Kinetics of HIV-1 Latency Reversal Quantified on the Single-Cell Level Using a Novel Flow-Based Technique.

UNLABELLED: HIV-1 establishes a pool of latently infected cells early following infection. New therapeutic approaches aiming at diminishing this persisting reservoir by reactivation of latently infected cells are currently being developed and tested. However, the reactivation kinetics of viral mRNA and viral protein production, and their respective consequences for phenotypical changes in infected cells that might enable immune recognition, remain poorly understood. We adapted a novel approach to assess the dynamics of HIV-1 mRNA and protein expression in latently and newly infected cells on the single-cell level by flow cytometry. This technique allowed the simultaneous detection of gagpol mRNA, intracellular p24 Gag protein, and cell surface markers. Following stimulation of latently HIV-1-infected J89 cells with human tumor necrosis factor alpha (hTNF-α)/romidepsin (RMD) or HIV-1 infection of primary CD4(+) T cells, four cell populations were detected according to their expression levels of viral mRNA and protein. gagpol mRNA in J89 cells was quantifiable for the first time 3 h after stimulation with hTNF-α and 12 h after stimulation with RMD, while p24 Gag protein was detected for the first time after 18 h poststimulation. HIV-1-infected primary CD4(+) T cells downregulated CD4, BST-2, and HLA class I expression at early stages of infection, proceeding Gag protein detection. In conclusion, here we describe a novel approach allowing quantification of the kinetics of HIV-1 mRNA and protein synthesis on the single-cell level and phenotypic characterization of HIV-1-infected cells at different stages of the viral life cycle. IMPORTANCE: Early after infection, HIV-1 establishes a pool of latently infected cells, which hide from the immune system. Latency reversal and immune-mediated elimination of these latently infected cells are some of the goals of current HIV-1 cure approaches; however, little is known about the HIV-1 reactivation kinetics following stimulation with latency-reversing agents. Here we describe a novel approach allowing for the first time quantification of the kinetics of HIV-1 mRNA and protein synthesis after latency reactivation or de novo infection on the single-cell level using flow cytometry. This new technique furthermore enabled the phenotypic characterization of latently infected and de novo-infected cells dependent on the presence of viral RNA or protein.

Changes in HIV-1 Capsid Stability Induced by Common Cytotoxic-T-Lymphocyte-Driven Viral Sequence Mutations.

UNLABELLED: HIV-1-infected individuals with protective HLA class I alleles exhibit better control of viremia and slower disease progression. Virus control in these individuals has been associated with strong and potent HIV-1-specific cytotoxic-T-lymphocyte (CTL) responses restricted by protective HLA alleles, but control of viremia also occurs in the presence of selected CTL escape mutations. CTL escape mutations restricted by protective HLA class I molecules are frequently located in the conserved p24 Gag sequence of HIV-1 that encodes the conical capsid core and have been suggested to reduce viral replication capacity. In this study, the consequences of well-described CTL-associated p24 Gag sequence mutations for HIV-1 capsid stability were assessed using a cyclosporine (CsA) washout assay. The frequently occurring HLA-B57- and HLA-B27-associated CTL escape mutations T242N and R264K resulted in delayed capsid uncoating, suggesting modulation of capsid stability. The described compensatory mutations L268M and S173A observed in R264K viruses reconstituted the capsid-uncoating half-time. Interestingly, capsid stability was correlated with infectivity. Taken together, these data demonstrate that CTL-driven escape mutations within p24 Gag restricted by protective HLA class I alleles have a significant impact on capsid stability that might contribute to the persistent control of viral replication observed despite viral escape from CTL responses. IMPORTANCE: Sequence mutations within p24 Gag selected by CTL responses restricted by protective HLA class I alleles have been associated with reduced viral fitness. However, the precise mechanisms underlying the reduced viral replication capacity and lower viral loads associated with these mutations remain unclear. Here, we demonstrate that dominant HLA-B27-associated CTL escape mutations within HIV-1 capsid lead to enhanced capsid rigidity, providing a possible mechanism for the reduced viral fitness of these variants.