mRNA vaccines - a new era in vaccinology.

mRNA vaccines represent a promising alternative to conventional vaccine approaches because of their high potency, capacity for rapid development and potential for low-cost manufacture and safe administration. However, their application has until recently been restricted by the instability and inefficient in vivo delivery of mRNA. Recent technological advances have now largely overcome these issues, and multiple mRNA vaccine platforms against infectious diseases and several types of cancer have demonstrated encouraging results in both animal models and humans. This Review provides a detailed overview of mRNA vaccines and considers future directions and challenges in advancing this promising vaccine platform to widespread therapeutic use.

Nucleoporin phosphorylation triggered by the encephalomyocarditis virus leader protein is mediated by mitogen-activated protein kinases.

Cardioviruses disrupt nucleocytoplasmic transport through the activity of their leader (L) protein. We have shown that hyperphosphorylation of nuclear pore proteins (nucleoporins or Nups), including Nup62, Nup153, and Nup214, is central to this L protein function and requires one or more cytosolic kinases. In this study, potential cellular enzymes involved in encephalomyocarditis virus (EMCV) L-directed Nup phosphorylation were screened with a panel of specific, cell-permeating kinase inhibitors. Extracellular signal-regulated receptor kinase (ERK) and p38 mitogen-activated protein kinase inhibitors (U0126 and SB203580) were sufficient to block Nup hyperphosphorylation in EMCV-infected or L-expressing cells. Recombinant L alone, in the absence of infection, triggered activation of ERK and p38, independent of their upstream signaling cascades. Conserved residues within the L zinc finger (Cys(19)) and acidic domain (Asp(48),(51),(52),(55)) were essential for this activation and for the phosphorylation of Nups, suggesting that the phenomena are linked. Analysis of the hyperphosphorylated Nup species revealed only phosphoserine and phosphothreonine residues. The sizes of the tryptic phosphopeptides derived from Nup62 were compatible with sites in the Phe/Gly repeat domain which display common consensus sequences for ERK and p38 substrates. The results provide strong evidence that ERK and p38 are the probable effector kinases required for L-dependent inhibition of nuclear trafficking.

Leader-induced phosphorylation of nucleoporins correlates with nuclear trafficking inhibition by cardioviruses.

Picornaviruses disrupt nucleocytoplasmic trafficking pathways during infection. Poliovirus and rhinovirus inhibit nuclear protein import/export through a series of 2A protease-dependent cleavages within nuclear pore proteins (nucleoporins [Nups]), including Nup62, Nup98, and Nup153. Cardioviruses lack the same protease and instead affect trafficking inhibition through an activity mapped to their leader (L) protein, a 67- to 76-amino acid (aa) polypeptide with no known enzymatic activity. We have shown that L from encephalomyocarditis virus (EMCV) binds and inhibits the activity of Ran-GTPase, a key regulator of nucleocytoplasmic transport. We now report that recombinant EMCV L triggers the unregulated efflux of protein cargo from preloaded HeLa cell nuclei in cell-free reactions dependent upon Xenopus egg cytosol or HeLa cell-derived cytosol. Recombinant L was the only viral protein necessary for this activity or for nuclear protein import inhibition. Mutational disruption of the L protein zinc finger domain (C(19)A) abrogated the inhibitory activity for both import and efflux in cell extracts, but mutations in the C-terminal acidic domain of L (aa 37 to 61) did not. Notably, HeLa cell nuclei treated with L, or those from EMCV-infected cells, showed reproducibly altered patterns of nucleoporin phosphorylation. Nup62, Nup153, and Nup214 each became hyperphosphorylated in an L-dependent manner. Staurosporine, a broad-spectrum kinase inhibitor, blocked this phosphorylation and rescued nuclear import/export activity from L-dependent inhibition. Therefore, cardioviruses target the same group of nucleoporins as enteroviruses, but the effector mechanism triggered by L (or L-Ran complexes) involves a unique cytosol-dependent phosphorylation cascade rather than proteolysis.

NMR structure of the mengovirus Leader protein zinc-finger domain.

The Leader protein is a defining feature of picornaviruses from the Cardiovirus genus. This protein was recently shown to inhibit cellular nucleocytoplasmic transport through an activity mapped to its zinc-binding region. Here we report the three-dimensional solution structure determined by nuclear magnetic resonance (NMR) spectroscopy of this domain (residues 5-28) from mengovirus. The domain forms a CHCC zinc-finger with a fold comprising a beta-hairpin followed by a short alpha-helix that can adopt two different conformations. This structure is divergent from those of other eukaryotic zinc-fingers and instead resembles motifs found in a group of DNA-binding proteins from Archaea.

A picornavirus protein interacts with Ran-GTPase and disrupts nucleocytoplasmic transport.

Active nucleocytoplasmic transport of protein and RNA in eukaryotes depends on the Ran-GTPase system to regulate cargo-receptor interactions. Several viruses, including the RNA picornaviruses, encode factors that alter nuclear transport with the aim of suppressing synthesis of antiviral factors and promoting viral replication. Picornaviruses in the cardiovirus genus express a unique 67-aa Leader protein (L), known to alter the subcellular distribution of IFN regulatory proteins targeted to the nucleus. We report here that L binds directly to Ran and blocks nuclear export of new mRNAs. In Xenopus egg extracts, recombinant L also inhibits mitotic spindle assembly, a RanGTP function crucial to cell-cycle progression. We propose that L inhibits nucleocytoplasmic transport during infection by disrupting the RanGDP/GTP gradient. This inhibition triggers an efflux of nuclear proteins necessary for viral replication and causes IFN suppression. To our knowledge, L is the first viral picornaviral protein to interact directly with Ran and modulate the Ran-dependent nucleocytoplasmic pathway.