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

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

Exploring the functional interaction between POSH and ALIX and the relevance to HIV-1 release

<p>Abstract</p> <p>Background</p> <p>The ALG2-interacting protein X (ALIX)/AIP1 is an adaptor protein with multiple functions in intracellular protein trafficking that plays a central role in the biogenesis of enveloped viruses. The ubiquitin E3-ligase POSH (plenty of SH3) augments HIV-1 egress by facilitating the transport of Gag to the cell membrane. Recently, it was reported, that POSH interacts with ALIX and thereby enhances ALIX mediated phenotypes in <it>Drosophila</it>.</p> <p>Results</p> <p>In this study we identified ALIX as a POSH ubiquitination substrate in human cells: POSH induces the ubiquitination of ALIX that is modified on several lysine residues <it>in vivo </it>and <it>in vitro</it>. This ubiquitination does not destabilize ALIX, suggesting a regulatory function. As it is well established that ALIX rescues virus release of L-domain mutant HIV-1, HIV-1Δ_PTAP, we demonstrated that wild type POSH, but not an ubiquitination inactive RING finger mutant (POSH^V14A), substantially enhances ALIX-mediated release of infectious virions derived from HIV-1Δ_PTAPL-domain mutant (YPX_nL-dependent HIV-1). In further agreement with the idea of a cooperative function of POSH and ALIX, mutating the YPX_nL-ALIX binding site in Gag completely abrogated augmentation of virus release by overexpression of POSH. However, the effect of the POSH-mediated ubiquitination appears to be auxiliary, but not necessary, as silencing of POSH by RNAi does not disturb ALIX-augmentation of virus release.</p> <p>Conclusion</p> <p>Thus, the cumulative results identified ALIX as an ubiquitination substrate of POSH and indicate that POSH and ALIX cooperate to facilitate efficient virus release. However, while ALIX is obligatory for the release of YPX_nL-dependent HIV-1, POSH, albeit rate-limiting, may be functionally interchangeable.</p

Tetherin-Driven Adaptation of Vpu and Nef Function and the Evolution of Pandemic and Nonpandemic HIV-1 Strains

Vpu proteins of pandemic HIV-1 M strains degrade the viral receptor CD4 and antagonize human tetherin to promote viral release and replication. We find that Vpus from SIVgsn, SIVmus and SIVmon infecting Cercopithecus primate species also degrade CD4 and antagonize tetherin. In contrast, SIVcpz, the immediate precursor of HIV-1, whose Vpu shares a common ancestry with SIVgsn/mus/mon Vpu, uses Nef rather than Vpu to counteract chimpanzee tetherin. Human tetherin, however, is resistant to Nef and thus poses a significant barrier to zoonotic transmission of SIVcpz to humans. Remarkably, Vpu from non-pandemic HIV-1 O strains are poor tetherin antagonists while those from the rare group N viruses do not degrade CD4. Thus, only HIV-1 M evolved a fully functional Vpu following the three independent cross-species transmissions that resulted in HIV-1 groups M, N, and O. This may explain why group M viruses are almost entirely responsible for the gobal HIV/AIDS pandemic

A Flow Cytometry-Based FRET Assay to Identify and Analyse Protein-Protein Interactions in Living Cells

Försters resonance energy transfer (FRET) microscopy is widely used for the analysis of protein interactions in intact cells. However, FRET microscopy is technically challenging and does not allow assessing interactions in large cell numbers. To overcome these limitations we developed a flow cytometry-based FRET assay and analysed interactions of human and simian immunodeficiency virus (HIV and SIV) Nef and Vpu proteins with cellular factors, as well as HIV Rev multimer-formation.Amongst others, we characterize the interaction of Vpu with CD317 (also termed Bst-2 or tetherin), a host restriction factor that inhibits HIV release from infected cells and demonstrate that the direct binding of both is mediated by the Vpu membrane-spanning region. Furthermore, we adapted our assay to allow the identification of novel protein interaction partners in a high-throughput format.The presented combination of FRET and FACS offers the precious possibility to discover and define protein interactions in living cells and is expected to contribute to the identification of novel therapeutic targets for treatment of human diseases

