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1.  Reassortment Complements Spontaneous Mutation in Influenza A Virus NP and M1 Genes To Accelerate Adaptation to a New Host 
Journal of Virology  2013;87(8):4330-4338.
Influenza A virus (IAV) infects a remarkably wide variety of avian and mammalian hosts. Evolution finely hones IAV genes to optimally infect and be transmitted in a particular host species. Sporadically, IAV manages to jump between species, introducing novel antigenic strains into the new host population that wreak havoc until herd immunity develops. IAV adaptation to new hosts typically involves reassortment of IAV gene segments from coinfecting virus strains adapted to different hosts in conjunction with multiple adaptive mutations in the various IAV genes. To better understand host adaptation between mammalian species in real time, we passaged mouse-adapted A/PR8/34 (PR8) in guinea pigs. Guinea pigs, unlike mice, support spontaneous and robust IAV transmission. For some IAV strains, including PR8, adaptation is required for a virus to attain transmissibility, providing an opportunity to understand the evolution of transmissibility in guinea pigs. Multiple guinea pig-adapted PR8 mutants generated by serial nasal wash passaging in independent lines replicated more efficiently and were transmitted by cocaging. All transmissible variants possessed one of two nonsynonymous mutations in M1, either alone or in combination with mutations in PB2, HA, NP, or NA. Rapid reassortment between independently selected variants combined beneficial mutations in NP and M1 to form the fittest virus capable of being transmitted. These findings provide further insight into genetic determinants in NP and M1 involved in PR8 IAV adaptation to be transmitted in a new host and clearly show the benefit of a segmented genome in rapidly generating optimal combinations of mutations in IAV evolution.
doi:10.1128/JVI.02749-12
PMCID: PMC3624365  PMID: 23365443
2.  Defining Influenza A Virus Hemagglutinin Antigenic Drift by Sequential Monoclonal Antibody Selection 
Cell host & microbe  2013;13(3):314-323.
SUMMARY
Human influenza A virus (IAV) vaccination is limited by “antigenic drift,” rapid antibody-driven escape reflecting amino acid substitutions in the globular domain of hemagglutinin (HA), the viral attachment protein. To better understand drift, we used anti-hemagglutinin monoclonal Abs (mAbs) to sequentially select IAV escape mutants. Twelve selection steps, each resulting in a single amino acid substitution in the hemagglutinin globular domain, were required to eliminate antigenicity defined by monoclonal or polyclonal Abs. Sequential mutants grow robustly, showing the structural plasticity of HA, although several hemagglutinin substitutions required an epistatic substitution in the neuraminidase glycoprotein to maximize growth. Selecting escape mutants from parental versus sequential variants with the same mAb revealed distinct escape repertoires, attributed to contextual changes in antigenicity and the mutation landscape. Since each hemagglutinin mutation potentially sculpts future mutation space, drift can follow many stochastic paths, undermining its unpredictability and underscoring the need for drift-insensitive vaccines.
doi:10.1016/j.chom.2013.02.008
PMCID: PMC3747226  PMID: 23498956
3.  Emetine optimally facilitates nascent chain puromycylation and potentiates the RiboPuromycylation Method (RPM) applied to inert cells 
Histochemistry and cell biology  2012;139(3):501-504.
We previously described the RiboPuromyclation method (RPM) to visualize and quantitate translating ribosomes in fixed and permeabilized cells by standard immunofluorescence. RPM is based on puromycylation of nascent chains bound to translating ribosomes followed by detection of puromycylated nascent chains with a puromycin-specific mAb. We now demonstrate that emetine optimally enhances nascent chain puromycylation, and describe a modified RPM protocol for identifying ribosome-bound nascent chains in metabolically inert permeabilized cells.
doi:10.1007/s00418-012-1063-8
PMCID: PMC3574230  PMID: 23229864
ribopuromycylation; puromycin; translation; ribosome
4.  Influenza A Virus Hemagglutinin Trimerization Completes Monomer Folding and Antigenicity 
Journal of Virology  2013;87(17):9742-9753.
Influenza A virus (IAV) remains an important human pathogen largely because of antigenic drift, the rapid emergence of antibody escape mutants that precludes durable vaccination. The most potent neutralizing antibodies interact with cognate epitopes in the globular “head” domain of hemagglutinin (HA), a homotrimeric glycoprotein. The H1 HA possesses five distinct regions defined by a large number of mouse monoclonal antibodies (MAbs), i.e., Ca1, Ca2, Cb, Sa, and Sb. Ca1-Ca2 sites require HA trimerization to attain full antigenicity, consistent with their locations on opposite sides of the trimer interface. Here, we show that full antigenicity of Cb and Sa sites also requires HA trimerization, as revealed by immunofluorescence microscopy of IAV-infected cells and biochemically by pulse-chase radiolabeling experiments. Surprisingly, epitope antigenicity acquired by HA trimerization persists following acid triggering of the globular domains dissociation and even after proteolytic release of monomeric heads from acid-treated HA. Thus, the requirement for HA trimerization by trimer-specific MAbs mapping to the Ca, Cb, and Sa sites is not dependent upon the bridging of adjacent monomers in the native HA trimer. Rather, complete antigenicity of HA (and, by inference, immunogenicity) requires a final folding step that accompanies its trimerization. Once this conformational change occurs, HA trimers themselves would not necessarily be required to induce a highly diverse neutralizing response to epitopes in the globular domain.
doi:10.1128/JVI.00471-13
PMCID: PMC3754138  PMID: 23824811
5.  Host versus flu: antibodies win a round? 
Structural and functional analyses of three neutralizing antibodies against influenza virus H2 HA may explain why this HA subtype has disappeared from circulation in the human population and point to a potential new avenue for antiflu therapeutics.
doi:10.1038/nsmb.2524
PMCID: PMC3762974  PMID: 23463305
6.  Monocytes, viruses and metaphors 
Cell Cycle  2012;11(9):1748-1749.
doi:10.4161/cc.20311
PMCID: PMC3372385  PMID: 22517432
dendritic cell; macrophage; monocyte; T cell; virus
7.  Anatomically restricted synergistic anti-viral activities of innate and adaptive immune cells in the skin 
Cell host & microbe  2013;13(2):155-168.
SUMMARY
Despite extensive ex vivo investigation, the spatiotemporal organization of immune cells interacting with virus-infected cells in tissues remains uncertain. To address this, we used intravital multiphoton microscopy to visualize immune cell interactions with virus-infected cells following epicutaneous vaccinia virus (VV) infection of mice. VV infects keratinocytes in epidermal foci, and numerous migratory dermal inflammatory monocytes outlying the foci. We observed Ly6G+ innate immune cells infiltrating and controlling foci, while CD8+ T cells remained on the periphery killing infected monocytes. Most antigen-specific CD8+ T cells in the skin did not interact with virus-infected cells. Blocking the generation of reactive nitrogen species relocated CD8+ T cells into foci, modestly reducing viral titers. Depletion of Ly6G+ and CD8+ cells dramatically increased viral titers, consistent with their synergistic but spatially segregated viral clearance activities. These findings highlight previously unappreciated differences in the anatomic specialization of antiviral immune cell subsets.
doi:10.1016/j.chom.2013.01.004
PMCID: PMC3591514  PMID: 23414756
9.  Distinct Pathways Generate Peptides from Defective Ribosomal Products for CD8+ T Cell Immunosurveillance 
To understand better the endogenous sources of MHC class I peptide ligands, we generated an antigenic reporter protein whose degradation is rapidly and reversibly controlled with Shield-1, a cell-permeant drug. Using this system, we demonstrate that defective ribosomal products (DRiPs) represent a major and highly efficient source of peptides and are completely resistant to our attempts to stabilize the protein. Although peptides also derive from nascent Shield-1–sensitive proteins and “retirees” created by Shield-1 withdrawal, these are much less efficient sources on a molar basis. We use this system to identify two drugs—each known to inhibit polyubiquitin chain disassembly—that selectively inhibit presentation of Shield-1–resistant DRiPs. These findings provide the initial evidence for distinct biochemical pathways for presentation of DRiPs versus retirees and implicate polyubiquitin chain disassembly or the actions of deubiquitylating enzymes as playing an important role in DRiP presentation.
doi:10.4049/jimmunol.1003096
PMCID: PMC3408966  PMID: 21228349
10.  From optical bench to cageside: intravital microscopy on the long road to rational vaccine design 
Immunological Reviews  2011;239(1):209-220.
