Functional genetic and biophysical analyses of membrane disruption by human adenovirus

The identification of the adenovirus (AdV) protein that mediates endosome penetration during infection has remained elusive. Several lines of evidence from previous studies suggest that the membrane lytic factor of AdV is the internal capsid protein VI. While these earlier results imply a role for protein VI in endosome disruption, direct evidence during cell entry has not been demonstrated. To acquire more definitive proof, we engineered random mutations in a critical N-terminal amphipathic α-helix of VI in an attempt to generate AdV mutants that lack efficient membrane penetration and infection. Random mutagenesis within the context of the AdV genome was achieved via the development of a novel technique that incorporates both error-prone PCR and recombineering. Using this system, we identified a single mutation, L40Q, that significantly reduced infectivity and selectively impaired endosome penetration. Furthermore, we obtained biophysical data showing that the lack of efficient endosomalysis is associated with reduced insertion of the L40Q mutation in protein VI (VI-L40Q) into membranes. Our studies indicate that protein VI is the critical membrane lytic factor of AdV during cellular entry and reveal the biochemical basis for its membrane interactions.     

Adenovirus type-5 entry and disassembly followed in living cells by FRET, fluorescence anisotropy, and FLIM

We have used fluorescence resonance energy transfer (FRET) to follow the process of capsid disassembly for adenovirus (Ad) serotype 5 (Ad5) in living CHO-CAR cells. Ad5 were weakly labeled on their capsid proteins with FRET donor and acceptor fluorophores. A progressive decrease in FRET efficiency recorded during Ad5 uptake revealed that the time course of Ad5 capsid disassembly has two sequential protein dissociation rates with half-times of 3 and 60 min. Fluorescence anisotropy measurements of the segmental motions of fluorophores on Ad5 indicate that the first rate is linked to the detachment from the capsid of the protruding, flexible fiber proteins. The second rate was shown to report on the combined dissociation of protein IX, penton base, and hexons, which form the rigid icosahedral capsid shell. Fluorescence lifetime imaging microscopy measurements using a pH-sensitive probe provided information on the pH of the microenvironment of Ad5 particles during intracellular trafficking, and confirmed that the fast fiber dissociation step occurred at the onset of endocytosis. The slower dissociation phase was shown to coincide with the escape of Ad5 from endocytic compartments into the cytosol, and its arrival at the nuclear membrane. These results demonstrate a rapid, quantitative live-cell assay for the investigation of virus-cell interactions and capsid disassembly.     

Localization of the N-terminus of minor coat protein IIIa in the adenovirus capsid

Minor coat protein IIIa is conserved in all adenoviruses (Ads) and is required for correct viral assembly, but its precise function in capsid organization is unknown. The latest Ad capsid model proposes that IIIa is located underneath the vertex region. To obtain experimental evidence on the location of IIIa and to further define its role, we engineered the IIIa gene to encode heterologous N-terminal peptide extensions. Recombinant Ad variants with IIIa encoding six-histidine (6His) tag, 6His, and FLAG peptides, or with 6His linked to FLAG with a (Gly₄Ser)₃ linker were rescued and analyzed for virus yield, capsid incorporation of heterologous peptides, and capsid stability. Longer extensions could not be rescued. Western blot analysis confirmed that the modified IIIa proteins were expressed in infected cells and incorporated into virions. In the Ad encoding the 6His-linker-FLAG-IIIa gene, the 6His tag was present in light particles, but not in mature virions. Immunoelectron microscopy of this virus showed that the FLAG epitope is not accessible to antibodies on the viral particles. Three-dimensional electron microscopy and difference mapping located the IIIa N-terminal extension beneath the vertex complex, wedged at the interface between the penton base and peripentonal hexons, therefore supporting the latest proposed model. The position of the IIIa N-terminus and its low tolerance for modification provide new clues for understanding the role of this minor coat protein in Ad capsid assembly and disassembly.     

A single point mutation in precursor protein VI doubles the mechanical strength of human adenovirus

Viruses are extensively studied as vectors for vaccine applications and gene therapies. For these applications, understanding the material properties of viruses is crucial for creating optimal functionality. Using atomic force microscopy (AFM) nanoindentation, we studied the mechanical properties of human adenovirus type 5 with the fiber of type 35 (Ad5F35) and compared it to viral capsids with a single point mutation in the protein VI precursor protein (pVI-S28C). Surprisingly, the pVI-S28C mutant turned out to be twice as stiff as the Ad5F35 capsids. We suggest that this major increase in strength is the result of the DNA crosslinking activity of precursor protein VII, as this protein was detected in the pVI-S28C mutant capsids. The infectivity was similar for both capsids, indicating that mutation did not affect the ability of protein VI to lyse the endosomal membrane. This study highlights that it is possible to increase the mechanical stability of a capsid even with a single point mutation while not affecting the viral life cycle. Such insight can help enable the development of more stable vectors for therapeutic applications.     

Characterization of Empty adenovirus particles assembled in the absence of a functional adenovirus IVa2 protein

The molecular mechanism for packaging of the adenovirus (Ad) genome into the capsid is likely similar to that of DNA bacteriophages and herpesviruses-the insertion of viral DNA through a portal structure into a preformed prohead driven by an ATP-hydrolyzing molecular machine. It is speculated that the IVa2 protein of adenovirus is the ATPase providing the power stroke of the packaging machinery. Purified IVa2 binds ATP in vitro and, along with a second Ad protein, the L4 22-kilodalton protein (L4-22K), binds specifically to sequences in the Ad genome that are essential for packaging. The efficiency of binding of these proteins in vitro was correlated with the efficiency of packaging in vivo. By utilizing a virus unable to express IVa2, pm8002, it was reported that IVa2 plays a role in assembly of the empty virion. We wanted to address the question of whether the ATP binding, and hence the putative ATPase activity, of IVa2 was required for its role in virus assembly. Our results show that ATPase activity was not required for the assembly of empty virus particles. In addition, we present evidence that particles were assembled in the absence of IVa2 by using two viruses null for IVa2-a deletion mutant virus, ΔIVa2, and the previously described mutant virus, pm8002. Empty virus particles produced by these IVa2 mutant viruses did not contain detectable viral DNA. We conclude that the major role of IVa2 is in viral DNA packaging. A characterization of the empty particles obtained from the IVa2 mutant viruses compared to wild-type empty particles is presented.     

Dissecting quasi-equivalence in nonenveloped viruses: membrane disruption is promoted by lytic peptides released from subunit pentamers, not hexamers

Nonenveloped viruses often invade membranes by exposing hydrophobic or amphipathic peptides generated by a proteolytic maturation step that leaves a lytic peptide noncovalently associated with the viral capsid. Since multiple copies of the same protein form many nonenveloped virus capsids, it is unclear if lytic peptides derived from subunits occupying different positions in a quasi-equivalent icosahedral capsid play different roles in host infection. We addressed this question with Nudaurelia capensis omega virus (NωV), an insect RNA virus with an icosahedral capsid formed by protein α, which undergoes autocleavage during maturation, producing the lytic γ peptide. NωV is a unique model because autocatalysis can be precisely initiated in vitro and is sufficiently slow to correlate lytic activity with γ peptide production. Using liposome-based assays, we observed that autocatalysis is essential for the potent membrane disruption caused by NωV. We observed that lytic activity is acquired rapidly during the maturation program, reaching 100% activity with less than 50% of the subunits cleaved. Previous time-resolved structural studies of partially mature NωV particles showed that, during this time frame, γ peptides derived from the pentamer subunits are produced and are organized in a vertical helical bundle that is projected toward the particle surface, while identical polypeptides in quasi-equivalent subunits are produced later or are in positions inappropriate for release. Our functional data provide experimental support for the hypothesis that pentamers containing a central helical bundle, observed in different nonenveloped virus families, are a specialized lytic motif.     

