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Jonathan W Yewdell

Publications and source records attributed to Jonathan W Yewdell.

46 records · Page 3Linked to original sources
PubMed2003

The influenza A virus PB1-F2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function.

The 11th influenza A virus gene product is an 87-amino-acid protein provisionally named PB1-F2 (because it is encoded by an open reading frame overlapping the PB1 open reading frame). A significant fraction of PB1-F2 localizes to the inner mitochondrial membrane in influenza A virus-infected cells. PB1-F2 appears to enhance virus-induced cell death in a cell type-dependent manner. For the present communication we have identified and characterized a region near the COOH terminus of PB1-F2 that is necessary and sufficient for its inner mitochondrial membrane localization, as determined by transient expression of chimeric proteins consisting of elements of PB1-F2 genetically fused to enhanced green fluorescent protein (EGFP) in HeLa cells. Targeting of EGFP to mitochondria by this sequence resulted in the loss of the inner mitochondrial membrane potential, leading to cell death. The mitochondrial targeting sequence (MTS) is predicted to form a positively charged amphipathic alpha-helix and, as such, is similar to the MTS of the p13(II) protein of human T-cell leukemia virus type 1. We formally demonstrate the functional interchangeability of the two sequences for mitochondrial localization of PB1-F2. Mutation analysis of the putative amphipathic helix in the PB1-F2 reveals that replacement of five basic amino acids with Ala abolishes mitochondrial targeting, whereas mutation of two highly conserved Leu to Ala does not. These findings demonstrate that PB1-F2 possesses an MTS similar to other viral proteins and that this MTS, when fused to EGFP, is capable of independently compromising mitochondrial function and cellular viability.

Amino Acid Sequence↗
PubMed2003

Heat-aggregated noninfectious influenza virus induces a more balanced CD8(+)-T-lymphocyte immunodominance hierarchy than infectious virus.

CD8(+)-T-cell (T(CD8+)) responses to infectious viruses are characterized by an immunodominance hierarchy in which the majority of T(CD8+) respond to one or a few immunodominant determinants, with a minority of T(CD8+) responding to a number of subdominant determinants. It is now well established that exogenous antigens are capable of inducing T(CD8+) to such immunodominant determinants, but the diversity of the response and the nature of the immunodominance hierarchy have not been examined. We addressed this issue by characterizing T(CD8+) responses to influenza virus preparations rendered inert by incubation for 10 min at 100 degrees C, as first reported by Speidel et al. (Eur. J. Immunol. 27:2391-2399, 1997). Extending these findings, we show that the primary T(CD8+) response to boiled virus can be sufficiently robust to be detected ex vivo by intracellular cytokine staining and that the response encompasses many of the peptides recognized by T(CD8+) induced by infectious virus. Importantly, the immunodominance hierarchy elicited was leveled, and we were unable to detect T(CD8+) that were specific for boiled virus. We used peritoneal exudate cells as antigen-presenting cells in vitro, and a number of observations indicated that boiled virus is processed via a phagocytic route that is likely to be endosomal in nature. These findings suggest that the repertoires of immunogenic peptides generated by endosomes and cytosolic processes overlap to a surprising degree. Furthermore, they demonstrate that the form of antigen administered can influence immunodominance hierarchies and that exogenous-antigen vaccines can induce broad and balanced T(CD8+) responses.

Animals↗
PubMed2002

Fusion proteins with COOH-terminal ubiquitin are stable and maintain dual functionality in vivo.

The ubiquitin (Ub) fusion degradation pathway functions to degrade fusion proteins containing a nonremovable Ub moiety at their NH(2) terminus (Johnson, E. S., Ma, P. C., Ota, I. M., and Varshavsky, A. (1995) J. Biol. Chem. 270, 17442-17456). Here we show that ubiquitin fusion degradation also targets proteins for proteasomal degradation when Ub is present in the middle of fusion proteins (X-Ub-Y), in a process that entails polyubiquitylation of Ub Lys(48). By contrast, fusion proteins bearing COOH-terminal Ub (X-Ub) are metabolically stable. Such fusion proteins, either newly biosynthesized or generated by Ub hydrolases, are reversibly conjugated to heterogeneous target proteins in a manner similar to wild-type Ub. Most importantly, the NH(2)-terminal fusion partner (X) can maintain its structure and function in the formed X-Ub conjugates as inferred from the fluorescence of green fluorescent protein-Ub conjugates and the incorporation of human immunodeficiency virus type 1 Gag-Ub into viral particles. These findings strongly suggest that 26S proteasomes exhibit spatial discrimination of Ub-conjugated proteins, sparing domains extended from the NH(2) terminus of Ub from unfolding and degradation. The multifunctionality of X-Ub fusion proteins opens the possibility for a number of novel practical applications, including the imaging of Ub conjugate formation in living cells.

Amino Acid Sequence↗
PubMed2002

Inhibitory effects of cytomegalovirus proteins US2 and US11 point to contributions from direct priming and cross-priming in induction of vaccinia virus-specific CD8(+) T cells.

The extent to which naive CD8(+) CTLs (T(CD8)(+)) are primed by APCs presenting endogenous Ags (direct priming) or Ags acquired from other infected cells (cross-priming) is a critical topic in basic and applied immunology. To examine the contribution of direct priming in the induction of VV-specific T(CD8)(+), we generated recombinant vaccinia viruses that express human CMV proteins (US2 and US11) that induce the destruction of newly synthesized MHC class I molecules. Expression of US2 or US11 was associated with a 24-63% decrease in numbers of primary or secondary VV-specific T(CD8)(+) responding to i.p. infection. Using HPLC-isolated peptides from VV-infected cells, we show that US2 and US11 selectively inhibit T(CD8)(+) responses to a subset of immunogenic VV determinants. Moreover, VV-US2 and lysates from VV-infected histoincompatible cells elicit T(CD8)(+) specific for a similar subset of VV determinants. These findings indicate that US2 and US11 can function in vivo to interfere with the activation of virus-specific T(CD8)(+). Furthermore, they suggest that 1) both cross-priming and direct priming contribute significantly to the generation of VV-specific T(CD8)(+), 2) the sets of immunogenic vaccinia virus determinants generated by cross-priming and direct priming are not completely overlapping, and 3) cross-priming overrides the effects of cis-acting viral interference with the class I Ag presentation pathway.

Animals↗
PubMed2002

Recycling CD1d1 molecules present endogenous antigens processed in an endocytic compartment to NKT cells.

Mouse CD1d1 molecules present endogenous glycolipids to NKT cells. Although glycolipid presentation requires CD1d1 transport through the endocytic pathway, the processing requirements for such endogenous Ag presentation by CD1d1 molecules are undefined. We examined CD1d1 Ag presentation to NKT cells by disrupting endocytic trafficking and function in cells expressing normal and mutated CD1d1 expressed by recombinant vaccinia viruses. Consistent with previous studies, we found that preventing CD1d1 localization to endosomes by altering its cytoplasmic targeting sequences abrogated recognition by Valpha14Jalpha281(+) NKT cells without affecting recognition by Valpha14(-) NKT cells. Increasing the pH of acidic compartments by incubating cells with chloroquine or bafilomycin A1 blocked CD1d1 recognition by Valpha14(+) (but not Valpha14(-)) NKT cells without reducing levels of cell surface CD1d1. Similar results were obtained with primaquine, which interferes with the recycling of cell surface glycoproteins. These results suggest that the loading of a subset of glycolipid ligands onto CD1d1 molecules entails the delivery of cell surface CD1d1 molecules and an acidic environment in the endocytic pathway.

Animals↗
PubMed2002

Visualizing priming of virus-specific CD8+ T cells by infected dendritic cells in vivo.

The rational design of vaccines that elicit CD8+ T cell responses requires knowledge of the identity of the antigen-presenting cell (APC), the location and time of presentation and the nature of the antigen presented by the APC. Here we address these questions for an antigen encoded by a recombinant vaccinia virus. We found that, following local infection, vaccinia virus infected macrophages and dendritic cells in draining lymph nodes. However, only the dendritic cells presented antigen to naïve CD8+ T cells, as determined by direct visualization of sectioned nodes by confocal microscopy. Presentation occurred as rapidly as 6 h after inoculation and quickly declined in parallel with the number of infected cells present in the nodes. These data provide direct evidence that virus-infected APCs prime naïve CD8+ T cells in vivo.

Animals↗
PubMed2002

Viral interference with antigen presentation.

CD8+ T cells play an important role in immunity to viruses. Just how important these cells are is demonstrated by the evolution of viral strategies for blocking the generation or display of peptide-major histocompatibility complex class I complexes on the surfaces of virus-infected cells. Here, we focus on viral interference with antigen presentation; in particular we consider the importance (and difficulty) of establishing the evolutionary significance (that is, the ability to enhance viral transmission) of viral gene products that interfere with antigen presentation in vitro.

Animals↗
PubMed2002

Mice deficient in perforin, CD4+ T cells, or CD28-mediated signaling maintain the typical immunodominance hierarchies of CD8+ T-cell responses to influenza virus.

CD8 T-cell (T(CD8+)) responses elicited by viral infection demonstrate the phenomenon of immunodominance: the numbers of T(CD8+) responding to different viral peptides vary over a wide range in a reproducible manner for individuals with the same major histocompatibility complex class I alleles. To better understand immunodominance, we examined T(CD8+) responses to multiple defined viral peptides following infection of mice with influenza virus. The immunodominance hierarchy of influenza virus-specific T(CD8+) was not greatly perturbed by the absence of either perforin or T-helper cells or by interference with B7 (CD80)-mediated signaling. These findings indicate that costimulation by antigen-presenting cells (APCs) or killing of APCs by T(CD8+) plays only a minor role in establishing the immunodominance hierarchy of antiviral T(CD8+) in this system. This points to intrinsic features of the T(CD8+) repertoire as major contributors to immunodominance.

Animals↗