Summary
No anti-viral vaccine is perfect. For some important pathogens, there are no effective vaccines. Many current vaccines are based on the working principles of Jenner and Pasteur, i.e. empiric administration of attenuated or inactivated forms of the pathogen. Tapping the full potential of vaccination requires a thorough understanding of the mechanism of immune activation by pathogens and their individual components. Though the rate of discovery continues to accelerate, the complexity of the immune system is daunting, particularly when integrated into the overall physiology of the host. Here, we review the application of multiphoton microscopy to examine host-pathogen interactions, focusing on our recent efforts to understand mouse CD8+ T-cell responses to viruses at the level of cellular interactions in lymph nodes draining the infection site. We also discuss our recent efforts to understand the influence of the sympathetic nervous system on antiviral immunity, with the ultimate goal of appreciating the traditional elements of immunity as just one facet of the total organismal response to infection and immunization.
doi:10.1111/j.1600-065X.2010.00973.x
PMCID: PMC3401942  PMID: 21198674
intravital microscopy; sympathetic nervous system; T cell; vaccine; virus
11.  RNA Polymerase II Inhibitors Dissociate Antigenic Peptide Generation from Normal Viral Protein Synthesis: A Role for Nuclear Translation in Defective Ribosomal Product Synthesis? 
Following viral infection, cells rapidly present peptides from newly synthesized viral proteins on MHC class I molecules, likely from rapidly degraded forms of nascent proteins. The nature of these defective ribosomal products (DRiPs) remains largely undefined. Using inhibitors of RNA polymerase II that block influenza A virus neuraminidase (NA) mRNA export from the nucleus and inhibit cytoplasmic NA translation, we demonstrate a surprising disconnect between levels of NA translation and generation of SIINFEKL peptide genetically inserted into the NA stalk. A 33-fold reduction in NA expression is accompanied by only a 5-fold reduction in Kb-SIINFEKL complex cell-surface expression, resulting in a net 6-fold increase in the overall efficiency of Ag presentation. Although the proteasome inhibitor MG132 completely blocked Kb-SIINFEKL complex generation, we were unable to biochemically detect a MG132-dependent cohort of NA DRiPs relevant for Ag processing, suggesting that a minute population of DRiPs is a highly efficient source of antigenic peptides. These data support the idea that Ag processing uses compartmentalized translation, perhaps even in the nucleus itself, to increase the efficiency of the generation of class I peptide ligands.
doi:10.4049/jimmunol.1002543
PMCID: PMC3398797  PMID: 21048111
12.  Frameshifting to PA-X Influenza 
Science (New York, N.Y.)  2012;337(6091):164-165.
The genome of influenza A virus encodes a newly discovered protein that diminishes its pathogenicity.
doi:10.1126/science.1225539
PMCID: PMC3777247  PMID: 22798590
13.  Don’t mess with ERAAP! 
Nature immunology  2012;13(6):526-528.
Immunosurveillance monitors subversion of the endoplasmic reticulum aminopeptidase ERAAP. ERAAP-deficient cells are killed by T cells that recognize nonclassical major histocompatibility complex class I Qa-1 molecules presenting peptides generated in the absence of ERAAP.
doi:10.1038/ni.2306
PMCID: PMC3777675  PMID: 22610241
14.  Most Influenza A Virions Fail To Express at Least One Essential Viral Protein 
Journal of Virology  2013;87(6):3155-3162.
