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 major core protein condenses DNA in clusters and bundles, modulating genome release and capsid internal pressure

Some viruses package dsDNA together with large amounts of positively charged proteins, thought to help condense the genome inside the capsid with no evidence. Further, this role is not clear because these viruses have typically lower packing fractions than viruses encapsidating naked dsDNA. In addition, it has recently been shown that the major adenovirus condensing protein (polypeptide VII) is dispensable for genome encapsidation. Here, we study the morphology and mechanics of adenovirus particles with (Ad5-wt) and without (Ad5-VII-) protein VII. Ad5-VII- particles are stiffer than Ad5-wt, but DNA-counterions revert this difference, indicating that VII screens repulsive DNA-DNA interactions. Consequently, its absence results in increased internal pressure. The core is slightly more ordered in the absence of VII and diffuses faster out of Ad5-VII– than Ad5-wt fractured particles. In Ad5-wt unpacked cores, dsDNA associates in bundles interspersed with VII-DNA clusters. These results indicate that protein VII condenses the adenovirus genome by combining direct clustering and promotion of bridging by other core proteins. This condensation modulates the virion internal pressure and DNA release from disrupted particles, which could be crucial to keep the genome protected inside the semi-disrupted capsid while traveling to the nuclear pore.

A Dual Laser Scanning Confocal and Transmission Electron Microscopy Analysis of the Intracellular Localization, Aggregation and Particle Formation of African Horse Sickness Virus Major Core Protein VP7

The bulk of the major core protein VP7 in African horse sickness virus (AHSV) self-assembles into flat, hexagonal crystalline particles in a process appearing unrelated to viral replication. Why this unique characteristic of AHSV VP7 is genetically conserved, and whether VP7 aggregation and particle formation have an effect on cellular biology or the viral life cycle, is unknown. Here we investigated how different small peptide and enhanced green fluorescent protein (eGFP) insertions into the VP7 top domain affected VP7 localization, aggregation, and particle formation. This was done using a dual laser scanning confocal and transmission electron microscopy approach in conjunction with analyses of the solubility, aggregation, and fluorescence profiles of the proteins. VP7 top domain modifications did not prevent trimerization, or intracellular trafficking, to one or two discrete sites in the cell. However, modifications that resulted in a misfolded and insoluble VP7-eGFP component blocked trafficking, and precluded protein accumulation at a single cellular site, perhaps by interfering with normal trimer–trimer interactions. Furthermore, the modifications disrupted the stable layering of the trimers into characteristic AHSV VP7 crystalline particles. It was concluded that VP7 trafficking is driven by a balance between VP7 solubility, trimer forming ability, and trimer–trimer interactions.

In Silico Studies on Development of Novel Virostatic Agents against Bluetongue Virus

The core of BTV is organized into three concentric structures of which VP7 protein forms the major core protein. The subcore consists of VP3 protein and the innermost part of the core is made of three minor proteins: VP1, VP4, and VP6. Earlier it was reported that core-like particles (CLPs) composed of viral VP7 and VP3 proteins were produced in order to study role of VP7 protein in intermolecular interactions in the BTV assembly process. Site specific mutational studies revealed that substitution of the single lysine residue of VP7 (Lys-255) by leucine abrogated CLP formation, indicating a critical role for this lysine. In the present study, homology modeling, mutagenesis, and docking studies were carried out in order to design potent leads in modulation of VP7 protein in abrogating CLP formation.