Influenza A Virus Negative Strand RNA Is Translated for CD8 T Cell Immunosurveillance.

Probing the limits of CD8 T cell immunosurveillance, we inserted the SIINFEKL peptide into influenza A virus (IAV)-negative strand gene segments. Although IAV genomic RNA is considered noncoding, there is a conserved, relatively long open reading frame present in segment 8, encoding a potential protein termed NEG8. The biosynthesis of NEG8 from IAV has yet to be demonstrated.

Correction: Influenza A virus hemagglutinin glycosylation compensates for antibody escape fitness costs.

[This corrects the article DOI: 10.1371/journal.ppat.

Influenza A virus hemagglutinin glycosylation compensates for antibody escape fitness costs.

Rapid antigenic evolution enables the persistence of seasonal influenza A and B viruses in human populations despite widespread herd immunity. Understanding viral mechanisms that enable antigenic evolution is critical for designing durable vaccines and therapeutics. Here, we utilize the primerID method of error-correcting viral population sequencing to reveal an unexpected role for hemagglutinin (HA) glycosylation in compensating for fitness defects resulting from escape from anti-HA neutralizing antibodies.

Biogenesis of influenza a virus hemagglutinin cross-protective stem epitopes.

Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines.

Radioactive Pulse-Chase Analysis and Immunoprecipitation.

Labeling of newly-synthesized polypeptides with radioactive amino acids followed by immunoprecipitation allows quantitative analysis of the fate of a given protein in a time-dependent manner. This biochemical approach is usually used to study a variety of processes, such as protein folding, co-translational modifications, intracellular transport, and even its rate of degradation. Here, I describe step by step a simple technique to both label newly-synthesized influenza A virus (IAV) hemagglutinin (HA) with [S]-methionine and then follow its maturation and transport through the secretory pathway by SDS-PAGE and fluorography (Magadan , 2013).

Zonal Sedimentation Analysis on Sucrose Gradients.

Zonal sedimentation analysis on sucrose gradients allows estimation of the molecular size of an individual protein or a protein complex by centrifugation at a constant speed under nondenaturing conditions. This method is particularly suitable for globular proteins like the influenza A virus (IAV) protein hemagglutinin (HA). Here, I describe step by step a protocol used to evaluate the oligomeric state of recombinant HA trimers (Magadan , 2013).

Influenza A virus hemagglutinin trimerization completes monomer folding and antigenicity.

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.

Anatomically restricted synergistic antiviral activities of innate and adaptive immune cells in the skin.

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 that outlie the foci.

Structural basis for the recognition of tyrosine-based sorting signals by the μ3A subunit of the AP-3 adaptor complex.

Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous μ1, μ2, μ3, and μ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on μ2 and μ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the μ family, we solved the crystal structure at 1.

Monitoring cotranslational protein folding in mammalian cells at codon resolution.

How the ribosome-bound nascent chain folds to assume its functional tertiary structure remains a central puzzle in biology. In contrast to refolding of a denatured protein, cotranslational folding is complicated by the vectorial nature of nascent chains, the frequent ribosome pausing, and the cellular crowdedness. Here, we present a strategy called folding-associated cotranslational sequencing that enables monitoring of the folding competency of nascent chains during elongation at codon resolution.

Adaptor protein 2-mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing.

The β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins.

Transmembrane domain determinants of CD4 Downregulation by HIV-1 Vpu.

The transmembrane domains (TMDs) of integral membrane proteins do not merely function as membrane anchors but play active roles in many important biological processes. The downregulation of the CD4 coreceptor by the Vpu protein of HIV-1 is a prime example of a process that is dependent on specific properties of TMDs. Here we report the identification of Trp22 in the Vpu TMD and Gly415 in the CD4 TMD as critical determinants of Vpu-induced targeting of CD4 to endoplasmic reticulum (ER)-associated degradation (ERAD).

Brucella alters endocytic pathway in J774 macrophages.

Brucella is a facultative intracellular bacterium which causes chronic infections in mammals by surviving and replicating within host cells. The putative role of the endoplasmic reticulum (ER) in the formation of the phagosome in non-professional phagocytes is supported by several research groups, but still leaves open the question of the fate of Brucella inside professional phagocytes and its resistance mechanisms therein. Macrophages are particularly important for the survival and spreading of Brucella during infection.

Ang2/fat-free is a conserved subunit of the Golgi-associated retrograde protein complex.

The Golgi-associated retrograde protein (GARP) complex mediates tethering and fusion of endosome-derived transport carriers to the trans-Golgi network (TGN). In the yeast Saccharomyces cerevisiae, GARP comprises four subunits named Vps51p, Vps52p, Vps53p, and Vps54p. Orthologues of the GARP subunits, except for Vps51p, have been identified in all other eukaryotes.

Structural basis for the wobbler mouse neurodegenerative disorder caused by mutation in the Vps54 subunit of the GARP complex.

The multisubunit Golgi-associated retrograde protein (GARP) complex is required for tethering and fusion of endosome-derived transport vesicles to the trans-Golgi network. Mutation of leucine-967 to glutamine in the Vps54 subunit of GARP is responsible for spinal muscular atrophy in the wobbler mouse, an animal model of amyotrophic lateral sclerosis. The crystal structure at 1.

Multilayered mechanism of CD4 downregulation by HIV-1 Vpu involving distinct ER retention and ERAD targeting steps.

A key function of the Vpu protein of HIV-1 is the targeting of newly-synthesized CD4 for proteasomal degradation. This function has been proposed to occur by a mechanism that is fundamentally distinct from the cellular ER-associated degradation (ERAD) pathway. However, using a combination of genetic, biochemical and morphological methodologies, we find that CD4 degradation induced by Vpu is dependent on a key component of the ERAD machinery, the VCP-UFD1L-NPL4 complex, as well as on SCF(beta-TrCP)-dependent ubiquitination of the CD4 cytosolic tail on lysine and serine/threonine residues.

Rab22a regulates the sorting of transferrin to recycling endosomes.

Rab22a is a member of the Rab family of small GTPases that localizes in the endocytic pathway. In CHO cells, expression of canine Rab22a (cRab22a) causes a dramatic enlargement of early endocytic compartments. We wondered whether transferrin recycling is altered in these cells.

Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus.

The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells.