The HIV 5' Gag Region Displays a Specific Nucleotide Bias Regulating Viral Splicing and Infectivity.

Alternative splicing and the expression of intron-containing mRNAs is one hallmark of HIV gene expression. To facilitate the otherwise hampered nuclear export of non-fully processed mRNAs, HIV encodes the Rev protein, which recognizes its intronic response element and fuels the HIV RNAs into the CRM-1-dependent nuclear protein export pathway. Both alternative splicing and Rev-dependency are regulated by the primary HIV RNA sequence. Here, we show that these processes are extremely sensitive to sequence alterations in the 5'coding region of the HIV genomic RNA. Increasing the GC content by insertion of either GFP or silent mutations activates a cryptic splice donor site in gag, entirely deregulates the viral splicing pattern, and lowers infectivity. Interestingly, an adaptation of the inserted GFP sequence toward an HIV-like nucleotide bias reversed these phenotypes completely. Of note, the adaptation yielded completely different primary sequences although encoding the same amino acids. Thus, the phenotypes solely depend on the nucleotide composition of the two GFP versions. This is a strong indication of an HIV-specific mRNP code in the 5' gag region wherein the primary RNA sequence bias creates motifs for RNA-binding proteins and controls the fate of the HIV-RNA in terms of viral gene expression and infectivity.

Assessing guideline adherence in patients with non-variceal upper gastrointestinal bleeding receiving antiplatelet and anticoagulant therapy.

Background & aims: Non-variceal upper gastrointestinal bleeding (NVUGIB) occurs frequently and is associated with a significant morbidity and mortality, especially in patients receiving antiplatelet or anticoagulant therapy (APT and ACT, respectively). We aimed to evaluate adherence to guideline recommendations published by European Society of Gastrointestinal Endoscopy (ESGE).Methods: Retrospective analysis of patients with NVUGIB und prior exposition to APT or ACT at a single university hospital between 01 January 2016 and 31 December 2017.Results: 270 patients were identified (70.4% male, median age 72 years). 6/17 (35.3%) patients receiving APT for primary cardiovascular prophylaxis, 39/71 (54.9%) and 35 (49.3%) patients receiving APT for secondary cardiovascular prophylaxis (using strict and liberal definition, respectively) and 13/25 (52%) patients receiving dual antiplatelet therapy (DAPT) were not managed according to current recommendations. Management of ACT for secondary thromboembolic prophylaxis did not follow guideline recommendations in 59/93 (63.4%) and 34/93 (36.6%) patients (using strict and liberal definition, respectively). 23.7% of patients with NVUGIB were exposed to combined APT and ACT for whom no guideline recommendations exist. Mortality for any reason was twice as high in patients who were not managed according to guideline recommendations (18.8% vs. 8% using strict definition, 20.5% vs. 10.2% using liberal definition), which did not remain significant after adjusting for comorbidities, whereas cardiovascular events were observed at similar rates.Conclusion: A significant number of patients with NVUGIB receiving APT or ACT is not managed according to current ESGE guideline recommendations. Strategies to implement these guidelines into daily practice need to be developed.

Site specific target binding controls RNA cleavage efficiency by the Kaposi's sarcoma-associated herpesvirus endonuclease SOX.

A number of viruses remodel the cellular gene expression landscape by globally accelerating messenger RNA (mRNA) degradation. Unlike the mammalian basal mRNA decay enzymes, which largely target mRNA from the 5' and 3' end, viruses instead use endonucleases that cleave their targets internally. This is hypothesized to more rapidly inactivate mRNA while maintaining selective power, potentially though the use of a targeting motif(s). Yet, how mRNA endonuclease specificity is achieved in mammalian cells remains largely unresolved. Here, we reveal key features underlying the biochemical mechanism of target recognition and cleavage by the SOX endonuclease encoded by Kaposi's sarcoma-associated herpesvirus (KSHV). Using purified KSHV SOX protein, we reconstituted the cleavage reaction in vitro and reveal that SOX displays robust, sequence-specific RNA binding to residues proximal to the cleavage site, which must be presented in a particular structural context. The strength of SOX binding dictates cleavage efficiency, providing an explanation for the breadth of mRNA susceptibility observed in cells. Importantly, we establish that cleavage site specificity does not require additional cellular cofactors, as had been previously proposed. Thus, viral endonucleases may use a combination of RNA sequence and structure to capture a broad set of mRNA targets while still preserving selectivity.

Neuromagnetic correlates of voice pitch, vowel type, and speaker size in auditory cortex.

Vowel recognition is largely immune to differences in speaker size despite the waveform differences associated with variation in speaker size. This has led to the suggestion that voice pitch and mean formant frequency (MFF) are extracted early in the hierarchy of hearing/speech processing and used to normalize the internal representation of vowel sounds. This paper presents a magnetoencephalographic (MEG) experiment designed to locate and compare neuromagnetic activity associated with voice pitch, MFF and vowel type in human auditory cortex. Sequences of six sustained vowels were used to contrast changes in the three components of vowel perception, and MEG responses to the changes were recorded from 25 participants. A staged procedure was employed to fit the MEG data with a source model having one bilateral pair of dipoles for each component of vowel perception. This dipole model showed that the activity associated with the three perceptual changes was functionally separable; the pitch source was located in Heschl's gyrus (bilaterally), while the vowel-type and formant-frequency sources were located (bilaterally) just behind Heschl's gyrus in planum temporale. The results confirm that vowel normalization begins in auditory cortex at an early point in the hierarchy of speech processing.

