Dynamic competition for hexon binding between core protein VII and lytic protein VI promotes adenovirus maturation and entry

Adenovirus minor coat protein VI contains a membrane-disrupting peptide that is inactive when VI is bound to hexon trimers. Protein VI must be released during entry to ensure endosome escape. Hexon:VI stoichiometry has been uncertain, and only fragments of VI have been identified in the virion structure. Recent findings suggest an unexpected relationship between VI and the major core protein, VII. According to the high-resolution structure of the mature virion, VI and VII may compete for the same binding site in hexon; and noninfectious human adenovirus type 5 particles assembled in the absence of VII (Ad5-VII-) are deficient in proteolytic maturation of protein VI and endosome escape. Here we show that Ad5-VII- particles are trapped in the endosome because they fail to increase VI exposure during entry. This failure was not due to increased particle stability, because capsid disruption happened at lower thermal or mechanical stress in Ad5-VII- compared to wild-type (Ad5-wt) particles. Cryoelectron microscopy difference maps indicated that VII can occupy the same binding pocket as VI in all hexon monomers, strongly arguing for binding competition. In the Ad5-VII- map, density corresponding to the immature amino-terminal region of VI indicates that in the absence of VII the lytic peptide is trapped inside the hexon cavity, and clarifies the hexon:VI stoichiometry conundrum. We propose a model where dynamic competition between proteins VI and VII for hexon binding facilitates the complete maturation of VI, and is responsible for releasing the lytic protein from the hexon cavity during entry and stepwise uncoating.

Dynamic competition for hexon binding between core protein VII and lytic protein VI promotes adenovirus maturation and entry

Adenovirus minor coat protein VI contains a membrane-disrupting peptide which is inactive when VI is bound to hexon trimers. Protein VI must be released during entry to ensure endosome escape. Hexon:VI stoichiometry has been uncertain, and only fragments of VI have been identified in the virion structure. Recent findings suggest an unexpected relationship between VI and the major core protein, VII. According to the high resolution structure of the mature virion, VI and VII may compete for the same binding site in hexon; and non-infectious human adenovirus type 5 particles assembled in the absence of VII (Ad5-VII-) are deficient in proteolytic maturation of protein VI and endosome escape. Here we show that Ad5-VII- particles are trapped in the endosome because they fail to increase VI exposure during entry. This failure was not due to increased particle stability, because capsid disruption happened at lower thermal or mechanical stress in Ad5-VII- compared to wildtype (Ad5-wt) particles. Cryo-EM difference maps indicated that VII can occupy the same binding pocket as VI in all hexon monomers, strongly arguing for binding competition. In the Ad5-VII- map, density corresponding to the immature amino-terminal region of VI indicates that in the absence of VII the lytic peptide is trapped inside the hexon cavity, and clarifies the hexon:VI stoichiometry conundrum. We propose a model where dynamic competition between proteins VI and VII for hexon binding facilitates the complete maturation of VI, and is responsible for releasing the lytic protein from the hexon cavity during entry and stepwise uncoating.Significance StatementCorrect assembly of an adenovirus infectious particle involves the highly regulated interaction of more than ten different proteins as well as the viral genome. Here we examine the interplay between two of these proteins: the major core protein VII, involved in genome condensation, and the multifunctional minor coat protein VI. Protein VI binds to the inner surface of adenovirus hexons (trimers of the major coat protein) and contains a lytic peptide which must be released during entry to ensure endosome rupture. We present data supporting a dynamic competition model between proteins VI and VII for hexon binding during assembly. This competition facilitates the release of the lytic peptide from the hexon cavity and ensures virus escape from the early endosome.

Adenovirus flow in host cell networks

Viruses are obligatory parasites that take advantage of intracellular niches to replicate. During infection, their genomes are carried in capsids across the membranes of host cells to sites of virion production by exploiting cellular behaviour and resources to guide and achieve all aspects of delivery and the downstream virus manufacturing process. Successful entry hinges on execution of a precisely tuned viral uncoating program where incoming capsids disassemble in consecutive steps to ensure that genomes are released at the right time, and in the right place for replication to occur. Each step of disassembly is cell-assisted, involving individual pathways that transmit signals to regulate discrete functions, but at the same time, these signalling pathways are organized into larger networks, which communicate back and forth in complex ways in response to the presence of virus. In this review, we consider the elegant strategy by which adenoviruses (AdVs) target and navigate cellular networks to initiate the production of progeny virions. There are many remarkable aspects about the AdV entry program; for example, the virus gains targeted control of a large well-defined local network neighbourhood by coupling several interacting processes (including endocytosis, autophagy and microtubule trafficking) around a collective reference state centred on the interactional topology and multifunctional nature of protein VI. Understanding the network targeting activity of protein VI, as well as other built-in mechanisms that allow AdV particles to be efficient at navigating the subsystems of the cell, can be used to improve viral vectors, but also has potential to be incorporated for use in entirely novel delivery systems.