Peptides released from reovirus outer capsid form membrane pores that recruit virus particles

Nonenveloped animal viruses must disrupt or perforate a cell membrane during entry. Recent work with reovirus has shown formation of size-selective pores in RBC membranes in concert with structural changes in capsid protein mu1. Here, we demonstrate that mu1 fragments released from reovirus particles are sufficient for pore formation. Both myristoylated N-terminal fragment mu1N and C-terminal fragment phi are released from particles. Both also associate with RBC membranes and contribute to pore formation in the absence of particles, but mu1N has the primary and sufficient role. Particles with a mutant form of mu1, unable to release mu1N or form pores, lack the ability to associate with membranes. They are, however, recruited by pores preformed with peptides released from wild-type particles or with synthetic mu1N. The results provide evidence that docking to membrane pores by virus particles may be a next step in membrane penetration after pore formation by released peptides.     

Pseudopackaging of adenovirus type 5 genomes into capsids containing the hexon proteins of adenovirus serotypes B, D, or E

Adenoviruses (Ad) show promise as a vector system for gene delivery in vivo. However, a major challenge in the development of Ad vectors is the circumvention of the host immune responses to Ad infection, including both the host cytotoxic T-cell response and the humoral response resulting in neutralizing antibodies. One method to circumvent the effect of neutralizing antibodies against an Ad vector is to use different Ad serotypes to deliver the transgene of interest. This approach has been demonstrated with Ad genomes of highly related members of subgroup C. However, it is not known whether an Ad5-based vector DNA molecule can be packaged into capsids of evolutionarily more divergent adenoviruses. The aim of these studies was to determine if capsids containing hexon proteins from other Ad subgroups could package the Ad5 genome. A genetic approach utilizing an Ad5 temperature-sensitive (ts) mutant with a mutation in the hexon protein was used. When grown at the nonpermissive temperature, Ad5 ts147 replicates normally, providing a source of Ad5 DNA for virus assembly, but does not produce virus particles due to the hexon protein mutation. Coinfection of Ad5 ts147 with a wild-type virus of other Ad serotypes (Ad3, Ad4, or Ad9), which supply functional hexon proteins, resulted in the pseudopackaging of the Ad5 DNA genome. Furthermore, the pseudopackaged Ad5 DNA virions obtained in the coinfections were infectious. Therefore, switching hexons did not impair the infectivity or uncoating process of the pseudopackaged virion. Since hexon protein is a major antigenic determinant of the Ad capsid, this approach may prove useful to reduce the antigenicity of therapeutic Ad vectors and allow repeated vector administration.     

Mechanism of adenovirus neutralization by Human alpha-defensins

Defensins are naturally occurring antimicrobial peptides that disrupt bacterial membranes and prevent bacterial invasion of the host. Emerging studies indicate that certain defensins also block virus infection; however, the mechanism(s) involved are poorly understood. We demonstrate that human alpha-defensins inhibit adenovirus infection at low micromolar concentrations, and this requires direct association of the defensin with the virus. Moreover, defensins inhibit virus disassembly at the vertex region, thereby restricting the release of an internal capsid protein, pVI, which is required for endosomal membrane penetration during cell entry. As a consequence, defensins hamper the release of adenovirus particles from endocytic vesicles, resulting in virion accumulation in early endosomes and lysosomes. Thus, defensins possess remarkably distinct modes of activity against bacteria and viruses, and their function may provide insights for the development of new antiviral strategies.     

