Klaus Früh

The ER-luminal domain of the HCMV glycoprotein US6 inhibits peptide translocation by TAP.

Human cytomegalovirus (HCMV) inhibits MHC class I antigen presentation by a sequential multistep process involving a family of unique short (US) region-encoded glycoproteins. US3 retains class I molecules, whereas US2 and US11 mediate the cytosolic degradation of heavy chains by the proteosomes. In US6-transfected cells, however, intracellular transport of class I molecules is impaired because of defective peptide translocation by transporters associated with antigen processing (TAP). Peptide transport is restored in HCMV mutants lacking US6. In contrast to the cytosolic herpes simplex virus protein ICP47, US6 interacts with TAP inside the endoplasmic reticulum lumen, as shown by US6 derivatives lacking the transmembrane and cytoplasmic domains and by the observation that US6 does not prevent peptides from binding to TAP. Thus, HCMV targets TAP for immune escape by a molecular mechanism different from that of herpes simplex virus.

Immune clearance of highly pathogenic SIV infection

Established infections with the human and simian immunodeficiency viruses (HIV and SIV, respectively) are thought to be permanent with even the most effective immune responses and antiretroviral therapies only able to control, but not clear, these infections. Whether the residual virus that maintains these infections is vulnerable to clearance is a question of central importance to the future management of millions of HIV-infected individuals. We recently reported that approximately 50% of rhesus macaques (RM; Macaca mulatta) vaccinated with SIV protein-expressing rhesus cytomegalovirus (RhCMV/SIV) vectors manifest durable, aviraemic control of infection with the highly pathogenic strain SIVmac239 (ref. 5). Here we show that regardless of the route of challenge, RhCMV/SIV vector-elicited immune responses control SIVmac239 after demonstrable lymphatic and haematogenous viral dissemination, and that replication-competent SIV persists in several sites for weeks to months. Over time, however, protected RM lost signs of SIV infection, showing a consistent lack of measurable plasma- or tissue-associated virus using ultrasensitive assays, and a loss of T-cell reactivity to SIV determinants not in the vaccine. Extensive ultrasensitive quantitative PCR and quantitative PCR with reverse transcription analyses of tissues from RhCMV/SIV vector-protected RM necropsied 69–172 weeks after challenge did not detect SIV RNA or DNA sequences above background levels, and replication-competent SIV was not detected in these RM by extensive co-culture analysis of tissues or by adoptive transfer of 60 million haematolymphoid cells to naive RM. These data provide compelling evidence for progressive clearance of a pathogenic lentiviral infection, and suggest that some lentiviral reservoirs may be susceptible to the continuous effector memory T-cell-mediated immune surveillance elicited and maintained by cytomegalovirus vectors.

Molecular mechanism and species specificity of tap inhibition by herpes simplex virus protein icp47

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)‐mediated translocation of antigen‐derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV‐infected cells by cytotoxic T‐lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species‐specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8‐azido‐ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP‐transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100‐fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate‐binding site of TAP.

Vpu Directs the Degradation of the Human Immunodeficiency Virus Restriction Factor BST-2/Tetherin via a βTrCP-Dependent Mechanism

ABSTRACT The primary roles attributed to the human immunodeficiency virus type 1 (HIV-1) Vpu protein are the degradation of the viral receptor CD4 and the enhancement of virion release. With regard to CD4 downregulation, Vpu has been shown to act as an adapter linking CD4 with the ubiquitin-proteasome machinery via interaction with the F-box protein βTrCP. To identify additional cellular βTrCP-dependent Vpu targets, we performed quantitative proteomics analyses using the plasma membrane fraction of HeLa cells expressing either wild-type Vpu or a Vpu mutant (S52N/S56N) that does not bind βTrCP. One cellular protein, BST-2 (CD317), was consistently underrepresented in the membrane proteome of cells expressing wild-type Vpu compared to the proteome of cells expressing the Vpu mutant. To verify the biological relevance of this phenotype for HIV pathogenesis, we showed that in T cells infected with HIV-1, BST-2 downregulation occurred in a Vpu-dependent manner. Recently, BST-2 has been identified as the interferon-inducible cellular factor Tetherin, which restricts HIV virion release in the absence of Vpu. We address here the unresolved mechanism of Vpu-mediated BST-2 downregulation. Our data show that the presence of wild-type Vpu reduced cell surface and total steady-state BST-2 levels, whereas that of the mutant Vpu had no effect. In addition, treatment of cells with the lysosome acidification inhibitor concanamycin A, but not treatment with the proteasome inhibitor MG132, reduced BST-2 downregulation by wild-type Vpu, thereby suggesting that the presence of Vpu leads to the degradation of BST-2 via an endosome-lysosome degradation pathway. The importance of βTrCP in this process was confirmed by demonstrating that in the absence of βTrCP, BST-2 levels were restored despite the presence of Vpu. Taken together, these data support the hypothesis that, in similarity to its role in CD4 degradation, Vpu acts as an adapter molecule linking BST-2 to the cellular ubiquitination machinery via βTrCP. However, in contrast to the proteasome-dependent degradation of CD4, which occurs in the endoplasmic reticulum, Vpu appears to interact with BST-2 in the trans-Golgi network or in early endosomes, leading to lysosomal degradation of BST-2. Via this action, Vpu could counter the tethering function of BST-2, resulting in enhanced HIV-1 virion release. Interestingly, although HIV-2 does not express Vpu, an isolate known to exhibit enhanced viral egress can downregulate surface BST-2 by an as-yet-unknown mechanism that does not appear to involve degradation. Understanding the molecular mechanisms of both Vpu-dependent and -independent mediated antagonism of BST-2 will be critical for therapeutic strategies that exploit this novel viral function.

Cytomegalovirus vectors violate CD8+ T cell epitope recognition paradigms

Introduction CD8+ T cell responses focus on a small fraction of total pathogen-encoded peptides, which are similar among individuals with shared major histocompatibility complex (MHC) alleles. This focus can limit immune control of genetically flexible pathogens, such as HIV and SIV, because CD8+ T cells in most infected subjects do not target sequences required for pathogen fitness, resulting in viral escape. Although a vaccine capable of broadening or redirecting CD8+ T cell epitope targeting to prevent viral escape would be highly advantageous, it remains unclear whether this targeting can be diverted from its default pattern during priming. Fibroblast-adapted RhCMV/gag vectors elicit MHC class II–restricted CD8+ T cells, greatly expanding the breadth of the response. (Top) Differential inhibition of SIVgag-specific CD8+ T cells from SIV+, fibroblast-adapted RhCMV/gag vector–vaccinated, and tropism-repaired RhCMV/gag vector–vaccinated rhesus macaques by MHC-I versus MHC-II blockade. (Bottom) Responses to consecutive SIVgag 15mer peptides in the indicated animals, classified by sensitivity to MHC-I versus MHC-II blockade. Methods We used intracellular cytokine analysis to compare the epitope targeting of SIV-specific CD8+ T cell responses in rhesus macaques with controlled SIV infection or after vaccination with either conventional SIV vaccines or rhesus cytomegalovirus (RhCMV) vectors. RhCMV vectors have been associated with stringent control of SIV challenge in the absence of protective MHC alleles. Results Fibroblast-adapted RhCMV/SIV vectors elicited SIV-specific CD8+ T cells that failed to target any canonical epitopes associated with SIV infection or conventional SIV vaccination. Instead, they recognized distinct epitopes characterized by extraordinary breadth (greater than that of conventional vaccines by a factor of >3), MHC class II (MHC-II) restriction (63% of epitopes), and high promiscuity (epitopes common to most or all responses in vaccinated macaques). These unconventionally targeted CD8+ T cell responses recognized autologous SIV-infected cells, indicating that processing and presentation of the unconventional epitopes is CMV-independent. However, CMV gene expression was responsible for directing epitope specificity of CD8+ T cells during priming. The induction of canonical SIV epitope–specific CD8+ T cell responses was specifically suppressed by expression of the Rh189/US11 gene, and the promiscuous MHC-I– and MHC-II–restricted CD8+ T cell responses occurred only in the absence of the Rh157.4–.6/UL128–131 genes involved in CMV tropism for nonfibroblasts. Discussion These findings suggest that CD8+ T cell recognition is more flexible than had been thought, and that the focused epitope recognition profiles of conventional CD8+ T cell responses may be primarily restricted by immunoregulation during priming (which can be subverted by CMV) rather than by intrinsic limitations in antigen processing/presentation or in T cell receptor repertoire. The ability of CMVs with different genetic modifications to differentially elicit CD8+ T cell responses with divergent patterns of epitope recognition raises the possibility of a CMV vector–based vaccine platform with programmable CD8+ T cell epitope targeting, including vectors that can selectively elicit CD8+ T cell responses targeting conventional or unconventional epitopes. Because the latter would be unaffected by escape mutations arising during natural infection, these vectors would be well suited for therapeutic vaccine applications. CMV Breaks All the Rules One vaccine strategy being pursued against HIV is to generate protection that is dependent on cell-mediated, rather than humoral, immune responses. A cytomegalovirus (CMV)–vectored vaccine that expresses simian immunodeficiency virus (SIV) antigens exhibits stringent and durable viral control upon SIV challenge in approximately half of vaccinated rhesus macaques. Hansen et al. (10.1126/science.1237874, see the Perspective by Goonetilleke and McMichael) sought to determine the basis for the protection and discovered that the CD8+ T cell response in vaccinated monkeys does not target canonical SIV epitopes, which SIV is known to escape, but rather generates a broad, promiscuous response. A vaccine that uses one virus to deliver components of a second virus elicits T cells that recognize noncanonical epitopes. [Also see Perspective by Goonetilleke and McMichael] CD8+ T cell responses focus on a small fraction of pathogen- or vaccine-encoded peptides, and for some pathogens, these restricted recognition hierarchies limit the effectiveness of antipathogen immunity. We found that simian immunodeficiency virus (SIV) protein–expressing rhesus cytomegalovirus (RhCMV) vectors elicit SIV-specific CD8+ T cells that recognize unusual, diverse, and highly promiscuous epitopes, including dominant responses to epitopes restricted by class II major histocompatibility complex (MHC) molecules. Induction of canonical SIV epitope–specific CD8+ T cell responses is suppressed by the RhCMV-encoded Rh189 gene (corresponding to human CMV US11), and the promiscuous MHC class I– and class II–restricted CD8+ T cell responses occur only in the absence of the Rh157.5, Rh157.4, and Rh157.6 (human CMV UL128, UL130, and UL131) genes. Thus, CMV vectors can be genetically programmed to achieve distinct patterns of CD8+ T cell epitope recognition.

