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Substantial increases in the conjugation of the main human SUMO paralogs, SUMO1, SUMO2, and SUMO3, are observed upon exposure to different cellular stressors, and such increases are considered important to facilitate cell survival to stress. Despite their critical cellular role, little is known about how the levels of the SUMO modifiers are regulated in the cell, particularly as it relates to the changes observed upon stress. Here we characterize the contribution of alternative splicing towards regulating the expression of the main human SUMO paralogs under normalcy and three different stress conditions, heat-shock, cold-shock, and Influenza A Virus infection. Our data reveal that the normally spliced transcript variants are the predominant mature mRNAs produced from the SUMO genes and that the transcript coding for SUMO2 is by far the most abundant of all. We also provide evidence that alternatively spliced transcripts coding for protein isoforms of the prototypical SUMO proteins, which we refer to as the SUMO alphas, are also produced, and that their abundance and nuclear export are affected by stress in a stress- and cell-specific manner. Additionally, we provide evidence that the SUMO alphas are actively synthesized in the cell as their coding mRNAs are found associated with translating ribosomes. Finally, we provide evidence that the SUMO alphas are functionally different from their prototypical counterparts, with SUMO1α and SUMO2α being non-conjugatable to protein targets, SUMO3α being conjugatable but targeting a seemingly different subset of proteins from those targeted by SUMO3, and all three SUMO alphas displaying different cellular distributions from those of the prototypical SUMOs. Thus, alternative splicing appears to be an important contributor to the regulation of the expression of the SUMO proteins and the cellular functions of the SUMOylation system.

Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1's roles in viral replication.

We report here the genome sequences of six newly isolated bacteriophages infecting Arthrobacter sp. ATCC 21022. All six have myoviral morphologies and have double-stranded DNA genomes with circularly permuted ends. The six phages are closely related with average nucleotide identities of 73.4 to 93.0% across genomes lengths of 49,797 to 51,347 bp.

SUMOylation, the post-translational conjugation of the Small Ubiquitin-like MOdifier (SUMO) to a target protein, regulates a wide array of cellular processes and plays important roles for numerous viruses during infection. However, the relevance of the cellular SUMOylation system for influenza virus infection remains mostly unexplored. We previously reported that the non-structural protein of influenza A virus NS1 is a bona fide SUMO target. Here we determine that at least four additional influenza virus proteins, namely PB1, NP, M1, and NS2, are also authentic SUMO targets, and provide data supporting that PB1, NP, and M1 are SUMOylated during viral infection. The functional relevance of SUMOylation for these proteins is supported by the observation that, despite no apparent changes in the cellular levels of the E1 and E2 SUMO enzymes, influenza viral infection leads to a global increase in cellular SUMOylation. This increase, characterized by the appearance of two new SUMOylated proteins of ∼70kDa and ∼52kDa of molecular weight, is dependent upon viral replication and cannot be recreated by interferon stimulation alone. Altogether, these observations indicate that influenza A virus interacts extensively with the cellular SUMOylation system during infection and suggest that SUMOylation plays an important role during influenza virus infection, potentially contributing to the functional diversity exhibited by influenza viral proteins.

Regulation of BCR signalling strength is crucial for B-cell development and function. Bright is a B-cell-restricted factor that complexes with Bruton's tyrosine kinase (Btk) and its substrate, transcription initiation factor-I (TFII-I), to activate immunoglobulin heavy chain gene transcription in the nucleus. Here we show that a palmitoylated pool of Bright is diverted to lipid rafts of resting B cells where it associates with signalosome components. After BCR ligation, Bright transiently interacts with sumoylation enzymes, blocks calcium flux and phosphorylation of Btk and TFII-I and is then discharged from lipid rafts as a Sumo-I-modified form. The resulting lipid raft concentration of Bright contributes to the signalling threshold of B cells, as their sensitivity to BCR stimulation decreases as the levels of Bright increase. Bright regulates signalling independent of its role in IgH transcription, as shown by specific dominant-negative titration of rafts-specific forms. This study identifies a BCR tuning mechanism in lipid rafts that is regulated by differential post-translational modification of a transcription factor with implications for B-cell tolerance and autoimmunity.