Nuclear targeting of adenovirus type 2 requires CRM1-mediated nuclear export

Incoming adenovirus type 2 (Ad2) and Ad5 shuttle bidirectionally along microtubules, biased to the microtubule-organizing center by the dynein/dynactin motor complex. It is unknown how the particles reach the nuclear pore complex, where capsids disassemble and viral DNA enters the nucleus. Here, we identified a novel link between nuclear export and microtubule-mediated transport. Two distinct inhibitors of the nuclear export factor CRM1, leptomycin B (LMB) and ratjadone A (RJA) or CRM1-siRNAs blocked adenovirus infection, arrested cytoplasmic transport of viral particles at the microtubule-organizing center or in the cytoplasm and prevented capsid disassembly and nuclear import of the viral genome. In mitotic cells where CRM1 is in the cytoplasm, adenovirus particles were not associated with microtubules but upon LMB treatment, they enriched at the spindle poles implying that CRM1 inhibited microtubule association of adenovirus. We propose that CRM1, a nuclear factor exported by CRM1 or a protein complex containing CRM1 is part of a sensor mechanism triggering the unloading of the incoming adenovirus particles from microtubules proximal to the nucleus of interphase cells.     

Morphogenesis of human adenovirus type 2 studied with fiber- and fiber and penton base-defective temperature-sensitive mutants.

The nature, polypeptide composition, and antigenic composition of the particles formed by six human adenovirus type 2 temperature-sensitive (ts) mutants were studied. ts115, ts116, and ts125 were phenotypically fiber-defective mutants, and ts103, ts104, and ts136 failed to synthesize detectable amounts of fiber plus penton base at 39.5 degrees C. The mutants belonged to five complementation groups, one group including ts116 and ts125. Except for ts103 and ts136, the other mutants were capable of producing particles at 39.5 degrees C. ts116 and ts125 accumulated light assembly intermediate particles (or top components) at nonpermissive temperatures, with few virus particles. The sodium dodecyl sulfate polypeptide pattern of ts116- or ts125-infected cells, intermediate particles, and virus particles showed that polypeptide IV (fiber) was smaller by a molecular weight of 2,000 than that in the wild-type virion and was glycosylated. In fiber plus penton base-defective ts104-infected cells, equivalent quantities of top components and viruses with a buoyant density (rho) of 1.345 g/ml (rho = 1.345 particles) were produced at 39.5 degrees C. These rho = 1.345 particles corresponded to young virions, as evidenced by the presence of uncleaved precursors to proteins VI, VIII, and VII. These young virions matured upon a shift down. Virus capsid vertex antigenic components underwent a phase of eclipse during their incorporation into mature virus particles. No antigenic penton base or IIa was detected in intermediate particles of all the ts mutants tested. Only hexon and traces of fiber antigens were found in ts104 young virions. Penton base and IIIa appeared as fully antigenically expressed capsid subunits in mature wild-type virions or ts104 virions after a shift down. The ts104 lesion is postulated to affect a regulatory function related in some way to penton base and fiber overproduction and the maturation processing of precursors PVI, PVII, and PVII.     

A Capsid-Encoded PPxY-Motif Facilitates Adenovirus Entry

Viruses use cellular machinery to enter and infect cells. In this study we address the cell entry mechanisms of nonenveloped adenoviruses (Ads). We show that protein VI, an internal capsid protein, is rapidly exposed after cell surface attachment and internalization and remains partially associated with the capsid during intracellular transport. We found that a PPxY motif within protein VI recruits Nedd4 E3 ubiquitin ligases to bind and ubiquitylate protein VI. We further show that this PPxY motif is involved in rapid, microtubule-dependent intracellular movement of protein VI. Ads with a mutated PPxY motif can efficiently escape endosomes but are defective in microtubule-dependent trafficking toward the nucleus. Likewise, depletion of Nedd4 ligases attenuates nuclear accumulation of incoming Ad particles and infection. Our data provide the first evidence that virus-encoded PPxY motifs are required during virus entry, which may be of significance for several other pathogens.     

