Fluorescent protein tagging of adenoviral proteins pV and pIX reveals 'late virion accumulation compartment'

The human adenovirus type 5 (HAdV5) causes disease of the upper and lower respiratory tract. The early steps of HAdV5 entry up to genome replication in the host nucleus have been extensively studied. However, late stages of infection remain poorly understood. Here, we set out to elucidate the spatiotemporal orchestration of late adenovirus nuclear remodeling in living cells. We generated virus mutants expressing fluorescently tagged protein IX (pIX) and protein V (pV), a capsid and viral genome associated protein, respectively. We found that during progeny virion production both proteins localize to a membrane-less, nuclear compartment, which is highly impermeable such that in immunofluorescence microscopy antibodies can hardly penetrate it. We termed this compartment 'late virion accumulation compartment' (LVAC). Correlation between light- and electron microscopy revealed that the LVAC contains paracrystalline arrays of viral capsids that arrange tightly packed within a honeycomb-like organization of viral DNA. Live-cell microscopy as well as FRAP measurements showed that the LVAC is rigid and restricts diffusion of larger molecules, indicating that capsids are trapped inside. Author summary Understanding the regulation of adenovirus morphogenesis is not only of interest to cell biologists but is also key to define novel drug targets as well as to optimize adenoviruses as tools for gene therapy. While early steps of the adenovirus 'life cycle' are well understood, it is currently debated how, when and where capsid components associate with viral DNA. Here we used a combination of imaging methods to detail virus-induced spatiotemporal changes at late stages of infection. We found that HAdV5 induces a structured, membrane-less nuclear compartment. In this compartment capsids are closely packed within a honeycomb-like organization of replicated DNA, such that the newly formed particles appear to be trapped and show very little motility. Interestingly, we found a clear discrepancy between immunostaining and fluorescent fusion tagging, indicating a limited penetration of immunostains into this compartment. Since other pathogens induce similar compartments during replication, interpretation of immunostaining data requires careful evaluation.