Abstract

Human immununodeficiency virus (HIV) infection is the leading cause of death due to viral infections worldwide. In the absence of an effective vaccine or consistent male condom use, there is a clear need for female-controlled preventatives such as topical vaginal microbicides. Recent attention has been focused on developing natural antimicrobial peptides, as anti-retroviral microbicides. Increasing evidence suggests that cationic antimicrobial peptides such as defensins are effective HIV-1 inhibitors. Human alpha- and beta-defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins – 18 residue cyclic peptides that are potent HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of thetadefensins in rhesus macaques involves the post-translational ligation of two nonapeptides, each derived from a 12-residue "demidefensin" precursor. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time, to our knowledge, that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human thetadefensin genes are transcribed, we used aminoglycosides to read-through the premature termination codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. The ability to reawaken retrocyclins genes from their 7 million years of slumber using aminoglycosides could provide a novel way to secure enhanced resistance to HIV-1 infection. Our studies on retrocyclin reveal that they are potential candidates to develop as topical vaginal microbicides to prevent sexual transmission of HIV-1. Mucosal surfaces of the vagina are the portals for heterosexual transmission of HIV-1 and therefore play a fundamental role in the pathogenesis of primary infection. In a search for direct biological evidence for the role of human vaginal fluid in innate host defense, we characterized the anti-HIV-1 function of cationic polypeptides within minimally manipulated vaginal fluid. In our studies, we revealed that vaginal fluid confers intrinsic anti-HIV-1 properties against both X4 and R5 strains of HIV-1, and could protect against HIV-1 infection and reduce proviral genome integration in organotypic cultures of human cervicovaginal tissue. The majority of this activity was contained in the cationic polypeptide fraction, and the depletion of cationic polypeptides using a selective cation-exchange resin ablated most of the intrinsic activity against HIV-1. By adding the cationic polypeptide fraction to depleted vaginal fluid, we were able to restore activity against HIV-1. Using a proteomic approach, we identified 18 cationic polypeptides within vaginal fluid, nearly all of which are either known antimicrobials or have other purported roles in host defense. Interestingly, physiologic concentrations of 13 of the cationic polypeptides were alone not active against HIV-1, yet in concert they partially restored the anti-HIV-1 activity of cation-depleted vaginal fluid. These results suggest that synergism between cationic polypeptides is complex and full anti-HIV-1 activity likely involves the aggregate of the cationic peptides and proteins in the acidic human vaginal fluid. Interestingly, retrocyclins retained complete anti-HIV-1 activity in the presence of human vaginal fluid. Therefore expression of retrocyclin peptides can help activate the natural defense mechanism against HIV-1. We next investigated the regulation of expression of retrocyclin (pseudo)gene. We identified a putative interferon response cluster upstream of the retrocyclin gene. The activity of this cluster was upregulated when treated with IFN-B although to a modest extent. Interestingly, the cluster also contained the binding site for an Interferon Consensus Sequence Binding Protein (ICSBP), a known repressor of the IFN inducible genes. Deletion of the ICSBP site or addition of a known inhibitor of ICSBP resulted in the increase in the activity of the cluster, indicating a role for ICSBP in the negative regulation of expression of retrocyclins. Collectively our data suggest that the expression of this ancestral gene is tightly regulated in both a positive and negative manner via the IFN response pathway.

Notes

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Graduation Date

2009

Semester

Summer

Advisor

Cole, Alexander M.

Degree

Doctor of Philosophy (Ph.D.)

College

College of Medicine

Department

Burnett School of Biomedical Sciences

Format

application/pdf

Identifier

CFE0002777

URL

http://purl.fcla.edu/fcla/etd/CFE0002777

Language

English

Release Date

July 2014

Length of Campus-only Access

None

Access Status

Doctoral Dissertation (Open Access)

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