Cryo-Electron Microscopy Structure of Adenovirus Type 2 Temperature-Sensitive Mutant 1 Reveals Insight into the Cell Entry Defect

The structure of the adenovirus type 2 temperature-sensitive mutant 1 (Ad2ts1) was determined to a resolution of 10 Å by cryo-electron microscopy single-particle reconstruction. Ad2ts1 was prepared at a nonpermissive temperature and contains the precursor forms of the capsid proteins IIIa, VI, and VIII; the core proteins VII, X (mu), and terminal protein (TP); and the L1-52K protein. Cell entry studies have shown that although Ad2ts1 can bind the coxsackievirus and Ad receptor and undergo internalization via αv integrins, this mutant does not escape from the early endosome and is targeted for degradation. Comparison of the Ad2ts1 structure to that of mature Ad indicates that Ad2ts1 has a different core architecture. The Ad2ts1 core is closely associated with the icosahedral capsid, a connection which may be mediated by preproteins IIIa and VI. Density within hexon cavities is assigned to preprotein VI, and membrane disruption assays show that hexon shields the lytic activity of both the mature and precursor forms of protein VI. The internal surface of the penton base in Ad2ts1 appears to be anchored to the core by interactions with preprotein IIIa. Our structural analyses suggest that these connections to the core inhibit the release of the vertex proteins and lead to the cell entry defect of Ad2ts1.Cryo-electron microscopy (cryo-EM) studies of adenovirus (Ad) combined with atomic resolution structures of component proteins (hexon, penton base, fiber, and protease) have led to a detailed structural model for the mature Ad virion (31). While the Ad protein capsid is icosahedral, the core does not follow the overall symmetry of the particle, and thus the core is not well represented in cryo-EM structures (43). The core is composed of the 36-kb double-stranded DNA (dsDNA) genome complexed with four viral proteins (V, VII, mu, and terminal protein [TP]) and the virally encoded cysteine protease. The core of the mature virion may also contain a few copies of the L1-52K protein (7), a possible scaffolding protein that is present in higher copy numbers in assembling virions (18).The capsid contains the major capsid proteins, hexon, penton base, and fiber, together with four minor capsid proteins (IIIa, VI, VIII, and IX). Cryo-EM difference mapping analyses have led to revised assignments for the locations of the minor capsid proteins, with protein IX on the exterior and the other three proteins on the inner capsid surface (9, 38). A scanning transmission EM study indicated that four trimers of protein IX stabilize the group of nine hexons in the center of each facet (11). However, more recent cryo-EM studies indicated that only the N-terminal domain of protein IX forms these trimeric assemblies (37, 38), while the C-terminal domain, which has a long predicted α-helix with strong propensity for coiled coil formation, associates in helical bundles at the facet edges (38). Two cryo-EM studies support the assignment of the tetrameric helical bundle on the capsid exterior to the C-terminal domain of protein IX (10, 23). Curiously, 12 monomers of protein IX per facet assemble into four trimers with their N-terminal domains and three tetramers with their C-terminal domains.The internal location for protein IIIa below the penton base and surrounding peripentonal hexons was confirmed by a study of virions with N-terminally tagged protein IIIa (39). Although the locations for proteins VI and VIII have not been experimentally confirmed, these proteins are more than likely on the internal side of the capsid, as there is no remaining unassigned cryo-EM density on the exterior of the capsid. In addition, proteins VI and VIII are two of the viral proteins that are produced in precursor form and cleaved by the viral protease during maturation of the assembled virion (22). The protease is presumed to be packaged within the interior of the virion, and therefore the assignment of proteins VI and VIII to the interior of the capsid where they would be accessible to the protease is logical. Density within the internal cavity of all 240 hexon trimers in the Ad capsid has been assigned to protein VI on the basis of biochemical and temperature sensitivity studies (38, 51).Ad cell entry begins with attachment of the Ad fiber to either coxsackievirus and Ad receptor (3) or CD46 (12), which serve as the primary attachment receptors for Ad on most cell types (31). Internalization via clathrin-mediated endocytosis is triggered by association of the Ad penton base with αv integrins (49). Escape from the endosome is facilitated by the membrane lytic activity of protein VI, which is released from the virion in the low-pH environment of the early endosome (50). The stepwise dismantling of the Ad virion during cell entry has been described biochemically (15) but has not been fully characterized structurally. After endosomal escape, the partially uncoated Ad virion is transported along microtubules (44) to the nucleus, where the viral genome is inserted into the nucleus via a nuclear pore complex.Propagation of an Ad2 temperature-sensitive mutant (Ad2ts1) at nonpermissive temperatures (>39°C) results in the synthesis of virions that have an uncoating defect (28, 30, 46). Although these Ad2ts1 particles are capable of interacting with coxsackievirus and Ad receptor and undergoing internalization via association with αv integrins, they are unable to escape the early endosome and thus are targeted for degradation in lysosomes (13, 14). The Ad2ts1 genetic defect is a point mutation (P137L) in protease that is linked to a defect in packaging into the virion (33). In wild-type Ad virions, the protease is activated inside nascent virions by the viral DNA as well as an 11-amino-acid peptide from the C-terminal end of protein VI (22). The Ad protease mediates the maturational cleavage of six structural proteins, i.e., IIIa, VI, VII, VIII, mu, and TP, as well as the presumed scaffolding protein L1-52K (26, 47, 48). In Ad2ts1 particles these cleavages do not occur. The presence of the precursor forms of these proteins in Ad2ts1 is associated with greater capsid stability (42, 50).Here we present a cryo-EM structural study of the Ad2ts1 particle that provides insight into the cell entry defect of this temperature-sensitive mutant. Comparison of the Ad2ts1 structure with that of a mature Ad virion indicates that the major differences are in the interior of the virion.     

