Conserved E1B-55K SUMOylation in Different Human Adenovirus Species Is a Potent Regulator of Intracellular Localization
ABSTRACT
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.
INTRODUCTION
The early 1B protein E1B-55K has been the focus of many recent investigations into the biology of human adenovirus species C type 5 (HAdV-C5, referred to as C5 in the manuscript). With its multiple functions, E1B-55K is an important regulator of various signaling pathways (1, 2).
SUMOylation is a reversible posttranslational modification (PTM). An enzymatic cascade covalently binds small ubiquitin-like modifier (SUMO) proteins to a substrate protein with various consequences like regulation of protein-protein interactions, protein stability, functional activation, and subcellular localization (3). It has previously been shown that HAdV-C5 E1B-55K SUMOylation at a consensus SUMO conjugation motif (SCM) at lysine 104 (K104) regulates numerous functions of this viral protein. E1B-55K SUMOylation regulates its own E3-SUMO-ligase activity, binding to p53 and repression of p53-dependent promoters (4–8). For example, SUMO-E1B-55K can induce Sp100A SUMOylation to inhibit its function as a tumor suppressor and an activator of p53-dependent transcription (9). Moreover, SUMOylation regulates E1B-55K-binding to and the subsequent degradation of Daxx, a PML nuclear body (PML-NB)-associated factor that inhibits adenoviral gene expression (10). SUMO-E1B-55K also interacts with PML IV and V, which are components of PML-NBs (11). Together with three cellular proteins (cullin 5 [Cul5], elongin B/C and RING-box protein 1 [Rbx1]), E1B-55K and E4orf6 form an E3-ubiquitin-ligase complex that recruits cellular proteins for proteasomal degradation (12–18). On the other hand, SUMOylation of E1B-55K is reduced by the cellular heterochromatin-associated transcription factor KAP1 through a still unknown mechanism (19). Finally, HAdV-C5 E1B-55K nuclear localization and intranuclear targeting are SUMOylation-dependent and SUMOylation occurs in close proximity to a nuclear export signal (NES) (20, 21).
To date, there are only very few reports that describe E1B-55K functions of other HAdV species. For instance, it has been shown that the formation of the E3-ubiquitin-ligase complex with E4orf6 is highly conserved among species. However, heterogeneity exists in the cullin proteins that form part of the complex and in the cellular targets that are ubiquitinated in the presence of different E1B-55K proteins (22). DNA ligase IV is efficiently degraded in the presence of all tested HAdV E1B-55K proteins and other cellular proteins like Mre11, integrin α-3 and BLM are only degraded by some HAdV species. Regarding p53-binding and -degradation, it has been shown that only HAdV-A12, C5 and F40 E1B-55Ks are able to both bind and degrade this tumor suppressor, while other species bind but do not efficiently degrade p53 (22, 23). Additionally, a previous report demonstrated that E1B-55K-binding to specific PML isoforms and colocalization with PML-NBs is a conserved function among HAdV species (24). The localization of E1B-55K in aggresomes (aggregations of misfolded proteins in eukaryotic cells), however, has only been described for HAdV-B16, C5, D9 and E4 (24, 25). Notably, E1B-55K proteins from different HAdV species differ in size ranging from 472 to 496 amino acid residues (1, 2). Nevertheless, we decided to keep the conventional nomenclature and use “E1B-55K” for the different proteins inspired by previous literature (1, 2).
In the present study, we characterize the SUMOylation of the E1B-55K proteins from HAdV species A12, B16 and B34, C5, D9, E4, and F40 as representative members of different HAdV types (1, 2, 26). We first identify the presence of functional SCMs and further investigate the consequences of SUMOylation on functions of these viral proteins. In addition, we show that a lysine residue at position 101 (K101) of HAdV-C5 E1B-55K affects SUMOylation and nucleo-cytoplasmic shuttling, most probably through regulation of the main SUMOylation-site K104.
RESULTS
A conserved SCM is present in the E1B-55K proteins from most HAdV species.
As shown previously, the multiple functions of HAdV-C5 E1B-55K are regulated by conjugation of SUMO-1 and SUMO-2 to the lysine residue at position K104 (K104). K104 has been described as the main E1B-55K SUMOylation-site and is part of a classical SCM (ΨKxE/D, where Ψ stands for a large hydrophobic amino acid, x for any amino acid and K, E and D for lysine, glutamic acid and aspartic acid, respectively), comprising amino acid residues 102 to 105 (5, 6, 9–11, 20, 21, 27). In order to investigate whether this motif is conserved in other HAdV species, we first performed amino acid sequence alignments of E1B-55K representatives from HAdV species A to F, comparing them to HAdV-C5 E1B-55K (Fig. 1).