Segmentation of the influenza A virus (IAV) genome enables rapid gene reassortment at the cost of complicating the task of assembling the full viral genome. By simultaneously probing for the expression of multiple viral proteins in MDCK cells infected at a low multiplicity with IAV, we observe that the majority of infected cells lack detectable expression of one or more essential viral proteins. Consistent with this observation, up to 90% of IAV-infected cells fail to release infectious progeny, indicating that many IAV virions scored as noninfectious by traditional infectivity assays are capable of single-round infection. This fraction was not significantly affected by target or producer cell type but varied widely between different IAV strains. These data indicate that IAV exists primarily as a swarm of complementation-dependent semi-infectious virions, and thus traditional, propagation-dependent assays of infectivity may drastically misrepresent the true infectious potential of a virus population.
doi:10.1128/JVI.02284-12
PMCID: PMC3592173  PMID: 23283949
15.  Defective Ribosomal Products Are the Major Source of Antigenic Peptides Endogenously Generated from Influenza A Virus Neuraminidase 
The defective ribosomal product (DRiP) hypothesis of endogenous Ag processing posits that rapidly degraded forms of nascent proteins are a major source of peptide ligands for MHC class I molecules. Although there is broad experimental support for the DRiP hypothesis, careful kinetic analysis of the generation of defined peptide class I complexes has been limited to studies of recombinant vaccinia viruses expressing genes derived from other organisms. In this study, we show that insertion of the SIIN-FEKL peptide into the stalk of influenza A virus neuraminidase (NA) does not detectably modify NA folding, degradation, transport, or sp. act. when expressed in its natural context of influenza A virus infection. Using the 25-D1.16 mAb specific for Kb-SIINFEKL to precisely quantitate cell surface complexes by flow cytometry, we demonstrate that SIINFEKL is generated in complete lockstep with initiation and abrogation of NA biosynthesis in both L-Kb fibroblast cells and DC2.4 dendritic/monocyte cells. SIINFEKL presentation requires active proteasomes and TAP, consistent with its generation from a cytosolic DRiP pool. From the difference in the shutoff kinetics of Kb-SIINFEKL complex expression following protein synthesis versus proteasome inhibition, we estimate that the t1/2 of the biosynthetic source of NA peptide is ~5 min. These observations extend the relevance of the DRiP hypothesis to viral proteins generated in their natural context.
doi:10.4049/jimmunol.0901907
PMCID: PMC2940057  PMID: 20038640
16.  Out with the Old, In with the New? Comparing Methods for Measuring Protein Degradation 
Cell biology international  2011;35(5):457-462.
Protein degradation is a key variable in controlling protein abundance in cells. Here, we compare classical methods to measure protein degradation rates with a novel GFP reporter library based method that characterizes degradation of thousands of individual proteins by flow cytometry. While no method is perfect, we conclude that chimeric gene reporter approaches should be applied cautiously due principally to GFP (or other tag) interference with organelle targeting or incorporation of chimeric proteins into macromolecular assemblies that results in spuriously high degradation rates.
doi:10.1042/CBI20110055
PMCID: PMC3727619  PMID: 21476986
17.  Cutting Edge: Sympathetic Nervous System Increases Proinflammatory Cytokines and Exacerbates Influenza A Virus Pathogenesis 
Although the sympathetic nervous system innervates the lung, little is known about its participation in host immunity to pulmonary pathogens. In this study, we show that peripheral sympathectomy reduces mouse morbidity and mortality from influenza A virus-induced pneumonia due to reduced inflammatory influx of monocytes, neutrophils, and NK cells. Mortality was also delayed by treating mice with an α-adrenergic antagonist. Sympathectomy diminished the immediate innate cytokine responses, particularly IL-1, which was profoundly reduced. These findings demonstrate an unexpected role for the sympathetic nervous system in innate antiviral immunity and in exacerbating the pathology of a virus of great significance to human and animal health.
