Marielle Cavrois

A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes

As an early event in the viral life cycle, the entry of enveloped viruses into target cells has received considerable attention. Viral fusion to cellular targets has been studied principally with fusion assays in which cells engineered to express the viral envelope are cultured with the target cells. These assays yield valuable information but do not fully recapitulate all of the variables governing the fusion of actual virions to their cellular targets. The virion membrane and the plasma membrane, for example, differ strikingly in their lipid and protein compositions. Two virion-based fusion assays have been described. One is based on the redistribution of a self-quenching fluorophore, whereas the second depends on photosensitized activation of a hydrophobic probe by a fluorescent lipid loaded into the target membrane. These assays are complex and have not been adapted to study fusion in complex cell populations. We have developed a simple, rapid assay allowing the detection of HIV-1 virion fusion to biologically relevant target cells, including primary CD4+ T lymphocytes. It is based on the incorporation of β-lactamase–Vpr chimeric proteins (BlaM-Vpr) into HIV-1 virions and their subsequent delivery into the cytoplasm of target cells as a result of virion fusion. This transfer is then detected by enzymatic cleavage of the CCF2 dye, a fluorescent substrate of β-lactamase (BlaM), loaded in the target cells. BlaM cleaves the β-lactam ring in CCF2, changing its fluorescence emission spectrum from green (520 nm) to blue (447 nm) and thereby allowing fusion to be detected by fluorescence microscopy, flow cytometry, or UV photometry.

Activation of NF-κB by the Human Herpesvirus 8 Chemokine Receptor ORF74: Evidence for a Paracrine Model of Kaposi's Sarcoma Pathogenesis

ABSTRACT Infection with human herpesvirus 8 (HHV-8), also known as Kaposis sarcoma (KS)-associated herpesvirus, is necessary for the development of KS. The HHV-8 lytic-phase gene ORF74 is related to G protein-coupled receptors, particularly interleukin-8 (IL-8) receptors. ORF74 activates the inositol phosphate/phospholipase C pathway and the downstream mitogen-activated protein kinases, JNK/SAPK and p38. We show here that ORF74 also activates NF-κB independent of ligand when expressed in KS-derived HHV-8-negative endothelial cells or primary vascular endothelial cells. NF-κB activation was enhanced by the chemokine GROα, but not by IL-8. Mutation of Val to Asp in the ORF74 second cytoplasmic loop did not affect ligand-independent signaling activity, but it greatly increased the response to GROα. ORF74 upregulated the expression of NF-κB-dependent inflammatory cytokines (RANTES, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor) and adhesion molecules (VCAM-1, ICAM-1, and E-selectin). Supernatants from transfected KS cells activated NF-κB signaling in untransfected cells and elicited the chemotaxis of monocytoid and T-lymphoid cells. Expression of ORF74 conferred on primary endothelial cells a morphology that was strikingly similar to that of spindle cells present in KS lesions. Taken together, these data, demonstrating that ORF74 activates NF-κB and induces the expression of proangiogenic and proinflammatory factors, suggest that expression of ORF74 in a minority of cells in KS lesions could influence uninfected cells or latently infected cells via autocrine and paracrine mechanisms, thereby contributing to KS pathogenesis.

Apolipoprotein (apo) E4 enhances HIV-1 cell entry in vitro, and the APOE ε4/ε4 genotype accelerates HIV disease progression

Originally recognized for their role in lipoprotein metabolism and cardiovascular disease, apolipoprotein (apo) E isoforms (apoE2, apoE3, and apoE4) have also been implicated to play a key role in several biological processes not directly related to their lipid transport function. For example, apoE4 contributes significantly to neurodegeneration in Alzheimers disease. However, the role of apoE in infectious diseases is less well defined. Here, by examining a large cohort of HIV+ European and African American subjects, we found that the APOE ε4/ε4 genotype is associated with an accelerated disease course and especially progression to death compared with the APOE ε3/ε3 genotype. However, an association between the ε4/ε4 genotype and HIV-associated dementia (HAD), a neurological condition with clinicopathological features similar to Alzheimers disease, was not detected. Consistent with the genotype–phenotype relationships observed, compared with recombinant apoE3, apoE4 enhanced HIV fusion/cell entry of both R5 and X4 HIV strains in vitro. These findings establish apoE as a determinant of HIV-AIDS pathogenesis and raise the possibility that current efforts to convert apoE4 to an “apoE3-like” molecule to treat Alzheimers disease might also have clinical applicability in HIV disease.