FIG 1
We found that the classical SCM was present in almost all HAdV species, with HAdV-A12 and F40 as exceptions (Fig. 1A, green box). Furthermore, using the prediction softwares GPS-SUMO and JASSA, we detected several nonconsensus motifs in F40 (28–30). Interestingly, a lysine at position 101 (K101) was found exclusively in E1B-55K proteins from species C HAdVs. E1B-55K proteins from other HAdV species carry an arginine (R) instead of a lysine at position 101 (Fig. 1A, blue box). Since K101 is in close proximity to the main SCM of HAdV-C5 E1B-55K, we decided to further investigate the impact of this residue on E1B-55K SUMOylation and function.
In contrast to the SCM, the leucine-rich NES appears to be conserved in E1B-55K proteins from species C HAdVs only (Fig. 1A, gray box). Most E1B-55K proteins displayed the leucine residues (L) corresponding to positions 83, 87 and 91 in HAdV-C5 E1B-55K, however, they were missing isoleucines (I) present at positions 90 and 93, which would reduce the strength of a functional NES (31). Moreover, HAdV-A12 and F40 E1B-55K proteins lacked both leucines and isoleucines, which would indicate the absence of a putative NES (31). These findings unequivocally demonstrate that E1B-55K proteins from most HAdV species possess a conserved SCM, while differences are apparent in their NES.
E1B-55K SUMOylation occurs at a consensus SCM and is conserved for SUMO-2, but not SUMO-1.
After we identified a conserved SCM in almost all E1B-55K proteins from different HAdV species, we set to investigate the functionality of this conjugation motif in comparison to the HAdV-C5 E1B-55K SCM. We generated different HA-tagged E1B-55K SCM-mutants from HAdV species A to F by exchanging the corresponding lysine (K) to an arginine (R) (Fig. 1B). Additionally, in order to elucidate the role of K101, which is unique to HAdV-C E1B-55K, we replaced K101 by an R (K101R). We transfected the wild type (wt) E1B-55K-expressing plasmids and their respective SCM-mutants into HeLa cells that are stably expressing His-tagged SUMO-1 or SUMO-2 and performed Ni-NTA pulldowns for His-tagged SUMO conjugates (Fig. 2).
FIG 2
As expected, all E1B-55K proteins with an SCM were modified by SUMO-2. HAdV-D9, B16, B34 and F40 E1B-55Ks were SUMOylated at higher levels compared to C5 E1B-55K, but SUMOylation of E4 E1B-55K appeared to be comparable to C5 E1B-55K (Fig. 2A, lanes 2, 4, 7, 11, 13 and 15). We only detected little to no high molecular weight material in HAdV-E4 transfections pointing toward a lack of certain additional PTMs of this protein which calls for further exploration (Fig. 2A and B, lanes 2 and 3). Although HAdV-F40 E1B-55K does not possess an SCM, we detected higher-migrating bands corresponding to SUMOylated forms of this protein (Fig. 2A, lane 15). These results suggest that SUMOylation might occur at a nonconsensus motif in HAdV species F E1B-55Ks. For HAdV-A12 E1B-55K, we only observed subtle SUMOylation, most probably because it lacks a conserved SCM (Fig. 2A, lane 9). Mutational inactivation of the SCM leads to the loss of SUMOylated E1B-55K in almost all HAdV species, indicating that the SCM found in our in silico alignment (Fig. 1) indeed is the SUMO conjugation site in HAdV E1B-55K proteins, similar to what is known for C5 (Fig. 2A, lanes 3, 5, 12, 14 and 16). The mutation of HAdV-D9 E1B-55K (K103R) only led to decreased SUMO-2 conjugation rather than a complete abrogation (Fig. 2A, lane 8), presumably due to the presence of an additional minor SUMOylation site in this protein. Moreover, there is no difference between wt HAdV-A12 E1B-55K and its putative SUMO-mutant, indicating that the SUMOylated bands could correspond to minor, nonconsensus SUMO-attachment domains in E1B-55K (Fig. 2A, lanes 9 and 10). Remarkably, SUMOylation levels of the HAdV-C5 K101R-mutant were substantially increased compared to the wt E1B-55K. In fact, SUMOylation of this mutant resembled E1B-55K proteins from other HAdV species with an arginine next to their SCM (Fig. 1A, blue box, and 2A, lane 6).
Similar to SUMO-2 conjugation, we also observed conjugation with SUMO-1 at the identified SCMs in all of the species tested (Fig. 2B). We saw SUMO-1 modifications of E1B-55Ks from HAdV-E4, C5, F40, A12, D9, B16 and B34, with the last three exhibiting the highest SUMOylation levels (Fig. 2B, lanes 2, 4, 7, 9, 11, 13 and 15). In contrast to SUMO-2 SUMOylation however, SUMO-1 conjugation to wt HAdV-F40 E1B-55K was rather weak (Fig. 2B, lane 15). Moreover, and in line with SUMO-2 SUMOylation, we did not see differences between wt HAdV-A12 E1B-55K and its putative SCM-mutant (Fig. 2B, lanes 9 and 10). Consistent with SUMO-2, SUMO-1 conjugation of the HAdV-C5 K101R-mutant increased compared to the wt protein (Fig. 2B, lanes 4 and 6). Interestingly, we detected higher-migrating bands in the immunoblot. These bands can be explained by the lack of an intrinsic SCM, which results in SUMO-1-mediated chain termination (32). Thus, SUMO-1 seems to be incorporated into SUMO-2 chains rather than being attached as a monomer (Fig. 2B).
Since we observed increased SUMOylation in the C5 K101R-mutant, we set to investigate if the SUMOylation of E1B-55K from other HAdV species, which already harbor an arginine at the respective site, is decreased when replaced by a lysine. Moreover, we assessed if mutation of the additional lysines around the E1B-55K SCM of A12 (K91R, K92R), F40 (K92R, K127R, K430R) and D9 (K157R, K302R) lead to decreased SUMOylation levels. However, this was not the case (data not shown). These results indicate that the specific role of K101 in species C HAdVs probably involves other, so far unknown factors that are necessary to regulate HAdV-C5 E1B-55K SUMOylation.
High SUMOylation levels correlate with nuclear localization of E1B-55K proteins from most HAdV species.
E1B-55K is actively shuttling between nucleus and cytoplasm via a CRM1-dependent NES (33). Additionally, E1B SUMOylation regulates its subcellular localization and vice versa. In the context of viral infection, the adenoviral protein E4orf6 has been shown to act as a negative regulator of HAdV-C5 E1B-55K SUMOylation and to control its nucleo-cytoplasmic and intranuclear localization (21, 34–36). Moreover, cellular proteins like KAP1 have been shown to control E1B-55K SUMOylation (19), highlighting the important role of this PTM in regulating E1B-55K subcellular localization.
To examine the effect of SUMOylation on the localization of E1B-55K proteins from different HAdV species, we performed immunofluorescence assays with HA-tagged E1B-55Ks or their respective SUMO-mutants (Fig. 3A, 4A, and 5A). Next, we assessed their nucleo-cytoplasmic localization by measuring the nuclear and cytoplasmic mean fluorescence intensities and calculating the mean nuclear intensity divided by the mean cytoplasmic intensity (Fig. 3B, 4B, and 5B) (37). We observed the trend that highly SUMOylated E1B-55Ks localize to the nucleus and that the loss of SUMOylation shifts protein localization to the cytoplasm, similar to what has been previously described for wt HAdV-C5 E1B-55K (Fig. 3A [panels j–r] and 3B [panels 2 and 3]). In our assays, wt HAdV-C5 E1B-55K was detected in the nucleus and the cytoplasm of the cells with subtle changes in its cellular localization with the SUMO-mutant K104R (Fig. 3A [panels j–l and p–r] and 3B [panel 3]). In contrast, transfections with the highly SUMOylated K101R-mutant led to a remarkable and highly significant increase of nuclear localization, further emphasizing the importance of SUMOylation for E1B-55K nuclear localization and shuttling (Fig. 3A [panels m–o] and 3B [panel 2]). In HAdV-E4, the wt protein was found in both cellular compartments with higher abundance in the nucleus (Fig. 3A [panels d–f] and 3B [panel 1]). The E4 K93R-mutant changed this phenotype with a significant decrease of its nuclear localization (Fig. 3A [panels g–i] and 3B [panel 1]). wt HAdV-D9 E1B-55K showed localization in both cellular compartments but with a more dominant nuclear localization pattern. In contrast, the K103R-mutant presented a significant shift toward a stronger cytoplasmic localization (Fig. 4A [panels a-f] and 4B [panel 1]). HAdV-B16, B34 and F40 wt E1B-55K proteins were present in the nucleus of most of the cells (Fig. 4A [panels m–o], 5A [panels a–c and g–i], 4B [panel 3], and 5B [panels 1 and 2]). Mutation of the E1B-55K SCM in these HAdV species led to relocalization of E1B-55K into the cytoplasm, thereby increasing the percentage of cells with E1B-55K in both subcellular compartments (Fig. 4A [panels p–r], 5A [panels d–f and j–-l], 4B [panel 3], and 5B [panels 1 and 2]). Interestingly, we observed similar localizations of wt HAdV-A12 E1B-55K and its K88R-mutant, both presenting a more dominant nuclear localization (Fig. 4A [panels g-–l] and 4B [panel 2]). These data are in concordance with the SUMOylation status of these two proteins (Fig. 2).
