CROI 2016 Abstract eBook

Abstract Listing

Oral Abstracts

138

Characterization of 2 CCR5 Tropic HIV-1 Viruses Exhibiting High Env Content Sean P. O’Brien 1 ;Terra M. Ireland 1 ; JulianW. Bess 1 ; James D. Roser 1 ; Elena N. Chertova 1 ; Gregory Q. Del Prete 2 ; Brandon Keele 2 ; Jeffrey Lifson 2 1 Leidos Biomed Rsr, Inc, Frederick, MD, USA; 2 Frederick Natl Lab, Frederick, MD, USA

Background: Generating high Env content HIV-1 virions with trimers in their native state would be useful for Env characterization and as a potential immunogen source for various vaccine strategies. While a truncation in the tail of the simian immunodeficiency virus greatly increases Env incorporation, attempts to transfer this approach to HIV have failed. Here we used a long-term culture approach to generate and characterize high Env HIV from the HIV-1 BaL and HIV-1 ADA viral lineages. Methods: The A66-R5 T-cell line was infected with HIV-1 BaL and HIV-1 ADA . Limiting dilution biological clones were generated at various time points post infection and virions derived from these clones were characterized for Env content. Gag and Env sequence analysis of the biological clones were used to identify potential molecular correlates of high-Env expression/incorporation. An infectious molecular clone (IMC) was generated for HIV-1 BaL , which was used to identify viral determinants of Env incorporation. Results: We isolated a biological clone of HIV-1 ADA constitutively producing virus with ~52 Env trimers per virion. Sequence analysis revealed a frame shift mutation causing a premature stop codon just past the membrane spanning domain of gp41. We also isolated a biological clone of HIV-1 BaL constitutively expressing virus with ~56 Env trimers per virion. Sequence analysis revealed an intact and full length Envelope but with three selected mutations in Gag (R15K, P66S, I75L). An infectious molecular clone (IMC) of this virus was generated and transfection produced virus was used to infect the A66-R5 cell line. Twenty-five limiting dilution biological clones were generated from the infected culture. Env content of the clones was highly heterogeneous ranging from~4 to ~64 trimers per virion and was stable over time in culture. Conclusions: We were able to develop two productively infected cell-lines producing CCR5 tropic HIV-1 BaL and HIV-1 ADA with levels of virion Env incorporation 4-8 fold higher than typical HIV-1 strains. Tail-truncation and possible Gag-Env interactions could explain the viral contributions to high-Env. Subsequent biological clones derived from cultures infected with an IMC of HIV-1 BaL exhibited a wide range in average trimers per virion, suggesting cellular parameters combine with viral genetics to significantly impact Env content. 139 Nuclear Entry of HIV Requires Reshaping of Integrase Multimers Frauke Christ 1 ; Lieve Dirix 2 ; Doortje Borrenberghs 2 ; Flore DeWit 1 ; Jolien Blokken 1 ; Susana Rocha 1 ; Jelle Hendrix 2 ; Zeger Debyser 1 ; Johan Hofkens 2 1 Katholieke Universiteit Leuven, Leuven, Belgium; 2 Katholieke Universiteit Leuven, Heverlee, Belgium Background: The detailed mechanism of HIV nuclear entry is only partially understood. Studies are typically limited to averaged information, whereas the heterogeneous pool of infecting virus particles requires single virus analysis. Technological limitations and the complexity of the process are the main obstacles to elucidate the nuclear import of viral particles. Using HIV trans-incorporating fluorescently labeled integrase (IN) we established complementary microscopy methods to visualize the dynamics of IN during nuclear entry of single PICs. Methods: We established state-of-the-art technology to accurately determine the fluorescence intensity of the single IN complex in the infected cell. The intensity directly relates to the number of IN molecules; hence the stoichiometry of each single complex can be studied in the context of its relative location in the cell. Furthermore, we used a complementary method that employs FRET via acceptor photobleaching to quantitatively study fluorescently labeled IN oligomers. The obtained FRET ratio is a measure of the interaction between the IN subunits and provides information about conformational changes. Through combination of both methods we investigated the dynamic interplay between the different IN subunits during HIV nuclear import. Results: Nuclear entry was associated with a significant reduction in the number of IN molecules in the PIC in HeLaP4 cells, a T cell line as well as in primary CD4+ T cells implying a general mechanism. Upon nuclear entry, but before chromatin tethering, the interaction with the host factor LEDGF/p75 induced an increase in the FRET ratio of the IN multimer. Addition of LEDGINs, small molecule inhibitors of the IN-LEDGF/p75 interaction, during virus production, prematurely increased the FRET ratio in the virions. Upon infection these pretreated viral particles were refractory to a reduction in the number of IN molecules per PIC and defective for nuclear entry. Conclusions: The composition of the PIC undergoes dynamic changes in both IN stoichiometry and affinity of IN-IN interaction. Our data indicate a role of the nuclear pore as a molecular filter, only tolerating PICs with a specific composition/size to pass through. Upon nuclear entry LEDGF/p75 induces rearrangements in the PIC complex allowing integration. In conclusion, the ability to study PIC composition at the single virus level provides the long-sought approach to unravel nuclear import of HIV-1 and to study the MOA of inhibitors such as LEDGINs. 140 1970s HIV-1 Genomes Reveal the Early History of the North American HIV/AIDS Epidemic Background: The precise origins of the western hemispheric HIV/AIDS pandemic remain contentious. Phylogenetic studies based upon HIV-1 sequences sampled after (in most cases long after) the recognition of AIDS in 1981 have suggested a lengthy cryptic period of circulation in the U.S. and Canada throughout the 1970s, and an even older presence in the Caribbean. However, no comprehensive evolutionary genomic analysis of HIV-1 in North America closer to the putative emergence of the virus in the 1970s has been possible since no non-African HIV-1 complete genomes from that era have been sequenced to date. Methods: Here, we combine approaches frommolecular biology, phylogenetics, and historical analysis to investigate the timing and geography of the emergence of HIV-1 group M subtype B in the U.S. We designed an approach to overcome the challenge of recovering HIV-1 genetic material from older serum specimens in which viral RNA template material is highly fragmented and at low concentration. We recovered near-full length HIV-1 genome sequences from eight U.S. patient serum samples from 1978-79 (comprising eight of the nine earliest HIV-1 group M genomes sequenced worldwide to date) and also from the individual known as ‘Patient 0’. We then used maximum likelihood and Bayesian ‘relaxed molecular clock’ phylogenetic and phylogenomic methods to infer timing and geography. Results: This early genomic ‘snapshot’ of HIV-1 reveals that by 1978-79 the HIV-1 epidemic in the U.S. already exhibited extensive genetic diversity – particularly in New York City (NYC) – having emerged around 1970 from a founder virus drawn from an older and more diverse subtype B epidemic in the Caribbean. Moreover, there is neither biological nor historical evidence that Patient 0 was the primary case in the U.S. or for subtype B as a whole. Conclusions: Our findings reveal a series of key founder events in the genesis of the subtype B epidemic, with a single virus moving from the African epicenter of HIV-1 group M to the Caribbean by 1967 [1964-1970], a single virus moving from the Caribbean to establish an epidemic in NYC by 1971 [1969-73], and yet another single virus moving from there to San Francisco by ~1975 (but with extensive geographical mixing in the U.S. and beyond shortly thereafter). We discuss, in the context of these insights into the early spread of HIV/ AIDS in North America, the genesis and persistence of beliefs about ‘Patient 0’ that are unsupported by scientific data. 141 Background: The primary cells in the Central Nervous System (CNS) infected with SIV and HIV are macrophages/microglia. It has been hypothesized that these cells serve as a reservoir of SIV and HIV infection within the CNS, but recent data suggest they can leave the CNS and join lymphatics in some case with virus. Understanding the biology of these cells and their traffic out of the CNS is critical to assess this hypothesis. Methods: To asses the fate of brain macrophages we introduced fluorescent Super Paramagnetic Iron Oxide Nanoparticles (SPIONs) into the cisterna magna which are taken up by phagocytic cells in the brain. This was done in six SIV infected and two normal macaques. We also trace labeled the CSF with dextran-red for short periods. We introduced the SPIONs and follow their fate detecting also their point of exit from the CNS. Michael Worobey 1 ;Thomas D.Watts 1 ; Richard A. McKay 2 ;Timothy Granade 3 ; Beryl A. Koblin 4 ;Walid Heneine 3 ; HaroldW. Jaffe 3 1 Univ of Arizona, Tucson, AZ, USA; 2 Univ of Cambridge, Cambridge, UK; 3 CDC, Atlanta, GA, USA; 4 New York Blood Cntr, New York, NY, USA SIV-Infected Brain Macrophages Leave the Central Nervous System Xavier Alvarez 1 ; Cecily Midkiff 1 ; Andrew Lackner 1 ; Kenneth C.Williams 2 1 Tulane Natl Primate Rsr Cntr, Covington, LA, USA; 2 Boston Coll, Chestnut Hill, MA, USA

Oral Abstracts

52

CROI 2016