Recent studies have demonstrated nuclear export by papillomavirus E1 proteins, but the requisite export sequence(s) for bovine papillomavirus (BPV) E1 were not defined. In this report we identify three functional nuclear export sequences (NES) present in BPV E1, with NES2 being the strongest in reporter assays. Nuclear localization of BPV1 E1 was modulated by over- or under-expression of CRM1, the major cellular exportin, and export was strongly reduced by the CRM1 inhibitor, Leptomycin B, indicating that E1 export occurs primarily through a CRM1-dependent process. Consistent with the in vivo functional results, E1 bound CRM1 in an in vitro pull-down assay. In addition, sumoylated E1 bound CRM1 more effectively than unmodified E1, suggesting that E1 export may be regulated by SUMO modification. Lastly, an E1 NES2 mutant accumulated in the nucleus to a greater extent than wild-type E1, yet was defective for viral origin replication in vivo. However, NES2 exhibited no intrinsic replication defect in an in vitro replication assay, implying that nucleocytoplasmic shuttling may be required to maintain E1 in a replication competent state.

The E1 protein, a papillomavirus (PV)-encoded origin-binding helicase essential for PV DNA replication, is post-translationally modified by sumoylation. As this modification is essential for the nuclear accumulation of the bovine PV E1 (BPV E1), factors modulating the sumoylation of E1 could ultimately alter the outcome of a papillomavirus infection. Therefore, we systematically tested the known sumoylation enhancing factors (E3 SUMO ligases), namely RanBP2 and PIAS family proteins, to determine their ability to bind to E1 and stimulate its sumoylation, using in vitro assays. We found that RanBP2 bound to BPV E1 but failed to bind to the E1 from a human PV (HPV11 E1), and lacked any sumoylation enhancing activity for both BPV E1 and HPV11 E1. In contrast, proteins of the PIAS family (except for PIASy) bound to both BPV E1 and HPV11 E1 and stimulated their sumoylation, with PIASxβ (Miz1) exerting the largest stimulatory effect. The structural integrity of the RING finger domain of the PIAS proteins was required for their E3 SUMO ligase activity on PV E1 sumoylation, but was dispensable for their PV E1 binding activity. Furthermore, the sumoylation enhancing activity exerted by the PIAS proteins on BPV E1 was more pronounced than on HPV11 E1, and appeared to favor SUMO1 versus SUMO2 as the SUMO modifier. Altogether, this study identifies PIAS proteins as possible modulators of PV E1 sumoylation during papillomavirus infections.

Post-translational modification by the conjugation of small ubiquitin-like modifiers is an essential mechanism to affect protein function. Currently, only a limited number of substrates are known for most of these modifiers, thus limiting our knowledge of their role and relevance for cellular physiology. Here, we report the development of a universal strategy for proteomic studies of ubiquitin-like modifiers. This strategy involves the development of stable transfected cell lines expressing a double-tagged modifier under the control of a tightly negatively regulated promoter, the induction of the expression and conjugation of the tagged modifier to cellular proteins, the tandem affinity purification of the pool of proteins covalently modified by the tagged-modifier, and the identification of the modified proteins by liquid chromatography and mass spectrometry. By applying this methodology to the proteomic analysis of SUMO-1 and SUMO-3 we determined that SUMO-1 and SUMO-3 are stable proteins exhibiting half-lives of over 20 h, demonstrated that sumoylation with both SUMO-1 and SUMO-3 is greatly stimulated by MG-132 and heat shock treatment, demonstrated the preferential usage of either SUMO-1 or SUMO-3 for some known SUMO substrates, and identified 122 putative SUMO substrates of which only 27 appeared to be modified by both SUMO-1 and SUMO-3. This limited overlapping in the subset of proteins modified by SUMO-1 and SUMO-3 supports that the SUMO paralogues are likely to be functionally distinct. Three of the novel putative SUMO substrates identified, namely the polypyrimidine tract-binding protein-associated splicing factor PSF, the structural microtubular component alpha-Tubulin, and the GTP-binding nuclear protein Ran, were confirmed as authentic SUMO substrates. The application of this universal strategy to the identification of the pool of cellular substrates modified by other ubiquitin-like modifiers will dramatically increase our knowledge of the biological role of the different ubiquitin-like conjugations systems in the cell.

