Ana J. Pérez-Berná

The Role of Capsid Maturation on Adenovirus Priming for Sequential Uncoating

Background: Adenovirus proteolytic maturation is required for correct uncoating in the cell. Results: Maturation makes the virion metastable and facilitates penton and peripheral core protein release, as well as cooperative genome ejection. Conclusion: Precursor proteins act as scaffolds favoring assembly. Maturation primes adenovirus for uncoating. Significance: Identifying the molecular determinants of virus stability and uncoating is key to understanding the infectious cycle. Adenovirus assembly concludes with proteolytic processing of several capsid and core proteins. Immature virions containing precursor proteins lack infectivity because they cannot properly uncoat, becoming trapped in early endosomes. Structural studies have shown that precursors increase the network of interactions maintaining virion integrity. Using different biophysical techniques to analyze capsid disruption in vitro, we show that immature virions are more stable than the mature ones under a variety of stress conditions and that maturation primes adenovirus for highly cooperative DNA release. Cryoelectron tomography reveals that under mildly acidic conditions mimicking the early endosome, mature virions release pentons and peripheral core contents. At higher stress levels, both mature and immature capsids crack open. The virus core is completely released from cracked capsids in mature virions, but it remains connected to shell fragments in the immature particle. The extra stability of immature adenovirus does not equate with greater rigidity, because in nanoindentation assays immature virions exhibit greater elasticity than the mature particles. Our results have implications for the role of proteolytic maturation in adenovirus assembly and uncoating. Precursor proteins favor assembly by establishing stable interactions with the appropriate curvature and preventing premature ejection of contents by tightly sealing the capsid vertices. Upon maturation, core organization is looser, particularly at the periphery, and interactions preserving capsid curvature are weakened. The capsid becomes brittle, and pentons are more easily released. Based on these results, we hypothesize that changes in core compaction during maturation may increase capsid internal pressure to trigger proper uncoating of adenovirus.

Structure and uncoating of immature adenovirus

Maturation via proteolytic processing is a common trait in the viral world and is often accompanied by large conformational changes and rearrangements in the capsid. The adenovirus protease has been shown to play a dual role in the viral infectious cycle: (a) in maturation, as viral assembly starts with precursors to several of the structural proteins but ends with proteolytically processed versions in the mature virion, and (b) in entry, because protease-impaired viruses have difficulties in endosome escape and uncoating. Indeed, viruses that have not undergone proteolytic processing are not infectious. We studied the three-dimensional structure of immature adenovirus particles as represented by the adenovirus type 2 thermosensitive mutant ts1 grown under non-permissive conditions and compared it with the mature capsid. Our three-dimensional electron microscopy maps at subnanometer resolution indicate that adenovirus maturation does not involve large-scale conformational changes in the capsid. Difference maps reveal the locations of unprocessed peptides pIIIa and pVI and help define their role in capsid assembly and maturation. An intriguing difference appears in the core, indicating a more compact organization and increased stability of the immature cores. We have further investigated these properties by in vitro disassembly assays. Fluorescence and electron microscopy experiments reveal differences in the stability and uncoating of immature viruses, both at the capsid and core levels, as well as disassembly intermediates not previously imaged.

Identification of the Membrane-Active Regions of the Severe Acute Respiratory Syndrome Coronavirus Spike Membrane Glycoprotein Using a 16/18-Mer Peptide Scan: Implications for the Viral Fusion Mechanism

ABSTRACT We have identified the membrane-active regions of the severe acute respiratory syndrome coronavirus (SARS CoV) spike glycoprotein by determining the effect on model membrane integrity of a 16/18-mer SARS CoV spike glycoprotein peptide library. By monitoring the effect of this peptide library on membrane leakage in model membranes, we have identified three regions on the SARS CoV spike glycoprotein with membrane-interacting capabilities: region 1, located immediately upstream of heptad repeat 1 (HR1) and suggested to be the fusion peptide; region 2, located between HR1 and HR2, which would be analogous to the loop domain of human immunodeficiency virus type 1; and region 3, which would correspond to the pretransmembrane region. The identification of these membrane-active regions, which are capable of modifying the biophysical properties of phospholipid membranes, supports their direct role in SARS CoV-mediated membrane fusion, as well as facilitating the future development of SARS CoV entry inhibitors.

