Identification of late assembly domains of the human endogenous retrovirus-K(HML-2)

Background: Late assembly (L)-domains are protein interaction motifs, whose dysfunction causes characteristic budding defects in enveloped viruses. Three different amino acid motifs, namely PT/SAP, PPXY and YPXnL have been shown to play a major role in the release of exogenous retroviruses. Although the L-domains of exogenous retroviruses have been studied comprehensively, little is known about these motifs in endogenous human retroviruses. Results: Using a molecular clone of the human endogenous retrovirus K113 that had been engineered to reverse the presumed non-synonymous postinsertional mutations in the major genes, we identified three functional L-domains of the virus, all located in the Gag p15 protein. A consensus PTAP tetrapeptide serves as the core of a main L-domain for the virus and its inactivation reduces virus release in HEK 293T cells by over 80%. Electron microscopy of cells expressing the PTAP mutant revealed predominantly late budding structures and budding chains at the plasma membrane. The fact that this motif determines subcellular colocalization with Tsg101, an ESCRT-I complex protein known to bind to the core tetrapeptide, supports its role as an L-domain. Moreover, two YPXnL motifs providing additional L-domain function were identified in the p15 protein. One is adjacent to the PTAP sequence and the other is in the p15 N-terminus. Mutations in either motif diminishes virus release and induces an L-domain phenotype while inactivation of all three L-domains results in a complete loss of particle release in HEK 293T cells. The flexibility of the virus in the use of L-domains for gaining access to the ESCRT machinery is demonstrated by overexpression of Tsg101 which rescues the release of the YPXnL mutants. Similarly, overexpression of Alix not only enhances release of the PTAP mutant by a factor of four but also the release of a triple mutant, indicating that additional cryptic YPXnL domains with a low affinity for Alix may be present. No L-domain activity is provided by the proline-rich peptides at the Gag C-terminus. Conclusions: Our data demonstrate that HERV-K(HML-2) release is predominantly mediated through a consensus PTAP motif and two auxiliary YPXnL motifs in the p15 protein of the Gag precursor

Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element

<p>Abstract</p> <p>Background</p> <p>The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown.</p> <p>Results</p> <p>By aligning the amino acid sequences encoded by the <it>gag-pro-pol </it>ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the <it>gag </it>gene product Pr74^Gagof HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C-terminally encoded glutamine- and proline-rich peptides, QP1 and QP2, spanning 23 and 19 amino acids, respectively.</p> <p>Conclusions</p> <p>Expression of reconstituted sequences of original HERV elements is an important tool for studying fundamental aspects of the biology of these ancient viruses. The analysis of HERV-K(HML-2) Gag processing and the nature of the mature Gag proteins presented here will facilitate further studies of the discrete functions of these proteins and of their potential impact on the human host.</p

Identification and functional characterization of L-domains of the human endogenous retrovirus K

Bis zum gegenwärtigen Zeitpunkt war die Knospung und Freisetzung der humanen endogen Retroviren der Familie K (HERV-K) nur wenig untersucht. Hingegen existierte eine Vielzahl von Untersuchungen über das Budding von exogenen Retroviren, bei denen „Late assembly“ (L)-Domänen die Abschnürung der Partikel maßgeblich steuern. Im Zuge der Arbeit konnten drei verschiedene L-Domänen von HERV-K(HML-2) identifiziert werden: PTAP, YPETLKL und YP(x6)L. Mutationen der in der p15-Domäne des Gag-Proteins lokalisierten Motive führten zu charakteristisch defekten Budding-Phänotypen, wodurch die Freisetzung der viralen Partikel stark beeinträchtigt wurde. Anhand von Messungen der Reversen Transkriptase-Aktivität in den Überständen von transfizierten HEK-293T- und Tera-1-Zellen war ein signifikanter Rückgang der Partikelfreisetzung aller Mutanten im Vergleich zum Wildtyp nachweisbar. Des Weiteren wiesen alle Mutanten reduzierte Mengen von prozessierten Gag-Proteinen in den ultrazentrifugierten Viruspellets auf. Besonders deutlich war dieser Effekt bei der Inaktivierung des hoch konservierten PTAP-Motivs ausgeprägt. HERV-K Partikel die sich durch ein mutiertes PTAP-Motiv auszeichneten waren häufig in einem späten Stadium der Knospung arretiert oder in Prokapsidketten über dünne Membranbrücken verbunden. Wie bei anderen Retroviren zeigte sich, dass die HERV-K L-Domänen die Virusfreisetzung zelltypabhängig begünstigen. Für ein weiteres, in der Kapsiddomäne des Gag-Proteins lokalisiertes Motiv, konnte keine Beteiligung an der HERV-K Knospung nachgewiesen werden. Ebenso wenig sind die QP1/2-Peptide an dem Freisetzungsprozess der endogenen Viren involviert. Damit die Viren an der Plasmamembran der Zelle knospen können, sind verschiedene Wirtsfaktoren des ESCRT („endosomal sorting complex required for transport“)-Systems notwendig. Eine besondere Rolle stellt hierbei das ESCRT-Protein Tsg101 dar, dessen Mitwirkung am HERV-K Budding belegt werden konnte. Dabei wird Tsg101 über die PTAP-Motive der Gag-Proteine zum Ort der Partikelabschnürung rekrutiert. Des Weiteren führt eine Überexpression von Tsg101 dazu, dass der Buddingdefekt der YPxnL-Mutanten teilweise aufgehoben wird. Neben Tsg101 konnte Alix, eine akzessorische Komponente der ESCRT- Maschinerie, als ein am HERV-K Budding beteiligtes Protein identifiziert werden. Wie Tsg101 fördert Alix das Budding von L-Domänen-Mutanten, woraufhin vermehrt Partikel an der Wirtszellmembran knospen können.To date, the budding and release of human endogenous retrovirus K has remained only poorly understood. In contrast, there are several studies describing the budding of exogenous retroviruses. These viruses exhibit late assembly (L)-domains that regulate the pinch-off of virus from the host cell membrane. During the course of this project, three different HERV-K(HML-2) L-domains were identified: PTAP, YPETLKL und YP(x6)L. Mutations of L-domain motifs within the p15 domain of the Gag protein resulted in characteristic phenotypes that showed defective budding and severely impaired particle release. By measuring the reverse transcriptase activity in the supernatants of transfected HEK 293T and Tera-1 cells, a significant reduction in the particle release of all mutants compared to the wild type was demonstrated. Furthermore, the level of processed Gag protein in virus pellets was shown to be decreased. However, the most drastic effects on virus release were observed by disrupting the single PTAP motif. Most of the PTAP mutant particles were arrested at a late budding stage or were organized in chain buds, in which different procapsids were connected by thin membrane stalks. As already shown for other retroviruses, the L-domains of human endogenous retrovirus K promote virus release in a cell type-dependent manner. Analysis of a further motif showed that this sequence, located in the capsid domain of the Gag protein, has no influence on HERV-K(HML-2) virus budding. Moreover, QP1/2 peptides play no role in the release of the endogenous viruses. Viruses are able to recruit cellular components of the "endosomal sorting complexes required for transport" (ESCRT) to bud from their host cell membrane. In this study, it was shown that Tsg101, a member of ESCRT system, plays a physiologically relevant role in HERV-K release and assembly. Therefore, the PTAP motif of Gag interacts with Tsg101 and recruits it to the site of particle budding. Furthermore, overexpression of Tsg101 restores the release of HERV-K(HML-2) mutants lacking YPxnL motifs. In addition, overexpression of Alix, an accessory component of ESCRT machinery also involved in HERV-K budding can, like Tsg101, markedly stimulate the budding of diverse HERV-K(HML-2) release mutants

RESEARCH Identification of late assembly domains of the human endogenous retrovirus-K(HML-2) Open Access

Background: Late assembly (L)-domains are protein interaction motifs, whose dysfunction causes characteristic budding defects in enveloped viruses. Three different amino acid motifs, namely PT/SAP, PPXY and YPXnL have been shown to play a major role in the release of exogenous retroviruses. Although the L-domains of exogenous retroviruses have been studied comprehensively, little is known about these motifs in endogenous human retroviruses. Results: Using a molecular clone of the human endogenous retrovirus K113 that had been engineered to reverse the presumed non-synonymous postinsertional mutations in the major genes, we identified three functional L-domains of the virus, all located in the Gag p15 protein. A consensus PTAP tetrapeptide serves as the core of a main L-domain for the virus and its inactivation reduces virus release in HEK 293T cells by over 80%. Electron microscopy of cells expressing the PTAP mutant revealed predominantly late budding structures and budding chains at the plasma membrane. The fact that this motif determines subcellular colocalization with Tsg101, an ESCRT-I complex protein known to bind to the core tetrapeptide, supports its role as an L-domain. Moreover, two YPXnL motifs providing additional L-domain function were identified in the p15 protein. One is adjacent to the PTAP sequence and the other is in the p15 N-terminus. Mutations in either motif diminishes virus release and induces an L-domain phenotype while inactivation of all three L-domains results in a complete loss of particl