CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation.

Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E-m(7)GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3(S10+) mitotic cell population having higher inactive p4E-BP1(T37/T46+) saturation levels than pH3(S10-) interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling.

Expanding the proteome of an RNA virus by phosphorylation of an intrinsically disordered viral protein.

The human proteome contains myriad intrinsically disordered proteins. Within intrinsically disordered proteins, polyproline-II motifs are often located near sites of phosphorylation. We have used an unconventional experimental paradigm to discover that phosphorylation by protein kinase A (PKA) occurs in the intrinsically disordered domain of hepatitis C virus non-structural protein 5A (NS5A) on Thr-2332 near one of its polyproline-II motifs. Phosphorylation shifts the conformational ensemble of the NS5A intrinsically disordered domain to a state that permits detection of the polyproline motif by using (15)N-, (13)C-based multidimensional NMR spectroscopy. PKA-dependent proline resonances were lost in the presence of the Src homology 3 domain of c-Src, consistent with formation of a complex. Changing Thr-2332 to alanine in hepatitis C virus genotype 1b reduced the steady-state level of RNA by 10-fold; this change was lethal for genotype 2a. The lethal phenotype could be rescued by changing Thr-2332 to glutamic acid, a phosphomimetic substitution. Immunofluorescence and transmission electron microscopy showed that the inability to produce Thr(P)-2332-NS5A caused loss of integrity of the virus-induced membranous web/replication organelle. An even more extreme phenotype was observed in the presence of small molecule inhibitors of PKA. We conclude that the PKA-phosphorylated form of NS5A exhibits unique structure and function relative to the unphosphorylated protein. We suggest that post-translational modification of viral proteins containing intrinsic disorder may be a general mechanism to expand the viral proteome without a corresponding expansion of the genome.

Hepatitis C virus nonstructural protein 5A: biochemical characterization of a novel structural class of RNA-binding proteins.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) exhibits a preference for G/U-rich RNA in vitro. Biological analysis of the NS5A RNA-binding activity and its target sites in the genome will be facilitated by a description of the NS5A-RNA complex. We demonstrate that the C-4 carbonyl of the uracil base and, by inference, the C-6 carbonyl of the guanine base interact with NS5A. U-rich RNA of 5 to 6 nucleotides (nt) is sufficient for high-affinity binding to NS5A. The minimal RNA-binding domain of NS5A consists of residues 2005 to 2221 (referred to as domain I-plus). This region of the protein includes the amino-terminal domain I as well as the subsequent linker that separates domains I and II. This linker region is the site of adaptive mutations. U-rich RNA-binding activity is not observed for an NS5A derivative containing only residues 2194 to 2419 (domains II and III). Mass spectrometric analysis of an NS5A-poly(rU) complex identified domains I and II as sites for interaction with RNA. Dimerization of NS5A was demonstrated by glutaraldehyde cross-linking. This dimerization is likely mediated by domain I-plus, as dimers of this protein are trapped by cross-linking. Dimers of the domain II-III protein are not observed. The monomer-dimer equilibrium of NS5A shifts in favor of dimer when U-rich RNA is present but not when A-rich RNA is present, consistent with an NS5A dimer being the RNA-binding-competent form of the protein. These data provide a molecular perspective of the NS5A-RNA complex and suggest possible mechanisms for regulation of HCV and cellular gene expression.

HCV resistance to cyclosporin A does not correlate with a resistance of the NS5A-cyclophilin A interaction to cyclophilin inhibitors.

BACKGROUND & AIMS: The cyclophilin (Cyp) inhibitors - cyclosporine A (CsA), NIM811, Debio 025, and SCY 635 - block HCV replication both in vitro and in vivo, and represent a novel class of potent anti-HCV agents. We and others showed that HCV relies on cyclophilin A (CypA) to replicate. We demonstrated that the hydrophobic pocket of CypA, where Cyp inhibitors bind, and which controls the isomerase activity of CypA, is critical for HCV replication. Recent studies showed that under Cyp inhibitor selection, mutations arose in the HCV nonstructural 5A (NS5A) protein. This led us to postulate that CypA assists HCV by acting on NS5A. METHODS: We tested this hypothesis by developing several interaction assays including GST pull-down assays, ELISA, and mammalian two-hybrid binding assays. RESULTS: We demonstrated that full-length NS5A and CypA form a stable complex. Remarkably, CsA prevents the CypA-NS5A interaction in a dose-dependent manner. Importantly, the CypA-NS5A interaction is conserved among genotypes and is interrupted by CsA. Surprisingly, the NS5A mutant protein, which arose in CsA-resistant HCV variants, behaves similarly to wild-type NS5A in terms of both CypA binding and CsA-mediated release from CypA. This latter finding suggests that HCV resistance to CsA does not correlate with a resistance of the CypA-NS5A interaction to Cyp inhibitors. Moreover, we found that CypA, devoid of its isomerase activity, fails to bind NS5A. CONCLUSIONS: Altogether these data suggest that CypA, via its isomerase pocket, binds directly to NS5A, and most importantly, that disrupting this interaction stops HCV replication.