Charakterisierung von Virus-Wirt Interaktionen welche die HIV-1 Replikation regulieren

Retroviruses like the human immunodeficiency virus type 1 (HIV-1) are obligate cellular parasites that depend on host cell factors for their replication. The aim of this study was to characterize two specific cases of these numerous virus-host interactions. First, the previously identified interaction between the cellular peptidyl-prolyl cis/trans isomerase cyclophilin A (CypA) and the HIV-1 accessory protein Vpr was investigated on the molecular level. HIV-1 Vpr induces G2 cell-cycle arrest and apoptosis in infected cells, and it facilitates the transport of the pre-integration complex into the nucleus of non-dividing cells. This multi-functional protein interacts with a variety of cellular factors, among them CypA. Although an N-terminal sequence in Vpr comprising proline residues in positions 5, 10, and 14 resembles the previously described CypA binding site in HIV-1 capsid, it was shown that the interaction between CypA and Vpr is regulated by a downstream proline in position 35 (Pro-35). To investigate the relevance of these proline residues for HIV-1 replication, individual mutations of all four proline residues were introduced in R5-tropic HIV-1 variants, which predominantly replicate in macrophages, and in X4-tropic viruses that primarily infect T cells. Comprehensive replication studies in human lymphoid tissue cultivated ex vivo revealed that only Pro-35 is required for efficient replication of R5-tropic variants, but not for X4-tropic viruses. In an attempt to clarify the cause of this phenomenon, it was observed that none of the proline mutations has an effect on the Vpr-mediated apoptosis and G2 cell-cycle arrest as well as on the expression and sub-cellular localization of Vpr. In contrast, however, the capacity for encapsidation into budding virions, which is mediated via binding of Vpr to the p6 domain of the Gag polyprotein, is significantly reduced by mutation of Pro-35. 1H-NMR spectroscopy revealed that mutation of Pro-35 results in a significant increase in the alpha-helical structure that will merge the two N-terminal helices of the full length molecule. Thus, Pro-35, potentially in concert with CypA or other host cell chaperones, regulates the conformation of the hydrophobic core of the molecule, whose integrity is required for encapsidation of Vpr and thus, is necessary for a productive infection of macrophages The second example of virus host interactions investigated in this thesis was the ubiquitination of ALIX (ALG-2 interacting protein X) by POSH (Plenty of SH3) and the functional relevance of this interaction for HIV-1 release. ALIX represents an ESCRT (endosomal sorting complex required for transport) associated adaptor protein with multiple functions in intracellular protein trafficking and plays a central role in the release of various enveloped viruses through interaction with the consensus YPXnL late (L)-domain motif. It was previously demonstrated by our collaboration partners that the ubiquitin E3 ligase POSH augments HIV-1 release by facilitating the transport of Gag to the cell membrane. Recently, it was reported, that POSH enhances ALIX mediated phenotypes in Drosophila. It was therefore a legitimate question to investigate of whether ALIX and POSH cooperate in terms of virus budding. In collaboration with our partners, it was found that POSH binds to ALIX in human cells. It was also observed that ALIX is an ubiquitination substrate of POSH. Wild type POSH, but not an ubiquitination inactive RING finger mutant (POSHV14A), induces formation of poly-ubiquitin chains on ALIX in vivo and in vitro. Yet, this poly-ubiquitination does not induce proteasomal degradation of ALIX. Further it was demonstrated that wild type POSH, but not the POSHV14A mutant, substantially enhances ALIX mediated Gag processing and release of infectious virions of an L-domain mutant HIV-1deltaPTAP variant. Additionally, the impact of POSH on an ALIX dependent HIV-1/EIAV (equine infectious anemia virus) hybrid (HIV-p9), in which the p6 domain in Gag was replaced by the corresponding p9 domain of EIAV was investigated. Consistent with the findings observed for HIV-1, POSH also augments the release of virus particles derived from this strictly ALIX-dependent hybrid-virus. In further agreement with the idea of a cooperative function of POSH and ALIX, silencing of ALIX by RNAi (RNA interference) or mutating the ALIX binding site in Gag completely abrogated augmentation of virus release by over-expression of POSH. However, the effect of the POSH mediated ubiquitination of ALIX appears to be auxiliary, but not necessary, as silencing of POSH by RNAi does not disturb ALIX mediated rescue of HIV-1 L-domain mutants. Thus, the cumulative results identified ALIX as an ubiquitination substrate of POSH and indicate that POSH and ALIX cooperate to facilitate efficient virus release in a process that may involve POSH-mediated