P04-41. Kinetics of Antibody Neutralization and Viral Evolution Following Envelope Vaccination in SIV-infected Rhesus Monkeys

Poster presentation. Conclusion: Our results indicate that env vaccination is associated with an accelerated development of autologous neutralizing antibodies. These antibodies were focused at least in part on the V1 region of Env, since there was selective pressure in this region of the envelope for the evolution of mutational changes

The triple combination of tenofovir, emtricitabine and efavirenz shows synergistic anti-HIV-1 activity in vitro: a mechanism of action study

<p>Abstract</p> <p>Background</p> <p>Tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and efavirenz (EFV) are the three components of the once-daily, single tablet regimen (Atripla) for treatment of HIV-1 infection. Previous cell culture studies have demonstrated that the double combination of tenofovir (TFV), the parent drug of TDF, and FTC were additive to synergistic in their anti-HIV activity, which correlated with increased levels of intracellular phosphorylation of both compounds.</p> <p>Results</p> <p>In this study, we demonstrated the combinations of TFV+FTC, TFV+EFV, FTC+EFV, and TFV+FTC+EFV synergistically inhibit HIV replication in cell culture and synergistically inhibit HIV-1 reverse transcriptase (RT) catalyzed DNA synthesis in biochemical assays. Several different methods were applied to define synergy including median-effect analysis, MacSynergy^®II and quantitative isobologram analysis. We demonstrated that the enhanced formation of dead-end complexes (DEC) by HIV-1 RT and TFV-terminated DNA in the presence of FTC-triphosphate (TP) could contribute to the synergy observed for the combination of TFV+FTC, possibly through reduced terminal NRTI excision. Furthermore, we showed that EFV facilitated efficient formation of stable, DEC-like complexes by TFV- or FTC-monophosphate (MP)-terminated DNA and this can contribute to the synergistic inhibition of HIV-1 RT by TFV-diphosphate (DP)+EFV and FTC-TP+EFV combinations.</p> <p>Conclusion</p> <p>This study demonstrated a clear correlation between the synergistic antiviral activities of TFV+FTC, TFV+EFV, FTC+EFV, and TFV+FTC+EFV combinations and synergistic HIV-1 RT inhibition at the enzymatic level. We propose the molecular mechanisms for the TFV+FTC+EFV synergy to be a combination of increased levels of the active metabolites TFV-DP and FTC-TP and enhanced DEC formation by a chain-terminated DNA and HIV-1 RT in the presence of the second and the third drug in the combination. This study furthers the understanding of the longstanding observations of synergistic anti-HIV-1 effects of many NRTI+NNRTI and certain NRTI+NRTI combinations in cell culture, and provides biochemical evidence that combinations of anti-HIV agents can increase the intracellular drug efficacy, without increasing the extracellular drug concentrations.</p

Abstract 104: ATF7IP does not alter the substrate specificity of the lysine methyltransferase SETDB1

Abstract The histone methyltransferase (HMT) SETDB1 is a strong candidate oncogene in melanoma and lung carcinomas. SETDB1 methylates lysine 9 of histone 3 (H3K9), utilizing S-adenosylmethionine (SAM) as the methyl donor. Its activity has been shown to be regulated by its partner protein ATF7IP, and here we examine the contribution of ATF7IP to the in vitro activity and substrate specificity of SETDB1. SETDB1 and ATF7IP were co-expressed in Sf21 insect cells and 1:1 stoichiometric complexes were purified for comparison against apo-SETDB1 enzyme. We employed both radiometric flashplate-based and MALDI mass spectrometry assays to follow methylation on histone 3 15-mer peptides where lysine 9 was either unmodified, mono-, or di-methylated. These two methods provide orthogonal readouts on H3K9 methylation: the radiometric flashplate-based assay employs 3H-labeled SAM and provides quantitation of labeled, methylated peptide. The MALDI mass spectrometry assay offers quantitation of consumption and accumulation of each individual H3K9 methylation state (H3K9me0, me1, me2, and me3). Results show that both apo-SETDB1 and SETDB1:ATF7IP complex catalyzed both monomethylation and dimethylation of H3K9 peptide substrates, but were unable to perform H3K9 trimethylation. While ATF7IP did not impact the substrate methylation profile, the activity of the complex was lower than apo-SETDB1 by a factor of 4. This difference was due to a decrease in the value of kcat as the substrate KM values were comparable between apo-SETDB1 and the SETDB1:ATF7IP complex. ATF7IP therefore does not alter SETDB1's substrate specificity. H3K9 monomethylation and dimethylation by SETDB1 occurred in a distributive manner and was unaffected by the presence of ATF7IP. This finding is important as H3K9 can be methylated by HMTs other than SETDB1 and a distributive mechanism would allow for interplay between multiple HMTs on H3K9me1 and H3K9me2. The results presented here indicate that ATF7IP does not alter substrate specificity of SETDB1 in vitro, and though it decreases SETDB1's catalytic activity, our data points to a nonenzymatic function of ATF7IP in regulating SETDB1 function. Citation Format: Aravind Basavapathruni, Jodi Gureasko, Margaret Porter Scott, P. Ann Boriack-Sjodin, Timothy J. Wigle, Thomas V. Riera, Robert A. Copeland. ATF7IP does not alter the substrate specificity of the lysine methyltransferase SETDB1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 104. doi:10.1158/1538-7445.AM2015-104</jats:p