Marek Widera

Genomic HEXploring allows landscaping of novel potential splicing regulatory elements

Effective splice site selection is critically controlled by flanking splicing regulatory elements (SREs) that can enhance or repress splice site use. Although several computational algorithms currently identify a multitude of potential SRE motifs, their predictive power with respect to mutation effects is limited. Following a RESCUE-type approach, we defined a hexamer-based ‘HEXplorer score’ as average Z-score of all six hexamers overlapping with a given nucleotide in an arbitrary genomic sequence. Plotted along genomic regions, HEXplorer score profiles varied slowly in the vicinity of splice sites. They reflected the respective splice enhancing and silencing properties of splice site neighborhoods beyond the identification of single dedicated SRE motifs. In particular, HEXplorer score differences between mutant and reference sequences faithfully represented exonic mutation effects on splice site usage. Using the HIV-1 pre-mRNA as a model system highly dependent on SREs, we found an excellent correlation in 29 mutations between splicing activity and HEXplorer score. We successfully predicted and confirmed five novel SREs and optimized mutations inactivating a known silencer. The HEXplorer score allowed landscaping of splicing regulatory regions, provided a quantitative measure of mutation effects on splice enhancing and silencing properties and permitted calculation of the mutationally most effective nucleotide.

An intronic G-run within HIV-1 intron 2 is critical for splicing regulation of vif-mRNA

ABSTRACT Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3′ splice site (3′ss) A1 but lack splicing at 5′ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3′ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3′ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5′ss D2. Here we show that an intronic G run (GI2-1) represses the use of a second 5′ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of GI2-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here.

Balanced splicing at the Tat-specific HIV-1 3'ss A3 is critical for HIV-1 replication

BackgroundThe viral regulatory protein Tat is essential for establishing a productive transcription from the 5′-LTR promoter during the early phase of viral gene expression. Formation of the Tat-encoding mRNAs requires splicing at the viral 3′ss A3, which has previously been shown to be both negatively and positively regulated by the downstream splicing regulatory elements (SREs) ESS2p and ESE2/ESS2. However, using the novel RESCUE-type computational HEXplorer algorithm, we were recently able to identify another splicing enhancer (ESE5807-5838, henceforth referred to as ESEtat) located between ESS2p and ESE2/ESS2. Here we show that ESEtat has a great impact on viral tat-mRNA splicing and that it is fundamental for regulated 3′ss A3 usage.ResultsMutational inactivation or locked nucleic acid (LNA)-directed masking of the ESEtat sequence in the context of a replication-competent virus was associated with a failure (i) to activate viral 3′ss A3 and (ii) to accumulate Tat-encoding mRNA species. Consequently, due to insufficient amounts of Tat protein efficient viral replication was drastically impaired. RNA in vitro binding assays revealed SRSF2 and SRSF6 as candidate splicing factors acting through ESEtat and ESE2 for 3′ss A3 activation. This notion was supported by coexpression experiments, in which wild-type, but not ESEtat-negative provirus responded to higher levels of SRSF2 and SRSF6 proteins with higher levels of tat-mRNA splicing. Remarkably, we could also find that SRSF6 overexpression established an antiviral state within provirus-transfected cells, efficiently blocking virus particle production. For the anti-HIV-1 activity the arginine-serine (RS)-rich domain of the splicing factor was dispensable.ConclusionsBased on our results, we propose that splicing at 3′ss A3 is dependent on binding of the enhancing SR proteins SRSF2 and SRSF6 to the ESEtat and ESE2 sequence. Mutational inactivation or interference specifically with ESEtat activity by LNA-directed masking seem to account for an early stage defect in viral gene expression, probably by cutting off the supply line of Tat that HIV needs to efficiently transcribe its genome.

The D-amino acid peptide D3 reduces amyloid fibril boosted HIV-1 infectivity

BackgroundAmyloid fibrils such as Semen-Derived Enhancer of Viral Infection (SEVI) or amyloid-β-peptide (Aβ) enhance HIV-1 attachment and entry. Inhibitors destroying or converting those fibrils into non-amyloidogenic aggregates effectively reduce viral infectivity. Thus, they seem to be suitable as therapeutic drugs expanding the current HIV-intervening repertoire of antiretroviral compounds.FindingsIn this study, we demonstrate that the small D-amino acid peptide D3, which was investigated for therapeutic studies on Alzheimer’s disease (AD), significantly reduces both SEVI and Aβ fibril boosted infectivity of HIV-1.ConclusionsSince amyloids could play an important role in the progression of AIDS dementia complex (ADC), the treatment of HIV-1 infected individuals with D3, that inhibits Aβ fibril formation and converts preformed Aβ fibrils into non-amyloidogenic and non-fibrillar aggregates, may reduce the vulnerability of the central nervous system of HIV patients for HIV associated neurological disorders.

