A novel influenza A virus mitochondrial protein that induces cell death.

While searching for alternative reading-frame peptides encoded by influenza A virus that are recognized by CD8+ T cells, we found an abundant immunogenic peptide encoded by the +1 reading frame of PB1. This peptide derives from a novel conserved 87-residue protein, PB1-F2, which has several unusual features compared with other influenza gene products in addition to its mode of translation. These include its absence from some animal (particularly swine) influenza virus isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation and mitochondrial localization. Exposure of cells to a synthetic version of PB1-F2 induces apoptosis, and influenza viruses with targeted mutations that interfere with PB1-F2 expression induce less extensive apoptosis in human monocytic cells than those with intact PB1-F2. We propose that PB1-F2 functions to kill host immune cells responding to influenza virus infection.

Cross-reactivity between hepatitis C virus and Influenza A virus determinant-specific cytotoxic T cells.

The cellular immune response contributes to viral clearance as well as to liver injury in acute and chronic hepatitis C virus (HCV) infection. An immunodominant determinant frequently recognized by liver-infiltrating and circulating CD8(+) T cells of HCV-infected patients is the HCV(NS3-1073) peptide CVNGVCWTV. Using a sensitive in vitro technique with HCV peptides and multiple cytokines, we were able to expand cytotoxic T cells specific for this determinant not only from the blood of 11 of 20 HCV-infected patients (55%) but also from the blood of 9 of 15 HCV-negative blood donors (60%), while a second HCV NS3 determinant was recognized only by HCV-infected patients and not by seronegative controls. The T-cell response of these healthy blood donors was mediated by memory T cells, which cross-reacted with a novel T-cell determinant of the A/PR/8/34 influenza A virus (IV) that is endogenously processed from the neuraminidase (NA) protein. Both the HCV NS3 and the IV NA peptide displayed a high degree of sequence homology, bound to the HLA-A2 molecule with high affinity, and were recognized by cytotoxic T lymphocytes with similar affinity (10(-8) M). Using the HLA-A2-transgenic mouse model, we then demonstrated directly that HCV-specific T cells could be induced in vivo by IV infection. Splenocytes harvested from IV-infected mice at the peak of the primary response (day 7 effector cells) or following complete recovery (day 21 memory cells) recognized the HCV NS3 peptide, lysed peptide-pulsed target cells, and produced gamma interferon. These results exemplify that host responses to an infectious agent are influenced by cross-reactive memory cells induced by past exposure to heterologous viruses, which could have important consequences for vaccine development.

Human cytomegalovirus protein US2 interferes with the expression of human HFE, a nonclassical class I major histocompatibility complex molecule that regulates iron homeostasis.

HFE is a nonclassical class I major histocompatibility complex (MHC) molecule that is mutated in the autosomal recessive iron overload disease hereditary hemochromatosis. There is evidence linking HFE with reduced iron uptake by the transferrin receptor (TfR). Using a panel of HFE and TfR monoclonal antibodies to examine human HFE (hHFE)-expressing cell lines, we demonstrate the expression of stable and fully glycosylated TfR-free and TfR-associated hHFE/beta2m complexes. We show that both the stability and assembly of hHFE complexes can be modified by the human cytomegalovirus (HCMV) viral protein US2, known to interfere with the expression of classical class I MHC molecules. HCMV US2, but not US11, targets HFE molecules for degradation by the proteasome. Whether this interference with the regulation of iron metabolism by a viral protein is a means of potentiating viral replication remains to be determined. The reduced expression of classical class I MHC and HFE complexes provides the virus with an efficient tool for altering cellular metabolism and escaping certain immune responses.

Multiple paths for activation of naive CD8+ T cells: CD4-independent help.

CD8(+) CTLs play a pivotal role in immune responses against many viruses and tumors. Two models have been proposed. The "three-cell" model focuses on the role of CD4(+) T cells, proposing that help is only provided to CTLs by CD4(+) T cells that recognize Ag on the same APC. The sequential "two-cell" model proposes that CD4(+) T cells can first interact with APCs, which in turn activate naive CTLs. Although these models provide a general framework for the role of CD4(+) T cells in mediating help for CTLs, a number of issues are unresolved. We have investigated the induction of CTL responses using dendritic cells (DCs) to immunize mice against defined peptide Ags. We find that help is required for activation of naive CTLs when DCs are used as APCs, regardless of the origin or MHC class I restriction of the peptides we studied in this system. However, CD8(+) T cells can provide self-help if they are present at a sufficiently high precursor frequency. The important variable is the total number of T cells responding, because class II-knockout DCs pulsed with two noncompeting peptides are effective in priming.

