HTLV-1 biofilm polarization maintained by tetraspanin CD82 is required for efficient viral transmission.

The human T-lymphotropic virus type 1 (HTLV-1) is an oncogenic retrovirus whose transmission relies primarily on cell-to-cell contacts as cell-free viruses are poorly infectious. Among the intercellular transmission routes described, HTLV-1 biofilms are adhesive structures polarized at the cell surface that confine virions in a protective environment, which is believed to promote their simultaneous delivery during infection. Here, we show that several tetraspanins are enriched in HTLV-1 biofilms and incorporated into the viral envelope. However, we report that only the tetraspanin CD82 interacts with HTLV-1 Gag proteins which initiates their polarization into viral biofilms. Also, we demonstrate that CD82 maintains HTLV-1 biofilm polarization and favors viral transmission, as its silencing induces a complete reorganization of viral clusters at the cell surface and reduces the ability of infected T-cells to transmit the virus. Our results highlight the crucial role of CD82 and its glycosylation state in the architectural organization of HTLV-1 biofilms and their subsequent transfer through intercellular contacts.IMPORTANCEIn the early stages of infection, human T-lymphotropic virus type 1 (HTLV-1) dissemination within its host is believed to rely mostly on cell-to-cell contacts. Past studies unveiled a novel mechanism of HTLV-1 intercellular transmission based on the remodeling of the host-cell extracellular matrix and the generation of cell-surface viral assemblies whose structure, composition, and function resemble bacterial biofilms. These polarized aggregates of infectious virions, identified as viral biofilms, allow the bulk delivery of viruses to target cells and may help to protect virions from immune attacks. However, viral biofilms' molecular and functional description is still in its infancy, although it is crucial to fully decipher retrovirus pathogenesis. Here, we explore the function of cellular tetraspanins (CD9, CD81, CD82) that we detect inside HTLV-1 particles within biofilms. Our results demonstrate specific roles for CD82 in the cell-surface distribution and intercellular transmission of HTLV-1 biofilms, which we document as two essential parameters for efficient viral transmission. At last, our findings indicate that N-glycosylation of cell-surface molecules, including CD82, is required for the polarization of HTLV-1 biofilms and for the efficient transmission of HTLV-1 between T-lymphocytes.

Quantifying membrane binding and diffusion with fluorescence correlation spectroscopy diffusion laws.

Many transient processes in cells arise from the binding of cytosolic proteins to membranes. Quantifying this membrane binding and its associated diffusion in the living cell is therefore of primary importance. Dynamic photonic microscopies, e.g., single/multiple particle tracking, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy (FCS), enable non-invasive measurement of molecular mobility in living cells and their plasma membranes. However, FCS with a single beam waist is of limited applicability with complex, non-Brownian, motions. Recently, the development of FCS diffusion laws methods has given access to the characterization of these complex motions, although none of them is applicable to the membrane binding case at the moment. In this study, we combined computer simulations and FCS experiments to propose an FCS diffusion law for membrane binding. First, we generated computer simulations of spot-variation FCS (svFCS) measurements for a membrane binding process combined to 2D and 3D diffusion at the membrane and in the bulk/cytosol, respectively. Then, using these simulations as a learning set, we derived an empirical diffusion law with three free parameters: the apparent binding constant KD, the diffusion coefficient on the membrane D2D, and the diffusion coefficient in the cytosol, D3D. Finally, we monitored, using svFCS, the dynamics of retroviral Gag proteins and associated mutants during their binding to supported lipid bilayers of different lipid composition or at plasma membranes of living cells, and we quantified KD and D2D in these conditions using our empirical diffusion law. Based on these experiments and numerical simulations, we conclude that this new approach enables correct estimation of membrane partitioning and membrane diffusion properties (KD and D2D) for peripheral membrane molecules.

Optimized production and fluorescent labeling of SARS-CoV-2 virus-like particles.