FIG 3
FIG 4
FIG 5
We still observed a fraction of the protein in the nucleus with almost all E1B-55K proteins from different HAdV species despite SCM inactivation. Having confirmed a complete loss of SUMOylation in these E1B-55K-mutants by immunoblotting, we further analyzed whether this loss of SUMOylation correlates with a decrease in colocalization between E1B-55K and SUMO-2 proteins (Fig. 5C). What we discovered was that E1B-55K and SUMO-2 colocalization indeed decreased upon SCM-inactivation in all HAdV species where the SCM mutation resulted in a severe reduction or a loss of SUMOylation. In HAdV-C5, the percentage of cells with a positive colocalization dropped from 41% to 6%, when comparing wt E1B-55K to the K104R-mutant (Fig. 5C).
As expected, the highly SUMOylated K101R-mutant exhibited high colocalization rates (74%), which were found to be much higher than for wt C5 E1B-55K (Fig. 5C). wt HAdV-E4 and B34 E1B-55K proteins colocalized with SUMO-2 with values of 50% or 80% and we saw a decrease down to 12% and 41% with their SUMO-mutants (Fig. 5C). A strong decrease of colocalization also resulted from mutational SCM-inactivation in HAdV-D9 E1B-55K (49% to 15%) and B16 E1B-55K (75% to 11%). Remarkably, colocalization of HAdV-F40 E1B-55K did not change much upon SCM-inactivation (38% to 43%) (Fig. 5C). Together, these data point toward a conserved correlation of E1B-55K SUMOylation and nuclear localization in most HAdV species.
SUMOylation of the different E1B-55K proteins regulates p53-mediated transcription.
E1B-55Ks from all HAdV species are able to repress p53-mediated transcription, with E4 as an exception (23). Moreover, HAdV-C5 E1B-55K SUMOylation regulates its function as a transcriptional repressor of p53-regulated promoters (27). To assess whether SUMOylation of the different E1B-55Ks also affects p53-regulated transcription, we performed a dual luciferase reporter gene assay using a CMV-promoter downstream of five p53 binding sites (38). In Fig. 6A, we provide the percentages of promoter activity in cells transfected with different E1B-55Ks and their respective SUMO-mutants, together with p53 and a p53-dependent promoter. As expected, wt E1B-55Ks from all species were able to repress p53-mediated transcription to a different extent (Fig. 6A) and overall, transcription inhibition did not correlate with E1B-55K levels (Fig. 6B). While repression by HAdV-B16, B34 and F40 E1B-55Ks was quite similar to C5 E1B-55K, repression by HAdV-E4 and A12 E1B-55Ks was not as effective (Fig. 6A).
FIG 6
Interestingly, repression by HAdV-D9 E1B-55K was the highest observed in our analysis (Fig. 6A). This nicely correlates with the highest SUMOylation levels of HAdV-D9 E1B-55K that we observed among all E1B-55Ks tested (Fig. 2A, lane 7; 2B, lane 7). Next, we assessed the SUMOylation-dependence of this phenomenon and observed an impaired p53 transcriptional repression with the HAdV-B16, E4 and F40 E1B-55K SUMO-mutants, comparable to levels of the C5 E1B-55K K104R-mutant (Fig. 6A). The HAdV-D9 K103R-mutant also showed a slight increase in p53-induced luciferase activity. However, it could still highly repress transcription compared to the negative control (Fig. 6A). On the other hand, and in concordance with its gain-of-function phenotype, the HAdV-C5 K101R-mutant highly repressed p53-dependent transcription, even at higher levels than the wt protein (Fig. 6A). Interestingly, neither the HAdV-B34 nor the A12 E1B-55K-mutants showed a difference in p53 transcriptional repression compared to their corresponding wt proteins (Fig. 6A). These results suggest that E1B-55K SUMOylation regulates its effect on p53 with a positive correlation between SUMO conjugation and p53 transcriptional repression. SUMO-mediated regulation was observed in most HAdV species whereas in others, E1B-55K uses SUMO-independent mechanisms to repress p53.
