Dataset Information

Reconstitution of the ancestral glycoprotein of human endogenous retrovirus k and modulation of its functional activity by truncation of the cytoplasmic domain.

ABSTRACT: 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.

SUBMITTER: Hanke K

PROVIDER: S-EPMC2786854 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Reconstitution of the ancestral glycoprotein of human endogenous retrovirus k and modulation of its functional activity by truncation of the cytoplasmic domain.

Hanke Kirsten K Kramer Philipp P Seeher Sandra S Beimforde Nadine N Kurth Reinhard R Bannert Norbert N

Journal of virology 20091007 24

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 ...[more]

PMID: 19812154

Similar Datasets

Project description:Herpesvirus membrane fusion depends on the core fusion machinery, comprised of glycoproteins B (gB) and gH/gL. Although gB structurally resembles autonomous class III fusion proteins, it strictly depends on gH/gL to drive membrane fusion. Whether the gH/gL complex needs to be membrane anchored to fulfill its function and which role the gH cytoplasmic (CD) and transmembrane domains (TMD) play in fusion is unclear. While the gH CD and TMD play an important role during infection, soluble gH/gL of herpes simplex virus 1 (HSV-1) seems to be sufficient to mediate cell-cell fusion in transient assays, arguing against an essential contribution of the CD and TMD. To shed more light on this apparent discrepancy, we investigated the role of the CD and TMD of the related alphaherpesvirus pseudorabies virus (PrV) gH. For this purpose, we expressed C-terminally truncated and soluble gH and replaced the TMD with a glycosylphosphatidylinositol (gpi) anchor. We also generated chimeras containing the TMD and/or CD of PrV gD or HSV-1 gH. Proteins were characterized in cell-based fusion assays and during virus infection. Although truncation of the CD resulted in decreased membrane fusion activity, the mutant proteins still supported replication of gH-negative PrV, indicating that the PrV gH CD is dispensable for viral replication. In contrast, PrV gH lacking the TMD, membrane-anchored via a lipid linker, or comprising the PrV gD TMD were nonfunctional, highlighting the essential role of the gH TMD for function. Interestingly, despite low sequence identity, the HSV-1 gH TMD could substitute for the PrV gH TMD, pointing to functional conservation.IMPORTANCE Enveloped viruses depend on membrane fusion for virus entry. While this process can be mediated by only one or two proteins, herpesviruses depend on the concerted action of at least three different glycoproteins. Although gB has features of bona fide fusion proteins, it depends on gH and its complex partner, gL, for fusion. Whether gH/gL prevents premature fusion or actively triggers gB-mediated fusion is unclear, and there are contradictory results on whether gH/gL function requires stable membrane anchorage or whether the ectodomains alone are sufficient. Our results show that in pseudorabies virus gH, the transmembrane anchor plays an essential role for gB-mediated fusion while the cytoplasmic tail is not strictly required.

Project description:This paper describes the preparation of giant unilamellar vesicles with reconstituted hamster P-glycoprotein (Pgp, ABCB1) for studying the transport activity of this efflux pump in individual liposomes using optical microscopy. Pgp, a member of ABC (ATP-binding cassette) transporter family, is known to contribute to the cellular multidrug resistance (MDR) against variety of drugs. The efficacy of many therapeutics is, thus, hampered by this efflux pump, leading to a high demand for simple and effective strategies to monitor the interactions of candidate drugs with this protein. Here, we applied small Pgp proteoliposomes to prepare giant Pgp-bearing liposomes via modified electroformation techniques. The presence of Pgp in the membrane of giant proteoliposomes was confirmed using immunohistochemistry. Assessment of Pgp ATPase activity suggested that this transporter retained its activity upon reconstitution into giant liposomes, with an ATPase specific activity of 439 ± 103 nmol/mg protein/min. For further confirmation, we assessed the transport activity of Pgp in these proteoliposomes by monitoring the translocation of rhodamine 123 (Rho123) across the membrane using confocal microscopy at various ATP concentrations (0-2 mM) and in the presence of Pgp inhibitors. Rate of change in Rho123 concentration inside the liposomal lumen was used to estimate the Rho123 transport rates (1/s) for various ATP concentrations, which were then applied to retrieve the Michaelis-Menten constant (Km) of ATP in Rho123 transport (0.42 ± 0.75 mM). Similarly, inhibitory effects of verapamil, colchicine, and cyclosporin A on Pgp were studied in this system and the IC50 values for these Pgp inhibitors were found 26.6 ± 6.1 ?M, 94.6 ± 47.6 ?M, and 0.21 ± 0.07 ?M, respectively. We further analyzed the transport data using a kinetic model that enabled dissecting the passive diffusion of Rho123 from its Pgp-mediated transport across the membrane. Based on this model, the permeability coefficient of Rho123 across the liposomal membrane was approximately 1.25×10-7 cm/s. Comparing the membrane permeability in liposomes with and without Pgp revealed that the presence of this protein did not have a significant impact on membrane integrity and permeability. Furthermore, we used this model to obtain transport rate constants for the Pgp-mediated transport of Rho123 (m3/mol/s) at various ATP and inhibitor concentrations, which were then applied to estimate values of 0.53 ± 0.66 mM for Km of ATP and 25.2 ± 5.0 ?M for verapamil IC50, 61.8 ± 34.8 ?M for colchicine IC50, and 0.23 ± 0.09 ?M for cyclosporin A IC50. The kinetic parameters obtained from the two analyses were comparable, suggesting a minimal contribution from the passive Rho123 diffusion across the membrane. This approach may, therefore, be applied for screening the transport activity of Pgp against potential drug candidates.

Dataset Information

Reconstitution of the ancestral glycoprotein of human endogenous retrovirus k and modulation of its functional activity by truncation of the cytoplasmic domain.

Publications

Reconstitution of the ancestral glycoprotein of human endogenous retrovirus k and modulation of its functional activity by truncation of the cytoplasmic domain.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets