Juan Fontana

Virus factories: associations of cell organelles for viral replication and morphogenesis

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 organelles but recruit specific cell organelles, building a unique structure. The formation of the viral factory involves a number of complex interactions and signalling events between viral and cell factors. Mitochondria, cytoplasmic membranes and cytoskeletal components frequently participate in the formation of viral factories, supplying basic and common needs for key steps in the viral replication cycle.

Structural Changes in Influenza Virus at Low pH Characterized by Cryo-Electron Tomography

ABSTRACT 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 postfusion state, dramatic changes in morphology are apparent: elongated particles are no longer observed, larger particles representing fused virions appear, the HA spikes become conspicuously disorganized, a layer of M1 matrix protein is no longer resolved on most virions, and the ribonucleoprotein complexes (RNPs) coagulate on the interior surface of the virion. To probe for intermediate states, preparations were imaged after 5 min at pH 4.9. These virions could be classified according to their glycoprotein arrays (organized or disorganized) and whether or not they have a resolved M1 layer. Employing subtomogram averaging, we found, in addition to the neutral-pH state of HA, two intermediate conformations that appear to reflect an outwards movement of the fusion peptide and rearrangement of the HA1 subunits, respectively. These changes are reversible. The tomograms also document pH-induced changes affecting the M1 layer that appear to render the envelope more pliable and hence conducive to fusion. However, it appears desirable for productive infection that fusion should proceed before the RNPs become coagulated with matrix protein, as eventually happens at low pH.

Visualization of proteins in intact cells with a clonable tag for electron microscopy

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 protein metallothionein (MT) as a tag for TEM in cells we have studied three MT-fusion proteins in Escherichia coli: AmiC, a component of the division ring, RecA, a DNA-binding protein, and a truncated cytoplasmic form of maltose-binding protein (MBP). Proteins fused to MT were expressed in E. coli. live cells treated with gold salts were processed by fast-freezing and freeze-substitution. Small electron-dense particles were detected in sections of bacteria expressing the MT-fusion proteins and immunogold labelling confirmed that these particles were associated to the fusion proteins. The distribution of the particles correlated with the functional locations of these proteins: MBP-MT3 concentrated in the cytoplasm, AmiC-MT1 in the bacterial division ring and RecA-MT1 in the nucleoid. The electron-dense tag was easily visualized by electron tomography and in frozen-hydrated cells.

Three-dimensional structure of Rubella virus factories

Abstract 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 transfer replicated RNA to assembly sites in Golgi membranes. We have studied the structure of RUBV factory in three dimensions by electron tomography and freeze-fracture. CPVs contain stacked membranes, rigid sheets, small vesicles and large vacuoles. These membranes are interconnected and in communication with the endocytic pathway since they incorporate endocytosed BSA-gold. RER and CPVs are coupled through protein bridges and closely apposed membranes. Golgi vesicles attach to the CPVs but no tight contacts with mitochondria were detected. Immunogold labelling confirmed that the mitochondrial protein p32 is an abundant component around and inside CPVs where it could play important roles in factory activities.

Novel replication complex architecture in rubella replicon-transfected cells

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 either in the presence or the absence of the RUB capsid (C) gene were used to study the structure of RCs in transfected cells. Confocal microscopy showed that the RUB replicase components P90 and P150 localized to CPVs, as did double‐stranded RNA (dsRNA), a marker for RNA synthesis. Electron microscopy (EM) showed that replicons generated CPVs containing small vesicles and large vacuoles, similar to CPVs from RUB‐infected cells and that the replicase proteins were sufficient for organelle recruitment. Some of these CPVs contained straight membranes. When cross‐sectioned, these rigid membranes appeared to be sheets of closely packed proteins. Immuno‐EM revealed that these sheets, apparently in contact with the cytosol, contained both P150 and P90, as well as dsRNA, and thus could be two‐dimensional arrays of functional viral replicases. Labelling of dsRNA after streptolysin‐O permeabilization showed that replication of viral genome takes place on the cytoplasmic side of CPVs. When present, C accumulated around CPVs. Mitochondrial protein P32 was detected within modified CPVs, the first demonstration of involvement of this protein, which interacts with C, with RCs.

A virus capsid‐like nanocompartment that stores iron and protects bacteria from oxidative stress

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. Here, we characterize the encapsulin nanocompartment from Myxococcus xanthus, which consists of a shell protein (EncA, 32.5 kDa) and three internal proteins (EncB, 17 kDa; EncC, 13 kDa; EncD, 11 kDa). Using cryo‐electron microscopy, we determined that EncA self‐assembles into an icosahedral shell 32 nm in diameter (26 nm internal diameter), built from 180 subunits with the fold first observed in bacteriophage HK97 capsid. The internal proteins, of which EncB and EncC have ferritin‐like domains, attach to its inner surface. Native nanocompartments have dense iron‐rich cores. Functionally, they resemble ferritins, cage‐like iron storage proteins, but with a massively greater capacity (~30,000 iron atoms versus ~3,000 in ferritin). Physiological data reveal that few nanocompartments are assembled during vegetative growth, but they increase fivefold upon starvation, protecting cells from oxidative stress through iron sequestration.

At low pH, Influenza virus matrix protein M1 undergoes a conformational change prior to dissociating from the membrane

ABSTRACT 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 cryo-electron tomography on the self-same specimens. At neutral pH, there were two kinds of complexes, corresponding to a lipid bilayer with embedded glycoproteins, with and without a closely associated, 4-nm-thick sheet of M1 protein. The reported discrepancies arose from differences in imaging conditions, i.e., in defocus and in whole-particle projections versus thin tomographic slices. Exposure of virions to low pH (as in the endosome) promotes membrane fusion, and previous work has shown that the M2 ion channel causes the virion interior to acidify also. We found that after 5 min at pH 4.9, the proportion of virions lacking an M1 layer increased from 10% to 50%. In virions retaining an M1 layer under these conditions, the M1-envelope complex exhibited two states, viz, the original, neutral-pH state and one in which the M1 layer appeared thinner and/or closer to the membrane. These observations extend previous indications that acidic pH causes the M1 layer to dissociate, leaving the envelope more pliable and, consequently, fusion compatible, and they show that dissociation is preceded by a conformational change in M1.

Recombinant HA1 produced in E. coli forms functional oligomers and generates strain-specific SRID potency antibodies for pandemic influenza vaccines

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 sheep immunized repeatedly with HA released and purified after bromelain-treatment of influenza virus grown in eggs. This approach was a major bottleneck for pandemic H1N1 (H1N1pdm09) potency reagent development in 2009. Alternative approaches are needed to make HA immunogens for generation of SRID reagents in the shortest possible time. In this study, we found that properly folded recombinant HA1 globular domain (rHA1) from several type A viruses including H1N1pdm09 and two H5N1 viruses could be produced efficiently using a bacterial expression system and subsequent purification. The rHA1 proteins were shown to form functional oligomers of trimers, similar to virus derived HA, and elicited high titer of neutralizing antibodies in rabbits and sheep. Importantly, the immune sera formed precipitation rings with reference antigens in the SRID assay in a dose-dependent manner. The HA contents in multiple H1N1 vaccine products from different manufacturers (and in several lots) as determined with the rHA1-generated sheep sera were similar to the values obtained with a traditionally generated sheep serum from NIBSC. We conclude that bacterially expressed recombinant HA1 proteins can be produced rapidly and used to generate SRID potency reagents shortly after new influenza strains with pandemic potential are identified.

