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.

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.

Leadership education in professional and graduate schools.

This article makes a case for the need of graduate leadership curricula, including professional programs such as health care, law, and engineering. Diversity, equity, and inclusion is focused on as a critical piece of this leadership curricula.

[Adaptation and validation in Chile of the patient identification instrument needing palliative care: NECPAL-CCOMS-ICO 3.1©]. / Adaptación y validación en Chile del instrumento de identificación de pacientes con necesidad de atención paliativa: NECPAL-CCOMS-ICO 3.1©.

OBJECTIVE: Adapt and validate the NECPAL instrument in Chile. DESIGN: Prospective, longitudinal, analytical study for validation of the instrument in 5 stages: cultural adaptation, content validation, pilot test, application, and statistical analysis. PLACE: Four primary care centers of the South East Metropolitan Health Service, in Santiago, Chile. PARTICIPANTS: Primary health care physicians and nurses for cultural adaptation and application, and palliative care experts for content validation. MAIN MEASUREMENTS: Cultural adaptation was carried out through cognitive interviews. Content validity was measured using Delphi method and the Lawshe content validity ratio (CVR) was obtained. In the pilot test, we measured stability (test-retest), inter judge harmony and application time in 14 chronic advanced patients (CAP). The test was applied to this same group, calculating the sample according to Nunally's recommendation. RESULTS: A sample of 118 CAP was obtained. The CVR was 0.75 and the average testing time was 6.7 min (SD = 4.01). The test-retest obtained a Kappa test concordance index between 0.632 and 1.0; and the interjudge harmony agreement between 0.192 and 0.692. The surprise question (PS) was positive in 20.3% of the sample. The main conditions associated with the disease-specific severity item, were fragility (23.7%), chronic heart disease (21.2%) and chronic lung disease (12.7%). The demand group and specific severity indicators obtained a greater predictive capacity of PS+, with an area under the curve of 0.808 (95% CI: 0.697-0.918). CONCLUSIONS: NECPAL is feasible to be used in Chile, has adequate psychometric properties and will allow early detection of patients in need of palliative care.

E1B-55K-Mediated Regulation of RNF4 SUMO-Targeted Ubiquitin Ligase Promotes Human Adenovirus Gene Expression.

Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4- and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention.

Human Adenovirus Core Protein V Is Targeted by the Host SUMOylation Machinery To Limit Essential Viral Functions.

Human adenoviruses (HAdV) are nonenveloped viruses containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear pore complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood. The core protein V is speculated to bridge the core and the surrounding capsid. It binds the genome in a sequence-independent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus, protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultaneously, the altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than that observed during wild-type infection. Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies.IMPORTANCE Many decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this concept needs expansion as the functions are more diverse. We showed that capsid protein VI regulates the antiviral response by modulation of the transcription factor Daxx during infection. Moreover, core protein VII interacts with SPOC1 restriction factor, which is beneficial for efficient viral gene expression. Here, we were able to show that core protein V also represents a novel substrate of the host SUMOylation machinery and contains several conserved SCMs; mutation of these consensus motifs reduced SUMOylation of the protein. Unexpectedly, we observed that introducing these mutations into HAdV promotes adenoviral replication. In conclusion, we offer novel insights into adenovirus core proteins and provide evidence that SUMOylation of HAdV factors regulates replication efficiency.

Does emotional disclosure about stress improve health in rheumatoid arthritis? Randomized, controlled trials of written and spoken disclosure.

Studies of the effects of disclosing stressful experiences among patients with rheumatoid arthritis (RA) have yielded inconsistent findings, perhaps due to different disclosure methods--writing or speaking--and various methodological limitations. We randomized adults with RA to a writing (n=88) or speaking (to a recorder) sample (n=93), and within each sample, to either disclosure or 1 of 2 control groups (positive or neutral events), which conducted four 20-minute, at-home sessions. Follow-up evaluations at 1, 3, and 6 months included self-reported, behavioral, physiological, and blinded physician-assessed outcomes. In both writing and speaking samples, the disclosure and control groups were comparably credible, and the linguistic content differed as expected. Covariance analyses at each follow-up point indicated that written disclosure had minimal effects compared with combined controls--only pain was reduced at 1 and 6 months, but no other outcomes improved. Spoken disclosure led to faster walking speed at 3 months, and reduced pain, swollen joints, and physician-rated disease activity at 6 months, but there were no effects on other outcomes. Latent growth curve modeling examined differences in the trajectory of change over follow-up. Written disclosure improved affective pain and walking speed; spoken disclosure showed only a marginal benefit on sensory pain. In both analyses, the few benefits of disclosure occurred relative to both positive and neutral control groups. We conclude that both written and spoken disclosure have modest benefits for patients with RA, particularly at 6 months, but these effects are limited in scope and consistency.

Alexithymia and pain in three chronic pain samples: comparing Caucasians and African Americans.

OBJECTIVE: African Americans often report greater pain than do Caucasians, but the factors responsible for this discrepancy are not known. We examined whether alexithymia-the trait of difficulty identifying and describing one's feelings and lacking introspection-may contribute to this ethnic group difference. We tested whether the mean level of alexithymia is higher, and whether alexithymia and pain are more highly correlated, among African Americans than among Caucasians in patients with chronic pain disorders. DESIGN: Three cross-sectional, correlational studies were conducted on three separate samples of patients with chronic pain. Analyses examined the full sample and then Caucasians and African Americans separately. SETTING AND PATIENTS: Patients were recruited primarily from treatment settings. Samples were patients with rheumatoid arthritis (N = 155), migraine headaches (N = 160), or systemic lupus erythematosus (N = 123), and each sample included only Caucasians or African Americans. MEASURES: The Toronto Alexithymia Scale-20 assessed global alexithymia and three alexithymia facets. Pain severity, functional disability, or symptoms were also measured on each sample. RESULTS: Similar findings occurred across all three samples. African Americans had only slightly higher mean alexithymia levels than did Caucasians, and this was partly accounted for by socioeconomic differences between groups. More importantly, alexithymia correlated only weakly with pain or symptom severity for each full sample, but the two ethnic groups showed different patterns. Alexithymia correlated positively with pain severity among African Americans, but was uncorrelated with pain among Caucasians, even after covarying for various socioeconomic variables. CONCLUSIONS: Alexithymia is more correlated with pain severity among African Americans with chronic pain disorders than among Caucasians, potentially contributing to the higher pain reports among African Americans.

Rapid identification of iron deficiency in blood donors with red cell indexes provided by Advia 120.

BACKGROUND: A new generation of automated hematology analyzers allows the rapid determination of various red cell (RBC) indexes, including the percentage of hypochromic mature RBCs (HYPOm) and the hemoglobin (Hb) content of reticulocytes (CHr). These indexes have not yet been validated as measures for the detection of iron deficiency in blood donors. STUDY DESIGN AND METHODS: Iron status was evaluated in a total of 1142 unselected prospective blood donors based on measurement of serum ferritin, soluble transferrin receptor, and Hb compared to RBC indexes provided by an automated hematology analyzer (Advia 120, Bayer HealthCare) including HYPOm and CHr. RESULTS: Assuming that the most precise measure for body iron storage is related to the logarithm of the ratio of soluble transferrin receptor to ferritin, the sensitivity of ferritin for the diagnosis of iron depletion was 89 percent compared to 57 percent for HYPOm and CHr, respectively, to 69 percent for the combination of both RBC indexes, and to 26 percent for Hb concentration. CONCLUSION: The RBC indexes HYPOm und CHr are significantly better screening measures for identification of iron depletion in blood donors than Hb.