Quantitative analysis of the formation of nucleoprotein complexes between HIV-1 Gag protein and genomic RNA using transmission electron microscopy.

In HIV, the polyprotein precursor Gag orchestrates the formation of the viral capsid. In the current view of this viral assembly, Gag forms low-order oligomers that bind to the viral genomic RNA triggering the formation of high-ordered ribonucleoprotein complexes. However, this assembly model was established using biochemical or imaging methods that do not describe the cellular location hosting Gag-gRNA complex nor distinguish gRNA packaging in single particles. Here, we studied the intracellular localization of these complexes by electron microscopy and monitored the distances between the two partners by morphometric analysis of gold beads specifically labeling Gag and gRNA. We found that formation of these viral clusters occurred shortly after the nuclear export of the gRNA. During their transport to the plasma membrane, the distance between Gag and gRNA decreases together with an increase of gRNA packaging. Point mutations in the zinc finger patterns of the nucleocapsid domain of Gag caused an increase in the distance between Gag and gRNA as well as a sharp decrease of gRNA packaged into virions. Finally, we show that removal of stem loop 1 of the 5'-untranslated region does not interfere with gRNA packaging, whereas combined with the removal of stem loop 3 is sufficient to decrease but not abolish Gag-gRNA cluster formation and gRNA packaging. In conclusion, this morphometric analysis of Gag-gRNA cluster formation sheds new light on HIV-1 assembly that can be used to describe at nanoscale resolution other viral assembly steps involving RNA or protein-protein interactions.

Hepatitis B Virus Core Protein Domains Essential for Viral Capsid Assembly in a Cellular Context.

Hepatitis B virus (HBV) core protein (HBc) is essential to the formation of the HBV capsid. HBc contains two domains: the N-terminal domain corresponding to residues 1-140 essential to form the icosahedral shell and the C-terminal domain corresponding to a basic and phosphorylated peptide, and required for DNA replication. The role of these two domains for HBV capsid assembly was essentially studied in vitro with HBc purified from mammalian or non-mammalian cell lysates, but their respective role in living cells remains to be clarified. We therefore investigated the assembly of the HBV capsid in Huh7 cells by combining fluorescence lifetime imaging microscopy/Förster's resonance energy transfer, fluorescence correlation spectroscopy and transmission electron microscopy approaches. We found that wild-type HBc forms oligomers early after transfection and at a sub-micromolar concentration. These oligomers are homogeneously diffused throughout the cell. We quantified a stoichiometry ranging from ~170 to ~230 HBc proteins per oligomer, consistent with the visualization of eGFP-containingHBV capsid shaped as native capsid particles by transmission electron microscopy. In contrast, no assembly was observed when HBc-N-terminal domain was expressed. This highlights the essential role of the C-terminal domain to form capsid in mammalian cells. Deletion of either the third helix or of the 124-135 residues of HBc had a dramatic impact on the assembly of the HBV capsid, inducing the formation of mis-assembled oligomers and monomers, respectively. This study shows that our approach using fluorescent derivatives of HBc is an innovative method to investigate HBV capsid formation.

Hepatitis B virus entry into HepG2-NTCP cells requires clathrin-mediated endocytosis.

Hepatitis B virus (HBV) is a leading cause of cirrhosis and hepatocellular carcinoma worldwide, with 250 million individuals chronically infected. Many stages of the HBV infectious cycle have been elucidated, but the mechanisms of HBV entry remain poorly understood. The identification of the sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor and the establishment of NTCP-overexpressing hepatoma cell lines susceptible to HBV infection opens up new possibilities for investigating these mechanisms. We used HepG2-NTCP cells, and various chemical inhibitors and RNA interference (RNAi) approaches to investigate the host cell factors involved in HBV entry. We found that HBV uptake into these cells was dependent on the actin cytoskeleton and did not involve macropinocytosis or caveolae-mediated endocytosis. Instead, entry occurred via the clathrin-mediated endocytosis pathway. HBV internalisation was inhibited by pitstop-2 treatment and RNA-mediated silencing (siRNA) of the clathrin heavy chain, adaptor protein AP-2 and dynamin-2. We were able to visualise HBV entry in clathrin-coated pits and vesicles by electron microscopy (EM) and cryo-EM with immunogold labelling. These data demonstrating that HBV uses a clathrin-mediated endocytosis pathway to enter HepG2-NTCP cells increase our understanding of the complete HBV life cycle.

Direct interaction between the hepatitis B virus core and envelope proteins analyzed in a cellular context.

Hepatitis B virus (HBV) production requires intricate interactions between the envelope and core proteins. Analyses of mutants of these proteins have made it possible to map regions involved in the formation and secretion of virions. Tests of binding between core and envelope peptides have also been performed in cell-free conditions, to study the interactions potentially underlying these mechanisms. We investigated the residues essential for core-envelope interaction in a cellular context in more detail, by transiently producing mutant or wild-type L, S, or core proteins separately or in combination, in Huh7 cells. The colocalization and interaction of these proteins were studied by confocal microscopy and co-immunoprecipitation, respectively. The L protein was shown to constitute a molecular platform for the recruitment of S and core proteins in a perinuclear environment. Several core amino acids were found to be essential for direct interaction with L, including residue Y132, known to be crucial for capsid formation, and residues L60, L95, K96 and I126. Our results confirm the key role of L in the tripartite core-S-L interaction and identify the residues involved in direct core-L interaction. This model may be valuable for studies of the potential of drugs to inhibit HBV core-envelope interaction.

BAY 41-4109-mediated aggregation of assembled and misassembled HBV capsids in cells revealed by electron microscopy.

HBc is a small protein essential for the formation of the icosahedral HBV capsid. Its multiple roles in the replication cycle make this protein a promising target for the development of antiviral molecules. Based on the structure of HBc, a series of HBV assembly inhibitors, also known as capsid assembly modulators, were identified. We investigated the effect of BAY 41-4109, a heteroaryldihydropyrimidine derivative that promotes the assembly of a non-capsid polymer. We showed, by confocal microscopy, that BAY 41-4109 mediated HBc aggregation, mostly in the cytoplasm of Huh7 cells. Image analysis revealed that aggregate size depended on BAY 41-4109 concentration and treatment duration. Large aggregates in the vicinity of the nucleus were enclosed by invaginations of the nuclear envelope. This deformation of the nuclear envelope was confirmed by transmission electron microscopy (TEM) and immuno-TEM. These two techniques also revealed that the HBc aggregates were accumulations of capsid-like shells with an electron-dense material consisting of HBV core fragments. These findings, shedding light on the ultrastructural organization of HBc aggregates, provide insight into the mechanisms of action of BAY 41-4109 against HBV and will serve as a basis for comparison with other HBV capsid assembly inhibitors.

TFPI-2 silencing increases tumour progression and promotes metalloproteinase 1 and 3 induction through tumour-stromal cell interactions.

Tissue factor pathway inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI-2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI-2 is frequently down-regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI-2 down-regulation in the National Cancer Institute (NCI)-H460 non-small cell lung cancer cell line using specific micro interfering micro-interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI-2 down-regulation enhanced cell adhesion to collagen IV and laminin via an increase in α(1) integrin on cell surface, and increased MMP expression (mainly MMP-1 and -3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI-2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP-1, -3 and -7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases.