Cellular SUMO-specific proteases regulate HAdV-C5 E1B-55K SUMOylation and virus-induced cell transformation.

Various viral proteins are post-translationally modified by SUMO-conjugation during the human adenovirus (HAdV) replication cycle. This modification leads to diverse consequences for target proteins as it influences their intracellular localization or cell transformation capabilities. SUMOylated HAdV proteins include the multifunctional oncoprotein E1B-55K. Our previous research, along with that of others, has demonstrated a substantial influence of yet another adenoviral oncoprotein, E4orf6, on E1B-55K SUMOylation levels. Protein SUMOylation can be reversed by cellular sentrin/SUMO-specific proteases (SENPs). In this study, we investigated the interaction of E1B-55K with cellular SENPs to understand deSUMOylation activities and their consequences for cell transformation mediated by this adenoviral oncoprotein. We show that E1B-55K interacts with and is deSUMOylated by SENP 1, independently of E4orf6. Consistent with these results, we found that SENP 1 prevents E1A/E1B-dependent focus formation in rodent cells. We anticipate these findings to be the groundwork for future studies on adenovirus-host interactions, the mechanisms that underlie E1B-55K SUMOylation, as well as the role of this major adenoviral oncoprotein in HAdV-mediated cell transformation.

Telomeric repeats in the commercial SB-1 vaccine facilitate viral integration and contribute to vaccine efficacy.

Marek's disease virus (MDV) integrates its genome into the telomeres of host chromosomes and causes fatal lymphomas in chickens. This integration is facilitated by telomeric repeat sequences (TMRs) at the ends of the viral genome, and is crucial for MDV-induced lymphomagenesis. The SB-1 vaccine virus is commonly used in commercial bivalent vaccines against MDV and also contains TMRs at its ends. Here, we demonstrate that SB-1 efficiently integrates its genome into the chromosomes of latently infected T cells. Deletion of the TMRs from the SB-1 genome did not affect virus replication, but severely impaired virus integration and genome maintenance in latently infected T cells and in chickens. Strikingly, the reduced integration and maintenance of latent SB-1 significantly impaired vaccine protection. Taken together, our data revealed that the TMRs facilitate SB-1 integration and that integration and/or maintenance of the latent viral genome is critical for vaccine protection.

A comparative review of adenovirus A12 and C5 oncogenes.

Oncogenic viruses contribute to 15% of global human cancers. To achieve that, virus-encoded oncoproteins deregulate cellular transcription, antagonize common cellular pathways, and thus drive cell transformation. Notably, adenoviruses were the first human viruses proven to induce cancers in diverse animal models. Over the past decades, human adenovirus (HAdV)-mediated oncogenic transformation has been pivotal in deciphering underlying molecular mechanisms. Key adenovirus oncoproteins, encoded in early regions 1 (E1) and 4 (E4), co-ordinate these processes. Among the different adenovirus species, the most extensively studied HAdV-C5 displays lower oncogenicity than HAdV-A12. A complete understanding of the different HAdV-A12 and HAdV-C5 oncoproteins in virus-mediated cell transformation, as summarized here, is relevant for adenovirus research and offers broader insights into viral transformation and oncogenesis.

Impact of viral telomeric repeat sequences on herpesvirus vector vaccine integration and persistence.

Marek's disease virus (MDV) vaccines were the first vaccines that protected against cancer. The avirulent turkey herpesvirus (HVT) was widely employed and protected billions of chickens from a deadly MDV infection. It is also among the most common vaccine vectors providing protection against a plethora of pathogens. HVT establishes latency in T-cells, allowing the vaccine virus to persist in the host for life. Intriguingly, the HVT genome contains telomeric repeat arrays (TMRs) at both ends; however, their role in the HVT life cycle remains elusive. We have previously shown that similar TMRs in the MDV genome facilitate its integration into host telomeres, which ensures efficient maintenance of the virus genome during latency and tumorigenesis. In this study, we investigated the role of the TMRs in HVT genome integration, latency, and reactivation in vitro and in vivo. Additionally, we examined HVT infection of feather follicles. We generated an HVT mutant lacking both TMRs (vΔTMR) that efficiently replicated in cell culture. We could demonstrate that wild type HVT integrates at the ends of chromosomes containing the telomeres in T-cells, while integration was severely impaired in the absence of the TMRs. To assess the role of TMRs in vivo, we infected one-day-old chickens with HVT or vΔTMR. vΔTMR loads were significantly reduced in the blood and hardly any virus was transported to the feather follicle epithelium where the virus is commonly shed. Strikingly, latency in the spleen and reactivation of the virus were severely impaired in the absence of the TMRs, indicating that the TMRs are crucial for the establishment of latency and reactivation of HVT. Our findings revealed that the TMRs facilitate integration of the HVT genome into host chromosomes, which ensures efficient persistence in the host, reactivation, and transport of the virus to the skin.

The adenovirus DNA-binding protein DBP.

Adenoviruses are a group of double-stranded DNA viruses that can mainly cause respiratory, gastrointestinal, and eye infections in humans. In addition, adenoviruses are employed as vector vaccines for combatting viral infections, including SARS-CoV-2, and serve as excellent gene therapy vectors. These viruses have the ability to modulate the host cell machinery to their advantage and trigger significant restructuring of the nuclei of infected cells through the activity of viral proteins. One of those, the adenovirus DNA-binding protein (DBP), is a multifunctional non-structural protein that is integral to the reorganization processes. DBP is encoded in the E2A transcriptional unit and is highly abundant in infected cells. Its activity is unequivocally linked to the formation, structure, and integrity of virus-induced replication compartments, molecular hubs for the regulation of viral processes, and control of the infected cell. DBP also plays key roles in viral DNA replication, transcription, viral gene expression, and even host range specificity. Notably, post-translational modifications of DBP, such as SUMOylation and extensive phosphorylation, regulate its biological functions. DBP was first investigated in the 1970s, pioneering research on viral DNA-binding proteins. In this literature review, we provide an overview of DBP and specifically summarize key findings related to its complex structure, diverse functions, and significant role in the context of viral replication. Finally, we address novel insights and perspectives for future research.

Identification of Adenovirus E1B-55K Interaction Partners through a Common Binding Motif.

The adenovirus C5 E1B-55K protein is crucial for viral replication and is expressed early during infection. It can interact with E4orf6 to form a complex that functions as a ubiquitin E3 ligase. This complex targets specific cellular proteins and marks them for ubiquitination and, predominantly, subsequent proteasomal degradation. E1B-55K interacts with various proteins, with p53 being the most extensively studied, although identifying binding sites has been challenging. To explain the diverse range of proteins associated with E1B-55K, we hypothesized that other binding partners might recognize the simple p53 binding motif (xWxxxPx). In silico analyses showed that many known E1B-55K binding proteins possess this amino acid sequence; therefore, we investigated whether other xWxxxPx-containing proteins also bind to E1B-55K. Our findings revealed that many cellular proteins, including ATR, CHK1, USP9, and USP34, co-immunoprecipitate with E1B-55K. During adenovirus infection, several well-characterized E1B-55K binding proteins and newly identified interactors, including CSB, CHK1, and USP9, are degraded in a cullin-dependent manner. Notably, certain binding proteins, such as ATR and USP34, remain undegraded during infection. Structural predictions indicate no conservation of structure around the proposed binding motif, suggesting that the interaction relies on the correct arrangement of tryptophan and proline residues.

Adenovirus E1B-55K controls SUMO-dependent degradation of antiviral cellular restriction factors.

IMPORTANCE: Human adenoviruses (HAdVs) generally cause mild and self-limiting diseases of the upper respiratory and gastrointestinal tracts but pose a serious risk to immunocompromised patients and children. Moreover, they are widely used as vectors for vaccines and vector-based gene therapy approaches. It is therefore vital to thoroughly characterize HAdV gene products and especially HAdV virulence factors. Early region 1B 55 kDa protein (E1B-55K) is a multifunctional HAdV-encoded oncoprotein involved in various viral and cellular pathways that promote viral replication and cell transformation. We analyzed the E1B-55K dependency of SUMOylation, a post-translational protein modification, in infected cells using quantitative proteomics. We found that HAdV increases overall cellular SUMOylation and that this increased SUMOylation can target antiviral cellular pathways that impact HAdV replication. Moreover, we showed that E1B-55K orchestrates the SUMO-dependent degradation of certain cellular antiviral factors. These results once more emphasize the key role of E1B-55K in the regulation of viral and cellular proteins in productive HAdV infections.

The human adenovirus E1B-55K oncoprotein coordinates cell transformation through regulation of DNA-bound host transcription factors.

The multifunctional adenovirus E1B-55K oncoprotein can induce cell transformation in conjunction with adenovirus E1A gene products. Previous data from transient expression studies and in vitro experiments suggest that these growth-promoting activities correlate with E1B-55K-mediated transcriptional repression of p53-targeted genes. Here, we analyzed genome-wide occupancies and transcriptional consequences of species C5 and A12 E1B-55Ks in transformed mammalian cells by combinatory ChIP and RNA-seq analyses. E1B-55K-mediated repression correlates with tethering of the viral oncoprotein to p53-dependent promoters via DNA-bound p53. Moreover, we found that E1B-55K also interacts with and represses transcription of numerous p53-independent genes through interactions with transcription factors that play central roles in cancer and stress signaling. Our results demonstrate that E1B-55K oncoproteins function as promiscuous transcriptional repressors of both p53-dependent and -independent genes and further support the model that manipulation of cellular transcription is central to adenovirus-induced cell transformation and oncogenesis.

Viral and cellular telomerase RNAs possess host-specific anti-apoptotic functions.

Human telomerase RNA (hTR) is overexpressed in many cancers and protects T cells from apoptosis in a telomerase-independent manner. The most prevalent cancer in the animal kingdom is caused by the highly oncogenic herpesvirus Marek's disease virus (MDV). MDV encodes a viral telomerase RNA (vTR) that plays a crucial role in MDV-induced tumorigenesis and shares all four conserved functional domains with hTR. In this study, we assessed whether hTR drives tumor formation in this natural model of herpesvirus-induced tumorigenesis. Therefore, we replaced vTR with hTR in the genome of a highly oncogenic MDV. Furthermore, we investigated the anti-apoptotic activity of vTR, hTR, and their counterpart in the chicken [chicken telomerase RNA (cTR)]. hTR was efficiently expressed and did not alter replication of the recombinant virus. Despite its conserved structure, hTR did not complement the loss of vTR in virus-induced tumorigenesis. Strikingly, hTR did not inhibit apoptosis in chicken cells, but efficiently inhibited apoptosis in human cells. Inverse host restriction has been observed for vTR and cTR in human cells. Our data revealed that vTR, cTR, and hTR possess conserved but host-specific anti-apoptotic functions that likely contribute to MDV-induced tumorigenesis. IMPORTANCE hTR is overexpressed in many cancers and used as a cancer biomarker. However, the contribution of hTR to tumorigenesis remains elusive. In this study, we assessed the tumor-promoting properties of hTR using a natural virus/host model of herpesvirus-induced tumorigenesis. This avian herpesvirus encodes a telomerase RNA subunit (vTR) that plays a crucial role in viral tumorigenesis and shares all conserved functional domains with hTR. Our data revealed that vTR and cellular TRs of humans and chickens possess host-specific anti-apoptotic functions. This provides important translational insights into therapeutic strategies, as inhibition of apoptosis is crucial for tumorigenesis.

In vitro infection of primary chicken lymphocytes with Marek's disease virus.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that infects immune cells and causes a deadly lymphoproliferative disease in chickens. Cytokines and monoclonal antibodies promote the survival of chicken lymphocytes in vitro. Here, we describe protocols for the isolation, maintenance, and efficient MDV infection of primary chicken lymphocytes and lymphocyte cell lines. This facilitates the investigation of key aspects of the MDV life cycle in the primary target cells of viral replication, latency, genome integration, and reactivation. For complete details on the use and execution of this protocol, please refer to Schermuly et al.,1 Bertzbach et al. (2019),2 and You et al.3 For a comprehensive background on MDV, please see Osterrieder et al.4 and Bertzbach et al. (2020).5.

A Special Issue on Marek's Disease Virus-The Editors' View.

Marek's disease virus (MDV), an Alphaherpesvirus belonging to the genus Mardivirus, causes T cell lymphomas in chickens and remains one of the greatest threats to poultry production worldwide [...].

Updates and New Perspectives on Adenoviral Gene Therapy and Vaccine Vectors.

Adenoviruses are commonly used as efficient high-capacity vectors and excellent gene delivery vehicles [...].

The adenoviral E4orf3/4 is a regulatory polypeptide with cell transforming properties in vitro.

The human adenovirus species C type 5 (HAdV-C5) early region 4 (E4) encodes several distinct polypeptides, defined as E4orf1 to E4orf6/7 according to the order and arrangement of the corresponding open reading frames (ORFs). All E4 gene products operate through a complex network of interactions with key viral and cellular regulatory proteins involved in transcription, apoptosis, cell cycle control, and DNA repair. Here, we generated a set of virus mutants carrying point mutations in the individual E4 genes. The phenotypic characterizations of these mutants revealed that mutations of these ORFs had no or only moderate effects on virus replication. Even a triple mutant that fails to produce E4orf3, E4orf4, and the yet uncharacterized alternatively spliced E4orf3/4 fusion protein, was replicating to wild type levels. The E4orf3/4 protein consists of the N-terminal 33 amino acid residues from E4orf3 and the C-terminal 28 amino acid residues from E4orf4. Intriguingly, we found that, similar to E4orf3, E4orf3/4 possesses properties that support the E1A/E1B-induced transformation of primary rodent cells. These results identify and functionally characterize E4orf3/4 and conclude that E4orf3/4 is another E4 region protein that is dispensable for virus replication but promotes the E1A/E1B-induced transformation of primary rodent cells.

Global Transcriptome Analyses of Cellular and Viral mRNAs during HAdV-C5 Infection Highlight New Aspects of Viral mRNA Biogenesis and Cytoplasmic Viral mRNA Accumulations.

It is well established that human adenoviruses such as species C, types 2 and 5 (HAdV-C2 and HAdV-C5), induce a nearly complete shutoff of host-cell protein synthesis in the infected cell, simultaneously directing very efficient production of viral proteins. Such preferential expression of viral over cellular genes is thought to be controlled by selective nucleocytoplasmic export and translation of viral mRNA. While detailed knowledge of the regulatory mechanisms responsible for the translation of viral mRNA is available, the viral or cellular mechanisms of mRNA biogenesis are not completely understood. To identify parameters that control the differential export of viral and cellular mRNAs, we performed global transcriptome analyses (RNAseq) and monitored temporal nucleocytoplasmic partitioning of viral and cellular mRNAs during HAdV-C5 infection of A549 cells. Our analyses confirmed previously reported features of the viral mRNA expression program, as a clear shift in viral early to late mRNA accumulation was observed upon transition from the early to the late phase of viral replication. The progression into the late phase of infection, however, did not result in abrogation of cellular mRNA export; rather, viral late mRNAs outnumbered viral early and most cellular mRNAs by several orders of magnitude during the late phase, revealing that viral late mRNAs are not selectively exported but outcompete cellular mRNA biogenesis.

The Diverse Major Histocompatibility Complex Haplotypes of a Common Commercial Chicken Line and Their Effect on Marek's Disease Virus Pathogenesis and Tumorigenesis.

The major histocompatibility complex (MHC) is crucial for appropriate immune responses against invading pathogens. Chickens possess a single predominantly-expressed class I molecule with strong associations between disease resistance and MHC haplotype. For Marek's disease virus (MDV) infections of chickens, the MHC haplotype is one of the major determinants of genetic resistance and susceptibility. VALO specific pathogen free (SPF) chickens are widely used in biomedical research and vaccine production. While valuable findings originate from MDV infections of VALO SPF chickens, their MHC haplotypes and associated disease resistance remained elusive. In this study, we used several typing systems to show that VALO SPF chickens possess MHC haplotypes that include B9, B9:02, B15, B19 and B21 at various frequencies. Moreover, we associate the MHC haplotypes to MDV-induced disease and lymphoma formation and found that B15 homozygotes had the lowest tumor incidence while B21 homozygotes had the lowest number of organs with tumors. Finally, we found transmission at variable levels to all contact birds except B15/B21 heterozygotes. These data have immediate implications for the use of VALO SPF chickens and eggs in the life sciences and add another piece to the puzzle of the chicken MHC complex and its role in infections with this oncogenic herpesvirus.

Marek's Disease Virus Virulence Genes Encode Circular RNAs.

Circular RNAs (circRNAs) are a recently rediscovered class of functional noncoding RNAs that are involved in gene regulation and cancer development. Next-generation sequencing approaches identified circRNA fragments and sequences underlying circularization events in virus-induced cancers. In the present study, we performed viral circRNA expression analysis and full-length sequencing in infections with Marek's disease virus (MDV), which serves as a model for herpesvirus-induced tumorigenesis. We established inverse PCRs to identify and characterize circRNA expression from the repeat regions of the MDV genome during viral replication, latency, and reactivation. We identified a large variety of viral circRNAs through precise mapping of full-length circular transcripts and detected matching sequences with several viral genes. Hot spots of circRNA expression included the transcriptional unit of the major viral oncogene encoding the Meq protein and the latency-associated transcripts (LATs). Moreover, we performed genome-wide bioinformatic analyses to extract back-splice junctions from lymphoma-derived samples. Using this strategy, we found that circRNAs were abundantly expressed in vivo from the same key virulence genes. Strikingly, the observed back-splice junctions do not follow a unique canonical pattern, compatible with the U2-dependent splicing machinery. Numerous noncanonical junctions were observed in viral circRNA sequences characterized from in vitro and in vivo infections. Given the importance of the genes involved in the transcription of these circRNAs, our study contributes to our understanding and complexity of this deadly pathogen. IMPORTANCE Circular RNAs (circRNAs) were rediscovered in recent years both in physiological and pathological contexts, such as in cancer. Viral circRNAs are encoded by at least two human herpesviruses, the Epstein Barr virus and the Kaposi's Sarcoma-associated herpesvirus, both associated with the development of lymphoma. Marek's disease virus (MDV) is a well-established animal model to study virus-induced lymphoma but circRNA expression has not been reported for MDV yet. Our study provided the first evidence of viral circRNAs that were expressed at key steps of the MDV lifecycle using genome-wide analyses of circRNAs. These circRNAs were primarily found in transcriptional units that corresponded to the major MDV virulence factors. In addition, we established a bioinformatics pipeline that offers a new tool to identify circular RNAs in other herpesviruses. This study on the circRNAs provided important insights into major MDV virulence genes and herpesviruses-mediated gene dysregulation.

