CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

Results: When comparing the gp120-CD4-R5 complex in R5 tropic virus to the gp120-CD4-X4 complex in X4 tropic virus, the higher charge on the V3 associated with X4 tropism led to greater thermodynamic stability via favorable electrostatic interactions in the gp120-CD4-X4 complex than V3 that was tropic to R5. Cross-sectional analysis of X4 tropic versus R5 tropic sequences identified a significant correlation between the BA of gp120 to CD4+X4 (p<0.001) but not between the BA of gp120 to CD4+R5 (p=0.35). In longitudinal analysis, switching tropism from R5 to X4 was associated with a significant decline in BA between gp120 to CD4+R5 by 52%-76% and a concomitant increase in BA between gp120 to CD4+X4 by 0.4%-29.3%. Conclusions: Structural modeling demonstrated that X4 tropism is highly associated with the BA of gp120 to CD4+X4, while R5 tropism is not associated with the BA between gp120 and CD4+R5. Further, when a viral population switches from R5 to X4 tropism, the thermodynamic stability of the gp120-CD4-X4 complex considerably increases while the thermodynamic stability of the gp120-CD4-R5 complex decreases. Together, these data provide new insights into the mechanisms of viral co-receptor interaction and co-receptor tropism switching. 220 Selective Cell-Free or Cell-to-Cell HIV-1 Infection by gp41 Cytoplasmic Tail Mutants Natasha D. Durham 1 ; Benjamin K. Chen 1 1 Icahn School of Medicine at Mount Sinai, New York, NY, US; 2 Icahn School of Medicine at Mount Sinai, New York, NY, US Background: The gp41 transmembrane subunit of the HIV-1 envelope (Env) has a cytoplasmic tail (CT) ~150 amino acids (aa) long. The CT has several functions during the viral life cycle, including the i) endocytosis of cell-surface Env ii) packaging Env into viral particles and iii) controlling the fusogenic potential of Env. Full truncation of the gp41 CT (∆CT144) does not directly impair viral fusion and can generate infectious virus in a cell-type dependent manner. Smaller deletions result in non-infectious virus particles, although Env is still expressed and packaged. Only the full truncation, ∆CT144, has been tested in the context of cell-to-cell infection. We therefore set out to systematically examine which domains of the CT are required for cell-to-cell infectivity in comparison to those needed for cell-free infection. Methods: We constructed a series of truncation mutants that remove three major structural motifs in the gp41 CT, LLP-1, LLP-3 and LLP-2. Mutants were examined for HIV-1 envelope expression levels on the surface of transfected T-cells and total Env levels packaged onto virus particles. We used flow-cytometry to assess the single-round infectivity of cell-free and cell-associated virus in MT4 and primary CD4+ T-cells. Results: Small truncations ≤ 43 aa that remove LLP-1 severely impaired the infectivity of cell-free virus, while cell-to-cell infection remained ~50% as infectious as WT HIV-1. Similar selective deficiency in cell-free but not cell-to-cell infection was observed with the LLP-3 point mutants. Conversely, large truncations ≥ 93 aa severely impaired cell-to-cell infectivity while maintaining infectious viral particles. Intermediate truncations (59-90 aa) showed profound impairment of both modes of infection. Conclusions: We identified gp41 CT mutants with striking selective deficiencies in cell-free but not cell-to-cell infectivity, or vice versa. Our results indicate that functional properties of Env required for cell-free and cell-to-cell infection are genetically distinct. These differences may reflect a different intrinsic regulatory influence of the CT on cell-associated versus particle-associated Env, or differential interaction with host or viral proteins. Our findings highlight the positive and negative potential of the gp41 CT in regulating cell-free and cell-to-cell HIV-1 infection. We will use these mutants in future studies to define the contributions of cell-free and cell-to-cell infection in vivo , and to study one mode of infection in isolation. 