DISCUSSION
In this study, we discovered that E1B-55K proteins from almost all HAdV species are highly SUMOylated at conserved SCMs, indicating that this PTM is not only important for HAdV-C5 E1B-55K but also for E1B-55Ks from other HAdV species (Fig. 1 and 2). Intriguingly, HAdV-F40 E1B-55K is highly SUMOylated at K90 and a consensus SCM is missing (Fig. 1 and 2). SUMOylation of this protein must occur at a nonconsensus site, as described for several proteins. Some examples are the cellular protein mouse double minute 2 homolog (Mdm2), the cAMP response element-binding protein (CREB) and the Epstein-Barr virus (EBV) transactivator protein BZLF1 (39–42). Furthermore, this nonconsensus SUMO conjugation site in HAdV-F40 E1B-55K aligns with the SCM in the other HAdV species (Fig. 1). Slight changes between our alignment and previously published E1B alignments are likely explained by the use of different software (24, 26). Since accessibility to the substrate plays an important role in SUMOylation, it is conceivable that the position of the lysine residue N-terminal of E1B-55K is critical for a successful modification of the protein (43). While the SCM is highly conserved among the different HAdV species, a lysine residue K101 and a leucine-rich NES are only found in HAdVs from species C, affecting the SUMOylation and nucleo-cytoplasmic shuttling of E1B-55K (Fig. 2 and 3). A previous report suggested a model that illustrates the mechanism of E1B-55K shuttling. In this model, HAdV-C5 E1B-55K is SUMOylated at K104 upon entry into the nucleus. There, K104 SUMOylation facilitates targeting of E1B-55K to viral replication centers (RCs) and PML-NBs, simultaneously obstructing CRM1-binding to the NES. At these subnuclear structures, E1B-55K is deSUMOylated via an unknown mechanism, probably involving E4orf6, enabling a CRM1-dependent nuclear export (21). In our study, we demonstrate that K101 is also involved in SUMOylation and shuttling of HAdV-C5 E1B-55K, but by a so far unknown mechanism. An explanation could be the nuclear localization of HAdV-C5 K101R that further enhances its SUMOylation and could therefore block nuclear export via the NES. Alternatively, SUMOylation could interfere with nuclear-cytoplasmic shuttling of E1B-55K and retain K101R in the nucleus. Despite the use of a CRM1-dependent export, HAdV-C5 E1B-55K is thought to have evolved another, CRM1-independent export pathway (21). We provide evidence that E1B-55K from most HAdV species exit the nucleus via a CRM1-independent pathway because CRM1 is an export receptor for leucine-rich NES (44) and the leucine-rich HAdV-C5 E1B-55K NES is only partially conserved and apparently absent in some HAdV species (A12 and F40) (Fig. 1). Nevertheless, we observed a shift from nuclear to cytoplasmic localizations upon mutational SCM inactivation in E1B-55K from other HAdV species (Fig. 3 to 5). Most interestingly, we also observed this change in HAdV-F40 E1B-55K, where the complete NES is presumably missing. Thus, it is likely that all tested E1B-55Ks evolved a CRM1-independent export pathway and only HAdV-C5 developed an additional CRM1-dependent mechanism to enable and facilitate nucleo-cytoplasmic shuttling of the protein. This suggests that E1B-55K SUMOylation in these species is regulating nuclear export. Furthermore, these results imply that high SUMOylation of E1B-55Ks from different HAdV species could block nuclear export and enhance nuclear retention, similar to what we observed with the HAdV-C5 K101R-mutant, our prototype mutant for high SUMOylation. It remains elusive whether E1B-55K from other HAdV species are able to actively shuttle between the nucleus and cytoplasm. Yet, our findings indicate that the mechanism is different from HAdV-C5. These remaining questions need to be addressed in follow-up studies with compounds targeting nuclear transport, ideally in the context of HAdV infection.
SUMOylation can have multiple effects on a protein and has been shown to alter protein-protein interactions, protein stability, localization, or activation (3). In viruses, SUMOylation can additionally serve as a switch between a latent and a lytic infection. The Kaposi’s sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is highly SUMOylated and by creating a SUMO-rich milieu required for chromatin condensation, the protein represses the expression of lytic KSHV genes (45–47). Another example is EBV BZLF1, which also is a target of the SUMO conjugation machinery (39). SUMOylation and deSUMOylation of BZLF1 are responsible for the onset of latency or the reactivation of the virus, respectively (48). Notably, both herpesviral proteins are functional homologs of C5 E1B-55K and they all are involved in the repression of gene transcription and inhibition of p53 (47, 49). We revealed that the lysine residue K101 is specific for HAdV-C and that SUMOylation of E1B-55K from this species is highly affected by K101 (Fig. 1 and 2). Moreover, wt HAdV-C5 E1B-55K is less SUMOylated compared to E1B-55Ks from most HAdV species (Fig. 2).