doi:10.4049/jimmunol.0903395
PMCID: PMC2941093  PMID: 20018617
18.  Efficient Cross-Priming of Antiviral CD8+ T Cells by Antigen Donor Cells Is GRP94 Independent1 
Cross-priming, the activation of naive CD8+ T cells by dendritic cells presenting Ags synthesized by other cells, is believed to play an important role in the generation of antiviral and antitumor responses. The molecular mechanism(s) underlying cross-priming remain poorly defined and highly controversial. GRP94 (gp96), an abundant endoplasmic reticulum chaperone with innate immune-activating capacity, has been widely reported to play a major role in cross-priming. In this study, we show that cells whose expression of GRP94 is silenced via transient or stable transfection with GRP94-directed small interfering RNAs demonstrate no reduction in their abilities to generate class I peptide complexes in cultured cells or to prime antiviral CD8+ T cell responses in vivo. In demonstrating the dispensability of GRP94, our finding points to the importance of alternative mechanisms for generation of class I peptide complexes from endogenous and exogenous Ags and immunogens.
doi:10.4049/jimmunol.0901828
PMCID: PMC2749969  PMID: 19752220
19.  Positional Bias of MHC Class I Restricted T-Cell Epitopes in Viral Antigens Is Likely due to a Bias in Conservation 
PLoS Computational Biology  2013;9(1):e1002884.
The immune system rapidly responds to intracellular infections by detecting MHC class I restricted T-cell epitopes presented on infected cells. It was originally thought that viral peptides are liberated during constitutive protein turnover, but this conflicts with the observation that viral epitopes are detected within minutes of their synthesis even when their source proteins exhibit half-lives of days. The DRiPs hypothesis proposes that epitopes derive from Defective Ribosomal Products (DRiPs), rather than degradation of mature protein products. One potential source of DRiPs is premature translation termination. If this is a major source of DRiPs, this should be reflected in positional bias towards the N-terminus. By contrast, if downstream initiation is a major source of DRiPs, there should be positional bias towards the C-terminus. Here, we systematically assessed positional bias of epitopes in viral antigens, exploiting the large set of data available in the Immune Epitope Database and Analysis Resource. We show a statistically significant degree of positional skewing among epitopes; epitopes from both ends of antigens tend to be under-represented. Centric-skewing correlates with a bias towards class I binding peptides being over-represented in the middle, in parallel with a higher degree of evolutionary conservation.
Author Summary
To defend the host from an infection, the immune system continuously scans cell surfaces for foreign objects. Specifically, a virus inside a cell exploits the host to make copies of its proteins; viral proteins are broken up into peptide fragments; and the fragments are displayed on the infected cell's surface, thereby allowing detection and cell-killing. How these peptide fragments for cell-surface presentation are generated remains unknown. An understanding of this step will lead to rational design of vaccines and insights into tumor immunosurveillance and autoimmunity. One possible mechanism is that the peptide fragments come from defective proteins missing either the beginning or end regions, which may result in a bias. Here, we analyzed locations of a large set of known viral epitopes, peptide fragments recognized by the immune system, within their proteins. We find that all regions of proteins are represented well by the immune system. However, there is a statistically significant bias in the central regions of proteins, which correlate with a pattern of conservation spanning the length of viral proteins. Our results suggest a combined effect of conservation and enhancement of immune responses through repeated exposures in shaping the distribution of known viral epitopes.
doi:10.1371/journal.pcbi.1002884
PMCID: PMC3554532  PMID: 23357871
20.  Vaccinia and influenza A viruses select rather than adjust tRNAs to optimize translation 
Nucleic Acids Research  2012;41(3):1914-1921.
