Juan Fontana

Basque Center for Biophysics (Biofisika), CSIC, UPV/EHU

Importance: HIV-1 maturation involves dissection of the Gag polyprotein by the viral protease, and assembly of a conical capsid enclosing the viral ribonucleoprotein. Maturation inhibitors (MIs) prevent the final cleavage step at the site between the capsid protein (CA) and the spacer peptide 1 (SP1), apparently by binding at this site and denying...

Unlabelled: During virion maturation, HIV-1 capsid protein assembles into a conical core containing the viral ribonucleoprotein (vRNP) complex, thought to be composed mainly of the viral RNA and nucleocapsid protein (NC). After infection, the viral RNA is reverse transcribed into double-stranded DNA, which is then incorporated into host chromosome...

Influenza virus, the causative agent of flu, enters the host cell by endocytosis. The low pH encountered inside endosomes triggers conformational changes in the viral glycoprotein hemagglutinin (HA), that mediate fusion of the viral and cellular membranes. This releases the viral genome into the cytoplasm of the infected cell, establishing the onse...

Living cells compartmentalize materials and enzymatic reactions to increase metabolic efficiency. While eukaryotes use membrane-bound organelles, bacteria and archaea rely primarily on protein-bound nanocompartments. Encapsulins constitute a class of nanocompartments widespread in bacteria and archaea whose functions have hitherto been unclear. Her...

The Arenaviridae family of segmented RNA viruses contains nearly 70 species with several associated with fatal haemorrhagic fevers, including Lassa, Lujo and Junin viruses. Lymphocytic choriomeningitis arenavirus (LCMV) is associated with fatal neurologic disease in humans and additionally represents a tractable model for studying arenavirus biolog...

Influenza A virus (IAV) is well known for its pandemic potential. While current surveillance and vaccination strategies are highly effective, therapeutic approaches are often short-lived due to the high mutation rates of IAV. Recently, monoclonal antibodies (mAbs) have emerged as a promising therapeutic approach, both against current strains and fu...

Translational regulation at the stage of initiation can impact the number of ribosomes translating each mRNA molecule. However, the translational activity of single 80S ribosomes (monosomes) on mRNA is less well understood, even though these 80S monosomes represent the dominant ribosomal complexes in vivo. Here, we used cryo-EM to determine the tra...

Lymphocytic choriomeningitis virus (LCMV) is an enveloped and segmented negative-sense RNA virus classified within the Arenaviridae family of the Bunyavirales order. LCMV is associated with fatal disease in immunocompromised populations and, as the prototypical arenavirus member, acts as a model for the many highly pathogenic members of the Arenavi...

Lymphocytic choriomeningitis virus (LCMV) is a bisegmented negative-sense RNA virus classified within the Arenaviridae family of the Bunyavirales order. LCMV is associated with fatal disease in immunocompromized populations, and as the prototypical arenavirus, acts as a model for the many serious human pathogens within this group. Here, we examined...

Following endocytosis, enveloped viruses employ the changing environment of maturing endosomes as cues to promote endosomal escape, a process often mediated by viral glycoproteins. We previously showed that both high [K⁺] and low pH promote entry of Bunyamwera virus (BUNV), the prototypical bunyavirus. Here, we use sub-tomogram averaging and AlphaF...

Influenza A virus (IAV) is well known for its pandemic potential. While current surveillance and vaccination strategies are highly effective, therapeutic approaches are short-lived due to the high mutation rates of IAV. Currently, monoclonal antibodies (mAbs) have emerged as a promising approach to tackle future IAV pandemics. Additionally, several...

The Arenaviridae family within the Bunyavirales order of segmented RNA viruses contains over 50 species grouped into four genera, Antennavirus , Hartmanivirus , Mammarenavirus and Reptarenavirus . Several mammarenaviruses are associated with fatal hemorrhagic fevers, including Lassa, Lujo and Junin viruses. The mammarenavirus member lymphocytic cho...