Studies of Ebola Virus Glycoprotein-Mediated Entry and Fusion by Using Pseudotyped Human Immunodeficiency Virus Type 1 Virions: Involvement of Cytoskeletal Proteins and Enhancement by Tumor Necrosis Factor Alpha

ABSTRACT The Ebola filoviruses are aggressive pathogens that cause severe and often lethal hemorrhagic fever syndromes in humans and nonhuman primates. To date, no effective therapies have been identified. To analyze the entry and fusion properties of Ebola virus, we adapted a human immunodeficiency virus type 1 (HIV-1) virion-based fusion assay by substituting Ebola virus glycoprotein (GP) for the HIV-1 envelope. Fusion was detected by cleavage of the fluorogenic substrate CCF2 by β-lactamase-Vpr incorporated into virions and released as a result of virion fusion. Entry and fusion induced by the Ebola virus GP occurred with much slower kinetics than with vesicular stomatitis virus G protein (VSV-G) and were blocked by depletion of membrane cholesterol and by inhibition of vesicular acidification with bafilomycin A1. These properties confirmed earlier studies and validated the assay for exploring other properties of Ebola virus GP-mediated entry and fusion. Entry and fusion of Ebola virus GP pseudotypes, but not VSV-G or HIV-1 Env pseudotypes, were impaired in the presence of the microtubule-disrupting agent nocodazole but were enhanced in the presence of the microtubule-stabilizing agent paclitaxel (Taxol). Agents that impaired microfilament function, including cytochalasin B, cytochalasin D, latrunculin A, and jasplakinolide, also inhibited Ebola virus GP-mediated entry and fusion. Together, these findings suggest that both microtubules and microfilaments may play a role in the effective trafficking of vesicles containing Ebola virions from the cell surface to the appropriate acidified vesicular compartment where fusion occurs. In terms of Ebola virus GP-mediated entry and fusion to various target cells, primary macrophages proved highly sensitive, while monocytes from the same donors displayed greatly reduced levels of entry and fusion. We further observed that tumor necrosis factor alpha, which is released by Ebola virus-infected monocytes/macrophages, enhanced Ebola virus GP-mediated entry and fusion to human umbilical vein endothelial cells. Thus, Ebola virus infection of one target cell may induce biological changes that facilitate infection of secondary target cells that play a key role in filovirus pathogenesis. Finally, these studies indicate that pseudotyping in the HIV-1 virion-based fusion assay may be a valuable approach to the study of entry and fusion properties mediated through the envelopes of other viral pathogens.

Nef Is Physically Recruited into the Immunological Synapse and Potentiates T Cell Activation Early after TCR Engagement

The HIV-1 protein Nef enhances viral pathogenicity and accelerates disease progression in vivo. Nef potentiates T cell activation by an unknown mechanism, probably by optimizing the intracellular environment for HIV replication. Using a new T cell reporter system, we have found that Nef more than doubles the number of cells expressing the transcription factors NF-κB and NFAT after TCR stimulation. This Nef-induced priming of TCR signaling pathways occurred independently of calcium signaling and involved a very proximal step before protein kinase C activation. Engagement of the TCR by MHC-bound Ag triggers the formation of the immunological synapse by recruiting detergent-resistant membrane microdomains, termed lipid rafts. Approximately 5–10% of the total cellular pool of Nef is localized within lipid rafts. Using confocal and real-time microscopy, we found that Nef in lipid rafts was recruited into the immunological synapse within minutes after Ab engagement of the TCR/CD3 and CD28 receptors. This recruitment was dependent on the N-terminal domain of Nef encompassing its myristoylation. Nef did not increase the number of cell surface lipid rafts or immunological synapses. Recently, studies have shown a specific interaction of Nef with an active subpopulation of p21-activated kinase-2 found only in the lipid rafts. Thus, the corecruitment of Nef and key cellular partners (e.g., activated p21-activated kinase-2) into the immunological synapse may underlie the increased frequency of cells expressing transcriptionally active forms of NF-κB and NFAT and the resultant changes in T cell activation.

Human Immunodeficiency Virus Fusion to Dendritic Cells Declines as Cells Mature

ABSTRACT The maturation of dendritic cells (DCs) is associated with a diminished ability to support human immunodeficiency virus (HIV) replication; however, the precise step in the HIV life cycle impaired by DC maturation remains uncertain. Using an HIV virion-based fusion assay, we now show that HIV fusion to monocyte-derived DCs (MDDCs) both decreases and kinetically slows when DCs are induced to mature with poly(I:C) and tumor necrosis factor alpha. Specifically, laboratory-adapted CCR5-tropic 81A virions fused with markedly lower efficiency to mature MDDCs than immature DCs. In contrast, fusion of NL4-3, the isogenic CXCR4-tropic counterpart of 81A, was low in both immature and mature MDDCs. Fusion mediated by primary HIV envelopes, including seven CCR5- and four CXCR4-tropic envelopes, also decreased with DC maturation. The kinetics of virion fusion were also altered by both the state of DC maturation and the coreceptor utilized. Fusion of 81A and NL4-3 virions was delayed in mature compared to immature MDDCs, and NL4-3 fused more slowly than 81A in both mature and immature MDDCs. Surprisingly, primary envelopes with CXCR4 tropism mediated fusion to immature MDDCs with efficiencies similar to those of primary CCR5-tropic envelopes. This result contrasted with the marked preferential fusion of the laboratory-adapted 81A over NL4-3 in immature MDDCs and in ex vivo Langerhans cells, indicating that these laboratory-adapted HIV strains do not fully recapitulate all of the properties of primary HIV isolates. In conclusion, our results demonstrate that the defect in HIV replication observed in mature MDDCs stems at least in part from a decline in viral fusion.

