CROI 2016 Abstract eBook
196 Capsid Plays a Distinct Role in HIV Infection of TCR or TLR2-Activated CD4+ T Cells Kimyata Valere 1 ; Jian Ding 2 ;Theresa Chang 1 1 Rutgers Univ, Newark, NJ, USA; 2 PH Rsr Inst, Newark, NJ, USA
Background: HIV-1 capsid (CA) proteins play a crucial role in multiple stages of HIV infection including nuclear import. However, the function of the CA is not fully understood in primary resting CD4+ T cells. We have previously shown that TLR2 activation enhances HIV infection by promoting nuclear import in resting CD4+ T cells. In this study, we examined the role of the HIV CA protein and their analogs in TLR2-mediated enhancement of HIV infection in resting CD4+ T cells. Methods: VSV-G pseudotyped luciferase reporter virus with wild-type (WT) CA or CA mutants were prepared by transfecting HEK293T cells. PBMCs were isolated from buffy coat of healthy donors by Histopaque gradient centrifugation. Primary resting CD4+ T cells were purified by negative selection from PBMCs, and exposed to pseudotyped virus for 2 h. Cells were stimulated with TLR2 ligand (Pam 3 CSK 4 ) or immobilized anti-CD3 Ab in the presence of IL2. HIV infection was determined by measuring luciferase activity 3 days after infection. Cell pellets were collected 12 and 48 h after infection. Total DNA was isolated and analyzed for HIV late reverse-transcribed (RT) and 2LTR products by quantitative real- time PCR. Results: E45A, E71A, and ΔcPPT mutations did not affect HIV infection of primary CD4+ T cells in response to TLR2 or TCR activation. Conversely, P38A, K70A, and L136D mutants were not infectious in TCR- or TLR2-activated CD4+ T cells. T54A/N57A, Q63A/Q67A, E128A/R132A, R143A, and Q219A mutants were infectious in CD4+ T cells in response to TCR activation, but not TLR2. The levels of late RT products were reduced in TLR2-activated CD4+ T cells infected by N74D mutant, but not other mutants. The levels of 2LTR circles, markers for HIV nuclear import, were also reduced in N74D mutant-infected cells. 2LTR circles were diminished in TLR2-activated CD4+ T cells with infection by P38A,T54A/N57A, Q63A/Q67A, K70A, E128A/R132A, L136D, and R143A mutants, suggesting a crucial role for these residues in nuclear import. Infection by Q219A led to greater levels of 2LTR circles compared to WT in TLR2-activated cells, suggesting that Q219 may be important for viral integration. Conclusions: Mutations in the CA have differential impacts on HIV infection of TLR2- and TCR-activated CD4+ T cells. CA mutations altered 2LTR production suggesting its role in HIV nuclear import in primary CD4+ T cells in response to TLR2 activation. Our study provides an insight for developing antiretroviral agents targeting HIV CA proteins. 197 Consequences of p24 Gag Mutations for Capsid Stability and Viral Fitness Philipp Schommers 1 ; Gloria Martrus 2 ; Urte Matschl 2 ;Tom J. Hope 3 ; Gerd Fätkenheuer 1 ; Marcus Altfeld 2 1 Univ of Cologne, Cologne, Germany; 2 Heinrich Pette Inst, Hamburg, Germany; 3 Northwestern Univ, Feinberg Sch of Med, Chicago, IL, USA
Background: HIV-1-infected individuals encoding for specific HLA alleles, such as B27 and B57, exhibit better control of viremia. This has been associated with strong and highly potent HIV-1-specific CTL responses restricted by these alleles, forcing the virus to evade immune pressure through the selection of CTL-escape mutations with reduced viral fitness. While immunodominant HIV-1-specific CTLs in HLA-B 57+ subjects are directed against the TW10 epitope within p24 Gag, CTLs in HLA-B27+ subjects are directed against the KK10 p24 Gag epitope, resulting in CTL escape mutations (B57+: T242N, B27+ R264K and L268M) in these epitopes. The goal of our studies was to determine the consequences of these mutations within HIV capsid for viral uncoating and fitness. Methods: An HIV-1 NL4-3 deltaEnv strain expressing GFP (HIV-GFP) in the place of