Membrane interactions and alignment of structures within the HIV-1 Vpu cytoplasmic domain: effect of phosphorylation of serines 52 and 56

AbstractThe cytoplasmic domain of the HIV-1 accessory protein Vpu is involved in the binding and degradation of the viral receptor CD4. In order to analyze previous structural models in the context of membrane environments, regions of VpuCYTO incorporating particular conformational features have been synthesized and labelled with 15N at selected backbone amides. Well-oriented proton-decoupled 15N solid-state NMR spectra with 15N chemical shifts at the most upfield position indicate that the amphipathic helix within [15N-Leu 45]-Vpu27–57 strongly interacts with mechanically aligned POPC bilayers and adopts an orientation parallel to the membrane surface. No major changes in the topology of this membrane-associated amphipathic helix were observed upon phosphorylation of serine residues 52 and 56, although this modification regulates biological function of Vpu. In contrast, [15N-Ala 62]-Vpu51–81 exhibits a pronounced 15N chemical shift anisotropy

The Host-Pathogen interaction of human cyclophilin A and HIV-1 Vpr requires specific N-terminal and novel C-terminal domains

<p>Abstract</p> <p>Background</p> <p>Cyclophilin A (CypA) represents a potential key molecule in future antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication. CypA interacts with the virus proteins Capsid (CA) and Vpr, however, the mechanism through which CypA influences HIV-1 infectivity still remains unclear.</p> <p>Results</p> <p>Here the interaction of full-length HIV-1 Vpr with the host cellular factor CypA has been characterized and quantified by surface plasmon resonance spectroscopy. A C-terminal region of Vpr, comprising the 16 residues ⁷⁵GCRHSRIGVTRQRRAR⁹⁰, with high binding affinity for CypA has been identified. This region of Vpr does not contain any proline residues but binds much more strongly to CypA than the previously characterized N-terminal binding domain of Vpr, and is thus the first protein binding domain to CypA described involving no proline residues. The fact that the mutant peptide Vpr^75-90R80A binds more weakly to CypA than the wild-type peptide confirms that Arg-80 is a key residue in the C-terminal binding domain. The N- and C-terminal binding regions of full-length Vpr bind cooperatively to CypA and have allowed a model of the complex to be created. The dissociation constant of full-length Vpr to CypA was determined to be approximately 320 nM, indicating that the binding may be stronger than that of the well characterized interaction of HIV-1 CA with CypA.</p> <p>Conclusions</p> <p>For the first time the interaction of full-length Vpr and CypA has been characterized and quantified. A non-proline-containing 16-residue region of C-terminal Vpr which binds specifically to CypA with similar high affinity as full-length Vpr has been identified. The fact that this is the first non-proline containing binding motif of any protein found to bind to CypA, changes the view on how CypA is able to interact with other proteins. It is interesting to note that several previously reported key functions of HIV-1 Vpr are associated with the identified N- and C-terminal binding domains of the protein to CypA.</p

Solution structure of the equine infectious anemia virus p9 protein: a rationalization of its different ALIX binding requirements compared to the analogous HIV-p6 protein

<p>Abstract</p> <p>Background</p> <p>The equine infection anemia virus (EIAV) p9 Gag protein contains the late (L-) domain required for efficient virus release of nascent virions from the cell membrane of infected cell.</p> <p>Results</p> <p>In the present study the p9 protein and N- and C-terminal fragments (residues 1-21 and 22-51, respectively) were chemically synthesized and used for structural analyses. Circular dichroism and ¹H-NMR spectroscopy provide the first molecular insight into the secondary structure and folding of this 51-amino acid protein under different solution conditions. Qualitative ¹H-chemical shift and NOE data indicate that in a pure aqueous environment p9 favors an unstructured state. In its most structured state under hydrophobic conditions, p9 adopts a stable helical structure within the C-terminus. Quantitative NOE data further revealed that this α-helix extends from Ser-27 to Ser-48, while the N-terminal residues remain unstructured. The structural elements identified for p9 differ substantially from that of the functional homologous HIV-1 p6 protein.</p> <p>Conclusions</p> <p>These structural differences are discussed in the context of the different types of L-domains regulating distinct cellular pathways in virus budding. EIAV p9 mediates virus release by recruiting the ALG2-interacting protein X (ALIX) via the YPDL-motif to the site of virus budding, the counterpart of the YPX_nL-motif found in p6. However, p6 contains an additional PTAP L-domain that promotes HIV-1 release by binding to the tumor susceptibility gene 101 (Tsg101). The notion that structures found in p9 differ form that of p6 further support the idea that different mechanisms regulate binding of ALIX to primary versus secondary L-domains types.</p

The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding

<p>Abstract</p> <p>Background</p> <p>Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur <it>in vitro </it>and <it>in vivo</it>. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined.</p> <p>Results</p> <p>Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl <it>cis</it>/<it>trans </it>isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding.</p> <p>Conclusions</p> <p>Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl <it>cis</it>/<it>trans </it>isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data. In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding. This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl <it>cis/trans </it>interconversion during binding to the RHFP³⁵RIW motif of N-terminal Vpr.</p