Lymphocytic choriomeningitis virus (LCMV) is a bisegmented negative-sense RNA virus classified within the Arenaviridae family of the Bunyavirales order. LCMV is associated with fatal disease in immunocompromised populations, and as the prototypical arenavirus, acts as a model for the many serious human pathogens within the Arenaviridae family. Here...

Lymphocytic choriomeningitis virus (LCMV) is a model arenavirus that causes fatalities within immunocompromised populations. To enter cells, the LCMV envelope fuses with endosomal membranes, for which two requirements are low pH and interaction between LCMV GP spike and receptor CD164. LCMV subsequently uncoats, where genome-associated NP separates...

Diarrhea, often severe, is a recognised and frequently early symptom during acute COVID-19 infection and may persist or develop for the first time in patients with long-COVID, with socio-economic consequences. Diarrheal mechanisms in these cases are poorly understood. There is evidence for disruption of intestinal epithelial barrier function, and a...

Following internalisation, viruses employ the changing environment of maturing endosomes as cues to promote endosomal escape, a process mediated by viral glycoproteins. Specifically, we previously showed that both high [K+] and low pH promote entry of Bunyamwera virus (BUNV), the prototypical bunyavirus. Here, we used sub-tomogram averaging combine...

The Bunyavirales order is the largest group of negative-sense RNA viruses, containing many lethal human pathogens for which approved anti-infective measures are not available. The bunyavirus genome consists of multiple negative-sense RNA segments enwrapped by the virus-encoded nucleocapsid protein (NP), which together with the viral polymerase form...

The Bunyavirales order of RNA viruses comprises emerging pathogens for which approved preventative or therapeutic measures for human use are not available. The genome of all Bunyavirales consists of negative-sense RNA segments wrapped by the virus-encoded nucleocapsid protein (NP) to form ribonucleoproteins (RNPs). RNPs represent the active templat...

Ribosomes have long been thought of as homogeneous macromolecular machines, but recent evidence suggests they are heterogeneous and could be specialised to regulate translation. Here, we have characterised ribosomal protein heterogeneity across 4 tissues of Drosophila melanogaster. We find that testes and ovaries contain the most heterogeneous ribo...

Ion channels play key roles in almost all facets of cellular physiology and have emerged as key host cell factors for a multitude of viral infections. A catalogue of ion channel-blocking drugs have been shown to possess antiviral activity, some of which are in widespread human usage for ion channel-related diseases, highlighting new potential for d...

: The family Hantaviridae within the Bunyavirales order comprises tri-segmented negative sense RNA viruses, many of which are rodent-borne emerging pathogens associated with fatal human disease. In contrast, hantavirus infection of corresponding rodent hosts results in inapparent or latent infections, which can be recapitulated in cultured cells th...

Ribosomes have long been thought of as homogeneous, macromolecular machines but recent evidence suggests they are heterogeneous and their specialisation can regulate translation. Here, we have characterised ribosomal protein heterogeneity across 5 tissues of Drosophila melanogaster. We find that testis and ovary contain the most heterogeneous ribos...

Influenza virus is the causative agent of the ‘flu’. According to the World Health Organisation, Influenza causes up to 5 million cases of severe flu and 500 000 deaths annually. To release its genome inside the cell and start an infection, Influenza virus must fuse its envelope with the endosomal membrane of the host, making this process an excell...

The Bunyavirales order of segmented negative sense RNA viruses includes over 500 isolates that infect insects, animals, and plants, and are often associated with severe and fatal disease in humans. To multiply and cause disease, bunyaviruses must transport their genomes from outside the cell into the cytosol, achieved by transit through the endocyt...

The herpesvirus family contains eight human pathogens that act as the causative agent of several human maladies. The herpesvirus family can be further subdivided alphaherpesviruses, betaherpesviruses, and the oncogenic gammaherpesviruses. Herpesvirus replication and capsid assembly occurs within the nucleus, from there capsids migrate into the cyto...

