A Bidirectional SF2/ASF- and SRp40-Dependent Splicing Enhancer Regulates Human Immunodeficiency Virus Type 1 rev, env, vpu, and nef Gene Expression

The integrated human immunodeficiency virus type 1 (HIV-1) genome is transcribed in a single pre-mRNA that is alternatively spliced into more than 40 mRNAs. We characterized a novel bidirectional exonic splicing enhancer (ESE) that regulates the expression of the HIV-1 env, vpu, rev, and nef mRNAs. The ESE is localized downstream of the vpu-, env-, and nef-specific 3′ splice site no. 5. SF2/ASF and SRp40 activate the ESE and are required for efficient 3′ splice site usage and binding of the U1 snRNP to the downstream 5′ splice site no. 4. U1 snRNP binding to the 5′ splice site no. 4 is required for splicing of the rev and nef mRNAs and to increase expression of the partially spliced env mRNA. Finally, our results indicate that this ESE is necessary for the recruitment of the U1 snRNP to the 5′ splice site no. 4, even when the 5′ splice site and the U1 snRNA have been mutated to obtain a perfect complementary match. The ESE characterized here is highly conserved in most viral subtypes

HIV-1 seroreversion in HIV-1-infected children: do genetic determinants play a role?

Background: HIV-1 seroreversion in infants with vertically transmitted HIV-1 infection who started ART in the first months of life has been reported in only a subset of patients. However, the reason why most infants remain seropositive despite similar treatment response is not understood. Here, we assessed whether HIV-1 seroreversion in maternally infected infants is associated with genetic determinants. Methods: HIV-1-infected infants with a history of documented HIV-1 seroreversion were identified throughout Germany using a standardized questionnaire. At study entry immune reconstitution and anti-HIV-1 antibody expression were monitored as clinical parameters. To search for genetic determinants high-resolution HLA genotyping was performed. In addition, the coding sequence of the chemokine receptor CCR5 was analyzed by Sanger sequencing regarding potential mutations. Results: Patients showed normal numbers and frequencies of lymphocyte subpopulations. Five out of eight patients still had seronegative HIV-1 antibody status at study entry. HLA genotyping revealed the enrichment of HLA-DQB1*03 and DQB1*06 alleles within the patient cohort. Only one patient was found to carry a 32 bp-deletion within the CCR5 gene. Conclusion: Our results indicate that the phenotype of HIV-1 seroreversion in infants might correlate with the presence of HLA class II alleles DQB1*03 and DQB1*06. This finding supports the idea of genetic predisposition determining HIV-1 seroreversion in vertically infected infants effectively treated with AR

A novel approach to describe a U1 snRNA binding site

RNA duplex formation between U1 snRNA and a splice donor (SD) site can protect pre-mRNA from degradation prior to splicing and initiates formation of the spliceosome. This process was monitored, using sub-genomic HIV-1 expression vectors, by expression analysis of the glycoprotein env, whose formation critically depends on functional SD4. We systematically derived a hydrogen bond model for the complementarity between the free 5′ end of U1 snRNA and 5′ splice sites and numerous mutations following transient transfection of HeLa-T4(+) cells with 5′ splice site mutated vectors. The resulting model takes into account number, interdependence and neighborhood relationships of predicted hydrogen bond formation in a region spanning the three most 3′ base pairs of the exon (–3 to –1) and the eight most 5′ base pairs of the intron (+1 to +8). The model is represented by an algorithm classifying U1 snRNA binding sites which can or cannot functionally substitute SD4 with respect to Rev-mediated env expression. In a data set of 5′ splice site mutations of the human ATM gene we found a significant correlation between the algorithmic classification and exon skipping (P = 0.018, χ(2)-test), showing that the applicability of the proposed model reaches far beyond HIV-1 splicing. However, the algorithmic classification must not be taken as an absolute measure of SD usage as it may be modified by upstream sequence elements. Upstream to SD4 we identified a fragment supporting ASF/SF2 binding. Mutating GAR nucleotide repeats within this site decreased the SD4-dependent Rev-mediated env expression, which could be balanced simply by artificially increasing the complementarity of SD4

Next-generation-sequencing-spectratyping reveals public T-cell receptor repertoires in pediatric very severe aplastic anemia and identifies a beta chain CDR3 sequence associated with hepatitis-induced pathogenesis

Krell P, Reuther S, Fischer U, et al. Next-generation-sequencing-spectratyping reveals public T-cell receptor repertoires in pediatric very severe aplastic anemia and identifies a beta chain CDR3 sequence associated with hepatitis-induced pathogenesis. Haematologica. 2013;98(9):1388-1396