Mechanism of Neutralization of Herpes Simplex Virus by Antibodies Directed at the Fusion Domain of Glycoprotein B

ABSTRACT 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 physical location of epitopes recognized by anti-gB neutralizing monoclonal antibodies (MAbs). Located in the base, FR1 contains two internal fusion loops (FLs) and is the site of gB-lipid interaction (the fusion domain). Many of the MAbs to FR1 are neutralizing, block cell-cell fusion, and prevent the association of gB with lipid, suggesting that these MAbs affect FL function. Here we characterize FR1 epitopes by using electron microscopy to visualize purified Fab-gB ectodomain complexes, thus confirming the locations of several epitopes and localizing those of MAbs DL16 and SS63. We also generated MAb-resistant viruses in order to localize the SS55 epitope precisely. Because none of the epitopes of our anti-FR1 MAbs mapped to the FLs, we hyperimmunized rabbits with FL1 or FL2 peptides to generate polyclonal antibodies (PAbs). While the anti-FL1 PAb failed to bind gB, the anti-FL2 PAb had neutralizing activity, implying that the FLs become exposed during virus entry. Unexpectedly, the anti-FL2 PAb (and the anti-FR1 MAbs) bound to liposome-associated gB, suggesting that their epitopes are accessible even when the FLs engage lipid. These studies provide possible mechanisms of action for HSV neutralization and insight into how gB FR1 contributes to viral fusion. IMPORTANCE For herpesviruses, such as HSV, entry into a target cell involves transfer of the capsid-encased genome of the virus to the target cell after fusion of the lipid envelope of the virus with a lipid membrane of the host. Virus-encoded glycoproteins in the envelope are responsible for fusion. Antibodies to these glycoproteins are important biological tools, providing a way of examining how fusion works. Here we used electron microscopy and other techniques to study a panel of anti-gB antibodies. Some, with virus-neutralizing activity, impair gB-lipid association. We also generated a peptide antibody against one of the gB fusion loops; its properties provide insight into the way the fusion loops function as gB transits from its prefusion form to an active fusogen.

Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-Inhibited Virions: a Role for Integrase in Maturation

ABSTRACT 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 chromosomes by integrase (IN) catalysis. Certain IN mutations (class II) and antiviral drugs (allosteric IN inhibitors [ALLINIs]) adversely affect maturation, resulting in virions that contain “eccentric condensates,” electron-dense aggregates located outside seemingly empty capsids. Here we demonstrate that in addition to this mislocalization of electron density, a class II IN mutation and ALLINIs each increase the fraction of virions with malformed capsids (from ∼12% to ∼53%). Eccentric condensates have a high NC content, as demonstrated by “tomo-bubblegram” imaging, a novel labeling technique that exploits the susceptibility of NC to radiation damage. Tomo-bubblegrams also localized NC inside wild-type cores and lining the spherical Gag shell in immature virions. We conclude that eccentric condensates represent nonpackaged vRNPs and that either genetic or pharmacological inhibition of IN can impair vRNP incorporation into mature cores. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with internal electron density and the infectivity of a class II IN deletion mutant virus. Moreover, the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose a role for IN in initiating core morphogenesis and vRNP incorporation into the mature core during HIV-1 maturation. IMPORTANCE Maturation, a process essential for HIV-1 infectivity, involves core assembly, whereby the viral ribonucleoprotein (vRNP, composed of vRNA and nucleocapsid protein [NC]) is packaged into a conical capsid. Allosteric integrase inhibitors (ALLINIs) affect multiple viral processes. We have characterized ALLINIs and integrase mutants that have the same phenotype. First, by comparing the effects of ALLINIs on several steps of the viral cycle, we show that inhibition of maturation accounts for compound potency. Second, by using cryoelectron tomography, we find that ALLINIs impair conical capsid assembly. Third, by developing tomo-bubblegram imaging, which specifically labels NC protein, we find that ALLINIs block vRNP packaging; instead, vRNPs form “eccentric condensates” outside the core. Fourth, malformed cores, typical of integrase-deleted virus, are partially replaced by conical cores when integrase is supplied in trans. Fifth, vRNA is necessary for ALLINI-induced eccentric condensate formation. These observations suggest that integrase is involved in capsid morphogenesis and vRNP packaging.