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

Adenoviruses are very efficient high-capacity vaccine vectors and are common gene delivery systems. Despite their extensive use in preclinical models and clinical trials over the past decades, adenoviral vectors still require optimization. To achieve that, more thorough characterizations of adenoviral genes and gene products, as well as pathogen-host interactions, are indispensable. The adenoviral DNA binding protein (DBP) is a key regulatory protein involved in various cellular and viral processes. Here, we show that single amino acid exchange mutations in human adenovirus C5 (HAdV-C5) DBP strongly influence adenoviral replication by altering interaction with the cellular ubiquitination machinery. Specifically, phenotypic analyses of DBP mutants demonstrate that single amino acid substitutions can regulate interactions with the cellular USP7 deubiquitinase, impede viral DNA synthesis, and completely abolish viral late protein expression and progeny production. Importantly, cells infected with the DBP mutant UBM5 consistently lack DBP-positive replication centers (RCs), which are usually formed during the transition from the early to the late phase of infection. Our findings demonstrate that DBP regulates a key step at the onset of the late phase of infection and that this activity is unambiguously linked to the formation and integrity of viral RCs. These data provide the experimental basis for future work that targets DBP and its interference with the formation of viral RCs during productive infection. Consequently, this work will have immediate impact on DNA virus and adenovirus research in general and, potentially, also on safety optimization of existing and development of novel adenoviral vectors and anti-adenoviral compounds. IMPORTANCE To further understand the biology of human adenoviruses (HAdVs) and to optimize HAdVs for use in prophylactic and therapeutic therapies, a thorough understanding of key viral proteins is paramount. As one of the essential HAdV proteins, the DNA binding protein DBP plays important roles in various steps of the viral replication cycle. In this work, we aimed at deciphering the role of single amino acid exchange mutations in the HAdV-C5 DBP on interaction with the cellular deubiquitinase USP7 and regulation of viral replication. We identify interaction with USP7, viral replication center formation, and viral progeny production as potently regulated steps of the viral life cycle that are affected by these few and distinct mutations in DBP.

Protein-Protein Interactions Facilitate E4orf6-Dependent Regulation of E1B-55K SUMOylation in HAdV-C5 Infection.

The human adenovirus type C5 (HAdV-C5) E1B-55K protein is a multifunctional regulator of HAdV-C5 replication, participating in many processes required for maximal virus production. Its multifunctional properties are primarily regulated by post-translational modifications (PTMs). The most influential E1B-55K PTMs are phosphorylation at highly conserved serine and threonine residues at the C-terminus, and SUMO conjugation to lysines 104 (K104) and 101 (K101) situated in the N-terminal region of the protein, which have been shown to regulate each other. Reversible SUMO conjugation provides a molecular switch that controls key functions of the viral protein, including intracellular trafficking and viral immune evasion. Interestingly, SUMOylation at SUMO conjugation site (SCS) K104 is negatively regulated by another multifunctional HAdV-C5 protein, E4orf6, which is known to form a complex with E1B-55K. To further evaluate the role of E4orf6 in the regulation of SUMO conjugation to E1B-55K, we analyzed different virus mutants expressing E1B-55K proteins with amino acid exchanges in both SCS (K101 and K104) in the presence or absence of E4orf6. We could exclude phosphorylation as factor for E4orf6-mediated reduction of E1B-55K SUMOylation. In fact, we demonstrate that a direct interaction between E1B-55K and E4orf6 is required to reduce E1B-55K SUMOylation. Additionally, we show that an E4orf6-mediated decrease of SUMO conjugation to K101 and K104 result in impaired co-localization of E1B-55K and SUMO in viral replication compartments. These findings indicate that E4orf6 inhibits E1B-55K SUMOylation, which could favor assembly of E4orf6-dependent E3 ubiquitin ligase complexes that are known to degrade a variety of host restriction factors by proteasomal degradation and, thereby, promote viral replication.

E1B-55K Is a Phosphorylation-Dependent Transcriptional and Posttranscriptional Regulator of Viral Gene Expression in Human Adenovirus C5 Infection.

