Search

The E2 protein encoded by human papillomaviruses (HPV) is a sequence-specific DNA-binding protein that recruits viral and cellular proteins. Bromodomain-containing protein 4 (BRD4) is a highly conserved interactor for E2 proteins that has been linked to E2’s functions as transcription modulator, activator of viral replication, and segregation factor for viral genomes. In addition to BRD4, a short form of BRD4 (BRD4S) is expressed from the BRD4 gene, which lacks the C-terminal domain of BRD4. E2 proteins interact with the C-terminal motif (CTM) of BRD4, but a recent study suggested that the phospho-dependent interaction domain (PDID) and the basic interaction domain (BID) in BRD4 also bind to E2. These domains are also present in BRD4S. We now find that HPV31 E2 interacts with the isolated PDID domain in living cells and also with BRD4S, which is present in detectable amounts in HPV-positive cell lines and is recruited into HPV31 E1- and E2-induced replication foci. Overexpression and knockdown experiments surprisingly indicate that BRD4S inhibits activities of E2. In line with that, the specific knockdown of BRD4S in the HPV31-positive CIN612-9E cell line induces mainly late viral transcripts. This occurs only in undifferentiated but not differentiated cells in which the productive viral replication cycle is induced. These data suggest that the BRD4S-E2 interaction is important to prevent HPV late gene expression in undifferentiated keratinocytes, which may contribute to immune evasion and HPV persistence. IMPORTANCE Human papillomaviruses (HPV) have coevolved with their host by using cellular factors like bromodomain-containing protein 4 (BRD4) to control viral processes, such as genome maintenance, gene expression, and replication. Here, we show that, in addition to the C-terminal motif in BRD4, the phospho-dependent interaction domain in BRD4 interacts with E2 proteins, which enable the recruitment of BRD4S, the short isoform of BRD4, to E2. Knockdown and overexpression of BRD4S reveal that BRD4S is a negative regulator of E2 activities. Importantly, the knockdown of BRD4S induces mainly L1 transcripts in undifferentiated CIN612-9E cells, which maintain replicating HPV31 genomes. Our study reveals an inhibitory role of BRD4S on HPV transcription, which may serve as an immune escape mechanism by the suppression of L1 transcripts and thus contribute to the establishment of persistent HPV infections.

Interleukin-21: A Potential Biomarker For Diagnosis and Predicting Prognosis in COVID-19 Patients Abstract Introduction COVID-19 patients have a wide spectrum of disease severity. Several biomarkers were evaluated as predictors for progression towards severe disease. IL-21 is a member of common ?-chain cytokine family and creates some specific effects during programming and maintenance of antiviral immunity. We aimed to assess IL-21 as a biomarker for diagnosis and outcome prediction in patients hospitalized with COVID-19. Method Patients with a preliminary diagnosis of COVID-19 and pneumonia other than COVID-19 admitted to a tertiary care hospital were included consecutively in this comparative study. Results The study population consisted of 51 patients with COVID-19 and 11 patients with non-COVID-19 pneumonia. Serum IL-21 concentration was markedly higher and serum CRP concentration was significantly lower in COVID-19 patients compared to non-COVID-19 pneumonia patients. Within COVID-19 patients 10 patients showed radiological and clinical progression. Patients with clinical worsening had lower lymphocyte count and haemoglobin. In addition to that deteriorating patients had higher urea, LDH levels and elevated concentration of both IL-6 and IL-21. The cut-off value of 106 ng/L for IL-21 has 80.0% sensitivity, %60.9 specificity for discriminating patients with clinical worsening. Multivariable analysis performed to define risk factors for disease progression identified IL-6 and IL-21 as independent predictors. Odds ratio for serum IL-6 concentrations ? 3.2 pg/mL was 8.07 (95% CI: 1.37-47.50, p=0.04) and odds ratio for serum IL-21 concentrations ? 106 ng/L was 6.24 (95% CI: 1.04 ? 37.3, p=0.02). Conclusion We identified specific differences in serum IL-21 between COVID-19 and non-COVID-19 pneumonia patients. Serum IL-21 measurement has promising predictive value for disease progression in COVID-19 patients. High serum IL-6 and IL-21 levels obtained upon admission are independent risk factors for clinical worsening.