Monitoring dynamics of human adenovirus disassembly induced by mechanical fatigue

The standard pathway for virus infection of eukaryotic cells requires disassembly of the viral shell to facilitate release of the viral genome into the host cell. Here we use mechanical fatigue, well below rupture strength, to induce stepwise disruption of individual human adenovirus particles under physiological conditions, and simultaneously monitor disassembly in real time. Our data show the sequence of dismantling events in individual mature (infectious) and immature (noninfectious) virions, starting with consecutive release of vertex structures followed by capsid cracking and core exposure. Further, our experiments demonstrate that vertex resilience depends inextricably on maturation, and establish the relevance of penton vacancies as seeding loci for virus shell disruption. The mechanical fatigue disruption route recapitulates the adenovirus disassembly pathway in vivo, as well as the stability differences between mature and immature virions.

Minimizing tip-sample forces in jumping mode atomic force microscopy in liquid.

Control and minimization of tip-sample interaction forces are imperative tasks to maximize the performance of atomic force microscopy. In particular, when imaging soft biological matter in liquids, the cantilever dragging force prevents identification of the tip-sample mechanical contact, resulting in deleterious interaction with the specimen. In this work we present an improved jumping mode procedure that allows detecting the tip-sample contact with high accuracy, thus minimizing the scanning forces (-100 pN) during the approach cycles. To illustrate this method we report images of human adenovirus and T7 bacteriophage particles which are prone to uncontrolled modifications when using conventional jumping mode.

Identification of the Membrane-active Regions of Hepatitis C Virus p7 Protein BIOPHYSICAL CHARACTERIZATION OF THE LOOP REGION

We have identified the membrane-active regions of the hepatitis C virus p7 protein by performing an exhaustive study of membrane rupture, hemifusion, and fusion induced by a p7-derived peptide library on model membranes having different phospholipid compositions. We report the identification in p7 of a highly membranotropic region located at the loop domain of the protein. Here, we have investigated the interaction of a peptide patterned after the p7 loop (peptide p7L), studying its binding and interaction with the lipid bilayer, and evaluated the binding-induced structural changes of the peptide and the phospholipids. We show that positively rich p7L strongly binds to negatively charged phospholipids and it is localized in a shallow position in the bilayer. Furthermore, peptide p7L exhibits a high tendency to oligomerize in the presence of phospholipids, which could be the driving force for the formation of the active ion channel. Therefore, our findings suggest that the p7 loop could be an attractive candidate for antiviral drug development, because it could be a target for antiviral compounds that may lead to new vaccine strategies.

Regulation of a viral proteinase by a peptide and DNA in one-dimensional space. IV. viral proteinase slides along DNA to locate and process its substrates

Background: The adenovirus proteinase and its precursor protein substrates are all sequence independent DNA binding proteins. Results: The proteinase slides along DNA to locate and process its substrates. Conclusion: Processing of precursor proteins by the adenovirus proteinase occurs on DNA. Significance: This is a new way an enzyme not involved in nucleic acid metabolism interacts with its substrates: sliding on DNA via one-dimensional diffusion. Precursor proteins used in the assembly of adenovirus virions must be processed by the virally encoded adenovirus proteinase (AVP) before the virus particle becomes infectious. An activated adenovirus proteinase, the AVP-pVIc complex, was shown to slide along viral DNA with an extremely fast one-dimensional diffusion constant, 21.0 ± 1.9 × 106 bp2/s. In principle, one-dimensional diffusion can provide a means for DNA-bound proteinases to locate and process DNA-bound substrates. Here, we show that this is correct. In vitro, AVP-pVIc complexes processed a purified virion precursor protein in a DNA-dependent reaction; in a quasi in vivo environment, heat-disrupted ts-1 virions, AVP-pVIc complexes processed five different precursor proteins in DNA-dependent reactions. Sliding of AVP-pVIc complexes along DNA illustrates a new biochemical mechanism by which a proteinase can locate its substrates, represents a new paradigm for virion maturation, and reveals a new way of exploiting the surface of DNA.

