Mysteries of adenovirus packaging

Abstract

It is conventionally held that most DNA viruses package their genomes by one of two fundamental mechanisms: described by the sequential or concurrent models of assembly and packaging. Sequential packaging involves the translocation of a viral genome into a pre-formed capsid, often referred to as the pro-capsid. In contrast, concurrent packaging does not require the assembly of a pro-capsid. Instead, the genome is condensed, and the capsid shell is formed around the genome. The accumulation of empty particles in adenovirus infected cells has led to the assumption that adenovirus packaging may be best described by the sequential model. However, existing models fail to adequately explain all experimental observations, leaving many mysteries of adenovirus genome packaging unresolved. In this review, we describe key findings in adenovirus assembly and packaging, and we discuss them in the context of the competing models of sequential versus concurrent packaging. We discuss recent findings that have redefined our understanding of adenovirus packaging, including the role of viral biomolecular condensates and visualization of viral assembly and packaging in situ. These advances have renewed interest in the concurrent model of packaging. We anticipate that lessons learned from adenovirus packaging will be highly valuable for the advancement of viral vectors and gene-delivery technologies. In reviewing this topic, we hope to stimulate discussion and facilitate future investigation that will ultimately resolve gaps in knowledge and expand our understanding of DNA virus genome packaging.

Keywords: adenoviruses; capsid assembly; delivery vectors; genome packaging.

Fig 1

Simplified schematic of proposed assembly and packaging mechanisms of DNA viruses. Assembly and packaging of most DNA viruses can be described by two fundamental models, sequential or concurrent. Viral DNA genomes may be single-stranded or double-stranded and may take different forms including circular or linear. For simplicity's sake, linear double-stranded DNA is shown. Sequential model: The capsid shell (pro-capsid) is first assembled and then subsequently packaged by translocation of the viral genome through the portal. The viral genome can be packaged as naked DNA or as a deoxyribonucleoprotein complex. The example shown is of a progeny particle containing a deoxyribonucleoprotein core. Concurrent model: The viral genome acts as a substrate around which the capsid assembles.

Fig 2

Model of coordinated assembly and packaging in the adenovirus-infected nucleus. (A) Viral replication compartments (pink) function as organizing hubs of viral genome replication, which provides viral genomes for packaging into progeny particles. Viral biomolecular condensates (green) form during the late stage of infection and are required for the assembly of packaged progeny particles. (B) The L1-52K packaging protein and capsid proteins including but not limited to IIIa, hexon, and fiber are enriched in viral biomolecular condensates. A nascent viral genome associates with viral core proteins, and the formation of the pre-core begins. Packaging occurs at the intersection of these pathways and involves the assembly of the capsid around the pre-core. The L1-52K facilitates this process by coupling the pre-core to the assembling capsid. Incorporation of the viral protease and its co-factors results in the proteolytic processing of structural proteins either during or after packaging, yielding the mature packaged particle. Empty capsids are assembled from capsid proteins in the nucleoplasm and are dead-end byproducts that result from capsid assembly that is not coordinated with the provision of viral genomes.