ubiquitination of ALIX.Retroviren wie das humane Immundefizienzvirus Typ 1 (HIV-1) sind Zellparasiten und für ihre Replikation auf die Interaktion mit Proteinen der Wirtszelle angewiesen. Das Ziel dieser Arbeit war die Charakterisierung zweier Beispiele dieser Virus- Wirt-Interaktionen. Zunächst wurde die bereits zuvor beschriebene Interaktion zwischen der Peptidyl-prolyl cis/trans Isomerase Cyclophilin A (CypA) und dem HIV-1 akzessorischen Protein Vpr auf molekularer Ebene untersucht. HIV-1 Vpr induziert in infizierten Zellen G2 Zellzyklus Arrest sowie Apoptose und erleichtert den Transport des viralen Prä-Integrations-Komplexes in den Zellkern von sich nicht teilenden Zellen. Obwohl die N-terminale Aminosäurensequenz von Vpr bestehend aus den Prolinen 5, 10 und 14, der bekannten CypA Bindestelle in HIV-1 Capsid ähnelt, wurde gezeigt, dass die Interaktion von CypA und Vpr von einem Prolin an Position 35 reguliert wird. Um die Bedeutung der Prolin Reste für die Replikation von HIV-1 zu untersuchen, wurden Mutationen aller Prolin Reste in R5-trophe HIV-1 Varianten, welche vornehmlich in Makrophagen replizieren, und X4-trophe Viren, welche hauptsächlich T-Zellen infizieren, eingefügt. Replikationsstudien in ex vivo kultivierten, human Lymphgewebe zeigten nun, dass ausschließlich Pro-35 wichtig ist für die Replikation von R5-trophen, nicht aber von X4-trophen HIV-1. Beim Versuch, die Ursache dafür zu klären wurde gefunden, dass keine der Prolin-Mutationen einen Einfluss auf die Vpr induzierte Apoptose und den G2 Zellzyklus Arrest hat. Ebenso werden die Expression und subzelluläre Lokalisation von Vpr nicht verändert. Im Gegensatz dazu jedoch war die Fähigkeit von Vpr in die sich bildenden Virionen eingebaut zu werden, welche durch die Bindung an die p6-Domäne des Gag Proteins vermittelt wird, bei einer Mutation von Pro-35 deutlich reduziert. NMR Spektroskopie zeigte, dass die Mutation von Pro-35 eine verstärkte alpha-helikale Struktur zur Folge hat, wodurch die beiden N-terminalen Helices des Moleküls fusionieren. Demnach reguliert Pro-35, möglicherweise im Zusammenspiel mit CypA, die Konformation des hydrophoben Kern-Bereichs des Moleküls, dessen Integrität wichtig ist für den Einbau von Vpr in Virionen, und daher notwendig ist für di Replikation in Makrophagen. Die zweite Virus-Wirt Interaktion, welche in dieser Arbeit untersucht wurde, war der Einfluss der Ubiquitinylierung von ALIX (ALG-2 interacting protein X) durch POSH (Plenty of SH3) auf die Freisetzung von HIV-1. ALIX ist ein mit dem ESCRT (endosomal sorting complex required for transport) assoziiertes Protein mit verschiedensten Funktionen im intrazellulären Proteintransport und spielt eine zentrale Rolle bei der Freisetzung Viren durch die Interaktion mit dem Konsensus YPXnL late (L)-Domänen Motiv. Schon vorher wurde von unseren Kooperationspartnern gezeigt, dass die E3 Ubiquitin-Ligase POSH die Freisetzung von HIV-1 erhöht. Vor kurzem wurde auch beschrieben, dass POSH ALIX abhängige Phänotypen in Drosophila verstärkt. Basierend auf dieser Entdeckung wurden Untersuchungen zur Klärung der Frage veranlasst, ob ALIX und POSH zum Zweck der Virus-Freisetzung interagieren. In Kooperation mit unseren Partnern wurde gefunden, dass ALIX ein Ubiquitinylierungs-Substrat von POSH ist. Wildtyp POSH, aber nicht eine Ubiquitinylierungs-defiziente RING-Finger Mutante (POSHV14A) induzierte die Bildung von Poly-Ubiquitin Ketten an ALIX in vivo und in vitro. Diese Ubiquitinylierung induzierte jedoch keinen proteasomalen Abbau von ALIX. Ferner konnte gezeigt werden, dass Wildtyp POSH, aber nicht die POSHV14A Mutante, die ALIX vermittelte Gag-Prozessierung und Virus-Freisetzung einer HIV-1deltaPTAP Mutante entscheidend verstärkt. Außerdem wurde der Einfluss von POSH auf ein ALIX abhängiges HIV-1/EIAV (equine infectious anemia virus) Hybrid-Virus (HIV-p9) untersucht, in welchem die p6 Domäne in Gag durch die entsprechende p9 Domäne von EIAV ersetzt wurde. Übereinstimmend mit den Ergebnissen für HIV-1 verstärkte POSH auch die Freisetzung dieses strikt von ALIX abhängigem Hybrid-Virus. In weiterer Übereinstimmung mit einer kooperativen Funktion von POSH und ALIX wurde die, durch POSH Überexpression induzierte, verstärkte Virus Freisetzung zum einen durch Herunterregulation der ALIX Expression mittels RNAi (RNA Interferenz), zum anderen durch Mutationen der ALIX-Bindestelle in Gag, komplett aufgehoben. Jedoch scheint der Effekt der Ubiquitinylierung von ALIX durch POSH unterstützend, aber nicht notwendig für die Funktion von ALIX, zu sein, da die Herunterregulation der POSH Expression mittels RNAi die ALIX vermittelten Freisetzung von HIV-1 L-Domänen Mutanten nicht verringert. Zusammenfassend wurde ALIX als Ubiquitinylierungs Substrat von POSH identifiziert und es wurden Hinweise darauf erbracht, dass POSH und ALIX in einem Prozess zusammenwirken, der eventuell die POSH- vermittelte Ubiquitinylierung von ALIX beinhaltet, um die effiziente Virusfreisetzung zu ermöglichen

