Assembly of infectious Kaposi's sarcoma-associated herpesvirus progeny requires formation of a pORF19 pentamer.

Herpesviruses cause severe diseases particularly in immunocompromised patients. Both genome packaging and release from the capsid require a unique portal channel occupying one of the 12 capsid vertices. Here, we report the 2.6 Å crystal structure of the pentameric pORF19 of the γ-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) resembling the portal cap that seals this portal channel. We also present the structure of its ß-herpesviral ortholog, revealing a striking structural similarity to its α- and γ-herpesviral counterparts despite apparent differences in capsid association. We demonstrate pORF19 pentamer formation in solution and provide insights into how pentamerization is triggered in infected cells. Mutagenesis in its lateral interfaces blocked pORF19 pentamerization and severely affected KSHV capsid assembly and production of infectious progeny. Our results pave the way to better understand the role of pORF19 in capsid assembly and identify a potential novel drug target for the treatment of herpesvirus-induced diseases.

Effect of clathrin light chains on the stiffness of clathrin lattices and membrane budding.

Clathrin-dependent transport processes require the polymerization of clathrin triskelia into polygonal scaffolds. Together with adapter proteins, clathrin collects cargo and induces membrane bud formation. It is not known to what extent clathrin light chains affect the structural and functional properties of clathrin lattices and the ability of clathrin to deform membranes. To address these issues, we have developed a novel procedure for analyzing clathrin lattice formation on rigid surfaces. We found that lattices can form on adaptor-coated convex-, planar- and even shallow concave surfaces, but the rate of formation and resistance to thermal dissociation of the lattice are greatly enhanced on convex surfaces. Atomic force microscopy on planar clathrin lattices demonstrates that the stiffness of the clathrin lattice is strictly dependent on light chains. The reduced stiffness of the lattice also compromised the ability of clathrin to generate coated buds on the surface of rigid liposomal membranes.

Viral modulation of type II interferon increases T cell adhesion and virus spread.

During primary varicella zoster virus (VZV) infection, infected lymphocytes drive primary viremia, causing systemic dissemination throughout the host, including the skin. This results in cytokine expression, including interferons (IFNs), which partly limit infection. VZV also spreads from skin keratinocytes to lymphocytes prior to secondary viremia. It is not clear how VZV achieves this while evading the cytokine response. Here, we show that VZV glycoprotein C (gC) binds IFN-γ and modifies its activity, increasing the expression of a subset of IFN-stimulated genes (ISGs), including intercellular adhesion molecule 1 (ICAM1), chemokines and immunomodulatory genes. The higher ICAM1 protein level at the plasma membrane of keratinocytes facilitates lymphocyte function-associated antigen 1-dependent T cell adhesion and expression of gC during infection increases VZV spread to peripheral blood mononuclear cells. This constitutes the discovery of a strategy to modulate IFN-γ activity, upregulating a subset of ISGs, promoting enhanced lymphocyte adhesion and virus spread.

Viral modulation of type II interferon increases T cell adhesion and virus spread.

During primary infection, varicella zoster virus (VZV) infects epithelial cells in the respiratory lymphoid organs and mucosa. Subsequent infection of lymphocytes, T cells in particular, causes primary viremia allowing systemic spread throughout the host, including the skin. This results in the expression of cytokines, including interferons (IFNs) which partly limit primary infection. VZV also spreads from skin keratinocytes to lymphocytes prior to secondary viremia. How VZV infects lymphocytes from epithelial cells while evading the cytokine response has not been fully established. Here, we show that VZV glycoprotein C (gC) binds IFN-γ and modifies its activity. Transcriptomic analysis revealed that gC in combination with IFN-γ increased the expression of a small subset of IFN-stimulated genes (ISGs), including intercellular adhesion molecule 1 (ICAM1), as well as several chemokines and immunomodulatory genes. The higher ICAM1 protein level at the plasma membrane of epithelial cells resulted in lymphocyte function-associated antigen 1 (LFA-1)-dependent T cell adhesion. This gC activity required a stable interaction with IFN-γ and signalling through the IFN-γ receptor. Finally, the presence of gC during infection increased VZV spread from epithelial cells to peripheral blood mononuclear cells. This constitutes the discovery of a novel strategy to modulate the activity of IFN-γ, inducing the expression of a subset of ISGs, leading to enhanced T cell adhesion and virus spread.

A CMV-induced adaptive human Vδ1+ γδ T cell clone recognizes HLA-DR.

The innate and adaptive roles of γδ T cells and their clonal γδ T cell receptors (TCRs) in immune responses are still unclear. Recent studies of γδ TCR repertoire dynamics showed massive expansion of individual Vδ1+ γδ T cell clones during viral infection. To judge whether such expansion is random or actually represents TCR-dependent adaptive immune responses, information about their cognate TCR ligands is required. Here, we used CRISPR/Cas9-mediated screening to identify HLA-DRA, RFXAP, RFX5, and CIITA as required for target cell recognition of a CMV-induced Vγ3Vδ1+ TCR, and further characterization revealed a direct interaction of this Vδ1+ TCR with the MHC II complex HLA-DR. Since MHC II is strongly upregulated by interferon-γ, these results suggest an inflammation-induced MHC-dependent immune response of γδ T cells.

CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups.

Tetraspanins exert a wide range of cellular functions of broad medical importance. Despite this, their biophysical characteristics are incompletely understood. Only two high-resolution structures of full-length tetraspanins have been solved. One is that of human CD81, which is involved in the infectivity of human pathogens including influenza, HIV, the malarial Plasmodium parasite and hepatitis C virus (HCV). The CD81 crystal structure identifies a cholesterol-binding pocket, which has been suggested to be important in the regulation of tetraspanin function. Here we investigate the use of styrene-maleic anhydride co-polymers (SMA) for the solubilisation and purification of CD81 within a lipid environment. When CD81 was expressed in the yeast Pichia pastoris, it could be solubilised and purified using SMA2000. This SMALP-encapsulated CD81 retained its native folded structure, as determined by the binding of two conformation-sensitive anti-CD81 antibodies. Analysis by size exclusion chromatography revealed two distinct populations of CD81, only one of which bound the HCV glycoprotein, E2. Optimization of expression and buffer conditions increased the proportion of E2-binding competent CD81 protein. Mass spectrometry analysis indicated that the lipid environment surrounding CD81 is enriched with negatively charged lipids. These results establish a platform to study the influence of protein-lipid interactions in tetraspanin biology.