Partial deletions within Autographa californica open reading frame 61 ( FP25K ) alter the expression and accumulation profile of several viral proteins and the transport of occlusion-derived virus (ODV)-E66 to intranuclear membranes during infection (S. C. Braunagel et al., J. Virol. 73:8559–8570, 1999). Here we show the effects of a full deletion and overexpression of FP25K on the transport and expression of two ODV envelope proteins, ODV-E66 (E66) and ODV-E25 (E25). Deletion and overexpression of FP25K substantially altered the levels of expression of E66 during infection. Compared with cells infected with wild-type (wt) virus, the levels of E66 were reduced fivefold in cells infected with a viral mutant lacking FP25K (ΔFP25K) and were slightly increased in cells infected with a viral mutant overexpressing FP25K (FP25K polh ). In contrast, no significant changes were observed in the levels of E25 among wt-, ΔFP25K-, and FP25K polh -infected cells. The changes observed in the levels of E66 among the different viral mutants were not accompanied by changes in either the time of synthesis, membrane association, protein turnover, or steady-state transcript abundance. Deletion of FP25K also substantially altered the transport and localization of E66 during infection. In cells infected with the ΔFP25K mutant virus, E66 accumulated in localized regions at the nuclear periphery and the outer nuclear membrane and did not traffic to intranuclear membranes. In contrast, in cells infected with the FP25K polh mutant virus E66 trafficked to intranuclear membranes. For comparison, E25 was normally transported to intranuclear membranes in both ΔFP25K- and FP25K polh -infected cells. Altogether these studies suggest that FP25K affects the synthesis of E66 at a posttranscriptional level, probably by altering the translation of E66; additionally, the block in transport of E66 at the nuclear envelope in ΔFP25K-infected cells suggests that the pathway of E66 trafficking to the inner nuclear membrane and intranuclear microvesicles is specifically regulated and must be influenced by factors that do not control the traffic of E25.

Previous reports indicate that mutations within the Autographa californica nucleopolyhedrosis virus FP25K gene (open reading frame 61) significantly reduce incorporation of enveloped nucleocapsids into viral occlusions. We report that FP25K is a nucleocapsid protein of both the budded virus (BV) and occluded virus (ODV), and we describe the effects of two FP25K mutations (480-1 [N-terminal truncation] and FP-βgal [C-terminal fusion]) on the expression and cellular localization of ODV-E66 and ODV-E25. Significantly decreased amounts of ODV-E66 are detected in cells infected with 480-1 or FP-βgal viral mutants, even though during FP-βgal infection, steady-state levels of ODV-E66 transcripts remain unchanged. While ODV-E66 is normally detected in intranuclear microvesicles and ODV envelopes by 24 h postinfection (p.i.), ODV-E66 remains cytosolic throughout infection in cells infected with 480-1 virus (up to 96 h p.i.), and its intranuclear localization is not detected until 96 h p.i. in cells infected with the FP-βgal mutant virus. The nuclear localization of ODV-E25 is not affected during infection by the FP-βgal mutant; however, its trafficking is significantly delayed during infection by the 480-1 mutant. Temporal Western blot analyses of cell lysates show that both 480-1 and FP-βgal mutant virus infections result in altered accumulation patterns of several structural proteins, including gp67, BV/ODV-E26, and the major capsid protein p39. In addition to BV/ODV-E26, ODV-E66 and gp67 may interact with FP25K, and ODV-E25 and p39 may also be components of a protein complex containing ODV-E66 and FP25K. Together, these data suggest that FP25K and its associated protein complex(es) may play an important role in the targeting and intracellular transport of viral proteins during infection.