The infectivity and lytic activity of minute virus of mice wild-type and derived vector particles are strikingly different

Gene therapy vectors have been developed from autonomous rodent parvoviruses that carry a therapeutic gene or a marker gene in place of the genes encoding the capsid proteins. These vectors are currently evaluated in preclinical experiments. The infectivity of the vector particles deriving from the fibroblastic strain of minute virus of mice (MVMp) (produced by transfection in human cells) was found to be far less (approximately 50-fold-less) infectious than that of wild-type virus particles routinely produced by infection of A9 mouse fibroblasts. Similarly, wild-type MVMp produced by transfection also had a low infectivity in mouse cells, indicating that the method and producer cells influence the infectivity of the virus produced. Interestingly, producer cells made as many full vector particles as wild-type particles, arguing against deficient packaging being responsible for the low infectivity of viruses recovered from transfected cells. The hurdle to infection with full particles produced through transfection was found to take place at an early step following entry and limiting viral DNA replication and gene expression. Infections with transfection or infection-derived virus stocks normalized for their replication ability yielded similar monomer and dimer DNA amplification and gene expression levels. Surprisingly, at equivalent replication units, the capacity of parvovirus vectors to kill tumor cells was lower than that of the parental wild-type virus produced under the same transfection conditions, suggesting that beside the viral nonstructural proteins, the capsid proteins, assembled capsids, or the corresponding coding region contribute to the lytic activity of these viruses.     

Features of reovirus outer capsid protein mu1 revealed by electron cryomicroscopy and image reconstruction of the virion at 7.0 Angstrom resolution

Reovirus is a useful model for addressing the molecular basis of membrane penetration by one of the larger nonenveloped animal viruses. We now report the structure of the reovirus virion at approximately 7.0 A resolution as obtained by electron cryomicroscopy and three-dimensional image reconstruction. Several features of the myristoylated outer capsid protein mu1, not seen in a previous X-ray crystal structure of the mu1-sigma3 heterohexamer, are evident in the virion. These features appear to be important for stabilizing the outer capsid, regulating the conformational changes in mu1 that accompany perforation of target membranes, and contributing directly to membrane penetration during cell entry.