Hemmung der Expression von HIV und porcinen endogenen Retroviren mittels RNA-Interferenz

Im Genom von Schweinen befinden sich provirale Sequenzen von porcinen endogenen Retroviren (PERVs), welche replikationskompetente Viren codieren. Die Sequenz eines A/C-rekombinanten PERV, bestehend aus dem gag/pol-Bereich von PERVC und dem env-Gen von PERV-A, wurde kloniert. Damit konnte zum ersten Mal das gesamte Genom dieses rekombinanten Subtyps sequenziert und analysiert werden. Zudem handelte es sich um ein auf humanen Zellen adaptiertes Virus, welches durch den Einbau zusätzlicher repetitiver Elemente in die LTR einen höheren Virustiter erreicht. In Zellen, die mit den Molekularklonen transfiziert wurden, konnten die VolllängenmRNA sowie die gespleißte env-mRNA nachgewiesen werden. Ebenfalls wurde das virale Gag-Protein und die reverse Transkriptase detektiert. Die Produktion infektiöser Partikel konnte bislang nicht nachgewiesen werden, die Ursachen werden analysiert. Um die Expression von PERV und HIV mittels RNA-Interferenz zu hemmen, wurden shRNA-Expressionskassetten gegen Target-Sequenzen innerhalb der viralen mRNAs in Vektoren kloniert. Es konnte gezeigt werden, das mittels RNA-Interferenz die Expression von HIV-1 unterdrückt wird. Expressionskassetten mit shRNAs gegen die HIV-1-Gene gag und rev wurden in konventionelle Expressionsvektoren kloniert. In HIV-1-infizierten HeLa-P4- und C8166-Zellen konnte mit diesen Konstrukten eine deutlich Reduktion der viralen Replikation erreicht werden. Auch mit den lentiviralen Konstrukten gegen HIV-1-rev und -tat war eine deutliche Hemmung der HIV-1-Expression messbar. Durch den Einbau von Fehlpaarungen in das 3´-Ende des Sense- Stranges konnte die Effizienz der shRNAs nicht gesteigert werden. Zur Hemmung der PERV-Expression wurden ebenfalls lentivirale Vektoren generiert, welche eine shRNA-Expressionskassette gegen eine im pol-Gen von PERV befindliche Sequenz trugen. Mit diesen Konstrukten wurden porcine Fibroblasten transduziert. Zum ersten Mal konnte eine Hemmung der Expression von PERV in primären Schweinezellen gezeigt werden. Mit diesen lentiviralen Konstrukten wäre es möglich, transgene Schweine zu erstellen, in deren Zellen die virale Expression herunterreguliert ist, wodurch diese Schweine eine erhöhte Sicherheit vor einer Infektion mit PERV bei einer Xenotransplantation bieten könnten