SARS-CoV-2 is an RNA enveloped virus responsible for the COVID-19 pandemic that conducted in 6 million deaths worldwide so far. SARS-CoV-2 particles are mainly composed of the 4 main structural proteins M, N, E and S to form 100 nm diameter viral particles. Based on productive assays, we propose an optimal transfected plasmid ratio mimicking the viral RNA ratio in infected cells. This allows SARS-CoV-2 Virus-Like Particle (VLPs) formation composed of the viral structural proteins M, N, E and mature S. Furthermore, fluorescent or photoconvertible VLPs were generated by adding a fluorescent protein tag on N or M mixing with unlabeled viral proteins and characterized by western blots, atomic force microscopy coupled to fluorescence and immuno-spotting. Thanks to live fluorescence and super-resolution microscopies, we quantified VLPs size and concentration. SARS-CoV-2 VLPs present a diameter of 110 and 140 nm respectively for MNE-VLPs and MNES-VLPs with a concentration of 10e12 VLP/ml. In this condition, we were able to establish the incorporation of the Spike in the fluorescent VLPs. Finally, the Spike functionality was assessed by monitoring fluorescent MNES-VLPs docking and internalization in human pulmonary cells expressing or not the receptor hACE2. Results show a preferential maturation of S on N(GFP) labeled VLPs and an hACE2-dependent VLP internalization and a potential fusion in host cells. This work provides new insights on the use of non-fluorescent and fluorescent VLPs to study and visualize the SARS-CoV-2 viral life cycle in a safe environment (BSL-2 instead of BSL-3). Moreover, optimized SARS-CoV-2 VLP production can be further adapted to vaccine design strategies.

[Illuminating the nanoscopic world of viruses by fluorescence super-resolution microscopy]. / Apport de la microscopie de fluorescence super-résolue : dans l'intimité des virus !

The recent revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and acquisition speed, led to the ability to visualize nano-scaled objects. Currently, the use of a new generation of super-resolution fluorescence microscopes coupled to improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus and molecule level. In this review, after a brief chronological description of these new approaches, we highlight several examples of super-resolution microscopies that have allowed to revisit our understanding of several human viruses and of host-pathogen interactions.

Illuminating the nanoscopic world of viruses by fluorescence super-resolution microscopy.

The recent revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and acquisition speed, led to the ability to visualize nano-scaled objects. Currently, the use of a new generation of super-resolution fluorescence microscopes coupled to improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus and molecule level. In this review, after a brief chronological description of these new approaches, we highlight several examples of super-resolution microscopies that have allowed to revisit our understanding of several human viruses and of host-pathogen interactions.

The ESCRT-0 Protein HRS Interacts with the Human T Cell Leukemia Virus Type 2 Antisense Protein APH-2 and Suppresses Viral Replication.

The divergent clinical outcomes of human T cell leukemia virus type 1 (HTLV-1) and HTLV-2 infections have been attributed to functional differences in their antisense proteins. In contrast to HTLV-1 bZIP factor (HBZ), the role of the antisense protein of HTLV-2 (APH-2) in HTLV-2 infection is poorly understood. In previous studies, we identified the endosomal sorting complex required for transport 0 (ESCRT-0) subunit HRS as a novel interaction partner of APH-2 but not HBZ. HRS is a master regulator of endosomal protein sorting for lysosomal degradation and is hijacked by many viruses to promote replication. However, no studies to date have shown a link between HTLVs and HRS. In this study, we sought to characterize the interaction between HRS and APH-2 and to investigate the impact of HRS on the life cycle of HTLV-2. We confirmed a direct specific interaction between APH-2 and HRS and showed that the CC2 domain of HRS and the N-terminal domain of APH-2 mediate their interaction. We demonstrated that HRS recruits APH-2 to early endosomes, possibly furnishing an entry route into the endosomal/lysosomal pathway. We demonstrated that inhibition of this pathway using either bafilomycin or HRS overexpression substantially extends the half-life of APH-2 and stabilizes Tax2B expression levels. We found that HRS enhances Tax2B-mediated long terminal repeat (LTR) activation, while depletion of HRS enhances HTLV-2 production and release, indicating that HRS may have a negative impact on HTLV-2 replication. Overall, our study provides important new insights into the role of the ESCRT-0 HRS protein, and by extension the ESCRT machinery and the endosomal/lysosomal pathway, in HTLV-2 infection.IMPORTANCE While APH-2 is the only viral protein consistently expressed in infected carriers, its role in HTLV-2 infection is poorly understood. In this study, we characterized the interaction between the ESCRT-0 component HRS and APH-2 and explored the role of HRS in HTLV-2 replication. HRS is a master regulator of protein sorting for lysosomal degradation, a feature that is manipulated by several viruses to promote replication. Unexpectedly, we found that HRS targets APH-2 and possibly Tax2B for lysosomal degradation and has an overall negative impact on HTLV-2 replication and release. The negative impact of interactions between HTLV-2 regulatory proteins and HRS, and by extension the ESCRT machinery, may represent an important strategy used by HTLV-2 to limit virus production and to promote persistence, features that may contribute to the limited pathogenic potential of this infection.