Nef was modified using site-directed mutagenesis to encode for the p24 Gag mutations T242N, R264K and L268M, respectively. The capsid stability was determined using a Cyclosporin A (CsA) washout assay. Owl-monkey-kindey (OMK) cells, in which Trim-Cylcophilin A (CypA) inhibits HIV-infection by stopping the uncoating of the intracellular capsid, were infected with HIV-GFP variants in the presence of CsA. CsA blocks Trim-CypA in OMK cells. Washing-out CsA in a time-dependent manner allowed quantification of HIV Gag uncoating kinetics. Viral fitness was assessed in parallel by infecting primary PBMCs with the described HIV-GFP variants. Results: The HIV p24 Gag mutations T242N, L268M and R264K resulted in significant reduced viral fitness. Compared to wildtype NL4-3, the T242N, L268M and R264K mutations showed an decrease of 35%, 17% and 85% of infected PBMCs, respectively (p=0.048, p=0.014 and p=0.028). Significant differences were observed in uncoating kinetics between WT NL4-3 and T242N mutants (mean: 115 min SEM+/- 6min vs. 132 min +/- 5 min, p=0.03) as well as between WT NL4-3 and R264K mutants (120 min: +/- 6 min vs. 164 min +/- 4 min, p=<0.0001). In contrast, no difference was observed between WT NL4-3 and L268Mmutants. (Fig.1) Conclusions: HIV-NL4-3 strains containing the HLA-B57- and HLA-B27-associated p24 Gag capsid mutations T242N and R264K uncoat significantly slower compared to the wildtype virus, indicating enhanced capsid rigidity and altered interactions with CypA. No significant differences in uncoating kinetics were observed for the L268Mmutation, in line with previous data indicating that this mutation restores the viral fitness of R264K viruses.
198 The HIV Env Signal Peptide Impacts the Glycosylation and Antigenicity of gp120 Jason Yolitz 1 ; Catherine Schwing 1 ; DonaldVan Ryk 1 ; DanlanWei 2 ; Fatima Nawaz 1 ; Katija Jelicic 1 ; Claudia Cicala 1 ; Anthony S. Fauci 1 ; James Arthos 3 1 NIAID, NIH, Bethesda, MD, USA; 2 NIAID, NIH, Bethesda, MD, USA; 3 NIH, Bethesda, MD, USA
Background: Transmission of HIV across mucosal tissues involves a complex yet inefficient series of events that are not yet fully understood. The HIV-1 envelope protein (Env) of early-transmitting viruses has been found to present distinct transmission signatures. One such signature involves a reduced number of potential N-linked glycosylation sites, underscoring the importance of post-translational modifications in transmission fitness. Recently, an additional transmission signature has been identified in the signal peptide of Env. This signature involves the over-representation of basic residues at a specific position in the signal peptide. We investigated the potential impact of this signal peptide signature on gp120 glycosylation and antigenicity. Methods: Two recombinant Envs were constructed, one derived from a chronic isolate that lacks the signal peptide signature and a second from a transmitting isolate that includes the signal peptide signature. Chimeric Envs were also constructed in which the two signal peptides were swapped. All four gp120s were probed with glycan-, structure- and interaction-specific probes in a surface plasmon resonance binding assay. Results: The signature found in the signal peptide of Env influences qualitative aspects of Env glycosylation that in turn affect the structure and antigenicity of Env in a major way. The signal peptide impacts Env’s affinity for DC-SIGN, a lectin receptor expressed on dendritic cells that is believed to be involved in mucosal transmission. Additionally, affinity for the monoclonal antibody 17b, which recognizes the CD4-induced confirmation of Env is also altered. Conclusions: We conclude that the HIV Env signal peptide is an important determinant of Env processing, folding and antigenicity. These observations may aid in the development of Env based HIV vaccines.
Made with FlippingBook - Online catalogs