virus-like particles (VLPs) obtained by expressing genes encoding the HCV structural proteins in mammalian cells can be used as an alternative model for studying HCV morphogenesis. In particular, this HCV-LP model has made it possible to demonstrate that HCV budding occurs at the ER membrane and that the core protein drives this process. The HCV-LP model opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions, which may make it possible to establish the long-awaited in vitro culture system for HCV.

Animals↗

Subtype B human immunodeficiency virus (HIV) type 1 mutant that escapes detection in a fourth-generation immunoassay for HIV infection.

We report a case of human immunodeficiency virus (HIV) type 1 infection not detected by a highly sensitive combined antigen-antibody assay. The virus was a subtype B strain harboring a unique sequence within the immunodominant epitope of the transmembrane glycoprotein. Immunochemical analysis indicated that this sequence was probably responsible for the failure to detect HIV antibodies.

Acquired Immunodeficiency Syndrome↗

Human choriocarcinoma cell resistance to natural killer lysis due to defective triggering of natural killer cells.

The trophoblast, the outermost layer of the human placenta, lacks expression of the classical human leukocyte antigen (HLA) class I molecules. This prevents allorecognition by T cells but raises the question of what protects the trophoblast from natural killer (NK) cells. In a previous study, we have shown that choriocarcinoma cell (CC) resistance to NK lysis was mainly independent of HLA class I molecules. In the present study, we postulated that CC may prevent activation of NK cells by failing to stimulate their triggering receptors (TR). To test this hypothesis, we evaluated the lysis of JAR and JEG-3 CC after effective cross-linking and activation of NK cells by means of lectins or antibodies. Our results show that NK-resistant CC were sensitive to lysis by unstimulated peripheral blood lymphocytes in the presence of phytohemagglutin (PHA), to antibody-dependent cell cytotoxicity in presence of anti-Tja antibodies, and to monoclonal antibody redirected killing using anti-TR antibodies anti-CD16 and anti-CD244/2B4. Finally, CC fail to express CD48, the ligand for CD244/2B4. These results indicate that the resistance of CC to lysis results primarily from defective NK cell activation, at least partially due to the lack of expression of ligands, such as CD48, involved in the triggering of NK cells.

Antibodies, Monoclonal↗

Hepatitis C virus-like particle budding: role of the core protein and importance of its Asp111.

In the absence of a hepatitis C virus (HCV) culture system, the use of a Semliki Forest virus replicon expressing genes encoding HCV structural proteins that assemble into HCV-like particles provides an opportunity to study HCV morphogenesis. Using this system, we showed that the HCV core protein constitutes the budding apparatus of the virus and that its targeting to the endoplasmic reticulum by means of the signal sequence of E1 protein is essential for budding. In addition, the aspartic acid at position 111 in the HCV core protein sequence was found to be crucial for virus assembly, demonstrating the usefulness of this system for mapping amino acids critical to HCV morphogenesis.

Animals↗

Development and evaluation of a DNA enzyme immunoassay method for env genotyping of subtypes A through G of human immunodeficiency virus type 1 group M, with discrimination of the circulating recombinant forms CRF01_AE and CRF02_AG.

The tools currently available for genetic subtyping of human immunodeficiency virus type 1 are laborious or can be used only for the analysis of a limited number of samples and/or subtypes. We developed and evaluated a molecular biology-based method using subtype-specific oligonucleotide probes for env genotyping of subtypes A through G, CRF01_AE, and CRF02_AG. DNA enzyme immunoassay (DEIA) genotyping is based on nested PCR amplification of the 5' end of the env gene (proviral DNA), followed by subtype-specific hybridization and immunoenzymatic detection on microplates. DEIA genotyping was validated with a large number of samples (n = 128) collected in Europe (France; n = 47), West-Central Africa (Cameroon; n = 36), and West Africa (Senegal; n = 45). Three different formats, depending on the distribution of subtypes in the three countries, were developed. The results were compared with those obtained by sequencing of the V3-V5 region and phylogenetic analysis or an env heteroduplex mobility assay. Additional sequencing and phylogenetic analyses of the DEIA region (the first codon of the env coding sequence to the middle of conserved region C1 of gp120) were performed to investigate the reasons for discrepancies. Intense and highly specific reactions between the oligonucleotide probes and the corresponding samples were observed. Overall, correct identification was achieved for 107 of 128 samples (83.6%). One sample was not amplified, 10 (8%) were nontypeable (NT), and 10 (8%) were misidentified. Six of the 10 discordant samples were further investigated by phylogenetic analysis, which indicated that these samples corresponded to recombinants involving the env 5' end and the V3 and V5 regions of the two parental clades. Sequencing of NT samples showed numerous differences between sample and probe sequences, resulting in a lack of hybridization, and revealed the limitations of the selected probes in terms of specificity and sensitivity. We demonstrated the feasibility of DEIA genotyping: six subtypes plus the two most prevalent circulating recombinant forms were discriminated by using the 5' end of the env gene. This method can be adapted to the local situation by including only probes that correspond to the prevalent strains.

DNA, Viral↗

Hepatitis C virus-like particle morphogenesis.

Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions.

Animals↗