Nonviral methods of gene delivery possess several advantages over that of viral-based vectors, including having increased safety. However, the ability to achieve effective transport of therapeutic molecules across host cell membranes via nonviral methods remains a significant goal. Cell-derived nanoparticles known as vaults have been proposed as novel candidate transfer vehicles for various foreign molecules. Recombinant vault particles enter cells via macropinocytosis or phagocytosis but lack demonstrable membrane penetrating activity. To explore the feasibility of improving vault penetration into target cells, we incorporated the membrane lytic domain of adenovirus protein VI (pVI) into the interior of recombinant vault particles via fusion to the vault poly(ADP-ribose) polymerase (VPARP) interaction domain. The membrane lytic activity of the pVI domain was retained upon incorporation into vault particles. Moreover, internalization of vault-pVI complexes into murine macrophages promoted co-delivery of a soluble ribotoxin or a cDNA plasmid encoding GFP. These findings indicate that vault particles can be modified to enhance cell transfer of selected biomolecules.Keywords: adenovirus; gene delivery; membrane penetration; nanoparticles; protein VI; ribotoxin; vault

Diverse virus families have evolved to exploit the acidification of endosomal compartments to gain entry into cells. We describe a supramolecular approach for selectively targeting and inhibiting viral infections through this central biochemical pathway. Using adenovirus as a model non-enveloped virus, we have determined that an eight-residue cyclic d,l-α-peptide, selected from a directed combinatorial library, can specifically prevent the development of low pH in endocytic vesicles, arrest the escape of virions from the endosome, and abrogate adenovirus infection without an apparent adverse effect on cell viability. The likely generality of this approach against other pH-dependent viral infections is supported by the inhibition of type-A influenza virus escape from endosomes in the presence of the same peptide. Our studies suggest that self-assembling cyclic d,l-α-peptides hold considerable potential as a new rational supramolecular approach toward the design and discovery of broad-spectrum antiviral agents.Using adenovirus as a model non-enveloped virus, we have determined that eight-residue cyclic d,l-α-peptides can specifically prevent the development of low pH in endocytic vesicles, arresting the escape of virions from the endosome and preventing gene delivery by the virus.

When searching for their favorite host tissues, animal viruses frequently attach to cell-surface receptors that have key roles in normal cell physiology. Integrins are prime examples of physiologically important receptors that have been usurped by nonenveloped and enveloped viruses for attachment and/or cell entry. This family of heterodimeric receptors mediates cell adhesion, cell migration, tumor metastasis and cell differentiation. Recent investigations have shed new light on integrin structure as well as on the underlying molecular features of their association with viral ligands. In this review, we discuss several examples of virus–integrin interactions that highlight recent advances in this field. The continuing improvements in virus and cell imaging techniques have helped to uncover the molecular basis of how integrins are recognized by such a wide range of microbial pathogens to invade host cells.

The internal capsid protein VI mediates adenovirus (AdV) endosome penetration during cell entry. Essential to this process is the release of protein VI from the AdV capsid and subsequent membrane targeting and insertion by the liberated VI molecules within the endocytic vesicle. In this study, we describe a human AdV (HAdV) substitution mutant (AdV VI-G48C) within the critical N-terminal amphipathic α-helical domain of protein VI. The VI-G48C virus displays altered capsid stability that impacts protein VI release, membrane disruption and virus infectivity. This is due in part to aberrant disulfide-bonding of protein VI molecules within the AdV particle. Our results provide insight into the structural organization of protein VI in the virus particle, as well as highlight the role of protein VI in cell entry.Highlights► AdV protein VI mediates endosome penetration during cell entry. ► A G48C mutation in the VI membrane lytic domain decreases virus infectivity. ► VI-G48C molecules undergo disulfide bond formation in the AdV capsid. ► VI-G48C dimers increase capsid stability and lower membrane lytic activity. ► Reduction of VI-G48C disulfide bonds enhances membrane disruption.

Replication-defective and conditionally replicating adenovirus (AdV) vectors are currently being utilized in ∼ 25% of human gene transfer clinical trials. Unfortunately, progress in vector development has been hindered by a lack of accurate structural information. Here we describe the crystallization and preliminary X-ray diffraction analysis of a HAdV5 vector that displays a short flexible fiber derived from HAdV35. Crystals of Ad35F were grown in 100 mM HEPES pH 7.0, 200 mM Ca(OAc)2, 14% PEG 550 MME, 15% glycerol in 100 mM Tris–HCl 8.5. Freshly grown crystals diffracted well to 4.5 Å resolution and weakly to 3.5 Å at synchrotron sources. HAdV crystals belong to space group P1 with unit cell parameters a = 854.03 Å, b = 855.17 Å, c = 865.24 Å, α = 119.57°, β = 91.71°, γ = 118.08° with a single particle in the unit cell. Self-rotation and locked-rotation function analysis allowed the determination of the particle orientation. Molecular replacement, density modification and phase-extension procedures are being employed for structure determination.

Human adenoviruses cause a significant number of acute respiratory, enteric and ocular infections, however they have also served as useful model systems for uncovering fundamental aspects of cell and molecular biology. In addition, replication-defective forms of adenovirus are being used in gene transfer and vaccine clinical trials. Over the past decade, steady advances in structural biology techniques have helped reveal important insights into the earliest events in the adenovirus life cycle as well as virus interactions with components of the host immune system. This review highlights the continuing use of structure-based approaches to uncover the molecular features of adenovirus–host interactions.

•The fate of cleavage products of pVI in human adenovirus is investigated.•The cleaved N-terminus of pVI, pVIn, is retained in mature virus particles.•Native mass spectrometry shows up to 3 copies of pVIn binding to hexons in a pH-dependent manner.•pVIn binds to the base of peripentonal hexons on the capsid interior.•pVIn is retained in mature adenovirus and binds specifically to hexon.Mature human adenovirus particles contain four minor capsid proteins, in addition to the three major capsid proteins (penton base, hexon and fiber) and several proteins associated with the genomic core of the virion. Of the minor capsid proteins, VI plays several crucial roles in the infection cycle of the virus, including hexon nuclear targeting during assembly, activation of the adenovirus proteinase (AVP) during maturation and endosome escape following cell entry. VI is translated as a precursor (pVI) that is cleaved at both N- and C-termini by AVP. Whereas the role of the C-terminal fragment of pVI, pVIc, is well established as an important co-factor of AVP, the role of the N-terminal fragment, pVIn, is currently elusive. In fact, the fate of pVIn following proteolytic cleavage is completely unknown. Here, we use a combination of proteomics-based peptide identification, native mass spectrometry and hydrogen–deuterium exchange mass spectrometry to show that pVIn is associated with mature human adenovirus, where it binds at the base of peripentonal hexons in a pH-dependent manner. Our findings suggest a possible role for pVIn in targeting pVI to hexons for proper assembly of the virion and timely release of the membrane lytic mature VI molecule.Download high-res image (129KB)Download full-size image