With the aim of developing new strategies in antiviral therapy and vaccination as well as the investigation of the role of the regulatory HIV-1 proteins p6, Vpr, and Vpu in the pathogenesis of HIV-1, our work has been based upon interdisciplinary cooperation and application of a broad spectrum of methods within the fields of retrovirology, molecular and cell biology, biochemistry, protein structure determination, as well as immunology. The overall research goal is to contribute to the development of novel anti-retroviral strategies by deciphering the structural constraints and molecular mechanisms governing the function of viral proteins and their cellular counterparts.
We are currently working on the following main research topics:
This major project focuses on the role of the HIV-1 p6 protein as part of the Gag precursor in the late processes of virus replication, especially assembly and budding.
Viruses like HIV-1 hijack a cellular system known as the endosomal sorting complex required for transport (ESCRT) in order to promote their release from the cell surface. This cellular machinery is involved in cytokinesis and in the vacuolar protein sorting of endocytosed plasma membrane proteins by regulating the abscission of membrane stalks.
The HIV-1 p6 Gag-protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (L)-domains. Although p6 originates from one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds to at least two cellular budding factors of the ESCRT-complex, Tsg101 (tumor susceptibility gene product 101) and ALIX (ALG-2 interacting protein X), via its highly conserved primary PTAP and secondary YPXnL L-domains, respectively. Moreover, p6 is substrate for phosphorylation, ubiquitination and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. We investigated the importance of the highly conserved serine residue at position 40 (S40) which is phosphorylated by the atypical phosphokinase C (aPKC) and was until now not assigned to any known function of p6.
By performing membrane flotation assays we found that in HIV-1 expressing cells the mutant S40F, but not the conservative mutation of S40 to Asp (S40D) or Asn (S40N), leads to an increased membrane association of Gag. This correlates with an enhanced Gag-ubiquitination which predominantly consists of K48-linked polyubiquitin chains. Consistently, the S40F mutation augments the entry of Gag into the UPS and thus enhances its MHC-I antigen presentation and Gag-mediated T-cell activation. Others could show that the membrane resident ubiquitin E3 ligase Cul7 is able to ubiquitinate Gag. This finding led us to the hypothesis that the S40F mutant might also be ubiquitinated by this or other E3 ligases due to prolonged membrane association.
As an explanation for the enhanced membrane association of Gag, we found by performing high resolution NMR analysis that S40F, together with Tyr-36, induces the formation of a hydrophobic patch within the C-terminal α-helix of p6 which might account for the augmentation in membrane association of Gag.
We further analyzed the role of p6 in membrane association and found that the mutation of the highly conserved glutamic acids within p6 to alanine (E0A), like the S40F mutant, leads to an enhanced polyubiquitination and subsequent entry of Gag into the UPS and thus an increased MHC-I antigen presentation of Gag derived epitopes. In addition, like for the S40F mutant, the CA-SP1 processing of the E0A mutant is impaired, also resulting in loss of infectivity and significantly decreased replication capacity. Furthermore, the E0A mutant displays defective virus release that could not be rescued by ALIX overexpression. Most strikingly, the E0A mutant exhibits elevated membrane association which might be due to removal of the negatively charged Glu residues. We hypothesized that in wt situation these negative charges might contribute to the repulsion of p6 from the plasma membrane. To verify this hypothesis, we created mutants where all glutamic acids within p6 were exchanged with Asp residues (E0D) that altered the side chain of the amino acid but maintained the overall charge of p6. Intriguingly, this mutant behaves like the wt in terms of ubiquitination, MHC-I antigen presentation and membrane association of Gag, leading to the assumption that the negative charges but not the nature of the side chains of the glutamic acids are important for the functionality of p6 during late steps of replication.
The cumulative data support a model in which p6, in addition to matrix, acts as a membrane targeting domain of Gag, either by hydrophobic (S40F) or electrostatic (E0A) interactions with the inner leaflet of the plasma membrane.
There is a significantly higher risk for type II diabetes in HIV-1 carriers, albeit the molecular mechanism for this HIV-related pathology remains enigmatic. The HIV-1 p6 Gag-protein is synthesized as the C-terminal part of the Gag polyprotein Pr55. In this context, p6 promotes virus release by its two L-domains, and facilitates the incorporation of the viral accessory protein Vpr. However, the function of p6 in its mature form, after proteolytic release from Gag, has not been investigated yet and was another main project in our lab.
We found that mature p6 represents the first known viral substrate of the ubiquitously expressed cytosolic metallo-endopeptidase insulin-degrading enzyme (IDE). IDE is sufficient and required for degradation of p6, and p6 is approximately 100-fold more efficiently degraded by IDE than its eponymous substrate insulin. This observation appears to be specific for HIV-1, as p6 proteins from HIV-2 and simian immunodeficiency virus, as well as the 51 amino acid p9 from equine infectious anaemia virus were insensitive to IDE degradation. The amount of virus-associated p6, as well as the efficiency of release and maturation of progeny viruses, does not depend on the presence of IDE in the host cells, as it was shown by CRISPR/Cas9 edited IDE KO cells. However, HIV-1 mutants harboring IDE insensitive p6 variants exhibit reduced virus replication capacity, a phenomenon that seems to depend on the presence of an X4-tropic Env. Furthermore, competing for IDE by exogenous insulin or inhibiting IDE by the highly specific inhibitor 6bK, also reduced virus replication. This effect could be specifically attributed to IDE since replication of HIV-1 variants coding for an IDE-insensitive p6 were inert towards IDE-inhibition.
Our cumulative data support a model in which removal of p6 by IDE during viral entry is important for virus replication, at least in the case of X4 tropic HIV-1.
However, it remains unclear to which extent the IDE mediated degradation is phylogenetically conserved among HIV-1. We analyzed this issue and found two HIV-1 isolates with IDE resistant p6 proteins. Sequence comparison allowed deducing one single amino acid regulating IDE sensitivity of p6. Exchange of the N-terminal leucine residue of p6 derived from the IDE sensitive isolate HIV-1NL4-3 with proline enhances its stability, while replacing Pro-1 of p6 from the IDE insensitive isolate SG3 with leucine restores susceptibility towards IDE. Phylogenetic analyses of this natural polymorphism revealed that the N-terminal leucine is characteristic for p6 derived from HIV-1 group M except for subtype A, which predominantly expresses p6 with an N-terminal proline. Accordingly, p6 peptides derived from subtype A are not degraded by IDE.
In conclusion, IDE mediated degradation of p6 is specific for HIV-1 group M isolates and not occasionally distributed among HIV-1.
In recent years it has been well established that two major constituent parts of the UPS – the proteasome holoenzyme and a number of ubiquitin ligases – play a crucial role, not only in virus replication but also in the regulation of the immunogenicity of HIV-1. However, the role in HIV-1 replication of the third major UPS component, the deubiquitinating enzymes (DUBs), has remained largely unknown. Thus, we studied in another project the role of DUBs in HIV-1 replication.
We could show that the DUB-inhibitors (DIs) P22077 and PR-619, specific for the DUBs USP7 and USP47, impair Gag processing and thereby reduce the infectivity of released virions without affecting viral protease activity. Furthermore, the replication capacity of X4- and R5-tropic HIV-1NL4-3 in human lymphatic tissue is decreased upon treatment with these inhibitors without affecting cell viability. Most strikingly, combinatory treatment with DIs and proteasome inhibitors synergistically blocks virus replication at concentrations where mono-treatment was ineffective, indicating that DIs can boost the anti-retroviral activity of proteasome inhibitors. In addition, P22077 and PR-619 increase the polyubiquitination of Gag and thus its entry into the UPS and MHC-I pathway.
In summary, our data point towards a model in which specific inhibitors of DUBs not only interfere with virus spread but also increase the immune recognition of HIV-1 expressing cells. Thus, DIs might offer multiple options in antiretroviral therapy.
Deciphering the molecular mechanism of small regulatory virus proteins provides knowledge about specific host-virus interactions and should open the path to the development of innovative antiviral strategies. The major focus of this long term project in cooperation with the lab of Dr. Ashok Balasubramanyam (Baylor College of Medicine, Houston, TX, USA) has been the functional characterization of the HIV-1 accessory protein Vpr. In addition we performed functional analysis of another HIV-1 accessory protein, Vpu.
In the last years, we intensively investigated the HIV-1 accessory protein Vpr (viral protein R) of HIV-1. The 96 amino acid Vpr has multiple functions in HIV-1 pathogenesis, including virion incorporation, nuclear translocation of the HIV-1 preintegration complex, induction of cell cycle arrest at the G2/M phase, and the regulation of apoptosis.
