CROI 2017 Abstract e-Book

Abstract eBook

Oral Abstracts

15 EARLY CYTOPLASMIC UNCOATING IS NECESSARY FOR INFECTIVITY OF HIV-1 João I. Mamede , Gianguido C. Cianci, Meegan R. Anderson, Thomas Hope Northwestern Univ, Chicago, IL, USA

Background: After cell fusion, HIV delivers its conical capsid into the cytoplasm. The disassembly of the capsid is termed uncoating and is critical to infection. The understanding of the kinetics, dynamics, and localization of uncoating of infectious particles has been eluded by the unavoidable presence of non-infectious particles. The timing of uncoating remains under discussion with some models proposing that uncoating happens early and other models suggest that the intact capsid docks at the nuclear pore. These different hypotheses formed from diverse assays, lack the information for the kinetics and localization of uncoating of productively infectious viral particles. Methods: We used live-cell fluorescent imaging of intravirion fluid phase markers to determine the integrity of the HIV conical capsid core. To visualize dynamic changes in capsid integrity and composition, we utilized the HIV-iGFP construct. During viral maturation of HIV-iGFP, the GFP is liberated from Gag. A minority population of the free GFP is trapped in the capsid, while the remaining free GFP is located outside of the capsid. With this technique, the loss of the fluid phase GFP occurs in two steps: with fusion and upon the loss of capsid core integrity. Live-cell microscopy of HIV-iGFP virions with a viral complex marker such as Vpr or Integrase allows for the timing of these two steps. Through viral challenge with less than one virion per cell we are able to connect viral particle phenotype to infection. Results: The time between fusion and capsid integrity loss, for both HIV and VSV-G mediated fusion, in tissue culture and primary cells (macrophages and T cells), is approximately 30 minutes. Also, capsid integrity loss occurs entirely in the cytoplasm and co-relates to a big loss of p24CA. With our lowMOI approach, we were able to image individual particle uncoating that produces a viable infection and differentiate between different rates of uncoating. This analysis revealed that all particles associated with cellular infection showed changes in capsid integrity ~29 minutes. We were also able to halt uncoating by blocking specific steps of reverse transcription, linking uncoating to the occurrence of the first-strand transfer step. Conclusion: Together, these observations validate the early cytoplasmic uncoating model. Our live-imaging assay has the ability to follow uncoating at the single infectious particle level providing unprecedented insights into the early steps of HIV infection. 16 ECCENTRIC VIRAL GENOMIC RNA AND INTEGRASE ARE PREMATURELY DEGRADED IN TARGET CELLS Michaela Madison 1 , Dana Q. Lawson 1 , Jennifer Elliott 1 , Ayse N. Ozanturk 2 , Pratibha Chowdary Koneru 3 , James R. Fuchs 3 , Mamuka Kvaratskhelia 3 , Sebla B. Kutluay 1 1 Washington Univ in St. Louis, St. Louis, MO, USA, 2 Bilkent Univ, Ankara, Turkey, 3 Ohio State Univ, Columbus, OH, USA Background: Recent evidence indicates that inhibition of HIV-1 integrase (IN) binding to the viral RNA genome yields aberrant particles, in which the viral ribonucleoprotein complexes (vRNPs) are eccentrically localized outside the protective capsid core. These particles are non-infectious and blocked at an early reverse transcription stage in target cells. However, the basis of this reverse transcription defect is unknown, given that eccentric particles appear to retain all components necessary for reverse transcription, i.e. a dimeric viral RNA genome primed with tRNA-Lys, functional RT and normal levels of NC-RNA complexes. In addition, apart from reverse transcription products, the fates of viral core components in cells infected with eccentric particles have not been studied to date. Methods: To determine why the eccentric virus particles, generated with class II IN mutants such as R269A/K273A or ALLINI treatments of the WT virus, fail to support reveres transcription in target cells, we have monitored the fates of eccentric particle components in infected cells. To this end, we took advantage of a previously developed elaborate approach in which we biochemically tracked multiple core components in infected cells. Results: In this study, we show that in target cells eccentrically localized vRNPs and IN are prematurely degraded, whereas reverse transcriptase remains active and stably associated with capsid cores. Importantly, we show that in addition to viral RNAs, IN protein is also mislocalized in particles. Remarkably, the aberrantly shaped capsid cores in the eccentric particles can efficiently saturate and be degraded by a restricting TRIM5 protein, suggesting that TRIM5 recognition does not require the presence of fully formed cores. Conclusion: We propose that IN-RNA interactions allow for packaging of both vRNPs and IN within the protective capsid cores to ensure subsequent reverse transcription and productive infection in target cells. 