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Journal of Virology, December 2005, p. 15074-15083, Vol. 79, No. 24
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.24.15074-15083.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Ubiquitination of the Prototype Foamy Virus Envelope Glycoprotein Leader Peptide Regulates Subviral Particle Release

Nicole Stanke,1 Annett Stange,1 Daniel Lüftenegger,1 Hanswalter Zentgraf,2 and Dirk Lindemann1*

Institut für Virologie, Medizinische Fakultät "Carl Gustav Carus," Technische Universität Dresden, Dresden,1 Deutsches Krebsforschungszentrum, Heidelberg, Germany2

Received 26 July 2005/ Accepted 13 September 2005


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ABSTRACT
 
Foamy virus (FV) particle egress is unique among retroviruses because of its essential requirement for Gag and Env coexpression for budding and particle release. The FV glycoprotein undergoes a highly unusual biosynthesis resulting in the generation of three particle-associated, mature subunits, leader peptide (LP), surface (SU), and transmembrane (TM), derived from a precursor protein by posttranslational proteolysis mediated by furin or furinlike proteases. Previously at least three LP products of different molecular weights were detected in purified FV particles. Here we demonstrate that the higher-molecular-weight forms gp28LP and gp38LP are ubiquitinated variants of the major gp18LP cleavage product, which has a type II membrane topology. Furthermore, we show that all five lysine residues located within the N-terminal 60-amino-acid cytoplasmic domain of gp18LP can potentially be ubiquitinated, however, there seems to be a preference for using the first three. Inactivation of ubiquitination sites individually resulted in no obvious phenotype. However, simultaneous inactivation of the first three or all five ubiquitination sites in gp18LP led to a massive increase in subviral particles released by these mutant glycoproteins that were readily detectable by electron microscopy analysis upon expression of the ubiquitination-deficient glycoprotein by itself or in a proviral context. Surprisingly, only the quintuple ubiquitination mutant showed a two- to threefold increase in single-cycle infectivity assays, whereas all other mutants displayed infectivities similar to that of the wild type. Taken together, these data suggest that the balance between viral and subviral particle release of FVs is regulated by ubiquitination of the glycoprotein LP.


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INTRODUCTION
 
Retroviral glycoproteins (Env proteins) are usually translated as precursor proteins and go through a series of modifications before reaching their mature functional state enabling them to interact with specific cellular receptors and to fuse viral and cellular lipid membranes. In general the Env proteins are cotranslationally inserted into the rough endoplasmic reticulum membrane. During their transport to the cell surface the precursor proteins oligomerize and are proteolytically cleaved by cellular proteases into at least two mature subunits, surface (SU), mainly involved in receptor recognition, and transmembrane (TM), harboring the membrane fusion machinery. In addition, the Env proteins undergo different kinds of posttranslational modifications during their biosynthesis and intracellular transport. For example, retroviral Env proteins are generally modified by attachment of N- and sometimes O-linked carbohydrate chains at asparagine and serine or threonine residues, respectively, although the extent and subunit distribution show variation between individual glycoproteins (reviewed in reference 40). Furthermore, for some retroviral Env proteins the addition of fatty acids via linkage to cysteine residues have been reported (reviewed in reference 31).

Incorporation of the viral glycoprotein at the budding step during the particle release process is one of the last steps in the replication cycle of envelope viruses. This assembly step has long been thought to occur exclusively at the plasma membrane. However, increasing evidence was collected that budding can also take place at intracellular compartments (i.e., the multivesicular body) and that budding sites may depend on the type of producer cell analyzed (reviewed in reference 26). While budding across cellular membranes retroviruses incorporate the viral glycoproteins but also cellular proteins by active or passive mechanisms. Passive incorporation of viral or cellular membrane proteins into retroviral particles occurs in the absence of specific interactions with the viral capsid proteins, as long as they are not sterically incompatible with the viral core and are present at the budding site. Active incorporation requires specific interactions between the viral capsid and viral or cellular membrane proteins at the budding site. At least for some retroviruses (i.e., human immunodeficiency virus [HIV] and Mason-Pfizer monkey virus [MPMV]), there is accumulating evidence for an active incorporation of the cognate envelope protein through Gag-Env interactions (3, 20, 35, 44).

Foamy viruses (FVs), the only genus of the retroviral subfamily Spumaretrovirinae, display a replication strategy with features distinct from that of orthoretroviruses but resembling in many aspects those of hepadnaviruses (reviewed in reference 32). The FV glycoprotein plays an important role in the assembly process since its coexpression with the Gag protein is essential for targeting of FV capsids, preassembled in the cytoplasm, to cellular membranes and for budding of viral particles (reviewed in reference 13). In addition, the FV glycoprotein has a highly unusual biosynthesis for a retroviral glycoprotein. It is translated as a full-length precursor protein into the rough endoplasmic reticulum and initially has a type III protein configuration with both its N and C termini located intracytoplasmically (Fig. 1A) (8, 14). Only during its transport to the cell surface is it posttranslationally processed by cellular, most likely furin-like, proteases and not the signal peptidase complex, into at least three subunits (5, 7).