221 Analysis of Viral Evolution in the Blood Reveals Potential Insight Into the Evolution of Macrophage Tropism Maria M. Bednar 1 ; LiHua Ping 1 ; Kathryn Arrildt 1 ; Christa Sturdevant 2 ; Sarah B. Joseph 1 ; Laura Kincer 1 ; Celia LaBranche 2 ; David Montefiori 2 ; Myron Cohen 3 ; Ronald Swanstrom 1 1 University of North Carolina, Durham, NC, US; 2 Duke University, Durham, NC, US; 3 University of North Carolina, Chapel Hill, NC, US Background: Understanding the evolution of entry phenotype of HIV-1 is essential for our understanding of HIV-1 pathogenesis, latency, and disease progression . The use of a more precise assay to define entry phenotype based on the ability of HIV-1 to use low levels of CD4 for efficient entry has clarified the relationship of viruses that have evolved to infect macrophages as distinct from R5 viruses that predominantly replicate in T cells. We have used this assay to identify infrequent examples of macrophage-tropic (M-tropic) viruses and partially M-tropic viruses (intermediate) in cerebral spinal fluid and genital tract. However the prevalence of M-tropic viruses in other compartments, such as the blood, remains unknown. We set out to determine if M-tropic viruses ever reach a point of systemic infection. Methods: Viral RNA was isolated from blood plasma samples from viremic subjects infected with either subtype B or subtype C HIV-1, and with CD4+ T cell counts of <100 cell/mm 3 . Individual env gene were isolated, cloned and analyzed for receptor usage using Affinofile cells, in order to determine the viral entry phenotype. Susceptibility of pseudoviruses to soluble CD4 was also evaluated. Results: 18 subtype B and 20 subtype C late-stage infected subjects were examined. As expected for late stage subjects, we identified X4 lineages in over 50% of the subjects. To date, no examples of efficient use of low CD4 for entry (M-tropic virus) have been found in the blood. However, we do occasionally see a level of infection on CD4-low cells that is marginally greater than that of the typical R5 T cell-tropic virus. This intermediate phenotype has also been seen in the CSF and genital tract. This new group of viruses showed increased sensitivity to soluble CD4 that is comparable to macrophage-tropic viruses. Conclusions: Using the definition of M-tropic virus as the ability to utilize low levels of CD4, leads to the conclusion that these variants are rare and are potentially limited to specific compartments of the body. The appearance of intermediate phenotypes in the blood argues that there may be some evolution toward macrophage tropism taking place in compartments that shed virus into the blood late in disease. The high level of sensitivity to soluble CD4 among intermediate viruses suggests that the evolution to M-tropism is at least a two-step process where the viruses first become more sensitive to interaction with CD4 followed by more efficient fusion. 222 Mechanistic Differences in Interactions of HIV-1 and HIV-2With Dendritic Cells Suzanne D. Kijewski ; Hisashi Akiyama; Caitlin Miller; Nora P. Ramirez; Rahm Gummuluru Boston University School of Medicine, Boston, MA, US Background: HIV-2 infection has been restricted predominantly to West Africa; in contrast HIV-1 has spread rapidly and accounts for 95% of all HIV infections globally. Interestingly, HIV-2 infected individuals maintain low viral loads and have lower mortality rates than HIV-1 infected individuals. The mechanistic basis for viral control and slower progression to AIDS in HIV-2 infected individuals remains unclear. We hypothesized that reduced interaction of HIV-2 with CD169, the primary HIV-1 attachment factor on myeloid dendritic cells (DCs) that targets captured virus particles to the trans infection pathway, plays an important role in its restricted pathogenesis. Methods: We constructed an HIV-2 proviral plasmid that encodes GFP between MA and CA and flanked by protease cleavage sites (HIV-2-iGFP), such that infectious virions contain GFP, similar to the previously described HIV-1-iGFP proviral clone ( J. Virol. 2007, 81:12596 ). To examine interactions of HIV-2 with CD169, THP1 cells constitutively expressing CD169 (THP1/CD169) were pulsed with HIV-1iGFP or HIV-2iGFP virions and virus capture in the presence or absence of α CD169 blocking antibodies was determined by flow cytometry. Intra-cellular localization of HIV-1-iGFP or HIV-2-iGFP within mature DCs was determined by immunofluorescence microscopy. To determine access of HIV-2 to the CD169 mediated trans infection pathway, mature DCs were exposed to GFP-expressing single-cycle of replication competent HIV-2 virus pseudotyped with HIV-2 Env, and then co-cultured with autologous CD4 + T cells.

Poster Abstracts

210

CROI 2015