Antigen presentation by MHC class I and its regulation by interferon γ

Antigen processing by MHC class I molecules begins with the generation of peptides by proteolytic breakdown of proteins. IFN-gamma upregulates gene expression of several proteasomal subunits as well as the proteasome regulator PA28; this implicated their role in antigen degradation. Crystallographic, mutational and biochemical studies contributed to our understanding of the basic principles of proteasomal protein degradation and the consequences of IFN-gamma induction for proteasome function. In addition, nonproteasomal mechanisms seem to be involved in antigen degradation. Leucine aminopeptidase, which is also upregulated by IFN-gamma, was shown to collaborate with the proteasome for epitope production and unknown proteases seem to compensate for the loss of proteasomal degradation in the presence of proteasome inhibitors. Thus, a rather complex picture emerges for the rules governing peptide production in the presence or absence of IFN-gamma.

Downregulation of Major Histocompatibility Complex Class I by Human Ubiquitin Ligases Related to Viral Immune Evasion Proteins

ABSTRACT Poxviruses and gamma-2 herpesviruses share the K3 family of viral immune evasion proteins that inhibit the surface expression of glycoproteins such as major histocompatibility complex class I (MHC-I), B7.2, ICAM-1, and CD95(Fas). K3 family proteins contain an amino-terminal PHD/LAP or RING-CH domain followed by two transmembrane domains. To examine whether human homologues are functionally related to the viral immunoevasins, we studied seven membrane-associated RING-CH (MARCH) proteins. All MARCH proteins located to subcellular membranes, and several MARCH proteins reduced surface levels of known substrates of the viral K3 family. Two closely related proteins, MARCH-IV and MARCH-IX, reduced surface expression of MHC-I molecules. In the presence of MARCH-IV or MARCH-IX, MHC-I was ubiquitinated and rapidly internalized by endocytosis, whereas MHC-I molecules lacking lysines in their cytoplasmic tail were resistant to downregulation. The amino-terminal regions containing the RING-CH domain of several MARCH proteins examined catalyzed multiubiquitin formation in vitro, suggesting that MARCH proteins are ubiquitin ligases. The functional similarity of the MARCH family and the K3 family suggests that the viral immune evasion proteins were derived from MARCH proteins, a novel family of transmembrane ubiquitin ligases that seems to target glycoproteins for lysosomal destruction via ubiquitination of the cytoplasmic tail.

Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: A newly discovered mechanism for modulating the multicatalytic proteinase complex

The degradation of cytoplasmic antigens to peptides presented by class I MHC molecules is thought to be mediated by the ubiquitin/proteasome pathway. Support for this view came from our observation that the subunit composition of proteasomes can be changed by interferon‐gamma (IFN‐gamma) treatment. Thereby two subunits, LMP2 and LMP7, which are encoded in the MHC class II region, are incorporated into the proteasomal complex, whereas other subunits disappear. In the experiments reported in this communication we studied the subunit changes occurring in cell lines where the expression of LMP2 or LMP7 can be regulated individually either by IFN‐gamma induction or by applying a new system to control the expression of transfected LMPs. In both situations LMP2 induction leads exclusively to the disappearance of housekeeping subunit 2, whereas LMP7 affects only subunit 10. Subunit 2 was found to be 76% homologous to LMP2. Since incorporation of LMP2 into the proteasomal complex prevents processing of the subunit 2 precursor, we conclude that LMP2 displaces subunit 2 during assembly. Subunit displacement is most likely a general mechanism to modulate the catalytic activity of the proteasomal complex without changing its structure. Furthermore, the controlled incorporation of transfected subunits into the complex offers a new approach to study proteasome function in vivo.

Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B

Human cytomegalovirus (HCMV) infection alters the expression of many cellular genes, including IFN-stimulated genes (ISGs) [Zhu, H., Cong, J.-P., Mamtora, G., Gingeras, T. & Shenk, T. (1998) Proc. Natl. Acad. Sci. USA 95, 14470–14475]. By using high-density cDNA microarrays, we show that the HCMV-regulated gene expression profile in fibroblasts does not differ substantially from the response generated by IFN. Furthermore, we identified the specific viral component triggering this response as the envelope glycoprotein B (gB). Cells treated with gB, but not other herpesviral glycoproteins, exhibited the same transcriptional profile as HCMV-infected cells. Thus, the interaction of gB with its as yet unidentified cellular receptor is the principal mechanism by which HCMV alters cellular gene expression early during infection. These findings highlight a pioneering paradigm for the consequences of virus–receptor interactions.

Molecular Mechanism of BST2/Tetherin Downregulation by K5/MIR2 of Kaposi's Sarcoma-Associated Herpesvirus

ABSTRACT K3/MIR1 and K5/MIR2 of Kaposis sarcoma-associated herpesvirus (KSHV) are viral members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family and contribute to viral immune evasion by directing the conjugation of ubiquitin to immunostimulatory transmembrane proteins. In a quantitative proteomic screen for novel host cell proteins downregulated by viral immunomodulators, we previously observed that K5, as well as the human immunodeficiency virus type 1 (HIV-1) immunomodulator VPU, reduced steady-state levels of bone marrow stromal cell antigen 2 (BST2; also called CD317 or tetherin), suggesting that BST2 might be a novel substrate of K5 and VPU. Recent work revealed that in the absence of VPU, HIV-1 virions are tethered to the plasma membrane in BST2-expressing HeLa cells. By targeting BST2, K5 might thus similarly overcome an innate antiviral host defense mechanism. Here we establish that despite its type II transmembrane topology and carboxy-terminal glycosylphosphatidylinositol (GPI) anchor, BST2 represents a bona fide target of K5 that is downregulated during primary infection by and reactivation of KSHV. Upon exit of the protein from the endoplasmic reticulum, lysines in the short amino-terminal domain of BST2 are ubiquitinated by K5, resulting in rapid degradation of BST2. Ubiquitination of BST2 is required for degradation, since BST2 lacking cytosolic lysines was K5 resistant and ubiquitin depletion by proteasome inhibitors restored BST2 surface expression. Thus, BST2 represents the first type II transmembrane protein targeted by K5 and the first example of a protein that is both ubiquitinated and GPI linked. We further demonstrate that KSHV release is decreased in the absence of K5 in a BST2-dependent manner, suggesting that K5 contributes to the evasion of intracellular antiviral defense programs.