The Bunyavirales order of segmented negative-sense RNA viruses includes > 500 isolates that infect insects, animals, and plants and are often associated with severe and fatal disease in humans. To multiply and cause disease, bunyaviruses must translocate their genomes from outside the cell into the cytosol, achieved by transit through the endocytic...

Late in the HIV-1 replication cycle, the viral structural protein Gag is targeted to virus assembly sites at the plasma membrane of infected cells. The capsid (CA) domain of Gag plays a critical role in the formation of the hexameric Gag lattice in the immature virion, and, during particle release, CA is cleaved from the Gag precursor by the viral...

Structural analyses highlight the constraint of virus evolution to a limited number of capsid protein folds and assembly strategies that result in a functional virion. We report the cryo-EM and cryo-electron tomography structures and the results of atomic force microscopy studies of the infectious bursal disease virus (IBDV), a double-stranded RNA...

Many enveloped viruses enter cells through the endocytic network, from which they must subsequently escape through fusion of viral and endosomal membranes. This membrane fusion is mediated by virus-encoded spikes that respond to the changing endosomal environment, which triggers conformational change in the spike that initiate the fusion process. S...

In order to multiply and cause disease a virus must transport its genome from outside the cell into the cytosol, most commonly achieved through the endocytic network. Endosomes transport virus particles to specific cellular destinations and viruses exploit the changing environment of maturing endocytic vesicles as triggers to mediate genome release...

All enveloped viruses, including herpesviruses, must fuse their envelope with the host membrane to deliver their genomes into target cells, making this essential step subject to interference by antibodies and drugs. Viral fusion is mediated by a viral surface protein that transits from an initial prefusion conformation to a final postfusion conform...

Primary Envelopment of the Herpes Simplex 1 Virion - Volume 23 Issue S1 - William W. Newcomb, Juan Fontana, Dennis C. Winkler, Naiqian Cheng, J. Bernard Heymann, Alasdair C. Steven

Applications of Bubblegram Imaging - Volume 23 Issue S1 - Weimin Wu, Naiqian Cheng, Alasdair C. Steven, Juan Fontana

Many viruses migrate between different cellular compartments for successive stages of assembly. The HSV-1 capsid assembles in the nucleus and then transfers into the cytoplasm. First, the capsid buds through the inner nuclear membrane, becoming coated with nuclear egress complex (NEC) protein. This yields a primary enveloped virion (PEV) whose enve...

HIV-1 maturation involves proteolysis of the precursor polyproteins, Gag and Gag-Pol, and assembly of a conical core containing the viral ribonucleoprotein (vRNP), comprised of the vRNA genome and associated proteins, notably nucleocapsid (NC). Allosteric integrase inhibitors (ALLINIs) are antiviral drugs initially thought to impede the viral integ...

Unlabelled: The herpes simplex virus 1 (HSV-1) capsid is a massive particle (~200 MDa; 1,250-Å diameter) with T=16 icosahedral symmetry. It initially assembles as a procapsid with ~4,000 protein subunits of 11 different kinds. The procapsid undergoes major changes in structure and composition as it matures, a process driven by proteolysis and expu...

div class="title">Exploiting the Susceptibility of HIV-1 Nucleocapsid Protein to Radiation Damage in Tomo-Bubblegram Imaging - Volume 21 Issue S3 - Juan Fontana, Kellie A. Jurado, Naiqian Cheng, Alan Engelman, Alasdair C. Steven

Unlabelled: Glycoprotein B (gB), the fusogen of herpes simplex virus (HSV), is a class III fusion protein with a trimeric ectodomain of known structure for the postfusion state. Seen by negative-staining electron microscopy, it presents as a rod with three lobes (base, middle, and crown). gB has four functional regions (FR), defined by the physica...