Transfer RNAs (tRNAs) are central to protein synthesis and impact translational speed and fidelity by their abundance. Here we examine the extent to which viruses manipulate tRNA populations to favor translation of their own genes. We study two very different viruses: influenza A virus (IAV), a medium-sized (13 kB genome) RNA virus; and vaccinia virus (VV), a large (200 kB genome) DNA virus. We show that the total cellular tRNA population remains unchanged following viral infection, whereas the polysome-associated tRNA population changes dramatically in a virus-specific manner. The changes in polysome-associated tRNA levels reflect the codon usage of viral genes, suggesting the existence of local tRNA pools optimized for viral translation.
doi:10.1093/nar/gks986
PMCID: PMC3561966  PMID: 23254333
21.  MF59 Adjuvant Enhances Diversity and Affinity of Antibody-Mediated Immune Response to Pandemic Influenza Vaccines 
Science translational medicine  2011;3(85):85ra48.
Oil-in-water adjuvants have been shown to improve immune responses against pandemic influenza vaccines as well as reduce the effective vaccine dose, increasing the number of doses available to meet global vaccine demand. Here, we use genome fragment phage display libraries and surface plasmon resonance to elucidate the effects of MF59 on the quantity, diversity, specificity, and affinity maturation of human antibody responses to the swine-origin H1N1 vaccine in different age groups. In adults and children, MF59 selectively enhanced antibody responses to the hemagglutinin 1 (HA1) globular head relative to the more conserved HA2 domain in terms of increased antibody titers as well as a more diverse antibody epitope repertoire. Antibody affinity, as inferred by greatly diminished (≥10-fold) off-rate constants, was significantly increased in toddlers and children who received the MF59-adjuvanted vaccine. Moreover, MF59 also improved antibody affinity maturation after each sequential vaccination against avian H5N1 in adults. For both pandemic influenza vaccines, there was a close correlation between serum antibody affinity and virus-neutralizing capacity. Thus, MF59 quantitatively and qualitatively enhances functional antibody responses to HA-based vaccines by improving both epitope breadth and binding affinity, demonstrating the added value of such adjuvants for influenza vaccines.
doi:10.1126/scitranslmed.3002336
PMCID: PMC3501657  PMID: 21632986
22.  DRiPs Solidify: Progress in Understanding Endogenous MHC Class I Antigen Processing 
Trends in immunology  2011;32(11):548-558.
Defective Ribosomal Products (DRiPs) are a subset of rapidly degraded polypeptides that provide peptide ligands for MHC class I molecules. Here, I review recent progress in understanding DRiP biogenesis. These findings place DRiPs at the center of the MHC class I antigen processing pathway, linking immunosurveillance of viruses and tumors to mechanisms of specialized translation and cellular compartmentalization. DRiPs enable the immune system to rapidly and sensitively detect alterations in cellular gene expression.
doi:10.1016/j.it.2011.08.001
PMCID: PMC3200450  PMID: 21962745
23.  Nuclear translation visualized by ribosome-bound nascent chain puromycylation 
The Journal of Cell Biology  2012;197(1):45-57.
A new method for visualizing translation in cells via standard immunofluorescence microscopy provides evidence for translation in the nucleoplasm and nucleolus.
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.
doi:10.1083/jcb.201112145
PMCID: PMC3317795  PMID: 22472439
24.  Viva la Revolución: Rethinking Influenza A Virus Antigenic Drift 
Current opinion in virology  2011;1(3):177-183.
Rapid antigenic evolution of the influenza A virus hemagglutinin has precluded developing vaccines that provide durable protection. The yearly costs of influenza (circa $1011 in the USA alone) easily justify investments in better understanding the interaction of influenza with antibodies and other inducible elements of the immune system that potentially limit or circumvent antigenic variation. Here, I summarize exciting new findings that offer the possibility of a quantum improvement in vaccine efficacy, focusing on studies clearly documenting robust neutralizing antibody responses to the conserved stem region of the hemagglutinin.
doi:10.1016/j.coviro.2011.05.005
PMCID: PMC3199585  PMID: 22034587
25.  Cross–dressers turn on T cells 
Nature  2011;471(7340):581-582.
Memory T cells remember viruses from previous infections, providing immunity by facilitating the killing of infected cells. For this, they exploit cross-dressing, the transfer of antigens between antigen-presenting cells.
doi:10.1038/471581a
PMCID: PMC3400133  PMID: 21455165

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