A novel assay for detecting virus-specific antibodies triggering activation of Fcγ receptors.

IgG responses are crucial in antiviral defence and instrumental for the serodiagnosis of infections. Fcγ receptors (FcγRs), which recognize the Fc-part of IgG, differ regarding their IgG binding affinity, IgG subclass preference, cellular expression profile and pathogen elimination mechanisms elicited upon activation. Assessing their activation in vitro is of fundamental importance, but technically difficult. Therefore, a novel assay for measuring antiviral IgG antibodies triggering activation of individual host Fcγ receptors was established. The assay comprises the co-cultivation of virus-infected target cells with immune IgG antibodies and mouse BW5147 hybridoma cells stably expressing chimeric FcγR-CD3ζ chain molecules consisting of the extracellular domain of human FcγRIIIA, FcγRIIA or FcγRI, respectively, fused to the transmembrane and intracellular domains of the mouse CD3ζ chain. Triggering of the chimeric FcγR receptors by immune complexes formed on the surface of IgG-opsonized virus-infected target cells resulted in FcγR activation leading to IL-2 secretion by BW5147 cells, which was quantified as a surrogate marker in an ELISA. Target cells infected with various human pathogenic viruses including herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), measles virus (MV), and respiratory syncytial virus (RSV) or displaying human immunodeficiency virus-1 (HIV-1) gp120 evoke dose-dependent IgG responses demonstrating the universal applicability of the assay. Taken together, a new reliable and simple tool for measuring antibodies triggering activation of Fcγ receptors was established. This assay will be instrumental for defining novel correlates of IgG immunity and the design of new therapeutic IgGs.

Analysis of Competing HIV-1 Splice Donor Sites Uncovers a Tight Cluster of Splicing Regulatory Elements within Exon 2/2b

ABSTRACT The HIV-1 accessory protein Vif is essential for viral replication by counteracting the host restriction factor APOBEC3G (A3G), and balanced levels of both proteins are required for efficient viral replication. Noncoding exons 2/2b contain the Vif start codon between their alternatively used splice donors 2 and 2b (D2 and D2b). For vif mRNA, intron 1 must be removed while intron 2 must be retained. Thus, splice acceptor 1 (A1) must be activated by U1 snRNP binding to either D2 or D2b, while splicing at D2 or D2b must be prevented. Here, we unravel the complex interactions between previously known and novel components of the splicing regulatory network regulating HIV-1 exon 2/2b inclusion in viral mRNAs. In particular, using RNA pulldown experiments and mass spectrometry analysis, we found members of the heterogeneous nuclear ribonucleoparticle (hnRNP) A/B family binding to a novel splicing regulatory element (SRE), the exonic splicing silencer ESS2b, and the splicing regulatory proteins Tra2/SRSF10 binding to the nearby exonic splicing enhancer ESE2b. Using a minigene reporter, we performed bioinformatics HEXplorer-guided mutational analysis to narrow down SRE motifs affecting splice site selection between D2 and D2b. Eventually, the impacts of these SREs on the viral splicing pattern and protein expression were exhaustively analyzed in viral particle production and replication experiments. Masking of these protein binding sites by use of locked nucleic acids (LNAs) impaired Vif expression and viral replication. IMPORTANCE Based on our results, we propose a model in which a dense network of SREs regulates vif mRNA and protein expression, crucial to maintain viral replication within host cells with varying A3G levels and at different stages of infection. This regulation is maintained by several serine/arginine-rich splicing factors (SRSF) and hnRNPs binding to those elements. Targeting this cluster of SREs with LNAs may lead to the development of novel effective therapeutic strategies.

Clinical course and core variability in HBV infected patients without detectable anti-HBc antibodies

BACKGROUND The presence of anti-HBc antibodies indicates direct encounter of the immune system with hepatitis B virus (HBV). OBJECTIVES Aim of our study was to seek for anti-HBc negative but HBV replicating patients and analyze their clinical course and preconditions. STUDY DESIGN From 1568 HBV-DNA positive patients, 29 patients (1.85%) tested negative for anti-HBc. The absence of anti-HBc could be confirmed in 19 patients using an alternative assay. In 16 of 19 cases, a partial or full HBV genome analysis was performed with NGS sequencing to evaluate if specific mutations were associated with anti-HBc absence. As a control group samples from 32 matched HBV infected patients with detectable anti-HBc were sequenced. RESULTS Patients with detectable HBV-DNA and sequenced HBV core region in the confirmed absence of anti-HBc were diagnosed with acute HBV infection (n=3), HBV reactivation (n=9) and chronic hepatitis B (n=4). Most patients (12/16) were immunosuppressed: 3/16 patients had an HIV coinfection, 7/16 patients suffered from a malignant disease and 4/16 patients underwent solid organ transplantation (from which 2/4 had a malignant disease). Compared to the control cohort, HBV variants from anti-HBc negative patients showed less variability in the core region. CONCLUSIONS In the absence of anti-HBc, HBV-DNA was most often found in immunocompromised hosts. Distinct mutations or deletions in the core region did not explain anti-HBc negativity. It would be advisable not to rely only on a single result of anti-HBc negativity to exclude HBV infection in immunocompromised hosts, but to measure anti-HBc repeatedly or with different methods.

An advanced BLT-humanized mouse model for extended HIV-1 cure studies

Objective: Although bone marrow, liver, thymus (BLT)-humanized mice provide a robust model for HIV-1 infection and enable evaluation of cure strategies dependent on endogenous immune responses, most mice develop graft versus host disease (GVHD), limiting their utility for extended HIV cure studies. This study aimed to: evaluate the GVHD-resistant C57 black 6 (C57BL/6) recombination activating gene 2 (Rag2)−/−&ggr;c−/−CD47−/− triple knockout (TKO)-BLT mouse as a model to establish HIV-1 latency. Determine whether TKO-BLT mice could be maintained on antiretroviral therapy (ART) for extended periods of time. Assess the rapidity of viral rebound following therapy interruption. Design: TKO-BLT mice were HIV-1 infected, treated with various ART regimens over extended periods of time and assayed for viral rebound following therapy interruption. Methods: Daily subcutaneous injection and oral ART-mediated suppression of HIV-1 infection was tested at various doses in TKO-BLT mice. Mice were monitored for suppression of viremia and cellular HIV-1 RNA and DNA prior to and following therapy interruption. Results: Mice remained healthy for 45 weeks posthumanization and could be treated with ART for up to 18 weeks. Viremia was suppressed to less than 200 copies/ml in the majority of mice with significant reductions in cellular HIV-1 RNA and DNA. Treatment interruption resulted in rapid viral recrudescence. Conclusion: HIV-1 latency can be maintained in TKO-BLT mice over extended periods on ART and rapid viral rebound occurs following therapy removal. The additional 15–18 weeks of healthy longevity compared with other BLT models provides sufficient time to examine the decay kinetics of the latent reservoir as well as observe delays in recrudescence in HIV-1 cure studies.

The PI3K pathway acting on alternative HIV-1 pre-mRNA splicing.

HIV-1 mediates pro-survival signals and prevents apoptosis via the phosphatidylinositol-3-kinase (PI3K) pathway. This pathway, however, also affects phosphorylation of serine-arginine (SR) proteins, a family of splicing regulatory factors balancing splice site selection. We now show that pharmacologic inhibition of PI3K signalling alters the HIV-1 splicing pattern of both minigene- and provirus-derived mRNAs. This indicates that HIV-1 might also promote PI3K signalling to balance processing of its transcripts by regulating phosphorylation of splicing regulatory proteins.

Mutational analysis of the internal membrane proximal domain of the HIV glycoprotein C-terminus.

This study focuses on the long stretch of highly conserved amino acids in the membrane proximal part of the HIV-1 cytoplasmic tail (Env amino acids (aa) 706-718) upstream of the overlap with the tat and rev second coding exons. Changes in Env aa 713 and 715, although they did not affect Env function, abrogated replicative spread. Other amino acid substitutions, i.e., 706-712, 714 and 716, despite their conservation, did not result in defective replicative phenotypes even in primary peripheral blood lymphocytes. Our results point to their involvement in presently unrecognized essential Env functions pertinent only in in vivo. Interestingly, changes in the codons for residues 717-718 as well as some mutations in residues 714-716 abrogated Gag expression but still allowed expression of functional Env in a rev-independent manner. This could be due to the inactivation of a rev-regulated negative element within the respective nucleotide sequence (8354-8368).