Computational Design of a PDZ Domain Peptide Inhibitor that Rescues CFTR Activity

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel mutated in patients with cystic fibrosis (CF). The most prevalent CFTR mutation, ΔF508, blocks folding in the endoplasmic reticulum. Recent work has shown that some ΔF508-CFTR channel activity can be recovered by pharmaceutical modulators (“potentiators” and “correctors”), but ΔF508-CFTR can still be rapidly degraded via a lysosomal pathway involving the CFTR-associated ligand (CAL), which binds CFTR via a PDZ interaction domain. We present a study that goes from theory, to new structure-based computational design algorithms, to computational predictions, to biochemical testing and ultimately to epithelial-cell validation of novel, effective CAL PDZ inhibitors (called “stabilizers”) that rescue ΔF508-CFTR activity. To design the “stabilizers”, we extended our structural ensemble-based computational protein redesign algorithm to encompass protein-protein and protein-peptide interactions. The computational predictions achieved high accuracy: all of the top-predicted peptide inhibitors bound well to CAL. Furthermore, when compared to state-of-the-art CAL inhibitors, our design methodology achieved higher affinity and increased binding efficiency. The designed inhibitor with the highest affinity for CAL (kCAL01) binds six-fold more tightly than the previous best hexamer (iCAL35), and 170-fold more tightly than the CFTR C-terminus. We show that kCAL01 has physiological activity and can rescue chloride efflux in CF patient-derived airway epithelial cells. Since stabilizers address a different cellular CF defect from potentiators and correctors, our inhibitors provide an additional therapeutic pathway that can be used in conjunction with current methods

The RNA-dependent RNA polymerase of the influenza A virus

The influenza A virus causes a highly contagious respiratory disease that significantly impacts our economy and health. Its replication and transcription is catalyzed by the viral RNA polymerase. This enzyme is also crucial for the virus, because it is involved in the adaptation of zoonotic strains. It is thus of major interest for the development of antiviral therapies and is being intensively studied. In this article, we will discuss recent advances that have improved our knowledge of the structure of the RNA polymerase and how mutations in the polymerase help the virus to spread effectively among new hosts

A mechanism for prime-realignment during influenza A virus replication

The influenza A virus genome consists of eight segments of single-stranded RNA. These segments are replicated and transcribed by a viral RNA-dependent RNA polymerase (RdRp) that is made up of the influenza virus proteins PB1, PB2 and PA. To copy the viral RNA (vRNA) genome segments and the complementary RNA (cRNA) segments, the replicative intermediate of viral replication, the RdRp must use two promoters and two different de novo initiation mechanisms. On the vRNA promoter, the RdRp initiates on the 3’ terminus, while on the cRNA promoter the RdRp initiates internally and subsequently realigns the nascent vRNA product to ensure that the template is copied in full. In particular the latter process, which is also used by other RNA viruses, is not understood. Here we provide mechanistic insight into prime-realignment during influenza virus replication and show that it is controlled by the priming loop and a helix-loop-helix motif of the PB1 subunit of the RdRp. Overall, these observations advance our understanding of how the influenza A virus initiates viral replication and amplifies the genome correctly.This work was funded by Wellcome Trust grants 098721/Z/12/Z and 206579/Z/17/Z (to A.J.W.T.V.), grant 825.11.029 from the Netherlands Organization for Scientific Research (to A.J.W.T.V.), and an Erasmus+ mobility grant (to J.O.)

The role of the PB2 627-domain in influenza A virus polymerase function

The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase composed of the subunits PA, PB1 and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding andthe PA endonuclease domains are both involved in cap-snatching, but the role of the auxiliary PB2 627-domain, implicated in host range restriction of influenza A viruses, is still poorly understood. Here we use structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627-domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerasewith PB1 and PA. Furthermore, we show that a recombinant viral polymerase lacking the PB2 627-domain is able to carry out cap-snatching, cap-dependent transcription initiation as well as cap-independent ApG dinucleotide extension in vitro, indicating that the PB2 627-domain of the influenza virus RNApolymerase is not involved in core catalytic functions of the polymerase. However, in a cellular context, the 627-domain is essential for both transcription and replication. In particular, we show that the PB2 627-domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627-domain in transcription and replication of the influenza virus RNA genome

The role of the PB2 627-domain in influenza A virus polymerase function

The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase composed of the subunits PA, PB1 and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding andthe PA endonuclease domains are both involved in cap-snatching, but the role of the auxiliary PB2 627-domain, implicated in host range restriction of influenza A viruses, is still poorly understood. Here we use structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627-domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerasewith PB1 and PA. Furthermore, we show that a recombinant viral polymerase lacking the PB2 627-domain is able to carry out cap-snatching, cap-dependent transcription initiation as well as cap-independent ApG dinucleotide extension in vitro, indicating that the PB2 627-domain of the influenza virus RNApolymerase is not involved in core catalytic functions of the polymerase. However, in a cellular context, the 627-domain is essential for both transcription and replication. In particular, we show that the PB2 627-domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627-domain in transcription and replication of the influenza virus RNA genome

Real-time analysis of single influenza virus replication complexes reveals large promoter-dependent differences in initiation dynamics

The viral RNA (vRNA) genome of influenza viruses is replicated by the RNA-dependent RNA polymerase (RNAP) via a complementary RNA (cRNA) intermediate. The vRNA promoter can adopt multiple conformations when bound by the RNAP. However, the dynamics, determinants, and biological role of these conformations are unknown; further, little is known about cRNA promoter conformations. To probe the RNA conformations adopted during initial replication, we monitored single, surface-immobilized vRNA and cRNA initiation complexes in real-time. Our results show that, while the 3′ terminus of the vRNA promoter exists in dynamic equilibrium between pre-initiation and initiation conformations, the cRNA promoter exhibited very limited dynamics. Two residues in the proximal 3′ region of the cRNA promoter (residues absent in the vRNA promoter) allowed the cRNA template strand to reach further into the active site, limiting promoter dynamics. Our results highlight promoter-dependent differences in influenza initiation mechanisms, and advance our understanding of virus replication

Real-time analysis of single influenza virus replication complexes reveals large promoter-dependent differences in initiation dynamics

The viral RNA (vRNA) genome of influenza viruses is replicated by the RNA-dependent RNA polymerase (RNAP) via a complementary RNA (cRNA) intermediate. The vRNA promoter can adopt multiple conformations when bound by the RNAP. However, the dynamics, determinants, and biological role of these conformations are unknown; further, little is known about cRNA promoter conformations. To probe the RNA conformations adopted during initial replication, we monitored single, surface-immobilized vRNA and cRNA initiation complexes in real-time. Our results show that, while the 3′ terminus of the vRNA promoter exists in dynamic equilibrium between pre-initiation and initiation conformations, the cRNA promoter exhibited very limited dynamics. Two residues in the proximal 3′ region of the cRNA promoter (residues absent in the vRNA promoter) allowed the cRNA template strand to reach further into the active site, limiting promoter dynamics. Our results highlight promoter-dependent differences in influenza initiation mechanisms, and advance our understanding of virus replication