SRSF1 acts as an IFN-I-regulated cellular dependency factor decisively affecting HIV-1 post-integration steps.

Efficient HIV-1 replication depends on balanced levels of host cell components including cellular splicing factors as the family of serine/arginine-rich splicing factors (SRSF, 1-10). Type I interferons (IFN-I) play a crucial role in the innate immunity against HIV-1 by inducing the expression of IFN-stimulated genes (ISGs) including potent host restriction factors. The less well known IFN-repressed genes (IRepGs) might additionally affect viral replication by downregulating host dependency factors that are essential for the viral life cycle; however, so far, the knowledge about IRepGs involved in HIV-1 infection is very limited. In this work, we could demonstrate that HIV-1 infection and the associated ISG induction correlated with low SRSF1 levels in intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs) during acute and chronic HIV-1 infection. In HIV-1-susceptible cell lines as well as primary monocyte-derived macrophages (MDMs), expression levels of SRSF1 were transiently repressed upon treatment with specific IFNα subtypes in vitro. Mechanically, 4sU labeling of newly transcribed mRNAs revealed that IFN-mediated SRSF1 repression is regulated on early RNA level. SRSF1 knockdown led to an increase in total viral RNA levels, but the relative proportion of the HIV-1 viral infectivity factor (Vif) coding transcripts, which is essential to counteract APOBEC3G-mediated host restriction, was significantly reduced. In the presence of high APOBEC3G levels, however, increased LTR activity upon SRSF1 knockdown facilitated the overall replication, despite decreased vif mRNA levels. In contrast, SRSF1 overexpression significantly impaired HIV-1 post-integration steps including LTR transcription, alternative splice site usage, and virus particle production. Since balanced SRSF1 levels are crucial for efficient viral replication, our data highlight the so far undescribed role of SRSF1 acting as an IFN-modulated cellular dependency factor decisively regulating HIV-1 post-integration steps.

A Novel, Broad-Acting Peptide Inhibitor of Double-Stranded DNA Virus Gene Expression and Replication.

Viral infections are a global disease burden with only a limited number of antiviral agents available. Due to newly emerging viral pathogens and increasing occurrence of drug resistance, there is a continuous need for additional therapeutic options, preferably with extended target range. In the present study, we describe a novel antiviral peptide with broad activity against several double-stranded DNA viruses. The 22-mer peptide TAT-I24 potently neutralized viruses such as herpes simplex viruses, adenovirus type 5, cytomegalovirus, vaccinia virus, and simian virus 40 in cell culture models, while being less active against RNA viruses. The peptide TAT-I24 therefore represents a novel and promising drug candidate for use against double-stranded DNA viruses.

Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry.

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals are currently not available, methods measuring the impact of potential antiviral agents are required. Here, a dual fluorescence reporter that allows the analysis of the BKPyV non-coding control-region (NCCR) driven early and late promoter activity was constructed to quantify the impact of potential antiviral drugs on viral gene expression via tdTomato and eGFP expression. In addition, by cloning BKPyV-NCCR amplicons which in this protocol have been exemplarily obtained from the blood-derived DNA of immunocompromised renal transplanted patients, the impact of NCCR-rearrangements on viral gene expression can be determined. Following cloning of the patient derived amplicons, HEK293T cells were transfected with the reporter-plasmids, and treated with potential antiviral agents. Subsequently, cells were subjected to FACS-analysis for measuring mean fluorescence intensities 72 h post transfection. To also test the analysis of drugs that have a potential cell cycle inhibiting effect, only transfected and thus fluorescent cells are used. Since this assay is performed in large T Antigen expressing cells, the impact of early and late expression can be analyzed in a mutually independent manner.

Impact of immune suppressive agents on the BK-Polyomavirus non coding control region.

BACKGROUND: Reactivation of the BK-Polyomavirus (BKPyV) can cause a polyomavirus associated nephropathy in approx. 10% of kidney transplant recipients. In these cases, current therapy is based on the reduction of immunosuppression. Since BKPyV-transcription is driven by the Non-Coding-Control-Region (NCCR) we were interested whether NCCR-activity is affected by immunosuppressive agents. METHODS: Plasma samples from 45 BKPyV-positive patients after renal transplantation were subjected to PCR-analysis. NCCR-amplicons were cloned into a plasmid that allows the quantification of early and late NCCR-activity by tdTomato and eGFP expression, respectively. HEK293T-cells were transfected with the reporter-plasmids, treated with immunosuppressive agents, and subjected to FACS-analysis. In addition, H727-cells were infected with patient derived BKPyV, treated with mTOR-inhibitors, and NCCR activity was analysed using qRT-PCR. RESULTS: While tacrolimus and cyclosporine-A did not affect NCCR-promoter-activity, treatment with mTOR1-inhibitor rapamycin resulted in the reduction of early, but not late-NCCR-promoter-activity. Treatment with dual mTOR1/2 inhibitors (INK128 or pp242) led to significant inhibition of early, however, concomitantly enhanced late-promoter-activity. In BKPyV infected cells both rapamycin and INK128 reduced early expression, however, INK128 resulted in higher late-mRNA levels when compared to rapamycin treatment. CONCLUSIONS: Our results demonstrate that mTOR1-inhibitors are able to reduce early-expression of wildtype and rearranged NCCRs, which might contribute to previously described inhibition of BKPyV-replication. Dual mTOR1/2-inhibitors, however, additionally might shift viral early into late-expression promoting synthesis of viral structural proteins and particle production.

Behind the scenes of HIV-1 replication: Alternative splicing as the dependency factor on the quiet.

Alternative splicing plays a key role in the HIV-1 life cycle and is essential to maintain an equilibrium of mRNAs that encode viral proteins and polyprotein-isoforms. In particular, since all early HIV-1 proteins are expressed from spliced intronless and late enzymatic and structural proteins from intron containing, i.e. splicing repressed viral mRNAs, cellular splicing factors and splicing regulatory proteins are crucial for the replication capacity. In this review, we will describe the complex network of cis-acting splicing regulatory elements (SREs), which are mainly localized in the neighbourhoods of all HIV-1 splice sites and warrant the proper ratio of individual transcript isoforms. Since SREs represent binding sites for trans-acting cellular splicing factors interacting with the cellular spliceosomal apparatus we will review the current knowledge of interactions between viral RNA and cellular proteins as well as their impact on viral replication. Finally, we will discuss potential therapeutic approaches targeting HIV-1 alternative splicing.