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RETROCYCLIN RC-101 OVERCOMES CATIONIC MUTATIONS ON THE HEPTAD REPEAT 2 OF HIV-1 GP41
Title
RETROCYCLIN RC-101 OVERCOMES CATIONIC MUTATIONS ON THE HEPTAD REPEAT 2 OF HIV-1 GP41.
Creator
Fuhrman, Christopher, Cole, Alexander, University of Central Florida
Abstract / Description
Retrocyclin RC-101, a θ-defensin with lectin-like properties, potently inhibits infection by many HIV-1 subtypes by binding to the heptad repeat (HR)-2 region of gp41 and preventing six-helix bundle formation. In the present study, we used in silico computational exploration to identify residues of HR2 that interacted with RC-101 and then analyzed the HIV-1 Sequence Database at LANL for residue variations in the HR1 and HR2 segments that could plausibly impart in vivo resistance. Docking...
Show moreRetrocyclin RC-101, a θ-defensin with lectin-like properties, potently inhibits infection by many HIV-1 subtypes by binding to the heptad repeat (HR)-2 region of gp41 and preventing six-helix bundle formation. In the present study, we used in silico computational exploration to identify residues of HR2 that interacted with RC-101 and then analyzed the HIV-1 Sequence Database at LANL for residue variations in the HR1 and HR2 segments that could plausibly impart in vivo resistance. Docking RC-101 to gp41 peptides in silico confirmed its strong preference for HR2 over HR1, and implicated residues crucial for its ability to bind HR2. We mutagenized these residues in pseudotyped HIV-1 JR.FL reporter viruses, and subjected them to single round replication assays in the presence of 1.25-10ug/ml RC-101. Except for one mutant that was partially resistant to RC-101, the other pseudotyped viruses with single-site cationic mutations in HR2 manifested absent or impaired infectivity or retained wild-type susceptibility to RC-101. Overall, these data suggest that most mutations capable of rendering HIV-1 resistant to RC-101 will also exert deleterious effects on the ability of HIV-1 to initiate infections - an interesting and novel property for a potential topical microbicide.
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Date Issued
2007
Identifier
CFE0001707, ucf:47333
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0001707
Antimicrobial Peptide Resistance and Immunomodulation by HIV-1 gp41
Title
Antimicrobial Peptide Resistance and Immunomodulation by HIV-1 gp41.
Creator
Wood, Matthew, Cole, Alexander, Chai, Karl, Teter, Kenneth, Parkinson, Christopher, University of Central Florida
Abstract / Description
Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide (ENF), are peptides designed to mimic, and thereby competitively inhibit, the viral fusion protein gp41. An exception to this is a class of cyclic, cationic, antimicrobial peptides known as ?-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity....
Show moreFusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide (ENF), are peptides designed to mimic, and thereby competitively inhibit, the viral fusion protein gp41. An exception to this is a class of cyclic, cationic, antimicrobial peptides known as ?-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the ?-defensin analog RC-101 is being developed as a microbicide to prevent sexual transmission of HIV-1. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development.Partial drug resistance due to genetic variability within HIV-1 presents a major hurdle in microbicide development. Drug-resistance mutations, whether naturally occurring or resulting from selection during treatment, often apply to many drugs in a particular class. Combining different drug classes into a single microbicide should provide greater protection against the growing variability observed in HIV. Our work has identified the beneficial effects of combining the fusion inhibitor RC-101 and the RT inhibitor CSIC to prevent transmission of clinically isolated and drug-resistant HIV-1.Several aspects of HIV-1 virulence and pathogenesis are mediated by the envelope protein gp41. Additionally, peptides derived from the gp41 ectodomain have been shown to induce chemotaxis in monocytes and neutrophils. While this chemotactic activity has been characterized, it is not known how these peptides could be produced under biological conditions. Our findings demonstrate that the epithelial serine protease matriptase efficiently cleaves the gp41 HR1 region at conserved residues into a chemotactic peptide.Here, we present evidence that advances our understanding of resistance to peptide entry inhibitors, reveals a potential benefit to combining specific drugs in an antiviral microbicide, and identifies a pathway by which HIV-1 may generate peptides to exploit host immunity. This work thereby facilitates improved methods in countering drug resistance and the development of new antiviral approaches to prevent HIV-1 transmission. Additionally, we have revealed basic mechanistic evidence that shed light on our current understanding of HIV-1 infection. Specifically, our focus on gp41 provides much needed insight into its role in membrane fusion, drug susceptibility, and modification of host responses.