Our pioneering studies on the interplay between the s mall u biquitin-like mo difier (SUMO) and influenza A virus identified the nonstructural protein NS1 as the first known SUMO target of influenza virus and one of the most abundantly SUMOylated influenza virus proteins. Here, we further characterize the role of SUMOylation for the A/Puerto Rico/8/1934 (PR8) NS1 protein, demonstrating that NS1 is SUMOylated not only by SUMO1 but also by SUMO2/3 and mapping the main SUMOylation sites in NS1 to residues K219 and K70. Furthermore, by using SUMOylatable and non-SUMOylatable forms of NS1 and an NS1-specific artificial SUMO ligase (ASL) that increases NS1 SUMOylation ∼4-fold, we demonstrate that SUMOylation does not affect the stability or cellular localization of PR8 NS1. However, NS1's ability to be SUMOylated appears to affect virus multiplication, as indicated by the delayed growth of a virus expressing the non-SUMOylatable form of NS1 in the interferon (IFN)-competent MDCK cell line. Remarkably, while a non-SUMOylatable form of NS1 exhibited a substantially diminished ability to neutralize IFN production, increasing NS1 SUMOylation beyond its normal levels also exerted a negative effect on its IFN-blocking function. This observation indicates the existence of an optimal level of NS1 SUMOylation that allows NS1 to achieve maximal activity and suggests that the limited amount of SUMOylation normally observed for most SUMO targets may correspond to an optimal level that maximizes the contribution of SUMOylation to protein function. Finally, protein cross-linking data suggest that SUMOylation may affect NS1 function by regulating the abundance of NS1 dimers and trimers in the cell.

X-linked inhibitor of apoptosis protein (XIAP) overexpression has been found to be associated with malignant cancer progression and aggression in individuals with many types of cancers. However, the molecular basis of XIAP in the regulation of cancer cell biological behavior remains largely unknown. In this study, we found that a deficiency of XIAP expression in human cancer cells by either knock-out or knockdown leads to a marked reduction in β-actin polymerization and cytoskeleton formation. Consistently, cell migration and invasion were also decreased in XIAP-deficient cells compared with parental wild-type cells. Subsequent studies demonstrated that the regulation of cell motility by XIAP depends on its interaction with the Rho GDP dissociation inhibitor (RhoGDI) via the XIAP RING domain. Furthermore, XIAP was found to negatively regulate RhoGDI SUMOylation, which might affect its activity in controlling cell motility. Collectively, our studies provide novel insights into the molecular mechanisms by which XIAP regulates cancer invasion and offer a further theoretical basis for setting XIAP as a potential prognostic marker and specific target for treatment of cancers with metastatic properties.

Lens epithelium-derived growth factor (LEDGF) proteins p75 and p52 are transcriptional coactivators that connect sequence-specific activators to the basal transcription machinery. We have found that these proteins are posttranslationally modified by SUMO (small ubiquitin-like modifier)-1 and SUMO-3. Three SUMOylation sites, K75, K250, and K254, were mapped on the shared N-terminal region of these molecules, while a fourth site, K364, was identified in the C-terminal part exclusive of LEDGF/p75. The N-terminal SUMO targets are located in evolutionarily conserved charge-rich regions that lack resemblance to the described consensus SUMOylation motif, whereas the C-terminal SUMO target is solvent exposed and situated in a typical consensus motif. SUMOylation did not affect the cellular localization of LEDGF proteins and was not necessary for their chromatin-binding ability, nor did it affect this activity. However, lysine to arginine mutations of the identified SUMO acceptor sites drastically inhibited LEDGF SUMOylation, extended the half-life of LEDGF/p75, and significantly increased its transcriptional activity on the heat shock protein 27 promoter, indicating a negative effect of SUMOylation on the transcriptional activity of LEDGF/p75. Considering that SUMOylation is known to negatively affect the transcriptional activity of all transcription factors known to transactivate heat shock protein 27 expression, these findings support the paradigm establishing SUMOylation as a global neutralizer of cellular processes upregulated upon cellular stress.