Immunoproteasomes shape immunodominance hierarchies of antiviral CD8(+) T cells at the levels of T cell repertoire and presentation of viral antigens.

Vertebrates express three cytokine-inducible proteasome subunits that are incorporated in the place of their constitutively synthesized counterparts. There is increasing evidence that the set of peptides generated by proteasomes containing these subunits (immunoproteasomes) differs from that produced by standard proteasomes. In this study, we use mice lacking one of the immunoproteasome subunits (LMP2) to show that immunoproteasomes play an important role in establishing the immunodominance hierarchy of CD8(+) T cells (T(CD8+)) responding to seven defined determinants in influenza virus. In LMP2(-/)- mice, responses to the two most dominant determinants drop precipitously, whereas responses to two subdominant determinants are greatly enhanced. Adoptive transfer experiments with naive normal and transgenic T(CD8+) reveal that the reduced immunogenicity of one determinant (PA(224-233)) can be attributed to decreased generation by antigen presenting cells (APCs), whereas the other determinant (NP(366-374)) is less immunogenic due to alterations in the T(CD8+) repertoire, and not, as reported previously, to the decreased capacity of LMP2(-/)- APCs to generate the determinant. The enhanced response to one of the subdominant determinants (PB1F2(62-70)) correlates with increased generation by LMP2(-)(/)- virus-infected cells. These findings indicate that in addition to their effects on the presentation of foreign antigens, immunoproteasomes influence T(CD8+) responses by modifying the repertoire of responding T(CD8+).

Cutting edge: recombinant adenoviruses induce CD8 T cell responses to an inserted protein whose expression is limited to nonimmune cells.

CD8 T cells (T(CD8+)) play a crucial role in immunity to viruses. Current understanding of activation of naive T cells entails Ag presentation by professional APCs (pAPCs). What happens, however, when viruses evolve to avoid infecting pAPCs? We have studied the consequences of this strategy by generating recombinant adenoviruses that express influenza A virus nucleoprotein under the control of tissue-specific promoters. We show that the immunogenicity of such viruses requires their delivery to organs capable of expressing nucleoprotein. This indicates that infection of pAPCs is not required for adenoviruses to elicit a T(CD8+) response, probably due to a cross-priming via pAPCs. While this bodes well for recombinant adenoviruses as vaccines, it dims their prospects as gene therapy vectors.

Multiple antigen-specific processing pathways for activating naive CD8+ T cells in vivo.

Current knowledge of the processing of viral Ags into MHC class I-associated ligands is based almost completely on in vitro studies using nonprofessional APCs (pAPCs). This is two steps removed from real immune responses to pathogens and vaccines, in which pAPCs activate naive CD8(+) T cells in vivo. Rational vaccine design requires answers to numerous questions surrounding the function of pAPCs in vivo, including their abilities to process and present peptides derived from endogenous and exogenous viral Ags. In the present study, we characterize the in vivo dependence of Ag presentation on the expression of TAP by testing the immunogenicity of model Ags synthesized by recombinant vaccinia viruses in TAP1(-/-) mice. We show that the efficiency of TAP-independent presentation in vitro correlates with TAP-independent activation of naive T cells in vivo and provide the first in vivo evidence for proteolytic processing of antigenic peptides in the secretory pathway. There was, however, a clear exception to this correlation; although the presentation of the minimal SIINFEKL determinant from chicken egg OVA in vitro was strictly TAP dependent, it was presented in a TAP-independent manner in vivo. In vivo presentation of the same peptide from a fusion protein retained its TAP dependence. These results show that determinant-specific processing pathways exist in vivo for the generation of antiviral T cell responses. We present additional findings that point to cross-priming as the likely mechanism for these protein-specific differences.

The export of major histocompatibility complex class I molecules from the endoplasmic reticulum of rat brown adipose cells is acutely stimulated by insulin.