From previous studies, we already know that not all E1B-55K functions are conserved throughout the HAdV species. Although they all form an E3-ubiquitin-ligase complex with E4orf6, they do not degrade the same cellular proteins (22, 23, 50). Importantly, p53 and Mre11 are only degraded by HAdV-A12, C5 and F40, but not by all HAdV species (20, 46). Moreover, E1B-55K accumulation in aggresomes near the nucleus, which appears to be inhomogeneous in terms of distribution and morphology (21, 23, 25, 51), is not conserved among HAdV species (24). Here, we show that SUMOylation of E1B-55K is another mechanism that is highly conserved among the different HAdV species. Still, the regulatory role of SUMOylation that we know from HAdV-C5 E1B-55K is not entirely conserved. For instance, HAdV-B34 E1B-55Ks evolved a SUMO-independent mechanism to repress p53-mediated transcription (Fig. 6). Why SUMOylation seems to regulate functions of E1B-55K from some but not all HAdV species could be due to differential phosphorylation (26) and is an interesting subject for further studies. Likewise, it is critical to verify the observed effects of SUMOylation of different E1B-55K proteins in the context of infection. That would allow to control for the impact of E1A and especially the E1A or E1B-55K binding to the SUMO-conjugating enzyme UBC9 (26, 52) as well as other viral proteins on the SUMOylation pathway. The use of viruses and virus mutants in infection along with thorough investigations of nuclear import and export pathways that are involved in the SUMO-dependent shuttling will be addressed in follow-up studies. That also applies for studies on the effect of E1B-55K SUMOylation on p53-mediated transcription to confirm the reporter assay findings.
In conclusion, we provide further evidence that the E1B-55K proteins from different HAdV species have conserved PTMs such as SUMOylation; however, E1B-55K SUMOylation in the different HAdV species might have different cellular consequences during infection. While HAdV-C5 still serves as a great molecular model, it is essential to continue investigating the functions of other E1B-55Ks in order to understand tissue tropism and pathogenesis of the different HAdV species.
MATERIALS AND METHODS
Cells.
H1299 cells (ATCC CRL-5803) and HeLa cells (ATCC CCL-2) stably expressing 6×His-SUMO-1 or 6×His-SUMO-2 (53) were grown in monolayer cultures in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, PAN-Biotech), 100 U penicillin and 100 μg streptomycin (PAN-Biotech) per ml in a 5% CO2 atmosphere at 37°C. HeLa cells expressing 6×His-SUMO were kept under 2 μM puromycin selection.
Plasmids and transient transfections.
The hemagglutinin (HA)-tagged E1B-55K proteins of the different HAdV species used in this study were expressed from pcDNA-3 as described previously (22). Mutations in the (putative) SUMO-conjugation motif (SCM) of the respective E1B-55K types (29, 30) were generated by site directed mutagenesis using primers shown in Table 1. For transient transfection, subconfluent cells were treated with a mixture of DNA and 25 kDa linear polyethylenimine (PEI; Polysciences Inc., Warrington, PA, USA) as described before (10).
TABLE 1
| Modification and directiona | Sequence (5′ → 3′) |
|---|---|
| HAdV-C5 K104R fwd | 5′-GGGCTAAAGGGGGTAAGGAGGGAGCGGGGG-3′ |
