Viral DNA Binding Protein SUMOylation Promotes PML Nuclear Body Localization Next to Viral Replication Centers

Abstract

Human adenoviruses (HAdVs) have developed mechanisms to manipulate cellular antiviral measures to ensure proper DNA replication, with detailed processes far from being understood. Host cells repress incoming viral genomes through a network of transcriptional regulators that normally control cellular homeostasis. The nuclear domains involved are promyelocytic leukemia protein nuclear bodies (PML-NBs), interferon-inducible, dot-like nuclear structures and hot spots of SUMO posttranslational modification (PTM). In HAdV-infected cells, such SUMO factories are found in close proximity to newly established viral replication centers (RCs) marked by the adenoviral DNA binding protein (DBP) E2A. Here, we show that E2A is a novel target of host SUMOylation, leading to PTMs supporting E2A function in promoting productive infection. Our data show that SUMOylated E2A interacts with PML. Decreasing SUMO-E2A protein levels by generating HAdV variants mutated in the three main SUMO conjugation motifs (SCMs) led to lower numbers of viral RCs and PML-NBs, and these two structures were no longer next to each other. Our data further indicate that SUMOylated E2A binds the host transcription factor Sp100A, promoting HAdV gene expression, and represents the molecular bridge between PML tracks and adjacent viral RCs. Consequently, E2A SCM mutations repressed late viral gene expression and progeny production. These data highlight a novel mechanism used by the virus to benefit from host antiviral responses by exploiting the cellular SUMO conjugation machinery. IMPORTANCE PML nuclear bodies (PML-NBs) are implicated in general antiviral defense based on recruiting host restriction factors; however, it is not understood so far why viruses would establish viral replication centers (RCs) juxtaposed to such “antiviral” compartments. To understand this enigma, we investigate the cross talk between PML-NB components and viral RCs to find the missing link connecting both compartments to promote efficient viral replication and gene expression. Taken together, the current concept is more intricate than originally believed, since viruses apparently take advantage of several specific PML-NB-associated proteins to promote productive infection. Simultaneously, they efficiently inhibit antiviral measures to maintain the viral infectious program. Our data provide evidence that SUMOylation of the viral RC marker protein E2A represents the basis of this virus-host interface and regulates various downstream events to support HAdV productive infection. These results are the basis of our current attempts to generate and screen for specific E2A SUMOylation inhibitors to constitute novel therapeutic approaches to limit and prevent HAdV-mediated diseases and mortality of immunosuppressed patients.

Nuclear body phase separation drives telomere clustering in ALT cancer cells

“…Similarly, protein sumoylation at other genomic loci may trigger PML association. Supporting this notion, a recent study finds that viral protein sumoylation is required for association of PML bodies with viral replication centers ( Stubbe et al , 2020 ).…”

The Role of ND10 Nuclear Bodies in Herpesvirus Infection: A Frenemy for the Virus?

“…These actions include the requirement of PML in EBV capsid assembly and in the viral productivity of HSV-1 and KSHV, the importance of Sp110 B in the EBV mRNA accumulation, and the involvement of ND10 in the maintenance of the latent HHV-6B genome. The pro-viral functions of ND10 have also been reported in many non-herpes viruses including adenoviruses and papillomaviruses [ 175 , 176 , 177 , 178 , 179 , 180 , 181 ], suggesting the possible common features of ND10 effects in DNA virus infections. HSV-1, with rather established culture systems and a clear protein functional profile, is an ideal model virus to lead the investigation into virus–ND10 interactions.…”

ATO (Arsenic Trioxide) Effects on Promyelocytic Leukemia Nuclear Bodies Reveals Antiviral Intervention Capacity

“…Indeed, the NiNTA pulldown in cells transfected with His 6 ‐tagged SUMO2 showed that SUMO2 modification of the viral DNA binding protein E2A was strongly reduced upon treatment with ATO. As PTM of E2A with SUMO2 is a prerequisite for the juxtaposition of HAdV replication centers to PML track‐like structures, in order to promote viral replication,45 the reduction in SUMOylation of E2A supposedly contributes to the observed decrease in replication center formation and also HAdV replication in general. In addition, recruitment of E1B‐55K to PML‐NBs, as well as the E3‐SUMO ligase function of the viral protein depends on E1B‐55K SUMOylation 34,35,103,91,104.…”

“…Additionally, it was shown that adenoviral DNA replication takes place at sites juxtaposed to PML‐NBs, and that the PML‐NB components Sp100A and Usp7, but not PML itself, are recruited to the replication centers 42–44. Recently, we provided evidence that targeting of the viral replication centers to sites of PML‐NBs before track formation is not dependent on interactions of PML‐NB components and the incoming viral genome, but is mediated by SUMOylation of the viral E2A protein promoting PML and Sp100A interaction with the viral DNA binding factor 45…”