The cellular SUMOylation system affects the function of numerous viral proteins. Hence, the identification of novel viral targets for the Small Ubiquitin-like MOdifier (SUMO) is key to our understanding of virus-host interactions. The data obtained in this study demonstrate that the non-structural influenza A viral protein NS1A is an authentic SUMO target through the use of a dicistronic expression plasmid containing SUMO (the modifier) and Ubc9 (the SUMO-conjugating enzyme) separated by an Internal Ribosomal Entry Site (IRES). This dual expression plasmid produces a robust increase in cellular SUMOylation, therefore facilitating the characterization of cellular and viral SUMO targets. The identification of NS1A as a bona fide SUMO target suggests, for the first time, a role for SUMOylation during influenza virus infection.

Regulation of the sumoylation system at the level of gene expression has not yet been explored. To begin to define transcriptional regulatory features, the promoter region for the SUMO1 gene was cloned from human genomic DNA and characterized. Initially, a 532 base pair fragment upstream of and including the predicted SUMO1 transcription start site (TSS) was cloned and shown to possess promoter activity. Subsequent deletion analysis showed that a smaller fragment containing 158 bp upstream of the TSS region exhibited basal promoter activity in both human and rodent cell lines. Within this basal promoter fragment, there were predicted binding sites for numerous transcription factors, including the nude mouse gene product, Whn (FoxN1). Electrophoretic mobility shift assays showed that Whn could bind to an ACGC motif adjacent to the TSR, and in transfection studies Whn stimulated a 3-fold increase in transcription from this cloned promoter in keratinocytes (HaCaT cells). Mutation of the ACGC motif abrogated both Whn binding and transcriptional activation, indicating that the Whn effect is likely due to direct interaction with this promoter element. Consistent with these observations on the cloned promoter region, Whn also modestly stimulated transcription from the endogenous, genomic SUMO1 promoter in HaCaT cells, consistent with Whn potentially playing a regulatory role for SUMO1 transcription in keratinocytes.

Spodoptera frugiperda Sf9 cells were found to possess an active endogenous sumoylation system. However, the endogenous sumoylation machinery did not efficiently modify exogenous proteins expressed by infection with recombinant baculoviruses. To overcome this limitation, mammalian sumoylation components were introduced by co-infection with recombinant baculoviruses expressing individual protein components of the sumoylation pathway. Expression of mammalian Ubc9 plus SUMO (either SUMO1 or SUMO3) was necessary and sufficient for active sumoylation of co-infected test proteins. This system provides a simple and convenient means to produce sumoylated mammalian proteins in a eukaryotic environment. Large-scale cultures should provide quantities of sumoylated proteins sufficient for potential purification.

SUMO modification regulates the activity of numerous transcription factors that have a direct role in cell-cycle progression, apoptosis, cellular proliferation, and development, but its role in differentiation processes is less clear. Keratinocyte differentiation requires the coordinated activation of a series of transcription factors, and as several crucial keratinocyte transcription factors are known to be SUMO substrates, we investigated the role of sumoylation in keratinocyte differentiation. In a human keratinocyte cell line model (HaCaT cells), Ca2+-induced differentiation led to the transient and coordinated transcriptional activation of the genes encoding crucial sumoylation system components, including SAE1, SAE2, Ubc9, SENP1, Miz-1 (PIASxβ), SUMO2 and SUMO3. The increased gene expression resulted in higher levels of the respective proteins and changes in the pattern of sumoylated substrate proteins during the differentiation process. Similarly to the HaCaT results, stratified human foreskin keratinocytes showed an upregulation of Ubc9 in the suprabasal layers. Abrogation of sumoylation by Gam1 expression severely disrupted normal HaCaT differentiation, consistent with an important role for sumoylation in the proper progression of this biological process.

