CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

176 FLOW VIROMETRY REVEALS PATIENT VARIATIONS IN HIV PROTEASE THAT CORRELATE WITH FITNESS Michał Bonar, Caroline Tabler, John Tilton Case Western Reserve University, Cleveland, OH, USA Background: Nearly all current methods for analyzing viruses interrogate large numbers of viruses, obscuring viral heterogeneity that may play a critical role in infection, immunity, and pathogenesis. Direct flow cytometric detection of viruses (“flow virometry”) enables high-throughput analysis of individual viruses. In this study, we wanted to evaluate HIV-1 protease activity within individual viruses and under physiological conditions of assembly and budding. Methods: We developed a Förster resonance energy transfer (FRET) construct consisting of the mUKG and mKOk fluorescent pair, separated by a protease cleavage site and linked to the viral accessory protein Vpr that is incorporated into virions via a non-covalent interaction with the Gag p6 protein. Viruses were monitored using a FACSAria II flow cytometer. Results: The FRET protease (PR) substrate is incorporated into viruses and undergoes cleavage in the presence of active protease, resulting in a colorimetric change that can be detected by flow virometry. Processing of the FRET PR substrate correlated extremely well (R2=0.93, p<0.0001) with processing of Gag by western blot over a wide range of protease inhibitor (PI) concentrations, indicating it is an accurate surrogate of protease activity within virions. Next, we generated viruses from patient-derived infectious molecular clones (IMCs) that incorporated the FRET PR reporter. We found that processing of the FRET PR reporter varies significantly in patients, with 35.0-59.8% of viruses demonstrating processing. Importantly, the extent of processing observed by flow virometry correlated with the infectivity of the viruses on JLTRG reporter cell lines (R2=0.29, p<0.0001). The FRET PR reporter also correctly identified PI drug resistance in 2 of 13 IMCs and was able to detect differences in budding efficiency for several Gag and PR mutant viruses. The assay is highly reproducible (Z-factor of 0.88) indicating it has robust sensitivity to probe mutant phenotypes or screen for drugs affecting the precursor or mature protease. Conclusion: Flow virometry represents a powerful technique for monitoring viral heterogeneity with important implications for immunity and pathogenesis. This study is the first demonstration that flow virometry can (1) monitor functional viral activities such as protease processing, (2) detect interpatient viral heterogeneity that correlates with fitness, (3) identify drug-resistant viruses, and (4) identify mutants resulting in alterations in viral budding or maturation.

to explore SAMHD1 levels in GALT. We found twofold higher median values of SAMHD1-mRNAs in PBMC compared to those measured in GALT paired samples (p=0.04). Moreover, SAMHD1 was expressed more strongly than MxB, HERC5, and IRF7 in virologically suppressed HIV-1-infected patients (p<0.0001 for all the analyses), and positive correlations were found between SAMHD1, MxB, HERC5, and IRF7, levels. Conclusion: Taken together these findings indicate that SAMHD1 is more strongly expressed than the classical IFN-related genes, increased during antiretroviral therapy and correlated with several ISGs in HIV-1-infected patients on HAART.

Poster Abstracts

175 SCHLAFEN 14 (SLFN14) INHIBITS TRANSCRIPTION OF HIV-1 BY TARGETING P-TEFB Chen Shumin , Shan Cen Chinese Academy of Sciences, Beijing, China

Background: SLFN14 belongs to Schlafen (SLFN) family involved in important functions, such as the control of cell proliferation, induction of immune responses, and the regulation of viral replication. Positive transcription elongation factor b (P-TEFb), which comprises cyclin-dependent kinase 9 (CDK9) kinase and cyclin T subunits, is an essential kinase complex for productive elongation of transcription of HIV genomes.We recently identified Schlafen 14 (SLFN14) that interacts with HIV-1 essential host cellular proteins complex P-TEFb and inhibits HIV-1 transcription. Methods: In our study, western blot, RT-PCR, Co-IP and ChIP-qPCR were performed to examine the effect of SLFN14 upon viral replication and transcription, and investigate its interaction with P-TEFb. Results: We found that over-expression of SLFN14 significantly reduced Gag expression and viral mRNA level, while silencing endogenous SLFN14 promoted viral replication. Using a HIV LTR reporter assay, we demonstrated that SLFN14 significantly impaired tat-mediated transcription activity of viral promoter, resulting in an inhibitory effect on productive elongation of viral mRNA transcription. Further mechanistic studies revealed that SLFN14 interact with P-TEFb dependent of N-terminus region of SLFN14. Furthermore, overexpression of SLFN14 reduced the phosphorylation of Ser2 (Ser2P) of RNA polymerase II (Pol II) CTD on HIV-1 promoter. Conclusion: This work provided evidence for the first time showing that SLFN14 interacts with P-TEFb and inhibits HIV-1 transcription. The study sheds a light on the role of SLFN14 in negative regulation of HIV-1 transcription, and may provide a novel strategy for treatment of HIV-1 infection.

177 THE HIV-1 ANTISENSE PROTEIN ASP IS A NOVEL STRUCTURAL PROTEIN OF THE VIRAL ENVELOPE Yvonne Affram, Juan C. Zapata, Marzena Pazgier, Maria Iglesias-Ussel, Krishanu Ray, Olga Latinovic, Fabio Romerio University of Maryland, Baltimore, MD, USA Background: The negative strand of the HIV-1 genome encodes a 189-aa, highly hydrophobic antisense protein (ASP) with no known homologs. Humoral and cellular immune responses against ASP in HIV-1 patients demonstrate that it is expressed in vivo, but its role in viral replication remains unknown. We


CROI 2019

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