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Date Issued
2014
Identifier
CFE0005560, ucf:50286
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005560
Evolutionary Relationships Among Staphylococci and the Prevention of Staphylococcus aureus Nasal Colonization
Title
Evolutionary Relationships Among Staphylococci and the Prevention of Staphylococcus aureus Nasal Colonization.
Creator
Lamers, Ryan, Cole, Alexander, Parkinson, Christopher, Chai, Xinqing, Moore, Sean, University of Central Florida
Abstract / Description
Staphylococcus is a significant cause of human infection and mortality, worldwide. Currently, there are greater than 60 taxa within Staphylococcus, and nearly all are pathogenic. The collective potential for virulence among species of Staphylococcus heightens the overall clinical significance of this genus and argues for a thorough understanding of the evolutionary relationships among species. Within Staphylococcus, aureus is the most common cause of human infection, where nasal carriage of...
Show moreStaphylococcus is a significant cause of human infection and mortality, worldwide. Currently, there are greater than 60 taxa within Staphylococcus, and nearly all are pathogenic. The collective potential for virulence among species of Staphylococcus heightens the overall clinical significance of this genus and argues for a thorough understanding of the evolutionary relationships among species. Within Staphylococcus, aureus is the most common cause of human infection, where nasal carriage of this bacterium is a known risk factor for autoinfection. The predisposition to infection by nasal carriers of S. aureus, and the ease with which strains are transferred between individuals, suggests that nasal carriage is a major vector for the transmission of virulent strains throughout the community. This hypothesis, however, has not been assessed in any great detail to identify the genetic relationships between clinical isolates of S. aureus and those strains being carried asymptomatically throughout the community. Also lacking within this field is a unified and robust estimate of phylogeny among species of Staphylococcus.Here, we report on a highly unified species phylogeny for Staphylococcus that has been derived using multilocus nucleotide data under multiple Bayesian and maximum likelihood approaches. Our findings are in general agreement with previous reports of the staphylococcal phylogeny, although we identify multiple previously unreported relationships. Regardless of methodology, strong nodal support and high topological agreement was observed with only minor variations in results between methods. Based on our phylogenetic estimates, we propose that Staphylococcus species can be evolutionarily clustered into 15 groups, and six species groups. In addition, our more defined phylogenetic analyses of S. aureus revealed strong genetic associations between both nasal carriage strains and clinical isolates. Genetic analyses of hypervariable regions from virulence genes revealed that not only do clinically relevant strains belong to identical genetic lineages as the nasal carriage isolates, but they also exhibited 100% sequence similarity within these regions. Our findings indicate that strains of S. aureus being carried asymptomatically throughout the community via nasal colonization are genetically related to those responsible for high levels of infection and mortality.Due to nasal carriage of S. aureus being a risk factor for autoinfection, standardized preoperative decolonization has become a major consideration for the prevention of nosocomial infection. Toward this end, we have identified the macrocyclic ?-defensin analogue RC-101 as a promising anti-S. aureus agent for nasal decolonization. RC-101 exhibited bactericidal effects against S. aureus in both epithelium-free systems, and ex vivo models containing human airway epithelia. Importantly, RC-101 exhibited potent anti-S. aureus activities against all strains tested, including USA300. Moreover, RC-101 significantly reduced the adherence, survival, and proliferation of S. aureus on human airway epithelia without any noted cellular toxicity or the induction of a proinflammatory response. Collectively, our findings identify RC-101 as a potential preventative of S. aureus nasal colonization.
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Date Issued
2011
Identifier
CFE0004124, ucf:49092
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004124
RETROCYCLIN, A POTENT HIV-1 ENTRY INHIBITOR.
Title
RETROCYCLIN, A POTENT HIV-1 ENTRY INHIBITOR.
Creator
Venkataraman, Nitya, Cole, Alexander, University of Central Florida
Abstract / Description
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...
Show moreHuman 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 theta-defensins 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 theta-defensin 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-β 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.
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Date Issued
2009
Identifier
CFE0002777, ucf:48104
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0002777