Sumoylation is a widespread posttranslational modification thought to affect primarily nuclear proteins, especially transcription factors for which sumoylation usually results in repression of their transactivational function. Recent proteomics studies have greatly expanded the cadre of known SUMO substrates, and an increasing number of cytoplasmic proteins have been identified as SUMO targets. However, very few of these cytosolic proteins have been evaluated for the functional consequences of sumoylation. Rajan et al . now demonstrate that the activity of an integral cytoplasmic membrane channel-forming protein, K2P1, is completely abrogated by sumoylation at a single lysine residue on the cytoplasmic tail. This is the first report of a plasma membrane protein as a SUMO substrate and explains the long-standing inability to demonstrate functionality of K2P1. Apparently, K2P1 is stoichiometrically sumoylated under most cellular conditions, so it is constitutively inactive until desumoylated. These observations raise several intriguing questions, including: How and where does K2P1 become sumoylated? Why, unlike most known substrates, is K2P1 so efficiently sumoylated? and, What are the signals and SUMO proteases that trigger K2P1 desumoylation? But most importantly, the report by Rajan et al . expands the functional roles attributed to sumoylation into the new arena of membrane protein functional regulation and suggests that similar mechanisms may regulate the function of other pore proteins.

Papillomaviruses are infectious agents for human and animal epithelial tissue, and nearly 100 distinct human types (HPVs) have been identified. When these viruses infect cutaneous or mucosal skin they can initially cause clinical warts or persistent infection with little or no visible manifestations. Warts, while usually benign, can be painful or cosmetically unacceptable and often require medical treatment. Furthermore, infection with certain specific HPV types, such as 16 or 18 (as well as several others), is the major risk factor for a woman's development of cervical cancer. In addition to cervical cancer, papillomaviruses have also been implicated in cancers of the skin and respiratory track though the evidence is not yet as conclusive. It is clear that prevention or elimination of papillomavirus infections would ultimately reduce the incidence of cervical cancer and possibly other epithelial cancers as well. Unfortunately, progress in vaccine development has been slow and no specific anti-papillomavirus agents are available. The rational development of effective anti-papillomaviral treatments will require a detailed understanding of how these viruses replicate and interact with the host cell, and much progress has been made in this area over the last 10 years. These viruses have small DNA genomes with limited coding capacity, and their complete array of viral protein products is known. This review will discuss the known functions of the viral proteins with a focus on strategies to interdict their biological activities as a possible means of specific therapy.

The occlusion derived form of baculovirus is specially adapted for primary infection of the host midgut epithelium. As such, the virion must contain the proteins essential for host range determination and initiation of infection. Because knowledge of virion composition is a prerequisite for functional investigation, this study used a combination of techniques to identify the proteins present within or associated with the occlusion-derived virus (ODV) virion. Thirty-one proteins, including proteins known to be essential for viral DNA replication, were identified with confidence. An additional 13 proteins were identified by using one of the three techniques. A comparison of gene conservation among the ODV proteins encoded in the 16 sequenced baculoviridae genomes is presented. With knowledge of the composition of ODV, it is now possible to target proteins and study their role(s) during primary infection.

orf101 is a late gene of Autographa californica nucleopolyhedrovirus (Ac M NPV). It encodes a protein of 42 kDa which is a component of the nucleocapsid of budded virus (BV) and occlusion-derived virus (ODV). To reflect this viral localization, the product of orf101 was named BV/ODV-C42 (C42). C42 is predominantly detected within the infected-cell nucleus: at 24 h postinfection (p.i.), it is coincident with the virogenic stroma, but by 72 h p.i., the stroma is minimally labeled while C42 is more uniformly located throughout the nucleus. Yeast two-hybrid screens indicate that C42 is capable of directly interacting with the viral proteins p78/83 ( 1629K ) and ODV-EC27 ( orf144 ). These interactions were confirmed using blue native gels and Western blot analyses. At 28 h p.i., C42 and p78/83 are detected in two complexes: one at approximately 180 kDa and a high-molecular-mass complex (500 to 600 kDa) which also contains EC27.