Characterisation of a human cell-adapted porcine endogenous retrovirus PERV-A/C.

Background: Porcine endogenous retroviruses (PERVs) pose a potential risk for xenotransplantation using pig cells, tissues or organs. A special threat comes from viruses generated by recombination between human-tropic PERV-A and ecotropic PERV-C. Serial passages of a recombinant PERV-A/C on human 293 cells resulted in increased infectious titers and a multimerization of transcription factor binding sites in the viral long terminal repeat (LTR). In contrast to the LTR, the sequence of the env gene did not change, indicating that the LTR represents the determinant of high infectivity. Material/Methods: The virus was further propagated on human cells and characterized by different methods (titration, sequencing, infection experiments, electron microscopy). Results: Further propagation on human 293 cells resulted in deletions and mutations in the LTR. In contrast to low-titer viruses, the high-titer virus was infectious for cells from non-human primates including chimpanzees. Scanning electron microscopy revealed clustering of budding virions at the cell surface of infected human cells and transmission electron microscopy indicated that the virus infects them via receptor-mediated endocytosis. Conclusions: After propagation of PERV on human cells without selection pressure, viruses with different LTR were generated. High titer PERV was shown to infect cells from non-human primates. The experiments performed here simulate the situation in vivo and give an extended characterization of human cell-adapted PERVs

Perinuclear localization of the HIV-1 regulatory protein Vpr is important for induction of G2-arrest

AbstractThe HIV-1 accessory protein Vpr induces G2 cell cycle arrest and apoptosis. Previous studies indicate that the induction of G2-arrest requires the localization of Vpr to the nuclear envelope. Here we show that treatment of Vpr-expressing HeLa cells with the caspase 3 inhibitor Z-DEVD-fmk induced accumulation of Vpr at the nuclear lamina, while other proteins or structures of the nuclear envelope were not influenced. Furthermore, Z-DEVD-fmk enhances the Vpr-mediated G2-arrest that even occurred in HIV-1NL4–3-infected T-cells. Mutation of Pro-35, which is important for the integrity of helix-α1 in Vpr, completely abrogated the Z-DEVD-fmk-mediated accumulation of Vpr at the nuclear lamina and the enhancement of G2-arrest. As expected, inhibition of caspase 3 reduced the induction of apoptosis by Vpr. Taken together, we could show that besides its role in Vpr-mediated apoptosis induction caspase 3 influences the localization of Vpr at the nuclear envelope and thereby augments the Vpr-induced G2-arrest

Ion channel activity of HIV-1 Vpu is dispensable for counteraction of CD317.

While the C-terminal domain of HIV-1 Vpu is critical for CD4 degradation, the transmembrane domain (TM) mediates ion channel activity, enhances virus release and is essential for counteracting CD317/Bst-2/Tetherin. Here we analyzed whether the ion channel activity of Vpu is required to antagonize CD317-mediated restriction of virion release. We examined TM-mutants of three conserved residues: the S23A mutation, which was previously shown to abrogate ion channel function, did not affect Vpu mediated augmentation of virus release. In contrast, the A14N and A18N mutation did not affect ion channel activity of Vpu, but substantially reduced its ability to support virus release and to down-regulate CD317 from the cell surface. Altogether, our data suggest that not the ion channel activity of Vpu, but its ability to remove CD317 from the cell surface is required to augment HIV-1 release