Multi-level Strategy for Identifying Proteasome-Catalyzed Spliced Epitopes Targeted by CD8+ T Cells during Bacterial Infection

Proteasome-catalyzed peptide splicing (PCPS) generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8+ T cells in L. monocytogenes-infected mice. While reacting to the spliced epitopes, these CD8+ T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8+ T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design

Highly conserved serine residue 40 in HIV-1 p6 regulates capsid processing and virus core assembly

Background: The HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L-) domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX), is a 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. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6. Results: Consistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF) reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA) and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site. Conclusions: Overall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively affects CA maturation and virus core formation, and consequently the infectivity of released virions

Thymulin gene therapy prevents the reduction in circulating gonadotropins induced by thymulin deficiency in mice

Integrity of the thymus during perinatal life is necessary for a proper maturation of the pituitarygonadal axis in mice and other mammalian species. Thus congenitally athymic (nude) female mice show significantly reduced levels of circulating gonadotropins, a fact that seems to be causally related to a number of reproductive derangements described in these mutants. Interestingly, a number of in vitro studies suggest that the thymic peptide thymulin may be involved in thymus-pituitary communication. To determine the consequences of low serum thymulin in otherwise normal animals, we induced short (8 days)- and long (33 days)-term thymulin deficiency in C57BL/6 mice by neonatally injecting (intraperitoneally) an anti-thymulin serum and assessed their circulating gonadotropin levels at puberty and thereafter. Control mice received an irrelevant antiserum. Gonadotropins were measured by radioimmunoassay and thymulin by bioassay. Both long- and short-term serum thymulin immunoneutralization resulted in a significant reduction in the serum levels of gonadotropins at 33 and 45 days of age. Subsequently, we injected (intramuscularly) an adenoviral vector harboring a synthetic DNA sequence (5′- ATGCAAGCCAAATCTCAAGGTGGATCCAACTAGTAG-3′) encoding a biologically active analog of thymulin, methionine-FTS, in newborn nude mice (which are thymulin deficient) and measured circulating gonadotropin levels when the animals reached 52 days of age. It was observed that neonatal thymulin gene therapy in the athymic mice restored their serum thymulin levels and prevented the reduction in circulating gonadotropin levels that typically emerges in these mutants after puberty. Our results indicate that thymulin plays a relevant physiological role in the thymuspituitary-gonadal axis.Instituto de Investigaciones Bioquímicas de La Plat

Single Nucleotide Polymorphisms in COX2 Is Associated with Persistent Primary Tooth and Delayed Permanent Tooth Eruption

Persistent primary tooth (PPT) is a prevalent clinical condition that occurs when a primary tooth is over-retained beyond the established period of its normal exfoliation time, remaining in the oral cavity. Many factors could be involved in the risk of PPT; therefore, the aim of this study was to evaluate if single nucleotide polymorphisms (SNPs) in the COX2 gene are associated with PPT. Children undergoing orthodontic treatment were screened. Orthopantomographs were assessed to evaluate PPT according to the Nolla stage of its permanent successor. The primary tooth was considered retained when its successor permanent tooth was in Nolla stage 8 and below the alveolar crypt, Nolla stage 9, or Nolla stage 10. A saliva sample from each child was collected and used for DNA extraction. A real-time PCR of two SNPs, rs689466 (−1195 G/A) and rs5275 (+665 T/C), was performed. A chi-square test was used to compare the allele and genotype distribution. Haplotype analysis was also performed. A total of 100 children were included in the study. Fifty-one had at least one PPT, while 49 children were classified as a control. The number of teeth persistent in the oral cavity ranged from 1 to 8. The genotype distribution was associated with PPT in the co-dominant model (p = 0.006) for SNP rs5275. The individuals that carry two T alleles (TT) compared with the individuals that carry at least one C allele (C + TC) had an almost three times higher chance of presenting with PPT (p = 0.012; OR = 2.99, CI95% 1.28 to 6.95–recessive model). The haplotype C-A for the SNPs rs5275 and rs689466, respectively, was significantly associated (p = 0.042). In conclusion, single nucleotide polymorphisms in the gene encoding for COX2 are associated with persistent primary tooth and may delay permanent tooth eruption

The 20S Proteasome Splicing Activity Discovered by SpliceMet

The identification of proteasome-generated spliced peptides (PSP) revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL) thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS). For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected

SIRT1 Regulates HIV Transcription via Tat Deacetylation

The human immunodeficiency virus (HIV) Tat protein is acetylated by the transcriptional coactivator p300, a necessary step in Tat-mediated transactivation. We report here that Tat is deacetylated by human sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent class III protein deacetylase in vitro and in vivo. Tat and SIRT1 coimmunoprecipitate and synergistically activate the HIV promoter. Conversely, knockdown of SIRT1 via small interfering RNAs or treatment with a novel small molecule inhibitor of the SIRT1 deacetylase activity inhibit Tat-mediated transactivation of the HIV long terminal repeat. Tat transactivation is defective in SIRT1-null mouse embryonic fibroblasts and can be rescued by expression of SIRT1. These results support a model in which cycles of Tat acetylation and deacetylation regulate HIV transcription. SIRT1 recycles Tat to its unacetylated form and acts as a transcriptional coactivator during Tat transactivation