Major histocompatibility complex class I (MHC-I) molecules have been implicated in several nonimmunological functions including the regulation and intracellular trafficking of the insulin-responsive glucose transporter GLUT4. We have used confocal microscopy to compare the effects of insulin on the intracellular trafficking of MHC-I and GLUT4 in freshly isolated rat brown adipose cells. We also used a recombinant vaccinia virus (rVV) to express influenza virus hemagglutinin (HA) as a generic integral membrane glycoprotein to distinguish global versus specific enhancement of protein export from the endoplasmic reticulum (ER) in response to insulin. In the absence of insulin, MHC-I molecules largely colocalize with the ER-resident protein calnexin and remain distinct from intracellular pools of GLUT4. Surprisingly, insulin induces the rapid export of MHC-I molecules from the ER with a concomitant approximately three-fold increase in their level on the cell surface. This ER export is blocked by brefeldin A and wortmannin but is unaffected by cytochalasin D, indicating that insulin stimulates the rapid transport of MHC-I molecules from the ER to the plasma membrane via the Golgi complex in a phosphatidyl-inositol 3-kinase-dependent and actin-independent manner. We further show that the effect of insulin on MHC-I molecules is selective, because insulin does not affect the intracellular distribution or cell-surface localization of rVV-expressed HA. These results demonstrate that in rat brown adipose cells MHC-I molecule export from the ER is stimulated by insulin and provide the first evidence that the trafficking of MHC-I molecules is acutely regulated by a hormone.

At the crossroads of cell biology and immunology: DRiPs and other sources of peptide ligands for MHC class I molecules.

CD8(+) T cells are a critical element of vertebrate immune responses to viruses and other intracellular parasites. They roam the body, monitoring cells for the presence of foreign peptides associated with MHC class I molecules of the major histocompatibility complex (MHC). Although it is clear that most of these peptides are generated through the action of proteasomes, the nature of the substrates degraded by proteasomes is an open question. Recent findings indicate that the major pool of substrates consists of a heterogeneous subset of proteins that are degraded within minutes of their synthesis. Evidence suggests that the fraction of newly synthesized proteins targeted for destruction is remarkably high - 30% or more, depending on cell type - possibly because they are defective in some way and cannot reach their intended conformation or location cellular in a time frame deemed appropriate by cells.

Cells adapted to the proteasome inhibitor 4-hydroxy- 5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone require enzymatically active proteasomes for continued survival.

The proteasome is the primary protease used by cells for degrading proteins and generating peptide ligands for class I molecules of the major histocompatibility complex. Based on the properties of cells adapted to grow in the presence of the proteasome inhibitor 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone (NLVS), it was proposed that proteasomes can be replaced by alternative proteolytic systems, particularly a large proteolytic complex with a tripeptidyl peptidase II activity. Here we show that NLVS-adapted cells retain sensitivity to a number of highly specific proteasome inhibitors with regard to antigenic peptide generation, accumulation of polyubiquitinated proteins, degradation of p53, and cell viability. In addition, we show that in the same assays (with a single minor exception), NLVS-adapted cells are about as sensitive as nonselected cells to Ala-Ala-Phe-chloromethylketone, a specific inhibitor of tripeptidyl peptidase II activity. Based on these findings, we conclude that proteasomes still have essential proteolytic functions in adapted cells that are not replaced by Ala-Ala-Phe-chloromethylketone-sensitive proteases.

Cut and trim: generating MHC class I peptide ligands.

Progress in understanding how peptide ligands are generated for MHC class I molecules took several interesting leaps and twists in the past year. Two independent lines of evidence suggest that most peptides are generated by proteasomal digestion of nascent proteins. The amino-terminally extended cytosolic precursors of an antigenic peptide were identified, bound to a mysterious carrier protein. Knowledge about the role of immunoproteasomes in antigen processing was fortified, cellular locales specialized for proteasomal degradation (and possibly antigenic-peptide production) were discovered and novel cytosolic proteases potentially involved in generating and trimming antigenic peptides were identified. The field is poised for quantitative analysis of the various pathways that contribute to the pool of peptides presented to the immune system by MHC class I molecules.

Proteasome inhibition interferes with gag polyprotein processing, release, and maturation of HIV-1 and HIV-2.

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6(gag), a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6(gag). Consistent with this, viruses with mutations in PR or p6(gag) were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.

Rapid degradation of a large fraction of newly synthesized proteins by proteasomes.

MHC class I molecules function to present peptides eight to ten residues long to the immune system. These peptides originate primarily from a cytosolic pool of proteins through the actions of proteasomes, and are transported into the endoplasmic reticulum, where they assemble with nascent class I molecules. Most peptides are generated from proteins that are apparently metabolically stable. To explain this, we previously proposed that peptides arise from proteasomal degradation of defective ribosomal products (DRiPs). DRiPs are polypeptides that never attain native structure owing to errors in translation or post-translational processes necessary for proper protein folding. Here we show, first, that DRiPs constitute upwards of 30% of newly synthesized proteins as determined in a variety of cell types; second, that at least some DRiPs represent ubiquitinated proteins; and last, that ubiquitinated DRiPs are formed from human immunodeficiency virus Gag polyprotein, a long-lived viral protein that serves as a source of antigenic peptides.

