CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

Conclusion: Our results demonstrate that exposed guanosines in the HIV-2 RNA leader are critical for genome packaging. Furthermore, not all guanosines in the RNA leader are equal; cumulative interactions between Gag and multiple specific sites direct genome packaging. 179 DETECTION AND SEQUENCING OF ASP TRANSCRIPTS DURING EARLY HIV-1 INFECTION Antonio Mancarella 1 , Brian T. Foley 2 , Giampietro Corradin 3 , Giuseppe Pantaleo 1 , Cecilia Graziosi 1 1 Lausanne University Hospital, Lausanne, Switzerland, 2 Los Alamos National Laboratory, Los Alamos, NM, USA, 3 University of Lausanne, Lausanne, Switzerland Background: The asp gene is an antisense ORF encoding for the putative HIV-1 AntiSense protein Asp. The existence of an asp gene product in HIV-infected individuals is still controversial. Detection of asp antisense RNA by conventional RT-PCR is hampered by RT non-specific priming, which does not allow to assess whether amplified products are from asp transcripts, env transcripts, or env genomic RNA. Thus, no information is yet available on the expression of this gene in patients. Using a modified RT-PCR methodology, we detected asp RNA in CD4+ T cells from patients during early infection, with high viremia and naïve to suppressive ART. We hereby report the first nucleotide sequences of asp transcripts in HIV-infected individuals. Methods: CD4+ T cells isolated from three patients infected with subtype B were stimulated with anti-CD3/CD28. Reverse transcription was performed using the biotinylated specific antisense primer, followed by cDNA purification by streptavidin-coated magnetic beads, PCR amplification with patient-specific asp primers, cloning and sequencing. Results: Expression of asp RNA was detected in CD4 + T cells from three HIV- infected individuals during early infection following stimulation with anti-CD3/ CD28. In contrast, no expression was detected in unfractionated PBMCs, either resting or stimulated, or in unstimulated CD4+ T cells. Sequence analysis of asp transcripts from cells (26 clones) and of the corresponding env on the plus strand in serum (30 clones) indicate that the dominant length variants in the asp RNA pool in cells are the same as those found in genomic env in serum. In asp RNA transcripts from cells, the complete canonical (i.e. as in HXB2) asp ORF was identified in 20% of the clones, in two of three patients. Clones carrying shorter or longer ORFs (non-canonical stop codons) were also identified, in regions that were either hypervariable or characterized by a variability to some extent in the corresponding env sequence on the plus strand. Conclusion: Our results show that asp RNA is easily detectable in stimulated CD4+ T cells isolated from untreated patients during early infection. Our data also represent the first nucleotide sequences obtained in patients for asp, demonstrating that it may well be expressed in those HIV-1 lineages in which the asp ORF is present. The finding of a new HIV antigen would represent an important step in our understanding of HIV pathogenesis and perhaps open new perspectives in the development of novel anti-HIV drugs and vaccines. 180 IDENTIFICATION OF HIV-1 ENV MUTATIONS THAT ENHANCE ENTRY INTO MACAQUE CELLS Jeremy I. Roop, Noah R. Cassidy , Jesse Bloom, Julie M. Overbaugh Fred Hutchinson Cancer Research Center, Seattle, WA, USA Background: Although rhesus macaques are a central animal model for HIV-1 vaccine development research, most transmission/founder (T/F) HIV-1 strains replicate poorly in macaque cells. This species-specific restriction has been attributed to the activity of host specific restriction factors, as well as a single nonsynonymous mutation in macaque CD4 that inhibits binding by HIV-1 Envelope (Env). Recent research efforts employing either laboratory evolution or structure guided design strategies have discovered several Env mutations in gp120 that enhance binding of macaque CD4 by T/F HIV-1 viruses. Additional screens have the potential to reveal mutations that further enhance HIV-1 infection of macaque cells, thereby facilitating the use of macaques for vaccine development. Methods: In order to identify additional Env mutations that promote infection of macaque cells, we utilized deep mutational scanning to screen thousands of Env point mutants for those that enhance usage of macaque receptors. Results: We identified many uncharacterized amino acid mutations in both the HR1 and HR2 regions of gp41 that enhance infection of macaque cells by up to 30-fold over wild type residues. These mutations also increased infection of cells bearing human CD4 and CCR5, suggesting a mechanism of action involving a general enhancement of infection kinetics, rather than a specific

