Flu's Tricky Tricks: After vaccination against influenza A virus, single-point mutations are selected in hemagglutinin (the virus molecule that binds to sialic acid molecules on the surface of host cells) that escape neutralization by polyclonal antibody responses. Hensley et al. (p. 734 ) have discovered that in mice these mutations increased the virus's avidity for sialic acid. Amino acid substitutions that occur during reiterations of immune escape and avidity modulation can thus drive antigenic variation. This constant evolution of influenza viruses requires us to change vaccine components annually, and, for equine influenza, Park et al. (p. 726 ) show that as the match between virus and vaccine strains drifts apart with time, the probability of becoming infected and the length of the infectious period increase to the point where outbreaks occur. Nevertheless, even imperfect vaccines may be of benefit to a population because increasing the proportion of vaccinated individuals can supply enough herd immunity to offset a poor antigenic match, especially if used in conjunction with antiviral drugs. For humans, Yang et al. (p. 729 , published online 10 September) estimate that the rate of transmission within U.S. households puts influenza A 2009 H1N1 (the current pandemic “swine flu”) in the higher range of transmissibility, compared to past seasonal and pandemic strains. Thus, to achieve mitigation this fall, children should be the first recipients of vaccine, followed by adults—aiming overall for 70% coverage of the population.

Whether protein translation occurs in the nucleus is contentious. To address this question, we developed the ribopuromycylation method (RPM), which visualizes translation in cells via standard immunofluorescence microscopy. The RPM is based on ribosome-catalyzed puromycylation of nascent chains immobilized on ribosomes by antibiotic chain elongation inhibitors followed by detection of puromycylated ribosome-bound nascent chains with a puromycin (PMY)-specific monoclonal antibody in fixed and permeabilized cells. The RPM correlates localized translation with myriad processes in cells and can be applied to any cell whose translation is sensitive to PMY. In this paper, we use the RPM to provide evidence for translation in the nucleoplasm and nucleolus, which is regulated by infectious and chemical stress.

For cells to function properly the process of translating RNA messengers into proteins needs to be accurate, on the whole. Yet work in HeLa cells now shows that about 1% of methionine residues used in protein synthesis are aminoacylated to 'textbook-incorrect' tRNAs. Surprisingly, the proportion of Met-misacylated tRNAs increases significantly when cells are under stress through viral infection or treatment with viral or bacterial Toll-like receptor ligands. Tests with other amino acids indicate that the phenomenon is limited to Met, and as Met residues are known to protect proteins against damage from reactive oxygen species, one possibility is that Met-misacylation is a natural protective response to cellular stress. Accurate transfer RNA (tRNA) aminoacylation is necessary for translational fidelity; however, the accuracy of tRNA aminoacylation in vivo is uncertain. In mammalian cells, approximately 1% of methionine residues used in protein synthesis are now shown to be aminoacylated to non-methionyl-tRNAs. Furthermore, misacylation of methionine increases up to tenfold upon exposing cells to viruses, toll-like receptor ligands or oxidative stress. Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 to 100,000 couplings1,2. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower3,4,5,6. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage7, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.

Antigenic epitopes differ in their immunogenicity. Yewdell and colleagues show that B cell and antibody responses to influenza A virus infection display reproducible dynamic immunodominance hierarchies to viral hemagglutinin epitopes. Immunodominance (ID) defines the hierarchical immune response to competing antigens in complex immunogens. Little is known regarding B cell and antibody ID despite its importance in immunity to viruses and other pathogens. We show that B cells and serum antibodies from inbred mice demonstrate a reproducible ID hierarchy to the five major antigenic sites in the influenza A virus hemagglutinin globular domain. The hierarchy changed as the immune response progressed, and it was dependent on antigen formulation and delivery. Passive antibody transfer and sequential infection experiments demonstrated 'original antigenic suppression', a phenomenon in which antibodies suppress memory responses to the priming antigenic site. Our study provides a template for attaining deeper understanding of antibody ID to viruses and other complex immunogens.