Charles E. Schmidt College of Medicine
Florida Atlantic University
777 Glades Road
Boca Raton, FL 33431
For the past two decades, our research focused on the study of the complex mechanisms that lead to the expression of cellular and viral genomes. We have characterized several key cellular factors and viral sequences required for efficient HIV-1 splicing and developed novel methodologies and reduced systems to study the transcription and regulation of the viral transcripts. At the same time, we have pioneered novel technologies to study RNA-RNA binding proteins interactions and worked at the characterization of key cellular RNA-RNA binding protein complexes. Currently, we are working on multiple projects, which aim at the understanding of how cellular and viral genes are expressed focusing on the role that RNA binding proteins have on transcription and splicing. We utilize a combination of genomic, cellular and biochemical approaches to characterize functions and pathways regulated by members of multiple families of RNA binding proteins. Other projects in our lab aim at the characterization of the mechanisms regulating the functions of T cells in response to viral infection. More recently, we have entered into multiple collaborative projects to develop diagnostic microfluidic chips for the detection of multiple viruses in bodily fluids. Other projects in the lab aim at the characterization of host-pathogen interactions in tropical and subtropical viruses such as Dengue, Zika, West Nile and Rift Valley Fever.
Summary of current projects:
Inhibition of HIV-1 replication by SRSF1 derived polypeptides. Transcription of the integrated HIV-1 proviral genome is tightly regulated by the interaction of the viral protein Tat with several cellular factors and the RNA Polymerase II complex. The transcribed viral pre-mRNA is spliced in multiple mRNAs to generate the nine different gene products required for viral replication. HIV has also developed a number of strategies to regulate splicing of its transcripts. Expression of the viral genome is dependent on the interactions between the viral promoter, RNA sequences, viral proteins and host cell factors. Alteration of the mechanisms regulating transcription and splicing of the viral messenger can dramatically affect viral infectivity and pathogenesis. Utilizing a combination of cell-based and biochemical approaches we have isolated a cellular RNA binding protein, SRSF1, which is an inhibitor of both viral transcription and splicing. SRSF1 exerts its activity by competing with the viral transcriptional transactivator Tat, thus reducing viral transcription, and by binding a series of sequences within the viral transcripts, which regulate the splicing of specific viral mRNAs. Over-expression of SRSF1 induces the disruption of both transcription and splicing mechanisms resulting in a strong inhibition of viral replication. The SRSF1 fragment retaining the highest antiviral activity is constituted by two RNA binding domains (RBDs), named RNA Recognition Motifs (RRMs) 1 and 2. Expression of a SRSF1 deletion mutant containing only RRM1 and 2 in stable cell lines can reduce the replication of a several viral strains up to 3000 fold without altering cell viability. We are now utilizing different approaches to deliver SRSF1 and its deletion mutants to HIV-1 infected T cells to inhibit HIV-1 replication.
SR proteins modulation of cellular gene transcription. We have investigated a possible role for SRSF1 and other members of the Serine argenine rich (SR family of splicing factors) in the transcription of cellular genes utilizing an RNA-Seq approach. We performed RNA-seq assays on HEK293 cells overexpressing SRSF1 at two time points, 15 and 48 hours post-transfection. We observed that a subset of cellular genes was regulated at both 15 and 48 hours following transient transfection of an SRSF1 expression plasmid. Since overexpression of the SRSF1 transcript is initially detectable at 12 hours after transfection the genes regulated at 15 hours (which expression is then confirmed at 48 hours) are likely primary transcriptional SRSF1 targets. We determined that the expression of genes that were transcriptionally activated by SRSF1 were also modulated by the 7SK snRNP particle, in agreement with previous work (Ji et al., Cell 2013). Nevertheless, over-expression of SRSF1 and knockdown of 7SK have a synergistic effect on transcription, suggesting that SRSF1 may regulate the transcription of certain genes via multiple mechanisms. We have also observed that a subset of members of the SR protein family (SRSF3,4,5,7) exhibits transcription activity similar to SRSF1. We are now working at elucidating the mechanisms underlying the transcriptional effects observed.
SRSF1 modulation of immune pathways. We analyzed the molecular and functional pathways affected by SRSF1 expression at 48 hours, thus included multiple genes which expression is modulated as a downstream result of SRSF1 functions in mRNA processing and metabolism. Pathways analysis revealed that this gene set was highly enriched in pathways involved in immune function (38 out 216 coding genes), more specifically TNF and IL-17 signaling pathways were enriched with a p≤1x10-5 and p≤1x10-8, respectively. We validated expression changes consistent with our RNASeq analysis in over 80% of the genes involved in immune functions. The proinflammatory cytokine TNF-α was strongly upregulated following SRSF1 expression (over 30 fold). Transient transduction of the cells with a TNF-α expression plasmid reproduced a gene expression pattern similar to the one observed with SRSF1 over-expression suggesting that regulation of TNF-α expression by SRSF1 is the key event leading to the differential expression of multiple genes with immune functions. Initial studies also show that SRSF1 can only partially activate the basal TNF-α promoter suggesting a role for long-range transcriptional enhancer/silencers in this mechanism.