| HAdV-C5 K104R rev | 5′-CCCCCGCTCCCTCCTTACCCCCTTTAGCCC-3′ |
| HAdV-C5 K101R fwd | 5′-GGGCAGGGGCTAAGGGGGGTAAAGAGGG-3′ |
| HAdV-C5 K101R rev | 5′-CCCTCTTTACCCCCCTTAGCCCCTGCCC-3′ |
| HAdV-E4 K93R fwd | 5′-CGAGTGGTCGGGAGAGGGGTATTAGGCGGGAGAGGC-3′ |
| HAdV-E4 K93R rev | 5′-GCCTCTCCCGCCTAATACCCCTCTCCCGACCACTCG-3′ |
| HAdV-D9 K103R fwd | 5′-CAGGGGAGTTAGGAGGGAGAG-3′ |
| HAdV-D9 K103R rev | 5′-CTCTCCCTCCTAACTCCCCTG-3′ |
| HAdV-D9 K157R fwd | 5′-GATAAATATGGCCTGGAGCAGATAAGAACCCATTGGTTGAACCCAG-3′ |
| HAdV-D9 K157R rev | 5′-CTGGGTTCAACCAATGGGTTCTTATCTGCTCCAGGCCATATTTATC-3′ |
| HAdV-D9 K302R fwd | 5′-CCAAGAGCGAGATGTCTGTGAGGCAGTGTGTGTTTGAGAAATGC-3′ |
| HAdV-D9 K302R rev | 5′-GCATTTCTCAAACACACACTGCCTCACAGACATCTCGCTCTTGG-3′ |
| HAdV-A12 K88R fwd | 5′-GCGCAGATGATAGAGATAGGCAGG-3′ |
| HAdV-A12 K88R rev | 5′-CCTGCCTATCTCTATCATCTGCGC-3′ |
| HAdV-A12 K91R fwd | 5′-GATAGAGATAAGCAGGAAAGAAAAGAAAGTTTAAAGG-3′ |
| HAdV-A12 K91R rev | 5′-CCTTTAAACTTTCTTTTCTTTCCTGCTTATCTCTATC-3′ |
| HAdV-A12 K92R fwd | 5′-GATAGAGATAAGCAGGAAAAAAGAGAAAGTTTAAAGG-3′ |
| HAdV-A12 K92R rev | 5′-CCTTTAAACTTTCTCTTTTTTCCTGCTTATCTCTATC-3′ |
| HAdV-B16 K101R fwd | 5′-CAGGACAGGGGCATTCGGAGGGAAAGGAATCC-3′ |
| HAdV-B16 K101R rev | 5′-GGATTCCTTTCCCTCCGAATGCCCCTGTCCTG-3′ |
| HAdV-B34 K103R fwd | 5′-GATAGGGGCGTTCGGAGGGAGAGGGC-3′ |
| HAdV-B34 K103R rev | 5′-GCCCTCTCCCTCCGAACGCCCCTATC-3′ |
| HAdV-F40 K90R fwd | 5′-CAAAGGGGGACAAGGAGAAAGATGG-3′ |
| HAdV-F40 K90R rev | 5′-CCATCTTTCTCCTTGTCCCCCTTTG-3′ |
| HAdV-F40 K92R fwd | 5′-GGACAAAGAGAAGGATGGAAAACGAGG-3′ |
| HAdV-F40 K92R rev | 5′-CCTCGTTTTCCATCCTTCTCTTTGTCC-3′ |
| HAdV-F40 K127R fwd | 5′-GATTTGGAAGATGAGTTTAGAAACGGTGAAATGAATTTG-3′ |
| HAdV-F40 K127R rev | 5′-CAAATTCATTTCACCGTTTCTAAACTCATCTTCCAAATC-3′ |
| HAdV-F40 K430R fwd | 5′-GGTGAGGTATGATGAGTCAAGGGTTCGTTGTCGCCCCTGTGAG-3′ |
| HAdV-F40 K430R rev | 5′-CTCACAGGGGCGACAACGAACCCTTGACTCATCATACCTCACC-3′ |
a
fwd = forward primer; rev = reverse primer.
Reporter gene assays.
For dual-luciferase assays, subconfluent cells were transfected as described above with 0.5 μg pRL-TK renilla under the control of the HSV-TK (herpes simplex virus thymidine kinase) promoter (Promega), 0.5 μg pRE Luc p53 promoter, 0.0015 μg p53 and 0.5 μg of the different E1B-constructs used in this study. Cells were lysed 24 h posttransfection and samples were measured and normalized using the Dual-Glo luciferase assay system (Promega) as described previously (54).
Antibodies and protein analysis.
Primary antibodies against cellular and ectopically expressed proteins included 6×His mouse MAb (631212; TaKaRa Bio Europe SAS), HA rat MAb 3F10, p53 rabbit pAb FL-393 (sc-6243; Santa Cruz), and β-Actin mouse MAb AC-15 (A5441; Merck).
For protein extracts, cells were resuspended in RIPA lysis buffer (50 mM Tris-HCl/pH 8.0, 150 mM NaCl, 5 mM EDTA/pH 8.0, 0.1% SDS, 1% NP-40, 0.5% sodiumdeoxycholate) freshly supplemented with 1% phenylmethylsulfonyl fluoride, 0.1% aprotinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 25 mM iodacetamide, and 25 mM N-ethylmaleimide. Proteins were boiled 5 min at 95°C in 5× Laemmli. For immunoblotting, equal amounts of protein lysate were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, incubated, and visualized as described previously (54).
Denaturing, purification, and analysis of SUMO-conjugates.