HIV patients manifest adipose dysfunction characterized by accelerated lipolysis, hepatosteatosis, dyslipidemia, insulin resistance, and hyperglycemia. However, the in vivo mechanisms whereby HIV infection induces those defects in human adipose disorders have not been reported. Thus, in cooperation with the lab of Dr. Ashok Balasubramanyam the pathogenic role of Vpr in HIV-associated adipose dysfunction was investigated. It could be shown that Vpr released from HIV-1 in tissue reservoirs, can disrupt PPAR/GR (peroxisome proliferator-activated receptor/ glucocorticoid receptor) co-regulation and cell cycle control to produce adipose dysfunction and hepatosteatosis. Moreover, we could show that Vpr broadly altered hepatic expression of LXR (liver X receptor) α-regulated lipid metabolic genes. Furthermore, Vpr diminishes hepatic fatty acid ß-oxidation which altogether contributes to the HIV-associated fatty liver disease.
Confirmation of these mechanisms in patients could pave the way for targeted treatment with small-molecule inhibitors of Vpr, GR antagonists, or PPAR agonists.
The HIV-1 accessory protein Vpu is an 81-amino-acid oligomeric type 1 integral membrane phosphoprotein, which is encoded exclusively in HIV-1 and related simian immunodeficiency viruses (SIV), but not in HIV-2. Vpu has been shown to induce degradation of the CD4 receptor by the ER-associated protein degradation (ERAD) pathway and to enhance virus particle release from the plasma membrane. Randomization of the transmembrane (TM) domain prevents Vpu`s ion channel formation and impairs its ability to regulate virus release. This suggested a causal relation between the ion channel activity of Vpu and its augmentation of virus release. Recently, it was shown that Vpu enhances HIV-1 virion release by counteracting a cellular restriction factor termed CD317 (also known as tetherin, BST-2 or HM1.24). Thus, in order to analyze whether ion channel activity of Vpu correlates with viral particle release, several TM mutants were generated. The highly conserved residues Ala-14 and Ala-18 in the TM domain of Vpu were analyzed for their ability to form ion channels and to counteract CD317.
Mutation of Ala-14 and Ala-18 to asparagine impairs the efficiency of surface down-regulation of CD317. However, both mutants still exhibit ion channel activity in 293T cells, indicating that channel activity of Vpu is not sufficient to support virus release. Furthermore, to assess its relevance in CD317 counteraction, we mutated Ser-23, which is essential for Vpu's ion channel activity, to alanine (S23A). Intriguingly, the S23A mutant still efficiently interacts with CD317, and thus supports virus release in the presence of CD317. Taken together, our data suggest that the ion channel activity of Vpu is not critical for counteraction of CD317.
Moreover, it was shown that Vpu also induces downregulation of the coactivating NK cell receptor, the NK, T-cell, B-cell antigen (NTB-A), from the cell surface in order to evade lysis of HIV-1 infected cells by NK cells. Pulse-chase analyses revealed that HIV-1 Vpu affects the glycosylation pattern of NTB-A by a mechanism that is distinct from the Vpu induced downregulation of CD4 and tetherin. In the presence of Vpu, only the high mannose form of NTB-A was detectable, suggesting that Vpu prevented the formation of the mature form of NTB-A. This phenomenon is associated with the ability of Vpu to downregulate cell surface NTB-A by retention of NTB-A within the Golgi-compartment. Furthermore, the Vpu-mediated effect on NTB-A glycosylation is highly conserved among Vpu proteins derived from HIV-1 and SIV and corresponds to the level of downregulation of NTB-A. Together, our results suggest that the reduction of NTB-A from the cell surface is associated with the Vpu-mediated effect on the glycosylation pattern of newly synthesized NTB-A molecules.
Moreover, it was shown that Vpu induces downregulation of cell surface CD155, a ligand for the DNAM-1 activating receptor of NK and CD8+ T-cells, to evade NK cell-mediated immune response. In order to analyze the effect of Vpu on the surface expression of CD155, Vpu TM and cytoplasmic deletion mutants were analyzed in HeLa cells, which constitutively express endogenous CD155. The results suggested that the TM domain of Vpu, particularly Ala-10, Ala-14 and Ala-18, is crucial for cell surface downregulation of CD155. Moreover, Vpu induces accumulation of CD155 in perinuclear compartments indicating that Vpu might inhibit trafficking of CD155 to the cell surface. Thus, Vpu seems to subvert NK cell responses against HIV-1 infected T-cells by modulation of multiple receptors necessary for NK cell activation.