The M1 matrix protein of influenza A virus, which plays multiple roles in virion assembly and infection, underlies the viral envelope. However, previous studies have given differing accounts of the number of layers in the M1/envelope complex and their thicknesses and compositions. To resolve this issue, we performed cryo-electron microscopy and cry...

Retrovirus infection starts with the binding of envelope glycoproteins to host cell receptors. Subsequently, conformational changes in the glycoproteins trigger fusion of the viral and cellular membranes. Some retroviruses, such as avian sarcoma/leukosis virus (ASLV), employ a two-step mechanism in which receptor binding precedes low-pH activation...

Recombinant hemagglutinin from influenza viruses with pandemic potential can be produced rapidly in various cell substrates. In this study, we compared the functionality and immunogenicity of bacterially produced oligomeric or monomeric HA1 proteins from H5N1 (A/Vietnam/1203/04) with those of the egg-based licensed subunit H5N1 (SU-H5N1) vaccine in...

of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Influenza virus enters host cells by endocytosis. The low pH of endosomes triggers conformational changes in hemagglutinin (HA) that mediate fusion of the viral and endosomal membranes. We have used cryo-electron tomography to visualize influenza A virus at pH 4.9, a condition known to induce fusogenicity. After 30 min, when all virions are in the...

Vaccine production and initiation of mass vaccination is a key factor in rapid response to new influenza pandemic. During the 2009-2010 H1N1 pandemic, several bottlenecks were identified, including the delayed availability of vaccine potency reagents. Currently, antisera for the single-radial immunodiffusion (SRID) potency assay are generated in sh...

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 01-06-09 Bibliografía

Viral factories are complex structures in the infected cell where viruses compartmentalize their life cycle. Rubella virus (RUBV) assembles factories by recruitment of rough endoplasmic reticulum (RER), mitochondria and Golgi around modified lysosomes known as cytopathic vacuoles or CPVs. These organelles contain active replication complexes that t...

Identification of proteins in 3D maps of cells is a main challenge in structural cell biology. For light microscopy (LM) clonable reagents such as green fluorescent protein represented a real revolution and equivalent reagents for transmission electron microscopy (TEM) have been pursued for a long time. To test the viability of the metal-binding pr...

Viral factories are novel structures built by viruses in infected cells. During their construction organelles are recruited and build a large scaffold for viral replication and morphogenesis. We have studied how a bunyavirus uses the Golgi to build the factory. With the help of confocal and 3D ultrastructural imaging together with molecular mapping...

Electron tomography allows the structures such as individual macromolecules, viruses and parts and even whole cells, to be reconstructed in their near-native state in three dimensions. In electron tomography, the transmission electron microscope is used to obtain the projection images of a sample from multiple angles, and those images are back-pro...

Viral factories are large structures built by cellular and viral components where viruses insert the macromolecular complexes needed for genome replication and morphogenesis of new viral particles. We are studying how factories are built and work with the assistance of a variety of electron microscopy (EM) methods. Our goal is to obtain three-dimen...

Rubella virus (RUB) assembles its replication complexes (RCs) in modified organelles of endo-lysosomal origin, known as cytopathic vacuoles (CPVs). These peculiar structures are key elements of RUB factories, where rough endoplasmic reticulum, mitochondria, and Golgi are recruited. Bicistronic RUB replicons expressing an antibiotic resistance gene...

Genome replication and assembly of viruses often takes place in specific intracellular compartments where viral components concentrate, thereby increasing the efficiency of the processes. For a number of viruses the formation of 'factories' has been described, which consist of perinuclear or cytoplasmic foci that mostly exclude host proteins and or...

Viral factories are large structures built by cellular and viral components where viruses insert the macromolecular complexes needed for genome replication and morphogenesis of new viral particles. Bunyaviruses build their factory around the Golgi complex and Togaviruses around modified lysosomes (1). Mitochondria and elements of the rough endoplas...