HeLa cells were transfected with the different HA-tagged E1B-55K plasmids. 48 h posttransfection, cells were lysed in 5 ml GuHCl-buffer (6 M guanidinium-HCl, 0.1 M Na2HPO4, 0.1 M NaH2PO4, 10 mM Tris-HCl/pH 8.0, 20 mM imidazole, 5 mM β-mercaptoethanol). 10% of the cells were lysed with RIPA lysis buffer for total protein analysis as described above. The remaining lysates were incubated overnight at 4°C with 50 μl Ni-nitrilotriacetic acid (Ni-NTA) beads prewashed with GuHCl-buffer. Next, beads were washed once with buffer A (8 M urea, 0.1 M Na2HPO4, 0.1 M NaH2PO4, 10 mM Tris-HCl/pH 8.0, 20 mM imidazole, 5 mM β-mercaptoethanol) and buffer B (8 M urea, 0.1 M Na2HPO4, 0.1 M NaH2PO4, 10 mM Tris-HCl/pH 6.3, 20 mM imidazole, 5 mM β-mercaptoethanol). Afterwards, proteins were eluted and separated by SDS-PAGE as described above.
Indirect immunofluorescence assay.
Cells were grown on glass-coverslips and transfected with the indicated expression vectors. Cells were then fixed with 4% paraformaldehyde (PFA) for 20 min at 4°C, permeabilized in PBS with 0.5% Triton X-100 for 10 min at room temperature (RT) and blocked in Tris-buffered saline-BG (TBS-BG; 5% wt/vol BSA and 5% wt/vol glycine) for 30 min at RT. Coverslips were afterwards incubated for 1 h at RT with the primary antibody diluted in PBS, then washed 3× with TBS-BG. Secondary antibodies were diluted in PBS and coverslips were incubated for 30 min at RT (Table 2). Coverslips were finally washed with TBS-BG and PBS and mounted in Glow medium. Images were obtained using a confocal spinning disk microscope (Nikon Eclipse Ti-E stand; Yokagawa CSU-W1 spinning disk; 2× Andor888 EM-CCD camera; Nikon 100× NA 1.49 objective) and an inverted fluorescent light microscope (Leica DMI 6000B). Images were analyzed with Fiji/ImageJ (NIH).
TABLE 2
| Name | Property | Source |
|---|---|---|
| 3F10 | Monoclonal rat α-HA-epitope antibody | Roche |
| SUMO-2/-3 | Monoclonal mouse α-SUMO-2/-3 antibody | MoBiTec |
| Cy3 α-rat IgG | Polyclonal Alexa 488-conjugated goat α-mouse IgG antibody | Invitrogen |
| Alexa Fluor 488 α-mouse IgG | Polyclonal Cy3-conjugated goat α-rat IgG antibody | Dianova |
Colocalization between HA-E1B-55K and SUMO-2 was quantified with a custom written Matlab app. The app allows to select single cells for which the Manders fractional overlap coefficients M1 and M2 (55) are calculated as measure of overlap between E1B-55K-foci (green signal) and SUMO-2 (red signal).
The ratio of the nuclear mean fluorescence to the cytoplasmic mean fluorescence was obtained in a three-step process: first, the nuclei were segmented using the U-Net based StarDist convolutional neural network (56, 57), more specifically the pretrained “versatile fluorescent nuclei” network. Here, the cytoplasmic area was defined by expanding the area of the nuclei and subtracting the original nuclear area – resulting in a small ring-shaped cytoplasmic area around the nuclei. Second, the transfected nuclei (and the corresponding cytoplasmic areas) were determined by thresholding in the red (HA-E1B-55K) channel. Third, the mean nuclear and cytoplasmic fluorescence intensities were measured and their ratios compared.
Statistical analyses.
Statistical analyses were performed using Graph-Pad Prism v9 (GraphPad Software Inc.). Mann-Whitney tests and one-way analysis of variance (ANOVA) with Tukey post hoc tests for multiple comparisons were used to compare E1B-55K intracellular localization, E1B-55K colocalization with SUMO and p53-transactivation. Data were considered significant if P ≤ 0.05.
ACKNOWLEDGMENTS
We thank Michael Tatham and Ron Hay at the University of Dundee for valuable input to this work.
The Leibniz Institute for Experimental Virology (HPI) is supported by the Freie und Hansestadt Hamburg and the Bundesministerium für Gesundheit.
The authors have no conflicts of interest to declare.
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Information & Contributors
Information
Published In
Journal of Virology
Volume 96 • Number 3 • 9 February 2022
eLocator: e00838-21
Editor: Lawrence Banks, International Centre for Genetic Engineering and Biotechnology
PubMed: 34787461
Copyright
© 2022 American Society for Microbiology. All Rights Reserved.
History
Received: 9 June 2021
Accepted: 9 November 2021
Accepted manuscript posted online: 17 November 2021
Published online: 9 February 2022
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Lawrence Banks
Editor
International Centre for Genetic Engineering and Biotechnology
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2022.
Conserved E1B-55K SUMOylation in Different Human Adenovirus Species Is a Potent Regulator of Intracellular Localization. J Virol
96:e00838-21.
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