Summary Distinct molecular pathways govern the differentiation of CD8+ effector T cells into memory or exhausted T cells during acute and chronic viral infection but these are not well-studied in humans. Here, we employed an integrative systems immunology approach to identify transcriptional commonalities and differences between virus-specific CD8+ T cells from patients with persistent and spontaneously resolving hepatitis C virus (HCV) infection during the acute phase. We observed dysregulation of metabolic processes during early persistent infection that were linked to changes in expression of genes related to nucleosomal regulation of transcription, T cell differentiation, and the inflammatory response and correlated with subject age, sex and the presence of HCV-specific CD4+ T cell populations. These early changes in HCV-specific CD8+ T cell transcription preceded the overt establishment of T cell exhaustion, making this signature a prime target in the search for the regulatory origins of T cell dysfunction in chronic viral infection.

While human leukocyte antigen B57 (HLA-B57) is associated with the spontaneous clearance of hepatitis C virus (HCV), the mechanisms behind this control remain unclear. Immunodominant CD8؉ T cell responses against the B57-restricted epitopes comprised of residues 2629 to 2637 of nonstructural protein 5B (NS5B 2629-2637 ) (KSKKTPMGF) and E2 541-549 (NTRPPLGNW) were recently shown to be crucial in the control of HCV infection. Here, we investigated whether the selection of deleterious cytotoxic T lymphocyte (CTL) escape mutations in the NS5B KSKKTPMGF epitope might impair viral replication and contribute to the B57-mediated control of HCV. Common CTL escape mutations in this epitope were identified from a cohort of 374 HCV genotype 1a-infected subjects, and their impact on HCV replication assessed using a transient HCV replicon system. We demonstrate that while escape mutations at residue 2633 (position 5) of the epitope had little or no impact on HCV replication in vitro, mutations at residue 2629 (position 1) substantially impaired replication. Notably, the deleterious mutations at position 2629 were tightly linked in vivo to upstream mutations at residue 2626, which functioned to restore the replicative defects imparted by the deleterious escape mutations. These data suggest that the selection of costly escape mutations within the immunodominant NS5B KSKKTPMGF epitope may contribute in part to the control of HCV replication in B57-positive individuals and that persistence of HCV in B57-positive individuals may involve the development of specific secondary compensatory mutations. These findings are reminiscent of the selection of deleterious CTL escape and compensatory mutations by HLA-B57 in HIV-1 infection and, thus, may suggest a common mechanism by which alleles like HLA-B57 mediate protection against these highly variable pathogens.

Ring-between-ring (RBR) E3 ligases have been implicated in autoimmune disorders and neurodegenerative diseases. The functions of many RBR E3s are poorly defined, and their regulation is complex, involving post-translational modifications and allosteric regulation with other protein partners. The functional complexity of RBRs, coupled with the complexity of the native ubiquitination reaction that requires ATP and E1 and E2 enzymes, makes it difficult to study these ligases for basic research and therapeutic purposes. To address this challenge, we developed novel chemical probes, ubiquitin C-terminal fluorescein thioesters UbMES and UbFluor, to qualitatively and quantitatively assess the activity of the RBR E3 ligase PARKIN in a simple experimental setup and in real time using fluorescence polarization. First, we confirmed that PARKIN does not require an E2 enzyme for substrate ubiquitination, lysine selection, and polyubiquitin chain formation. Second, we confirmed that UbFluor quantitatively detects naturally occurring activation states of PARKIN caused by Ser phosphorylation (pPARKIN) and phosphorylated ubiquitin (pUb). Third, we showed that both pUb and the ubiquitin-accepting substrate contribute to maximal pPARKIN ubiquitin conjugation turnover. pUb enhances the transthiolation step, whereas the substrate clears the pPARKIN∼Ub thioester intermediate. Finally, we established that UbFluor can quantify activation or inhibition of PARKIN by structural mutations. These results demonstrate the feasibility of using UbFluor for quantitative studies of the biochemistry of RBR E3s and for high-throughput screening of small-molecule activators or inhibitors of PARKIN and other RBR E3 ligases.

HECT E3 ubiquitin ligases (~28 known) are associated with many phenotypes in eukaryotes, and are important drug targets. However, assays used to screen for small molecule inhibitors of HECT E3s are complex and require ATP, Ub, E1, E2, and HECT E3 enzymes, producing three covalent thioester enzyme intermediates E1~Ub, E2~Ub, and HECT E3~Ub (~ indicates thioester bond), and mixtures of polyubiquitin chains. To reduce the complexity of the assay, we developed a novel class of fluorescent probes UbFluor that act as mechanistically relevant pseudosubstrates of HECT E3s. These probes undergo a direct transthiolation reaction with the catalytic cysteine of HECT E3s, producing the catalytically active HECT E3~Ub thioester accompanied by fluorophore release. Thus, a fluorescence polarization assay can continuously monitor UbFluor consumption by HECT E3s, and changes in UbFluor consumption rendered by biochemical point mutations or small molecule modulation of HECT E3 activity.