A Second SARS-CoV S2 Glycoprotein Internal Membrane-Active Peptide. Biophysical Characterization and Membrane Interaction†

The severe acute respiratory syndrome coronavirus (SARS-CoV) envelope spike (S) glycoprotein, a class I viral fusion protein, is responsible for the fusion between the membranes of the virus and the target cell. The S2 domain of protein S has been suggested to have two fusion peptides, one located at its N-terminus, downstream of the furin cleavage, and another, more internal, located immediately upstream of the HR1. Therefore, we have carried out a study of the binding and interaction with model membranes of a peptide corresponding to segment 873-888 of the SARS-CoV S glycoprotein, peptide SARS IFP, as well as the structural changes taking place in both the phospholipid and the peptide induced by the binding of the peptide to the membrane. We demonstrate that SARS IFP peptide binds to and interacts with phospholipid model membranes and shows a higher affinity for negatively charged phospholipids than for zwitterionic ones. SARS IFP peptide specifically decreases the mobility of the phospholipid acyl chains of negatively charged phospholipids and adopts different conformations in the membrane depending upon their composition. These data support its role in SARS-mediated membrane fusion and suggest that the regions where this peptide resides might assist the fusion peptide and/or the pretransmembrane segment of the SARS-CoV spike glycoprotein in the fusion process.

Processing of the L1 52/55k Protein by the Adenovirus Protease: a New Substrate and New Insights into Virion Maturation

ABSTRACT Late in adenovirus assembly, the viral protease (AVP) becomes activated and cleaves multiple copies of three capsid and three core proteins. Proteolytic maturation is an absolute requirement to render the viral particle infectious. We show here that the L1 52/55k protein, which is present in empty capsids but not in mature virions and is required for genome packaging, is the seventh substrate for AVP. A new estimate on its copy number indicates that there are about 50 molecules of the L1 52/55k protein in the immature virus particle. Using a quasi-in vivo situation, i.e., the addition of recombinant AVP to mildly disrupted immature virus particles, we show that cleavage of L1 52/55k is DNA dependent, as is the cleavage of the other viral precursor proteins, and occurs at multiple sites, many not conforming to AVP consensus cleavage sites. Proteolytic processing of L1 52/55k disrupts its interactions with other capsid and core proteins, providing a mechanism for its removal during viral maturation. Our results support a model in which the role of L1 52/55k protein during assembly consists in tethering the viral core to the icosahedral shell and in which maturation proceeds simultaneously with packaging, before the viral particle is sealed.

Distribution of DNA-condensing protein complexes in the adenovirus core

Genome packing in adenovirus has long evaded precise description, since the viral dsDNA molecule condensed by proteins (core) lacks icosahedral order characteristic of the virus protein coating (capsid). We show that useful insights regarding the organization of the core can be inferred from the analysis of spatial distributions of the DNA and condensing protein units (adenosomes). These were obtained from the inspection of cryo-electron tomography reconstructions of individual human adenovirus particles. Our analysis shows that the core lacks symmetry and strict order, yet the adenosome distribution is not entirely random. The features of the distribution can be explained by modeling the condensing proteins and the part of the genome in each adenosome as very soft spheres, interacting repulsively with each other and with the capsid, producing a minimum outward pressure of ∼0.06 atm. Although the condensing proteins are connected by DNA in disrupted virion cores, in our models a backbone of DNA linking the adenosomes is not required to explain the experimental results in the confined state. In conclusion, the interior of an adenovirus infectious particle is a strongly confined and dense phase of soft particles (adenosomes) without a strictly defined DNA backbone.