Dissecting the multifactorial causes of immunodominance in class I-restricted T cell responses to viruses.

Following influenza virus infection, the numbers of mouse TCD8+ cells responding to five different determinants vary more than 50-fold in primary responses but less so in secondary responses. Surprisingly, each determinant elicits a highly diverse and highly sensitive TCD8+ response. Inefficient antigen processing by virus-infected cells accounts for the poor immunogenicity of just one of the subdominant determinants. Overexpressing class I-peptide complexes using vaccinia virus revealed that the poor immunogenicity of two subdominant determinants reflects limitations in T cell responses unrelated to TCR diversity or sensitivity. Despite greatly enhanced expression, the immunodominant determinant is actually less immunogenic when overexpressed by vaccinia virus. Immunodominance is also modulated by determinant-specific variations in the capacity of TCD8+ to suppress responses to other determinants.

Mechanisms of viral interference with MHC class I antigen processing and presentation.

Viruses are ubiquitous and dangerous obligate intracellular parasites. To facilitate recognition of virus-infected cells by the immune system, vertebrates evolved a system that displays oligopeptides derived from viral proteins on the surface of cells in association with class I molecules of the major histocompatibility complex. Here we review the mechanisms counter-evolved by viruses to interfere with the generation of viral peptides, their intracellular trafficking, or the cell surface expression of class I molecules bearing viral peptides. This topic is important in its own right because the viruses that encode these proteins represent medically important pathogens, are potential vectors for vaccines or gene therapy, and provide strategies and tools for blocking immune recognition in transplantation, autoimmunity, and gene therapy. In addition, studies on viral interference provide unique insights into unfettered antigen processing and normal cellular functions that are exploited and exaggerated by viruses.

High peptide affinity for MHC class I does not correlate with immunodominance.

Cytotoxic T (Tc)-cell responses against influenza virus infection in BALB/c (H-2d) mice are dominated by Tc clones reactive to the viral nucleoprotein (NP). Here, we report investigations using recombinant vaccinia viruses (VV) encoding major histocompatibility complex (MHC) class I H-2Kd molecules differing by a single amino acid from glutamine (wild-type, Kdw) to histidine (mutant, Kdm) at position 114 located in the floor of the peptide-binding groove. Influenza-infected target cells expressing Kdw were strongly lysed by Kd-restricted Tc cells against A/WSN influenza virus or the immunodominant peptide of viral NP (NPP147-155), whereas infected Kdm-expressing targets gave little or no lysis, respectively, thus showing the immunodominance of NPP147-155. Kdm-expressing target cells saturated with synthetic NPP147-155 (10-5 M) were lysed similarly to Kdw-expressing targets by NPP147-155-specific Tc cells. Thus the defect in influenza-infected Kdm-expressing targets was quantitative; insufficient Kdm-peptide complexes were expressed. Tc-cell responses against four other viruses or alloantigens showed no effect of Kdm. When peptide transport-defective cells were infected with VV-Kdw or VV-Kdm and co-infected with a recombinant VV encoding an endoplasmic reticulum-targeted viral peptide, two influenza haemaglutinin peptides caused higher expression of Kdw than NPP147-155 indicating their higher affinity for Kdw. These results are inconsistent with the hypothesis that immunodominance in the anti-influenza response reflects high affinity of the immunodominant peptide, but are consistent with skewing of the Tc-cell receptor repertoire.

Intracellular localization of proteasomal degradation of a viral antigen.

To better understand proteasomal degradation of nuclear proteins and viral antigens we studied mutated forms of influenza virus nucleoprotein (NP) that misfold and are rapidly degraded by proteasomes. In the presence of proteasome inhibitors, mutated NP (dNP) accumulates in highly insoluble ubiquitinated and nonubiquitinated species in nuclear substructures known as promyelocytic leukemia oncogenic domains (PODs) and the microtubule organizing center (MTOC). Immunofluorescence revealed that dNP recruits proteasomes and a selective assortment of molecular chaperones to both locales, and that a similar (though less dramatic) effect is induced by proteasome inhibitors in the absence of dNP expression. Biochemical evidence is consistent with the idea that dNP is delivered to PODs/MTOC in the absence of proteasome inhibitors. Restoring proteasome activity while blocking protein synthesis results in disappearance of dNP from PODs and the MTOC and the generation of a major histocompatibility complex class I-bound peptide derived from dNP but not NP. These findings demonstrate that PODs and the MTOC serve as sites of proteasomal degradation of misfolded dNP and probably cellular proteins as well, and imply that antigenic peptides are generated at one or both of these sites.