Study of global gene expression in CD4 T cell following HIV-1 infection. We utilized an RNAseq approach to study the expression and splicing of cellular genes in CD4+ T cells in 3 conditions: naïve, activated and activated-HIV-1 infected. The data from the RNASeq experiment were analyzed utilizing a pipeline we have developed. This analysis identified 292 protein coding genes that were differently expressed following HIV-1 infection. Of the 292 genes 94 were also differentially expressed following activation of Naïve (unstimulated) CD4+ T cells with αCD3/αCD28 antibodies. 83 of the 94 genes in common (88%) exhibited apposite changes in the two datasets. Of these 83 genes 8 coded for transcription factors known to modulate T cell functions, activation and proliferation suggesting that HIV infection drives CD4+ T cells toward an inactive/latent state. Since a population of inactive/memory latently infected T cells is the main viral reservoir in infected individual undergoing anti-retroviral therapy (ART), our finding might be key in understanding how this reservoir of latently infected T cells is created.
HIV Viral Load Assay for Point-of-Care Settings. Although antiretroviral therapy (ART) is effective in saving AIDS patients' lives, the implementation of ART worldwide has been drastically hampered by the lack of treatment monitoring diagnostics and disease management. According to the World Health Organization (WHO), the coverage of ART is still only 34% of 29 million ART-eligible people in low- and middle-income countries, despite ART being affordable or even freely available in these countries. Expanding ART to all people living with HIV and improving disease management strategies can help avert 21 million AIDS-related deaths by 2030, as predicted by the WHO. One of the fundamental challenges to reduce HIV burden and its prevalence is the absence of point-of-care (POC) assays for viral load in resource-constrained areas lacking trained technicians, and modern lab infrastructure. To increase access to HIV care and to improve treatment outcomes, there is an urgent need to develop a reliable and inexpensive device for viral load quantification. Our goal is to develop a portable and low-cost (~$1) microchip technology for rapid quantification of HIV-1 viral load from plasma/whole blood at POC settings. In developed countries, HIV-1 viral load is regularly used to closely monitor and assess a patient’s response to ART, to ensure drug adherence and to stage disease progression. In contrast, developing countries are using CD4+ T lymphocyte count and clinical symptoms to guide ART, following the WHO guidelines with the exception of infants, where viral load assays are required. This is because HIV-1 viral load assays are expensive ($50-200 per test), and technically complex (RT-PCR based), requiring trained technicians. Studies have shown that a CD4+ cell counting strategy cannot detect early virological failure, which leads to an accumulation of drug-resistant strains in infected individuals and reduces ART efficacy. In collaboration with the laboratory of Dr. Waseem Asghar (FAU, College of Engineering and Computer Science), we are developing a highly sensitive and disposable HIV-1 viral load microfluidic chip to detect and quantify HIV-1 viral load from bodily fluids.
Development of a portable platform for Zika and Dengue virus detection. Zika virus (ZIKV) is a mosquito-borne virus that belongs to the genus flavivirus. As of May 2, 2018, the CDC has reported more than 42,000 ZIKV disease cases in the United States and US territories, with South Florida being the most severely affected region in the continental USA. Cases of microcephaly have increased by a factor of over 20 fold among newborns from women infected during pregnancy, which indicated a strong association between ZIKV infection in early pregnancy and fetal malformations. Additionally, ZIKV has been found in blood, fueling concerns about the risk of transfusion-transmission especially in at-risk transfusion recipient populations, such as pregnant women. Current ZIKV diagnosis assays are based on measuring early antibody immunoglobulin (Ig) M using enzyme-linked immunosorbent assay (Zika-MAC ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Current IgM antibody-based ELISA assays cannot reliably distinguish between ZIKV and other flaviviruses (Dengue, Chikungunya, etc.). Therefore, an IgM positive result in a dengue or Zika IgM ELISA test should be considered solely indicative of a recent flavivirus infection. Molecular diagnostic methods such as RT-qPCR, although highly specific, also require multiple labor-intensive processing steps, hence are not suitable for rapid testing at point-of-care (POC) centers. Currently no ZIKV viral load technology exists at the POC. Similarly, to the approach that we will utilize for the HIV-1 microfluidic chip, we are collaborating with the laboratory of Dr. Waseem Asghar (FAU, College of Engineering and Computer Science) to develop a disposable Zika/Dengue viral load microfluidic chip to detect and differentiate Zika and Dengue viruses from bodily fluids.