studied ASP expression in chronically infected myeloid and lymphoid cell lines, and its impact on viral fitness. Methods: For intracellular and nuclear staining, we used BD Cytofix/Cytoperm and eBioscience FoxP3 kits. Data were acquired on Millipore Guava flow cytometer and analyzed with FlowJo, or Zeiss LSM 800 confocal microscope and analyzed with Zen Blue. Virion-capture assays used antibodies immobilized on Protein G Dynabeads. For Fluorescence Correlation Spectroscopy (FCS) we used ISS Q2 confocal microscope and ISS VistaVision. Results: We analyzed two myeloid and seven lymphoid HIV-1 infected cell lines using a monoclonal antibody (324.6) against an epitope mapping between two putative transmembrane domains of ASP, and we detected ASP in the nucleus of all infected cell lines. Confocal microscopy evidenced a polarized nuclear distribution of ASP, preferentially in areas with low content of suppressive epigenetic marks. Reactivation of HIV-1 with PMA led to translocation of ASP to the cytoplasm and cell membrane. Cell surface detection of ASP without cell permeabilization shows extracellular exposure of the ASP epitope recognized by 324.6. Surface staining with antibodies to ASP and gp120 showed that the two proteins co-localize (Manders overlap coefficient 76%), suggesting that ASP might be incorporated in the membrane of budding virions. Indeed, 324.6 captured HIV-1 particles with efficiency similar to anti-gp120 VRC01. Also, FCS showed that the binding efficiency of 324.6 to single virions in solution was ~28%. Thus, these two assays demonstrate the presence of ASP on the surface of mature HIV-1 virions. Finally, we produced HIV-1NL4-3-derived viruses with single-nucleotide mutations that introduce early stop codons in the ASP open reading frame without changing the amino acid sequence of Env on the opposite strand. ASP-deficient viruses displayed a ~50% reduction in replication rate compared to wildtype virus. Conclusion: ASP is expressed on the surface of productively infected cells, and is a structural protein found in the envelope of mature HIV-1 virions. Further, ASP expression promotes viral replication. Thus, ASP may represent a new target for therapeutic or preventive vaccines. 178 UNPAIRED GUANOSINES IN THE HIV LEADER RNA DIRECT HIV GENOMIC RNA PACKAGING Chijioke Umunnakwe , Sheikh A. Rahman, Alice Duchon, Yang Liu, Olga A. Nikolaitchik, Jianbo Chen, Wei-Shau Hu, Vinay K. Pathak National Cancer Institute, Frederick, MD, USA Background: HIV-1 and HIV-2, the causative agents of AIDS, package two copies of their RNA genome into one viral particle. It remains unclear how the viral protein Gag specifically selects viral RNA from a large pool of cellular mRNAs. HIV-1 Gag protein has been shown to bind exposed guanosines in the leader region of its RNA and these interactions are thought to be important for packaging. Currently, little is known about HIV-2 RNA packaging mechanisms. To test the hypothesis that exposed guanosines in the HIV-2 leader RNA play a key role in RNA packaging, we mutated guanosines predicted to be exposed in nine regions of the leader RNA and examined the effects of these mutations on genome packaging. Methods: We visualized HIV-2 RNA in individual viral particles using single- virion analysis, an assay developed in our lab that can detect viral genomes at single RNA sensitivity. In this system, viral particles are visualized by tagging Gag proteins with cerulean fluorescent protein (CeFP), whereas RNA is visualized based on specific interactions between bacterial protein BglG tagged with yellow fluorescent protein (YFP) and RNA stem loop sequences (BSL) engineered into the HIV-2 constructs. RNA packaging efficiency is determined by quantifying the proportion of Gag particles (CeFP signal) that contain HIV RNA (YFP signal). Results: HIV-2 RNA with wild-type sequences were packaged efficiently: ~95% of viral particles contained viral RNA. In contrast, mutating guanosines in all nine regions of the HIV-2 leader RNA resulted in loss of RNA packaging: only ~10% of viral particles contained viral RNA. Thus, exposed guanosines are critical for HIV-2 RNA packaging. To identify specific regions crucial for RNA packaging, we examined additional mutants in which individual regions or multiple regions were mutated. We identified one primary region and three secondary regions that are important for RNA packaging. Mutation of any individual region did not significantly affect genome packaging. However, mutating the primary region together with any of the secondary regions caused defects in genome packaging and we identified the specific set of guanosines that were responsible for the most severe defect.

Poster Abstracts


CROI 2019

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