Evidence That the Adenovirus Single-Stranded DNA Binding Protein Mediates the Assembly of Biomolecular Condensates to Form Viral Replication Compartments

Abstract

A common viral replication strategy is characterized by the assembly of intracellular compartments that concentrate factors needed for viral replication and simultaneously conceal the viral genome from host-defense mechanisms. Recently, various membrane-less virus-induced compartments and cellular organelles have been shown to represent biomolecular condensates (BMCs) that assemble through liquid-liquid phase separation (LLPS). In the present work, we analyze biophysical properties of intranuclear replication compartments (RCs) induced during human adenovirus (HAdV) infection. The viral ssDNA-binding protein (DBP) is a major component of RCs that contains intrinsically disordered and low complexity proline-rich regions, features shared with proteins that drive phase transitions. Using fluorescence recovery after photobleaching (FRAP) and time-lapse studies in living HAdV-infected cells, we show that DBP-positive RCs display properties of liquid BMCs, which can fuse and divide, and eventually form an intranuclear mesh with less fluid-like features. Moreover, the transient expression of DBP recapitulates the assembly and liquid-like properties of RCs in HAdV-infected cells. These results are of relevance as they indicate that DBP may be a scaffold protein for the assembly of HAdV-RCs and should contribute to future studies on the role of BMCs in virus-host cell interactions.

Herpesvirus Replication Compartments: Dynamic Biomolecular Condensates?

“…With ongoing viral replication, increased concentration of viral DNA that acts as long molecular scaffold will cause molecular crowding inside the compartment, forcing disordered proteins to engage in more stable bonds with surrounding molecules and creating more complex entanglement ( Figure 1 C). Similar mechanisms have been shown for replication compartments of Adenovirus and Rotavirus viral factories, which show liquid properties when they arise and convert to a more viscous state with the progression of infection [ 64 , 70 ]. Right now, data are lacking on the properties of very late herpesvirus RCs.…”

A Single Amino Acid Switch in the Adenoviral DNA Binding Protein Abrogates Replication Center Formation and Productive Viral Infection

“…The HAdV serotype 5 (HAdV-C5) DBP is a 529-amino-acid product of the E2A gene and is expressed early and late in infection, regulated by different promoters ( 20 , 23 – 25 ). At early time points postinfection, DBP diffusely localizes in the nucleus, condenses into small foci toward the end of the early stage of infection, and accumulates in spherical liquid biomolecular condensates during the late phase of infection ( 20 , 23 , 24 , 26 , 27 ). These membrane-less structures provide a hub for several viral and cellular proteins and correspond to viral replication centers (RCs), which are not only hot spots for viral DNA replication, late gene expression, and virus assembly but also for the sequestration and inactivation of host restriction factors and the recruitment of proviral factors that facilitate efficient viral transcription ( 26 , 28 – 31 ).…”

The guardian of the genome meets a viral master gene regulator at a biomolecular condensate

“…Emerging common strategies to eukaryotic gene function involve the formation of transcription factories through the assembly of phase-separated condensates as super-enhancers or repressors (Peng et al, 2020; Safari et al, 2019). Likewise, evidence of DNA viral replication compartments of liquid-like nature (Hidalgo et al, 2021; Seyffert et al, 2021) might impact the virus-host interaction landscape. Replication was shown to take place through the formation of viral replication centers (VRCs) or foci containing the required components in different double stranded DNA viruses (Charman and Weitzman, 2020), and these were described in detail for papillomaviruses (Khurana et al, 2021; Sakakibara et al, 2011).…”