Denys Brand

Hepatitis C Virus-Like Particle Morphogenesis

ABSTRACT 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.

Frequency of early in utero HIV-1 infection : a blind DNA polymerase chain reaction study on 100 fetal thymuses

Objective: To estimate the prevalence of in utero transmission of HIV‐1 through the second trimester. Material and methods: One hundred consecutive, unselected, intact fetuses, beyond 15 weeks gestational age (mean, 22.4 weeks) were studied. These were obtained following spontaneous intrauterine deaths (n = 4), miscarriages (n = 4), and elective mid‐trimester terminations (n = 92), eight of which were fetuses with malformations from HIV‐1‐positive pregnancies. Coded DNA extracts from the fetal thymuses were tested blindly by polymerase chain reaction in three laboratories using a total of six different primer pairs. Results: Two thymuses tested positive [95% confidence interval (Cl), 0.2‐7]. Results from the three laboratories were consistent in all 100 cases. The two fetuses with HIV in the thymus both tested positive in other organs, demonstrating systemic HIV infection. The first fetus, whose mother had advanced AIDS, had died in utero and had diffuse toxoplasmosis. The second died following extremely premature delivery in a pregnancy complicated by repeated bleeding. HIV infection was observed in none of the 92 fetuses that resulted from elective mid‐trimester terminations (95% Cl, 0‐4). Conclusion: The frequency of early in utero HIV infection appears to be low, compared with transmission rates in infants born to HIV‐1‐infected mothers, suggesting that transmission occurs mostly later in pregnancy and/or at delivery. Specific risk factors may have implications in the occurrence of early as opposed to late transmission. AIDS 1995, 9:359‐366

Monitoring the Dynamics of the HIV Epidemic Using Assays for Recent Infection and Serotyping among New HIV Diagnoses: Experience after 2 Years in France

BACKGROUND New tools to better monitor dynamics of human immunodeficiency virus (HIV) transmission are needed. METHODS National surveillance of newly HIV diagnoses included the collection of dried serum spots to identify both recent infections (<6 months) and HIV serotypes. Multivariate analyses were used to identify factors associated with recent infection and infection with non-B subtypes. RESULTS Between 2003 and March 2005, 7902 new HIV diagnoses were reported. The overall proportion of recent infections was 24.9% (95% confidence interval, 23.8%-26.0%) and was highest among men who have sex with men (MSM; 46%). Recent infection was associated with being an MSM, <30 years old, of French nationality, and living in Paris. Nearly half of newly HIV-1 diagnoses were with non-B subtypes. The highest proportion of non-B infections was reported among African heterosexual persons (81%), but important proportions were reported among French heterosexual persons (34%) and MSM (12%). Being infected by a non-B subtype was independently associated with being African, <30 years old, heterosexual, and living in Paris. The proportion of HIV-2 infection was 1.9%, and 11 cases of HIV-1 group O infection were identified, mainly among West Africans. CONCLUSIONS There is evidence of high levels of HIV transmission among MSM in France and transmission of non-B subtypes within the indigenous French population.

Hepatitis C Virus-Like Particle Budding: Role of the Core Protein and Importance of Its Asp111

ABSTRACT 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.

Dendritic cells loaded with HIV-1 p24 proteins adsorbed on surfactant-free anionic PLA nanoparticles induce enhanced cellular immune responses against HIV-1 after vaccination.

Biodegradable nanoparticles with surface adsorbed antigens represent a promising method for in vivo delivery of vaccines targeting a wide range of infectious diseases or cancers. We investigated the feasibility of loading dendritic cells with a vaccine antigen, HIV p24 protein, on the surface of surfactant-free anionic (d,l-lactic acid, PLA) nanoparticles. The p24 protein had a high affinity for the nanoparticles and the antigenicity and immunogenicity of the p24 protein on the nanoparticle was well preserved after immunization. p24-coated nanoparticles were efficiently taken up by mouse dendritic cells (DCs), inducing DC maturation by increasing MHC-I, MHC-II, CD40, CD80 and CD86 surface expression and secreting IL-12 (p70) and IL-4. We evaluated the ability of DCs pulsed with p24-coated nanoparticles to elicit an optimal humoral and cellular immune response in the blood and intestine. DCs pulsed with p24-nanoparticles induced high seric and mucosal antibody production and elicited strong systemic and local lymproliferative responses, correlated with a Th1/Th2-type response, and systemic CTL responses in mice. Thus, DCs pulsed with antigen-loaded PLA nanoparticles may provide a novel delivery tool for cell therapy vaccination against chronic infectious diseases.

Core protein domains involved in hepatitis C virus-like particle assembly and budding at the endoplasmic reticulum membrane.

Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon, self‐assembles into HCV‐like particles (HCV‐LPs) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV particle morphogenesis by electron microscopy. Various mutated HCV core proteins with engineered internal deletions were expressed with this system, to identify core domains required or dispensable for HCV‐LP assembly. The HCV core protein sequence was compared with its counterpart in GB virus B (GBV‐B), the virus most closely related to HCV, to identify conserved domains. GBV‐B and HCV display similar tropism for liver hepatocytes and their core proteins are organized similarly into three main domains (I, II and III), although GBV‐B core is smaller and lacks ∼35 amino acids (aa) in domain I. The deletion of short hydrophobic domains (aa 133–152 and 153–167 in HCV core) that appear highly conserved in domain II of both GBV‐B and HCV core proteins resulted in loss of HCV core ER anchoring and self‐assembly into HCV‐LPs. The deletion of short domains found within domain I of HCV core protein but not in the corresponding domain of GBV‐B core according to sequence alignment had contrasting effects. Amino acids 15–28 and 60–66 were shown to be dispensable for HCV‐LP assembly and morphogenesis, whereas aa 88–106 were required for this process. The production of GBV‐B core protein from a recombinant SFV vector was associated with specific ER ultrastructural changes, but did not lead to the morphogenesis of GBV‐B‐LPs, suggesting that different budding mechanisms occur in members of the Flaviviridae family.

Sequential biogenesis of host cell membrane rearrangements induced by hepatitis C virus infection

Like most positive-strand RNA viruses, hepatitis C virus (HCV) forms a membrane-associated replication complex consisting of replicating RNA, viral and host proteins anchored to altered cell membranes. We used a combination of qualitative and quantitative electron microscopy (EM), immuno-EM, and the 3D reconstruction of serial EM sections to analyze the host cell membrane alterations induced by HCV. Three different types of membrane alteration were observed: vesicles in clusters (ViCs), contiguous vesicles (CVs), and double-membrane vesicles (DMVs). The main ultrastructural change observed early in infection was the formation of a network of CVs surrounding the lipid droplets. Later stages in the infectious cycle were characterized by a large increase in the number of DMVs, which may be derived from the CVs. These DMVs are thought to constitute the membranous structures harboring the viral replication complexes in which viral replication is firmly and permanently established and to protect the virus against double-stranded RNA-triggered host antiviral responses.

Hepatitis C virus ultrastructure and morphogenesis.

Abstract Details of the ultrastructure of hepatitis C virus (HCV) 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.

Role of the HIV Type 1 Glycoprotein 120 V3 Loop in Determining Coreceptor Usage

Macrophage (M)-tropic HIV-1 isolates use the beta-chemokine receptor CCR5 as a coreceptor for entry, while T cell line-adapted (TCLA) strains use CXCR4 and dual-tropic strains can use either CCR5 or CXCR4. To investigate the viral determinants involved in choice of coreceptor, we used a fusion assay based on the infection of CD4+ HeLa cells that express one or both coreceptors with Semliki Forest virus (SFV) recombinants expressing the native HIV-1 gp160 of a primary M-tropic isolate (HIV-1BX08), a TCLA isolate (HIV-1LAI), or a dual-tropic strain (HIV-1MN). We examined whether the V3 region of these glycoproteins interacts directly with the corresponding coreceptors by assaying coreceptor-dependent cell-to-cell fusion mediated by the different recombinants in the presence of various synthetic linear peptides. Synthetic peptides corresponding to different V3 loop sequences blocked syncytium formation in a coreceptor-specific manner. Synthetic V2 peptides were also inhibitory for syncytium formation, but showed no apparent coreceptor specificity. A BX08 V3 peptide with a D320 --> R substitution retained no inhibitory capacity for BX08 Env-mediated cell-to-cell fusion, but inhibited LAI Env-mediated fusion as efficiently as the homologous LAI V3 peptide. The same mutation engineered in the BX08 env gene rendered it able to form syncytia on CD4+CXCR4+CCR5-HeLa cells and susceptible to inhibition by SDF-1alpha and MIP-1beta. Other substitutions tested (D320 --> Q/D324 --> N or S306 --> R) exhibited intermediate effects on coreceptor usage. These results underscore the importance of the V3 loop in modulating coreceptor choice and show that single amino acid modifications in V3 can dramatically modify coreceptor usage. Moreover, they provide evidence that linear V3 loop peptides can compete with intact cell surface-expressed gp120/gp41 for CCR5 or CXCR4 interaction.

IFITM proteins are incorporated onto HIV-1 virion particles and negatively imprint their infectivity

BackgroundInterferon induced transmembrane proteins 1, 2 and 3 (IFITMs) belong to a family of highly related antiviral factors that have been shown to interfere with a large spectrum of viruses including Filoviruses, Coronaviruses, Influenza virus, Dengue virus and HIV-1. In all these cases, the reported mechanism of antiviral inhibition indicates that the pool of IFITM proteins present in target cells blocks incoming viral particles in endosomal vesicles where they are subsequently degraded.ResultsIn this study, we describe an additional mechanism through which IFITMs block HIV-1. In virus-producing cells, IFITMs coalesce with forming virions and are incorporated into viral particles. Expression of IFITMs during virion assembly leads to the production of virion particles of decreased infectivity that are mostly affected during entry in target cells. This mechanism of inhibition is exerted against different retroviruses and does not seem to be dependent on the type of Envelope present on retroviral particles.ConclusionsThe results described here identify a novel mechanism through which IFITMs affect HIV-1 infectivity during the late phases of the viral life cycle. Put in the context of data obtained by other laboratories, these results indicate that IFITMs can target HIV at two distinct moments of its life cycle, in target cells as well as in virus-producing cells. These results raise the possibility that IFITMs could similarly affect distinct steps of the life cycle of a number of other viruses.