Humane Papillomviren (HPV) sind die ätiologische Ursache sowohl für gut- als auch bösartige Erkrankungen der Haut und Schleimhaut. Dabei ist der Lebenszyklus von HPV insbesondere von zellulären Faktoren der infizierten Zelle abhängig. Ein wichtiger Faktor ist das Bromodomain-containing Protein 4 (Brd4), der mit den E2 Proteinen von Papillomviren (PV) interagiert, um den Erhalt des viralen Genoms, als auch die virale Transkription und Replikation zu gewährleisten. In der vorliegenden Arbeit konnte gezeigt werden, dass es neben der konservierten Interaktionsstelle, welche die Transaktivierungsdomäne von E2 und den C-Terminus von Brd4 umfasst, eine von Wu et al. (2016) postulierte zweite Interaktionsstelle in Brd4 in vivo gibt. Diese wird als phosphorylation dependent interaction domain, kurz PDID, bezeichnet. Weiterführend konnte gezeigt werden, dass das HPV31 E2 Protein neben der langen Isoform von Brd4 auch die kurze Isoform Brd4S über die PDID binden kann. Dabei deutete die Überexpression als auch der Knockdown von Brd4S, im Zusammenhang E2-abhängiger Aktivitäten, auf eine repressive Funktion von Brd4S hin. Darüber hinaus wiesen undifferenzierte CIN612-9E-Zellen, in welchen das HPV31 Genom episomal vorliegt, im Zusammenhang mit reduzierten Brd4S-Mengen eine erhöhte L1-Transkriptmenge auf. Im Gegensatz dazu konnte in differenzierten CIN612-9E-Zellen gezeigt werden, dass eine verminderte Brd4S-Menge keine Veränderung der viralen Transkriptmengen bewirkt. Diese Ergebnisse könnten darauf hindeuten, dass Brd4S in undifferenzierten Zellen inhibierend auf die Expression des immunogenen L1 Proteins wirkt. Dies könnte HPV dazu verhelfen eine mögliche Erkennung durch das Immunsystem zu umgehen, um auf diese Weise eine persistente Infektion zu etablieren.

Orf virus (ORFV) represents a suitable vector for the generation of efficient, prophylactic antiviral vaccines against different pathogens. The present study investigated for the first time the therapeutic application of ORFV vector-based vaccines against tumors induced by cottontail rabbit papillomavirus (CRPV). ORFV-CRPV recombinants were constructed expressing the early CRPV gene E1, E2, E7, or LE6. In two independent experiments we used in total 23 rabbits which were immunized with a mixture of the four ORFV-CRPV recombinants or empty ORFV vector as a control 5 weeks after the appearance of skin tumors. For the determination of the therapeutic efficacy, the subsequent growth of the tumors was recorded. In the first experiment, we could demonstrate that three immunizations of rabbits with high tumor burden with the combined four ORFV-CRPV recombinants resulted in significant growth retardation of the tumors compared to the control. A second experiment was performed to test the therapeutic effect of 5 doses of the combined vaccine in rabbits with a lower tumor burden than in nonimmunized rabbits. Tumor growth was significantly reduced after immunization, and one vaccinated rabbit even displayed complete tumor regression until the end of the observation period at 26 weeks. Results of delayed-type hypersensitivity (DTH) skin tests suggest the induction of a cellular immune response mediated by the ORFV-CRPV vaccine. The data presented show for the first time a therapeutic potential of the ORFV vector platform and encourage further studies for the development of a therapeutic vaccine against virus-induced tumors. IMPORTANCE Viral vectors are widely used for the development of therapeutic vaccines for the treatment of tumors. In our study we have used Orf virus (ORFV) strain D1701-V for the generation of recombinant vaccines expressing cottontail rabbit papillomavirus (CRPV) early proteins E1, E2, LE6, and E7. The therapeutic efficacy of the ORFV-CRPV vaccines was evaluated in two independent experiments using the outbred CRPV rabbit model. In both experiments the immunization achieved significant suppression of tumor growth. In total, 84.6% of all outbred animals benefited from the ORFV-CRPV vaccination, showing reduction in tumor size and significant tumor growth inhibition, including one animal with complete tumor regression without recurrence.

The papillomavirus (PV) E2 protein is a nuclear, sequence-specific DNA-binding protein that regulates transcription and nuclear retention of viral genomes. E2 also interacts with the viral E1 protein to replicate the viral genome. E2 residue K111 is highly conserved among PV and has been implicated in contributing to nuclear transport, transcription, and replication. Cottontail rabbit (Sylvilagus floridanus) PV (CRPV or SfPV1) E2 K111R, A, or Q mutations are transcription deficient and localized to the cytoplasm, comparable to other PV types. The addition of a nuclear localization signal (NLS) resulted in nuclear E2 K111 mutant proteins but did not restore transcriptional activation, and this is most likely due to an impaired binding to the cellular Brd4 protein. Surprisingly, coexpression of E1 with E2 K111 mutations resulted in their nuclear localization and, for K111A and R mutations, the activation of an E1/E2-dependent reporter construct. Interestingly, the nuclear localization of E2 K111Q mutant protein was independent from the presence of the conserved bipartite NLS in E1 and the direct interaction between E1 and E2. On the other hand, the cytoplasmic E1 NLS mutation could be targeted to the nucleus by wild-type E2, and this was dependent upon an interaction between E1 and E2. In summary, our studies have uncovered that E1 and E2 control each other's subcellular localization: direct binding of E2 to E1 can direct E1 to the nucleus independently from the E1 NLS, and E1 can direct E2 to the nucleus without an intact NLS or direct binding to E2. IMPORTANCE Papillomaviruses encode the DNA-binding E1 and E2 proteins, which form a complex and are essential for genome replication. Both proteins are targeted to the nucleus via nuclear localization signals. Our studies have uncovered that cytoplasmic mutant E1 or E2 proteins can be localized to the nucleus when E1 or E2 is also present. An interaction between E1 and E2 is necessary to target cytoplasmic E1 mutant proteins to the nucleus, but cytoplasmic E2 mutant proteins can be targeted to the nucleus without a direct interaction, which points to a novel function of E1.