Native and engineered tropism of vectors derived from a rare species D adenovirus serotype 43

Unique molecular properties of species D adenoviruses (Ads)-the most diverse yet underexplored group of Ads-have been used to develop improved gene vectors. The low seroprevalence in humans of adenovirus serotype 43 (Ad43), an otherwise unstudied species D Ad, identified this rare serotype as an attractive new human gene therapy vector platform. Thus, in this study we wished to assess biological properties of Ad43 essential to its vectorization. We found that (1) Ad43 virions do not bind blood coagulation factor X and cause low random transduction upon vascular delivery; (2) they clear host tissues more quickly than do traditionally used Ad5 vectors; (3) Ad43 uses CD46 as primary receptor; (4) Ad43 can use integrins as alternative primary receptors. As the first step toward vectorization of Ad43, we demonstrated that the primary receptor specificity of the Ad43 fiber can be altered to achieve infection via Her2, an established oncotarget. Whereas this modification required use of the Ad5 fiber shaft, the presence of this domain in chimeric virions did not make them susceptible for neutralization by anti-Ad5 antibodies

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

Structure of a Cell Entry Defective Human Adenovirus Provides Insights into Precursor Proteins and Capsid Maturation : Cryo-EM structure of ts1 virion of an adenovirus

Funding Information: We would like to thank Dr. Francisco Asturias for his advice on electron microscopy experiments and Dr. J.C. Ducom for installing Scipion and cisTEM packages on the HPC cluster and computational support in general. This work was supported by the NIH grant R21 AI146644 to V.S.R. Publisher Copyright: © 2021 Elsevier LtdMaturation of adenoviruses is distinguished by proteolytic processing of several interior minor capsid proteins and core proteins by the adenoviral protease and subsequent reorganization of adenovirus core. We report the results derived from the icosahedrally averaged cryo-EM structure of a cell entry defective form of adenovirus, designated ts1, at a resolution of 3.7 Å as well as of the localized reconstructions of unique hexons and penton base. The virion structure revealed the structures and organization of precursors of minor capsid proteins, pIIIa, pVI and pVIII, which are closely associated with the hexons on the capsid interior. In addition to a well-ordered helical domain (a.a. 310–397) of pIIIa, highlights of the structure include the precursors of VIII display significantly different structures near the cleavage sites. Moreover, we traced residues 4–96 of the membrane lytic protein (pVI) that includes an amphipathic helix occluded deep in the hexon cavity suggesting the possibility of co-assembly of hexons with the precursors of VI. In addition, we observe a second copy of pVI ordered up to residue L40 in the peripentonal hexons and a few fragments of density corresponding to 2nd and 3rd copies of pVI in other hexons. However, we see no evidence of precursors of VII binding in the hexon cavity. These findings suggest the possibility that differently bound pVI molecules undergo processing at the N-terminal cleavage sites at varying efficiencies, subsequently creating competition between the cleaved and uncleaved forms of VI, followed by reorganization, processing, and release of VI molecules from the hexon cavities.Peer reviewe

Biodistribution and inflammatory profiles of novel penton and hexon double-mutant serotype 5 adenoviruses

The use of adenovirus serotype 5 (Ad5) vectors in the clinical setting is severely hampered by the profound liver tropism observed after intravascular delivery coupled with the pronounced inflammatory and innate immune response elicited by these vectors. Liver transduction by circulating Ad5 virions is mediated by a high-affinity interaction between the capsid hexon protein and blood coagulation factor X (FX), whilst penton-α(v)integrin interactions are thought to contribute to the induction of anti-Ad5 inflammatory and innate immune responses. To overcome these limitations, we sought to develop and characterise for the first time novel Ad5 vectors possessing mutations ablating both hexon:FX and penton:integrin interactions. As expected, intravascular administration of the FX binding-ablated Ad5HVR5*HVR7*E451Q vector (AdT*) resulted in significantly reduced liver transduction in vivo compared to Ad5. In macrophage-depleted mice, increased spleen uptake of AdT* was accompanied by an elevation in the levels of several inflammatory mediators. However ablation of the penton RGD motif in the AdT* vector background (AdT*RGE) resulted in a significant 5-fold reduction in spleen uptake and attenuated the antiviral inflammatory response. A reduction in spleen uptake and inflammatory activation was also observed in animals after intravascular administration of Ad5RGE compared to the parental Ad5 vector, with reduced co-localisation of the viral beta-galactosidase transgene with MAdCAM-1+ sinus-lining endothelial cells. Our detailed assessment of these novel adenoviruses indicates that penton base RGE mutation in combination with FX binding-ablation may be a viable strategy to attenuate the undesired liver uptake and pro-inflammatory responses to Ad5 vectors after intravascular deliver