The multifunctional adenoviral E1B-55K phosphoprotein is a major regulator of viral replication and plays key roles in virus-mediated cell transformation. While much is known about its function in oncogenic cell transformation, the underlying features and exact mechanisms that implicate E1B-55K in the regulation of viral gene expression are less well understood. Therefore, this work aimed to unravel basic intranuclear principles of E1B-55K-regulated viral mRNA biogenesis using wild-type human adenovirus C5 (HAdV-C5) E1B-55K, a virus mutant with abrogated E1B-55K expression, and a mutant that expresses a phosphomimetic E1B-55K. By subnuclear fractionation, mRNA, DNA, and protein analyses as well as luciferase reporter assays, we show that (i) E1B-55K promotes the efficient release of viral late mRNAs from their site of synthesis in viral replication compartments (RCs) to the surrounding nucleoplasm, (ii) E1B-55K modulates the rate of viral gene transcription and splicing in RCs, (iii) E1B-55K participates in the temporal regulation of viral gene expression, (iv) E1B-55K can enhance or repress the expression of viral early and late promoters, and (v) the phosphorylation of E1B-55K regulates the temporal effect of the protein on each of these activities. Together, these data demonstrate that E1B-55K is a phosphorylation-dependent transcriptional and posttranscriptional regulator of viral genes during HAdV-C5 infection. IMPORTANCE Human adenoviruses are useful models to study basic aspects of gene expression and splicing. Moreover, they are one of the most commonly used viral vectors for clinical applications. However, key aspects of the activities of essential viral proteins that are commonly modified in adenoviral vectors have not been fully described. A prominent example is the multifunctional adenoviral oncoprotein E1B-55K that is known to promote efficient viral genome replication and expression while simultaneously repressing host gene expression and antiviral host responses. Our study combined different quantitative methods to study how E1B-55K promotes viral mRNA biogenesis. The data presented here propose a novel role for E1B-55K as a phosphorylation-dependent transcriptional and posttranscriptional regulator of viral genes.

Conserved E1B-55K SUMOylation in Different Human Adenovirus Species Is a Potent Regulator of Intracellular Localization.

Over the past decades, studies on the biology of human adenoviruses (HAdVs) mainly focused on the HAdV prototype species C type 5 (HAdV-C5) and revealed fundamental molecular insights into mechanisms of viral replication and viral cell transformation. Recently, other HAdV species are gaining more and more attention in the field. Reports on large E1B proteins (E1B-55K) from different HAdV species showed that these multifactorial proteins possess strikingly different features along with highly conserved functions. In this work, we identified potential SUMO-conjugation motifs (SCMs) in E1B-55K proteins from HAdV species A to F. Mutational inactivation of these SCMs demonstrated that HAdV E1B-55K proteins are SUMOylated at a single lysine residue that is highly conserved among HAdV species B to E. Moreover, we provide evidence that E1B-55K SUMOylation is a potent regulator of intracellular localization and p53-mediated transcription in most HAdV species. We also identified a lysine residue at position 101 (K101), which is unique to HAdV-C5 E1B-55K and specifically regulates its SUMOylation and nucleo-cytoplasmic shuttling. Our findings reveal important new aspects on HAdV E1B-55K proteins and suggest that different E1B-55K species possess conserved SCMs while their SUMOylation has divergent cellular effects during infection. IMPORTANCE E1B-55K is a multifunctional adenoviral protein and its functions are highly regulated by SUMOylation. Although functional consequences of SUMOylated HAdV-C5 E1B-55K are well studied, we lack information on the effects of SUMOylation on homologous E1B-55K proteins from other HAdV species. Here, we show that SUMOylation is a conserved posttranslational modification in most of the E1B-55K proteins, similar to what we know about HAdV-C5 E1B-55K. Moreover, we identify subcellular localization and regulation of p53-dependent transcription as highly conserved SUMOylation-regulated E1B-55K functions. Thus, our results highlight how HAdV proteins might have evolved in different HAdV species with conserved domains involved in virus replication and differing alternative functions and interactions with the host cell machinery. Future research will link these differences and similarities to the diverse pathogenicity and organ tropism of the different HAdV species.