The KSHV RNA regulator ORF57: target specificity and its role in the viral life cycle.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which enhances the expression of intron-less KSHV genes on multiple post-transcriptional levels mainly affecting RNA stability and export to the cytoplasm. Yet, it remains elusive how ORF57 recognizes viral RNAs and discriminates them from cellular messenger RNAs (mRNAs). Although one common binding motif on three separate KSHV RNAs has been described, most other lytic genes lack this sequence element. In this article we will review the sequence requirements for ORF57 to enhance RNA expression and discuss a model how ORF57 achieves specificity for viral RNAs. Finally, the role of ORF57 is integrated into the viral life cycle as a complex interplay with other viral and host factors and with implications for cellular gene expression.

ORF57 overcomes the detrimental sequence bias of Kaposi's sarcoma-associated herpesvirus lytic genes.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which enhances the expression of intronless KSHV genes on multiple posttranscriptional levels. However, it remains elusive how ORF57 recognizes viral RNAs. Here, we demonstrate that ORF57 also increases the expression of the multiple intron-containing K15 gene. The nucleotide bias of the K15 cDNA revealed an unusual high AT content. Thus, we optimized the K15 cDNA by raising the frequency of GC nucleotides, yielding an ORF57-independent version. To further prove the importance of the sequence bias of ORF57-dependent RNAs, we grouped KSHV mRNAs according to their AT content and found a correlation between AT-richness and ORF57 dependency. More importantly, latent genes, which have to be expressed in the absence of ORF57, have a low AT content and are indeed ORF57 independent. The nucleotide composition of K15 resembles that of HIV gag, which cannot be expressed unless RNA export is facilitated by the HIV Rev protein. Interestingly, ORF57 can partially rescue HIV Gag expression. Thus, the KSHV target RNAs of ORF57 and HIV gag RNA may share certain motifs based on the nucleotide bias. A bioinformatic comparison between wild-type and sequence-optimized K15 revealed a higher density for hnRNP-binding motifs in the former. We speculate that binding of particular hnRNPs to KSHV lytic transcripts is the prerequisite for ORF57 to enhance their expression. IMPORTANCE: The mostly intronless genes of KSHV are only expressed in the presence of the viral regulator protein ORF57, but how ORF57 recognizes viral RNAs remains elusive. We focused on the multiple intron-containing KSHV gene K15 and revealed that its expression is also increased by ORF57. Moreover, sequences in the K15 cDNA mediate this enhancement. The quest for a target sequence or a response element for ORF57 in the lytic genes was not successful. Instead, we found the nucleotide bias to be the critical determinant of ORF57 dependency. Based on the fact that ORF57 has only a weak affinity for nucleic acids, we speculate that a cellular RNA-binding protein provides the sequence preference for ORF57. This study provides evidence that herpesviral RNA regulator proteins use the sequence bias of lytic genes and the resulting composition of the viral mRNP to distinguish between viral and cellular mRNAs.

Protective effects of sphingosine-1-phosphate receptor agonist treatment after myocardial ischaemia-reperfusion.

AIMS: Several experimental studies have demonstrated protection against cardiac ischaemia-reperfusion injury achieved by pre-treatment with exogenous sphingosine-1-phosphate (S1P). We tested the hypothesis that pharmacological S1P receptor agonists improve recovery of function when applied with reperfusion. METHODS AND RESULTS: Isolated rat cardiomyocytes were stimulated with exogenous S1P, the selective S1P1 receptor agonist SEW2871, or the S1P1/3 receptor agonist FTY720. Western blot analysis was performed to analyse downstream signalling pathways. Ischaemia-reperfusion studies were conducted in rat cardiomyocytes, isolated Langendorff-perfused rat hearts, and in human myocardial muscle strip preparations to evaluate the effect of S1P receptor agonists on cell death and recovery of mechanical function. All S1P receptor agonists were able to activate Akt. This was associated with transactivation of the epidermal growth factor receptor. In isolated cardiomyocytes, selective stimulation of the S1P1 receptor by SEW2871 induced protection against cell death when administered either before or after ischaemia-reperfusion. In isolated rat hearts, treatment with FTY720 during reperfusion attenuated the rise in left ventricular end-diastolic pressure (LVEDP) and improved the recovery of left ventricular developed pressure without limiting infarct size. However, selective S1P1 receptor stimulation did not improve functional recovery but rather increased LVEDP. Additional experiments employing a human myocardial ischaemia-reperfusion model also demonstrated improved functional recovery induced by FTY720 treatment during reperfusion. CONCLUSION: Pharmacological S1P receptor agonists have distinct effects on ischaemia-reperfusion injury. Their efficacy when applied during reperfusion makes them potential candidates for pharmaceutical postconditioning therapy after cardiac ischaemia.