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

Lack of evidence for xenotropic murine leukemia virus-related virus(XMRV) in German prostate cancer patients

Background: A novel gammaretrovirus named xenotropic murine leukemia virus-related virus (XMRV) has been recently identified and found to have a prevalence of 40% in prostate tumor samples from American patients carrying a homozygous R462Q mutation in the RNaseL gene. This mutation impairs the function of the innate antiviral type I interferon pathway and is a known susceptibility factor for prostate cancer. Here, we attempt to measure the prevalence of XMRV in prostate cancer cases in Germany and determine whether an analogous association with the R462Q polymorphism exists. Results: 589 prostate tumor samples were genotyped by real-time PCR with regard to the RNaseL mutation. DNA and RNA samples from these patients were screened for the presence of XMRV-specific gag sequences using a highly sensitive nested PCR and RT-PCR approach. Furthermore, 146 sera samples from prostate tumor patients were tested for XMRV Gag and Env antibodies using a newly developed ELISA assay. In agreement with earlier data, 12.9% (76 samples) were shown to be of the QQ genotype. However, XMRV specific sequences were detected at neither the DNA nor the RNA level. Consistent with this result, none of the sera analyzed from prostate cancer patients contained XMRV-specific antibodies. Conclusion: Our results indicate a much lower prevalence (or even complete absence) of XMRV in prostate tumor patients in Germany. One possible reason for this could be a geographically restricted incidence of XMRV infections

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

Functional and molecular characterization of the Human Endogenous Retrovirus K113

Das Humane Endogene Retrovirus K113 ist das vollständigste HERV, welches derzeit bekannt ist. Es besitzt offene Leserahmen für alle viralen Proteine und liegt in ca. 15 % der menschlichen Bevölkerung integriert auf Chromosom 19 vor. Um dieses Provirus molekular und funktionell zu charakterisieren, wurde das komplette Genom in einen eukaryontischen Plasmidvektor kloniert. Durch die substantiell vorhandene LTR-Promotoraktivität des Provirus konnte die Transkription genauer untersucht werden. Zusätzlich war es möglich in mit dem Molekularklon transfizierten Zellen einzelne virale Proteine nachzuweisen. HERV-K113 ist nicht replikationskompetent, unter anderem besitzt das Provirus inaktivierende Mutationen in dem Reverse Transkriptase (RT) Gen. Die RT kann zwar durch Reversion dieser Mutationen wieder aktiviert werden, jedoch ist dies nicht ausreichend, um die Replikationskompetenz des Virus wieder herzustellen. Ebenfalls besitzt das Provirus ein nicht-funktionelles Oberflächenprotein. Durch Rückmutationen von postinsertionellen Aminosäure- Änderungen, identifiziert durch Sequenzvergleiche mit anderen Proviren der HERV-K(HML-2) Familie, konnte hier jedoch das ursprüngliche HERV-K113 Oberflächenprotein rekonstituiert werden. Unterstützend durch eine Kodonoptimierung konnte gezeigt werden, dass das rekonstruierte Env seine Fähigkeit wiedererlangt hat in Partikel eingebaut zu werden und den Eintritt des Virus in die Zelle initialisiert. Zusätzlich wurden die Verbesserung des Einbaus bei der Pseudotypisierung von lentiviralen Vektoren und die Erhöhung der Fusogenität durch carboxyterminale Verkürzungen des Env festgestellt. Für Replikationsuntersuchungen eines weiteren endogenen Retrovirus, dem XMRV (Xenotropic Murine Leukemia Virus-related Virus), welches als exogenes Virus offenbar den Menschen infizieren kann, wurde zunächst eine Prävalenz-Studie in deutschen Prostatakrebspatienten durchgeführt. Jedoch konnten weder auf DNA- und RNA-Ebene XMRV-spezische Sequenzen noch in getesteten Serum-Proben spezifische Antikörper detektiert werden.The human endogenous retrovirus is the most complete HERV known to date. It contains open reading frames for all viral proteins and is integrated on chromosome 19 in about 15% of the human population. For the molecular and functional characterization of this provirus, the complete genome was cloned into a small plasmid vector. Via the substantial LTR-promoter activity the transcription of the provirus could be shown. In addition, it was possible to detect nearly all viral proteins in cells transfected with the molecular clone. HERV-K113 is not replication competent. It carries inactivating mutations in the reverse transcriptase gene. These mutations can be reversed to reconstitute the active enzyme, but the reversion is not sufficient to reconstitute replication capacity of the virus. Additionally, the virus carries a non-functional surface protein. By back mutating putative postinsertional amino acid changes, which were identified via sequence analysis with other proviruses of the HERV-K(HML-2) family, the ancestral envelope protein could be reconstituted. Aided by codon-optimized expression, it could be shown that this reconstituted Env regained its ability to be incorporated into retroviral particles and to mediate entry. For replication analysis of another endogenous retrovirus, the XMRV (Xenotropic Murine Leukemia Virus-related Virus), which is obviously able to infect human cells, a prevalence study of German prostate cancer patients was done. However, XMRV specific sequences were detected at neither the DNA nor the RNA level. Consistent with this result, none of the sera analyzed from prostate cancer patients contained XMRV-specific antibodies

Reconstitution of the Ancestral Glycoprotein of Human Endogenous Retrovirus K and Modulation of Its Functional Activity by Truncation of the Cytoplasmic Domain▿ †

Endogenous retroviruses present in the human genome provide a rich record of ancient infections. All presently recognized elements, including the youngest and most intact proviruses of the human endogenous retrovirus K(HML-2) [HERV-K(HML-2)] family, have suffered postinsertional mutations during their time of chromosomal residence, and genes encoding the envelope glycoprotein (Env) have not been spared these mutations. In this study, we have, for the first time, reconstituted an authentic Env of a HERV-K(HML-2) provirus by back mutation of putative postinsertional amino acid changes of the protein encoded by HERV-K113. Aided by codon-optimized expression, we demonstrate that the reconstituted Env regained its ability to be incorporated into retroviral particles and to mediate entry. The original ancient HERV-K113 Env was synthesized as a moderately glycosylated gp95 precursor protein cleaved into surface and transmembrane (TM) subunits. Of the nine N-linked oligosaccharides, four are part of the TM subunit, contributing 15 kDa to its apparent molecular mass of 41 kDa. The carbohydrates, as well as the cytoplasmic tail, are critical for efficient intracellular trafficking, processing, stability, and particle incorporation. Whereas deletions of the carboxy-terminal 6 residues completely abrogated cleavage and virion association, more extensive truncations slightly enhanced incorporation but dramatically increased the ability to mediate entry of pseudotyped lentiviruses. Although the first HERV-K(HML-2) elements infected human ancestors about 30 million years ago, our findings indicate that their glycoproteins are in most respects remarkably similar to those of classical contemporary retroviruses and can still mediate efficient entry into mammalian cells

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