The achilles heel of the trojan horse model of HIV-1 trans-infection.

To ensure their survival, microbial pathogens have evolved diverse strategies to subvert host immune defenses. The human retrovirus HIV-1 has been proposed to hijack the natural endocytic function of dendritic cells (DCs) to infect interacting CD4 T cells in a process termed trans-infection. Although DCs can be directly infected by certain strains of HIV-1, productive infection of DCs is not required during trans-infection; instead, DCs capture and internalize infectious HIV-1 virions in vesicles for later transmission to CD4 T cells via vesicular exocytosis across the infectious synapse. This model of sequential endocytosis and exocytosis of intact HIV-1 virions has been dubbed the “Trojan horse” model of HIV-1 trans-infection. While this model gained rapid favor as a strong example of how a pathogen exploits the natural properties of its cellular host, our recent studies challenge this model by showing that the vast majority of virions transmitted in trans originate from the plasma membrane rather than from intracellular vesicles. This review traces the experimental lines of evidence that have contributed to what we view as the “rise and decline” of the Trojan horse model of HIV-1 trans-infection.

Host Sequences Flanking the Human T-Cell Leukemia Virus Type 1 Provirus In Vivo

ABSTRACT Human pathogenic retroviruses do not have common loci of integration. However, many factors, such as chromatin structure, transcriptional activity, DNA-protein interaction, CpG methylation, and nucleotide composition of the target sequence, may influence integration site selection. These features have been investigated by in vitro integration reactions or by infection of cell lines with recombinant retroviruses. Less is known about target choice for integration in vivo. The present study was conducted in order to assess the characteristics of cellular sequences targeted for human T-cell leukemia virus type 1 (HTLV-1) integration in vivo. Sequencing integration sites from ≥200 proviruses (19 kb of sequence) isolated from 29 infected individuals revealed that HTLV-1 integration is not random at the level of the nucleotide sequence. The virus was found to integrate in A/T-rich regions with a weak consensus sequence at positions within and without of the hexameric repeat generated during integration. These features were not associated with a preference for integration near active regions or repeat elements of the host chromosomes. Most or all of the regions of the genome appear to be accessible to HTLV-1 integration. As with integration in vitro, integration specificity in vivo seems to be determined by local features rather than by the accessibility of specific regions.

Fluorescence Resonance Energy Transfer-Based HIV-1 Virion Fusion Assay

The fluorescence resonance energy transfer (FRET)-based HIV-1 virion fusion assay exploits the incorporation of beta-lactamase-Vpr chimeric proteins into HIV-1 virions and their subsequent delivery into the cytoplasm of target cells as a marker of fusion. This transfer can be monitored by the enzymatic cleavage of the CCF2-AM dye, a fluorescent substrate of beta-lactamase (BlaM), loaded into the target cells. BlaM cleavage of the beta-lactam ring in CCF2-AM prevents the FRET between the coumarin and fluorescein moieties of the dye. This cleavage changes the fluorescence emission spectrum of CCF2-AM from green (520 nm) to blue (447 nm), and thus permits detection of fusion by fluorescence microscopy, flow cytometry, or UV photometry. This assay is simple and rapid to perform, and exhibits high sensitivity and specificity. Importantly, it can be applied to study HIV-1 virion fusion in primary cells and can be combined with immunostaining for subset discrimination in heterogeneous target cell populations. Finally, the assay can also be adapted to study fusion mediated by the envelope proteins from other viruses through the construction of HIV-1 pseudotypes.

Enhanced Fusion and Virion Incorporation for HIV-1 Subtype C Envelope Glycoproteins with Compact V1/V2 Domains

ABSTRACT In infected people, the HIV-1 envelope glycoprotein (Env) constantly evolves to escape the immune response while retaining the essential elements needed to mediate viral entry into target cells. The extensive genetic variation of Env is particularly striking in the V1/V2 hypervariable domains. In this study, we investigated the trade-off, in terms of fusion efficiency, for encoding V1/V2 domains of different lengths. We found that natural variations in V1/V2 length exert a profound impact on HIV-1 entry. Variants encoding compact V1/V2 domains mediated fusion with higher efficiencies than related Envs encoding longer V1/V2 domains. By exchanging the V1/V2 domains between Envs of the same infected person or between two persons linked by a transmission event, we further demonstrated that V1/V2 domains critically influence both Env incorporation into viral particles and fusion to primary CD4 T cells and monocyte-derived dendritic cells. Shortening the V1/V2 domains consistently increased Env incorporation and fusion, whereas lengthening the V1/V2 domains decreased Env incorporation and fusion. Given that in a new host transmitted founder viruses are distinguished by compact Envs with fewer glycosylation sites, our study points to fusion and possibly Env incorporation into virions as limiting steps for transmission of HIV-1 to a new host and suggests that the length and/or the N-glycosylation profile of the V1/V2 domain influences these early steps in the HIV life cycle.