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- Title
- THE ANTI-HIV-1 ACTIVITY OF HUMAN SEMINAL PLASMA.
- Creator
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Martellini, Julie, Cole, Alexander, University of Central Florida
- Abstract / Description
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Human immunodeficiency virus (HIV) has become a global pandemic over the past few decades, with new infections and related deaths in the millions each year. There is no cure in sight for HIV-1 infection, and there has been little progress in developing an efficacious vaccine. Heterosexual transmission of HIV-1 remains the principal mode of transmission throughout the world and thus measures, such as topical vaginal microbicides, to prevent infection of the female reproductive tract are...
Show moreHuman immunodeficiency virus (HIV) has become a global pandemic over the past few decades, with new infections and related deaths in the millions each year. There is no cure in sight for HIV-1 infection, and there has been little progress in developing an efficacious vaccine. Heterosexual transmission of HIV-1 remains the principal mode of transmission throughout the world and thus measures, such as topical vaginal microbicides, to prevent infection of the female reproductive tract are actively being explored. Recent trials of topical vaginal microbicides have shown that their interaction with the mucosal surfaces of the female reproductive tract as well as semen can hinder microbicide effectiveness against HIV-1 infection. Therefore, understanding the role these fluids play in HIV transmission would be critical towards developing effective antiviral prophylaxes. A recent study from our group demonstrated that human cervicovaginal secretions contained numerous cationic antimicrobial peptides and proteins, which collectively inhibited HIV-1 infection of target cells and tissues. To ascertain if human seminal plasma (SP), the main vector responsible for transmitting HIV-1, exhibited antiviral activity we utilized several anti-HIV assays in the presence or absence of minimally manipulated SP. The majority of the intrinsic anti-HIV-1 activity of SP resided in the cationic polypeptide fraction. Antiviral assays utilizing luciferase reporter cells and lymphocytic cells revealed the ability of whole SP to prevent HIV-1 infection, even when SP was diluted 3200-fold. Subsequent fractionation by continuous flow acid-urea (AU)-PAGE and antiviral testing revealed that cationic polypeptides within SP were responsible for the majority of anti-HIV-1 activity. A proteomic approach was utilized to resolve and identify 52 individual cationic polypeptides that contribute to the aggregate anti-HIV-1 activity of SP. One peptide fragment of semenogelin I, termed SG-1, was purified from SP by a multi-step chromatographic approach, protein sequenced, and determined to exhibit anti-HIV-1 activity against HIV-1. Anti-HIV-1 activity was transient, as whole SP incubated for prolonged time intervals exhibited a proportional decrease in anti-HIV-1 activity that was directly attributed to the degradation of semenogelin I peptides. Collectively, these results indicate that the cationic polypeptide fraction of SP is active against HIV-1, and that semenogelin-derived peptides contribute to the intrinsic anti-HIV-1 activity of SP. Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called "SEVI" capable of enhancing HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity. Our studies demonstrate the overall antiviral activity of human SP, but there is still a critical need for effective topical vaginal microbicides that can prevent HIV-1 transmission. The synthetic human retrocyclins are cyclic antimicrobial peptides that are remarkably active against HIV-1, and are being developed as topical vaginal microbicides. Herein, we assessed whether the putative proviral SEVI was able to adversely affect the anti-HIV-1 activity of the retrocyclin analog RC-101. While SEVI alone enhanced viral infection, this effect was completely negated in the presence of RC-101. Retrocyclins such as RC-101 are inhibitors of HIV-1 entry, by preventing gp41-mediated viral fusion. Interestingly, using an HIV-1 reverse transcriptase (RT) specific assay, we also determined that RC-101 directly inhibited the activity of RT in a dose dependent manner, suggesting a secondary mechanism of viral inhibition. Our group has determined that RC-101 induces only a modest level of resistance in HIV, which may be due in part to RC-101's dual mechanisms of viral inhibition.
Show less - Date Issued
- 2011
- Identifier
- CFE0003583, ucf:48916
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0003583
- Title
- STRUCTURE-FUNCTION ANALYSIS OF THE DROSOPHILA STUBBLE TYPE II TRANSMEMBRANE SERINE PROTEASE.
- Creator
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Morgan, Rachel, von Kalm, Laurence, University of Central Florida
- Abstract / Description
-
Hormonally-triggered regulatory hierarchies play a major role in organismal development. Disruption of a single member of such a hierarchy can lead to irregular development and disease. Therefore, knowledge of the members involved and the mechanisms controlling signaling through such pathways is of great importance in understanding how resulting developmental defects occur. Type II transmembrane serine proteases (TTSPs) make up a family of cell surface-associated proteases that play important...
Show moreHormonally-triggered regulatory hierarchies play a major role in organismal development. Disruption of a single member of such a hierarchy can lead to irregular development and disease. Therefore, knowledge of the members involved and the mechanisms controlling signaling through such pathways is of great importance in understanding how resulting developmental defects occur. Type II transmembrane serine proteases (TTSPs) make up a family of cell surface-associated proteases that play important roles in the development and homeostasis of a number of mammalian tissues. Aberrant expression of TTSPs is linked to several human disorders, including deafness, heart and respiratory disease and cancer. However, the mechanism by which these proteases function remains unknown. The ecdysone-responsive Stubble TTSP of Drosophila serves as a good model in which to study the functional mechanism of the TTSP family. The Stubble protease interacts with the intracellular Rho1 (RhoA) pathway to control epithelial development in imaginal discs. The Rho1 signaling pathway regulates cellular behavior via control of gene expression and actin cytoskeletal dynamics. However, the mechanism by which the Stubble protease interacts with the Rho1 pathway to control epithelial development, in particular leg imaginal disc morphogenesis, has yet to be elucidated. The Stubble protein consists of several conserved domains. One approach to a better understanding of the mechanism of action of Stubble in regulating Rho1 signaling is to define which of the conserved domains within the protease are required for proper function. Sequence analysis of twelve recessive Stubble mutant alleles has revealed that the proteolytic domain is essential for proper function. Alleles containing mutations which disrupt regions of the protease domain necessary for protease activation or substrate binding, as well as those with deletions or truncations that remove some portion of the proteolytic domain, result in defective epithelial development in vivo. In contrast, mutations in other regions of the Stubble protein, including the disulfide-knotted and cytoplasmic domains, were not observed. Another important step for defining the connection between Stubble and Rho1 signaling is to identify a Stubble target that acts as an upstream regulator of the Rho1 pathway. We performed a genetic screen in which 97 of the 147 Drosophila non-olfactory and non-gustatory G-protein-coupled receptors (GPCRs), a family of proteins that has been shown to be protease-activated and to activate Rho1 signaling, were tested for interactions with a mutant allele of Stubble. We found 4 genomic regions uncovering a total of 7 GPCRs that interact genetically when in heterozygous combination with a Stubble mutant. Further analysis of these genes is necessary to determine if any of these GPCRs is targeted by Stubble during activation of the Rho1 pathway.
Show less - Date Issued
- 2008
- Identifier
- CFE0002285, ucf:47875
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0002285