17 DYNAMICS OF NUCLEAR ENVELOPE ASSOCIATION AND NUCLEAR IMPORT OF HIV-1 COMPLEXES Ryan C. Burdick , Jianbo Chen, Jaya Sastri, Wei-Shau Hu, Vinay K. Pathak NCI, Frederick, MD, USA Background: During productive infection, HIV-1 must enter the nucleus to integrate its DNA into host genome. However, many aspects of nuclear import process are poorly understood because they have been difficult to study using biochemical or imaging assays, and have not been visualized in living cells. Methods: To elucidate critical HIV-1 post-entry events, we analyzed viral complexes labeled with yellow fluorescent protein-tagged APOBEC3F (A3F-YFP), a virion-incorporated host restriction factor, or Vpr-integrase-YFP fusion protein (IN-YFP) using live-cell imaging. Results: We first examined the association between HIV-1 viral complexes and nuclear envelope (NE) by live-cell imaging, and observed that most contacts are transient (<5 sec) and very few are stable (>20 min), suggesting only a subset of viral complexes is competent to stably dock with the NE. Most HIV-1 complexes forming transient interactions with the NE may be encountering regions that did not have any nuclear pore complexes (NPCs). We found that HIV-1-NE stable association is compromised when capsid is mutated to form viral cores that are unstable (K203A) or hyperstable (E128A/R132A), or when host protein Nup358 is knocked down. These findings indicate that HIV-1 capsid and host Nup358 play critical roles in forming the stable associations. To better understand the process of nuclear import, we captured for the first time the translocation of 21 HIV-1 complexes from the cytoplasm to the nucleus and their nuclear movements. Viral complexes labeled with A3F-YFP or IN-YFP behaved similarly, suggesting that the fluorescently tagged proteins did not influence their movements. They exhibited similar long and variable residence times at the NE (1.7 ± 1.7 hours), indicating that the viral core may be undergoing extensive dissociation and/or conformational rearrangements which require an extended period of association with the NPC prior to nuclear import. After import, the viral complexes exhibited a fast phase (<9 min) as they moved away from the point of entry, followed by a slow phase for the rest of the observation time, suggesting that they quickly associate with chromatin and/or other nuclear macromolecules. The viral complexes moved away from the nuclear point of entry, but remained in the nuclear periphery. Conclusion: The tracking of individual HIV-1 complexes provides insights into the dynamics of HIV-1 NE association, nuclear import, and movement inside the nucleus. 18 CYPA REGULATES HIV-1 ACCESS TO AN FG-GUIDED NUCLEAR ENTRY PATHWAY Guangai Xue 1 , Vineet KewalRamani 1 , Shih Lin Goh 2 , Hyun Jae Yu 1 , Anna Gres 3 , KyeongEun Lee 1 , Stefan G. Sarafianos 3 , Jeremy Luban 2 1 NCI, Frederick, MD, USA, 2 Univ of Massachusetts, Worcester, MA, USA, 3 Univ of Missouri, Columbia, MO, USA Background: The choreography of virus infection at the cellular level involves a successive series of host factor interactions to drive the viral replication program. The role of HIV-1 capsid (CA) in the early steps of replication is includes cytoplasmic trafficking, regulation of reverse transcription, interaction with the nuclear pore complex (NPC), and chromatin access during integration. Two host factor interfaces within CA, the CPSF6 and Cyclophilin A (CypA) binding sites, are critical to these steps. The CA binding site for CPSF6 shows selective interaction with protein motifs containing an FG-dipeptide, which is abundant in one-third of nucleoporins. FG-nucleoporins (FG-Nups) maintain the nuclear diffusion barrier and provide docking sites for nuclear transport receptors (NTRs). Notably, Nup153, a makes specific contacts with the same pocket in CA via an FG-containing motif. We sought to understand the respective roles of CPSF6 or CypA binding sites in CA in regulating nuclear entry. Methods: We performed a small interfering RNA (siRNA) screen targeting all known human nucleoporins to assess effects on Wild-type (WT) versus N74D and P90A HIV-1 infection. N74D and P90A are mutations that respectively impair either CPSF6 or CypA binding to CA. We used commercially available pools of siRNAs in the screening. Host factor specificity was confirmed by restoration of expression with siRNA-resistant isoforms of mRNA. Reverse transcription products were measured via qPCR.

Oral Abstracts

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CROI 2017