Differential regulation of cell motility and invasion by FAK

Cell migration and invasion are fundamental components of tumor cell metastasis. Increased focal adhesion kinase (FAK) expression and tyrosine phosphorylation are connected with elevated tumorigenesis. Null mutation of FAK results in embryonic lethality, and FAK−/− fibroblasts exhibit cell migration defects in culture. Here we show that viral Src (v-Src) transformation of FAK−/− cells promotes integrin-stimulated motility equal to stable FAK reexpression. However, FAK−/− v-Src cells were not invasive, and FAK reexpression, Tyr-397 phosphorylation, and FAK kinase activity were required for the generation of an invasive cell phenotype. Cell invasion was linked to transient FAK accumulation at lamellipodia, formation of a FAK–Src-p130Cas–Dock180 signaling complex, elevated Rac and c-Jun NH2-terminal kinase activation, and increased matrix metalloproteinase expression and activity. Our studies support a dual role for FAK in promoting cell motility and invasion through the activation of distinct signaling pathways

J Virol

In this study, we characterized the molecular basis for binding of adenovirus (AdV) to the cytoplasmic face of the nuclear pore complex (NPC), a key step during delivery of the viral genome into the nucleus. We used RNA interference (RNAi) to deplete cells of either Nup214 or Nup358, the two major Phe-Gly (FG) repeat nucleoporins localized on the cytoplasmic side of the NPC, and evaluated the impact on hexon binding and AdV infection. The accumulation of purified hexon trimers or partially disassembled AdV at the nuclear envelope (NE) was observed in digitonin-permeabilized cells in the absence of cytosolic factors. Both in vitro hexon binding and in vivo nuclear import of the AdV genome were strongly reduced in Nup214-depleted cells but still occurred in Nup358-depleted cells, suggesting that Nup214 is a major binding site of AdV during infection. The expression of an NPC-targeted N-terminal domain of Nup214 in Nup214-depleted cells restored the binding of hexon at the NE and the nuclear import of protein VII (pVII), indicating that this region is sufficient to allow AdV binding. We further narrowed the binding site to a 137-amino-acid segment in the N-terminal domain of Nup214. Together, our results have identified a specific region within the N terminus of Nup214 that acts as a direct NPC binding site for AdV. AdVs, which have the largest genome of nonenveloped DNA viruses, are being extensively explored for use in gene therapy, especially in alternative treatments for cancers that are refractory to traditional therapies. In this study, we characterized the molecular basis for binding of AdV to the cytoplasmic face of the NPC, a key step for delivery of the viral genome into the nucleus. Our data indicate that a 137-amino-acid region of the nucleoporin Nup214 is a binding site for the major AdV capsid protein, hexon, and that this interaction is required for viral DNA import. These findings provide additional insight on how AdV exploits the nuclear transport machinery for infection. The results could promote the development of new strategies for gene transfer and enhance understanding of the nuclear import of other viral DNA genomes, such as those of papillomavirus or hepatitis B virus that induce specific cancers

Humoral immune response to adenovirus induce tolerogenic bystander dendritic cells that promote generation of regulatory T cells.

Following repeated encounters with adenoviruses most of us develop robust humoral and cellular immune responses that are thought to act together to combat ongoing and subsequent infections. Yet in spite of robust immune responses, adenoviruses establish subclinical persistent infections that can last for decades. While adenovirus persistence pose minimal risk in B-cell compromised individuals, if T-cell immunity is severely compromised reactivation of latent adenoviruses can be life threatening. This dichotomy led us to ask how anti-adenovirus antibodies influence adenovirus T-cell immunity. Using primary human blood cells, transcriptome and secretome profiling, and pharmacological, biochemical, genetic, molecular, and cell biological approaches, we initially found that healthy adults harbor adenovirus-specific regulatory T cells (Tregs). As peripherally induced Tregs are generated by tolerogenic dendritic cells (DCs), we then addressed how tolerogenic DCs could be created. Here, we demonstrate that DCs that take up immunoglobulin-complexed (IC)-adenoviruses create an environment that causes bystander DCs to become tolerogenic. These adenovirus antigen loaded tolerogenic DCs can drive naïve T cells to mature into adenovirus-specific Tregs. Our study reveals a mechanism by which an antiviral humoral responses could, counterintuitively, favor virus persistence