Atomic Structures of Minor Proteins VI and VII in Human Adenovirus

ABSTRACT Human adenoviruses (Ad) are double-stranded DNA (dsDNA) viruses associated with infectious diseases, but they are better known as tools for gene delivery and oncolytic anticancer therapy. Atomic structures of Ad provide the basis for the development of antivirals and for engineering efforts toward more effective applications. Since 2010, atomic models of human Ad5 have been derived independently from photographic film cryo-electron microscopy (cryo-EM) and X-ray crystallography studies, but discrepancies exist concerning the assignment of cement proteins IIIa, VIII, and IX. To clarify these discrepancies, we employed the technology of direct electron counting to obtain a cryo-EM structure of human Ad5 at 3.2-Å resolution. Our improved structure unambiguously confirms our previous cryo-EM models of proteins IIIa, VIII, and IX and explains the likely cause of conflict in the crystallography models. The improved structure also allows the identification of three new components in the cavity of hexon—the cleaved N terminus of precursor protein VI (pVIn), the cleaved N terminus of precursor protein VII (pVIIn2), and mature protein VI. The binding of pVIIn2—and, by extension, that of genome-condensing pVII—to hexons is consistent with the previously proposed dsDNA genome-capsid coassembly for adenoviruses, which resembles that of single-stranded RNA (ssRNA) viruses but differs from the well-established mechanism of pumping dsDNA into a preformed protein capsid exemplified by tailed bacteriophages and herpesviruses. IMPORTANCE Adenovirus is a double-edged sword to humans: it is a widespread pathogen but can be used as a bioengineering tool for anticancer and gene therapies. The atomic structure of the virus provides the basis for antiviral and application developments, but conflicting atomic models for the important cement proteins IIIa, VIII, and IX from conventional/film cryo-EM and X-ray crystallography studies have caused confusion. Using cutting-edge cryo-EM technology with electron counting, we improved the structure of human adenovirus type 5 and confirmed our previous models of cement proteins IIIa, VIII, and IX, thus clarifying the inconsistent structures. The improved structure also reveals atomic details of membrane-lytic protein VI and genome-condensing protein VII and supports the previously proposed genome-capsid coassembly mechanism for adenoviruses.

A Single Maturation Cleavage Site in Adenovirus Impacts Cell Entry and Capsid Assembly

ABSTRACTProteolytic maturation drives the conversion of stable, immature virus particles to a mature, metastable state primed for cell infection. In the case of human adenovirus, this proteolytic cleavage is mediated by the virally encoded protease AVP. Protein VI, an internal capsid cement protein and substrate for AVP, is cleaved at two sites, one of which is near the N terminus of the protein. In mature capsids, the 33 residues at the N terminus of protein VI (pVIn) are sequestered inside the cavity formed by peripentonal hexon trimers at the 5-fold vertex. Here, we describe a glycine-to-alanine mutation in the N-terminal cleavage site of protein VI that profoundly impacts proteolytic processing, the generation of infectious particles, and cell entry. The phenotypic effects associated with this mutant provide a mechanistic framework for understanding the multifunctional nature of protein VI. Based on our findings, we propose that the primary function of the pVIn peptide is to mediate interactions between protein VI and hexon during virus replication, driving hexon nuclear accumulation and particle assembly. Once particles are assembled, AVP-mediated cleavage facilitates the release of the membrane lytic region at the amino terminus of mature VI, allowing it to lyse the endosome during cell infection. These findings highlight the importance of a single maturation cleavage site for both infectious particle production and cell entry and emphasize the exquisite spatiotemporal regulation governing adenovirus assembly and disassembly.IMPORTANCEPostassembly virus maturation is a cornerstone principle in virology. However, a mechanistic understanding of how icosahedral viruses utilize this process to transform immature capsids into infection-competent particles is largely lacking. Adenovirus maturation involves proteolytic processing of seven precursor proteins. There is currently no information for the role of each independent cleavage event in the generation of infectious virions. To address this, we investigated the proteolytic maturation of one adenovirus precursor molecule, protein VI. Structurally, protein VI cements the outer capsid shell and links it to the viral core. Functionally, protein VI is involved in endosome disruption, subcellular trafficking, transcription activation, and virus assembly. Our studies demonstrate that the multifunctional nature of protein VI is largely linked to its maturation. Through mutational analysis, we show that disrupting the N-terminal cleavage of preprotein VI has major deleterious effects on the assembly of infectious virions and their subsequent ability to infect host cells.

The Amphipathic Helix of Adenovirus Capsid Protein VI Contributes to Penton Release and Postentry Sorting

ABSTRACTNuclear delivery of the adenoviral genome requires that the capsid cross the limiting membrane of the endocytic compartment and traverse the cytosol to reach the nucleus. This endosomal escape is initiated upon internalization and involves a highly coordinated process of partial disassembly of the entering capsid to release the membrane lytic internal capsid protein VI. Using wild-type and protein VI-mutated human adenovirus serotype 5 (HAdV-C5), we show that capsid stability and membrane rupture are major determinants of entry-related sorting of incoming adenovirus virions. Furthermore, by using electron cryomicroscopy, as well as penton- and protein VI-specific antibodies, we show that the amphipathic helix of protein VI contributes to capsid stability by preventing premature disassembly and deployment of pentons and protein VI. Thus, the helix has a dual function in maintaining the metastable state of the capsid by preventing premature disassembly and mediating efficient membrane lysis to evade lysosomal targeting. Based on these findings and structural data from cryo-electron microscopy, we suggest a refined disassembly mechanism upon entry.IMPORTANCEIn this study, we show the intricate connection of adenovirus particle stability and the entry-dependent release of the membrane-lytic capsid protein VI required for endosomal escape. We show that the amphipathic helix of the adenovirus internal protein VI is required to stabilize pentons in the particle while coinciding with penton release upon entry and that release of protein VI mediates membrane lysis, thereby preventing lysosomal sorting. We suggest that this dual functionality of protein VI ensures an optimal disassembly process by balancing the metastable state of the mature adenovirus particle.