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  		FIG. 1. Schematic illustration of PFV Env membrane topology and expressions constructs. (A) Schematic illustration of the membrane topology of the PFV Env precursor protein as suggested from previous analysis (14, 17). (B) Schematic organization of the PFV Env protein. The N-terminal region spanning the complete leader peptide and partial sequences of the adjacent surface domain is enlarged. Individual lysine residues in the cytoplasmic domain of the LP and the asparagine residues of the first three potential N-glycosylation sites are indicated for the wild-type constructs (wt, EM002 or EM015). N-glycosylation sites used are indicated with a Y. For the individual mutant constructs amino acid sequences deviating at specific positions from the wild-type sequence are indicated. LP: leader peptide; SU: surface subunit; TM: transmembrane subunit; h: hydrophobic region of the LP; FP: fusion peptide; MSD: membrane-spanning domain; N: N terminus; C: C terminus; N1 to N3: potential N-glycosylation sites 1 to 3.

The N-terminal signal or leader peptide (LP) has a type II conformation, whereas the C-terminal TM subunit has a type I conformation. The internal SU subunit presumably associates with extracellular domains of TM on the luminal side (14, 43). Image reconstruction analysis from negative-stained electron microscopy pictures of the characteristic, prominent Env spike structures on FV particles indicates that the Env glycoprotein, as reported for other viral glycoproteins, forms trimeric complexes containing three copies of each of the three individual subunits in every Env spike structure (42). For the FV budding process, two essential interactions between Env and Gag are required (14, 27). One involves the N-terminal region of the putative membrane-spanning domain of the TM subunit (27). The more important, second essential interaction, however, is the contact of the N-terminal cytoplasmic region of the FV Env LP, the so-called budding domain, containing an essential, conserved WXXW sequence motif, with the N terminus of the FV Gag protein (14, 42). Most probably due to the crucial interaction between capsid and the FV Env LP, this cleavage product is particle associated. However, it also suggests that the LP may have additional functions in the FV replication cycle and FV Env function.

We have previously observed at least three different LP products in purified FV particles (14). In the present study we intended to characterize the nature of these different LP forms in more detail and examine their role in glycoprotein function and FV replication.


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MATERIALS AND METHODS
 
Cells. The human kidney cell line 293T (4), the human fibrosarcoma cell line HT1080 (30), and the FV indicator cell line BHK/LTR(HFV)lacZ (33) were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and antibiotics.

Expression constructs. The replication-deficient prototype foamy virus (PFV; formerly known as human foamy virus [HFV]) vector pDWP01 is a variant of the previously described PFV Gag/Pol/Env and enhanced green fluorescent protein (EGFP)-neo fusion protein expressing vector pDL01 (28) having most of the env open reading frame (ORF) deleted and all translation initiation codons of the env ORF overlapping the pol ORF mutated to ACG mutations (5). Thereby the Pol amino acid sequence remained unchanged but expression of residual Env peptides was completely prevented. The glycoprotein expression constructs used in this study are shown schematically in Fig. 1B. The PFV Env expression constructs pczHFVenvEM002 (15); EM015, (5) yielding wild-type proteins; and the constructs pczHFVenvEM058 ({Delta}N1), EM077 ({Delta}N2), and EM078 ({Delta}N3), expressing mutant glycoproteins having the first, second, or third potential N-glycosylation site inactivated by an N->Q exchange, respectively (14), were described earlier. The ubiquitination site mutants pczHFVenvEM134 to -EM140, based on the pczHFVenvEM015 wild-type constructs, were generated by recombinant PCR techniques. The individual cloning strategies and mutagenesis primers are available on request.

In constructs pczHFVenvEM135 ({Delta}Ubi1: K14R), EM136 ({Delta}Ubi2: K15R), EM137 ({Delta}Ubi3: K18R), EM138 ({Delta}Ubi4: K34R), and EM139 ({Delta}Ubi5: K53R) individual potential ubiquitination sites have been inactivated, whereas in pczHFVenvEM134 ({Delta}Ubi1-3: K14R, K15R, and K18R) the first three and in pczHFVenvEM140 ({Delta}Ubi1-5: K14R, K15R, K18R, K34R, and K53R) all five potential ubiquitination sites were mutated. The parental human cytomegalovirus immediate-early promoter-driven infectious proviral clone pcHSRV2 has been described previously (19). The mutant pcHSRV2EM026 proviral construct displays a wild-type replication phenotype and has a BsmBI restriction site introduced by silent mutagenesis into the pol ORF immediately upstream of the env translation start to facilitate introduction of env mutants into the proviral backbone. The pcHSRV2EM140 ({Delta}Ubi1-5) mutant provirus construct was generated by replacing a BsmBI/Kpn2I fragment of pcHSRV2EM026 with the same fragment of the pczHFVenvEM140 expression construct. The expression constructs for hemagglutinin (HA)-tagged or wild-type ubiquitin pSG5 HA-Ubi and pSG5 Ubi were a kind gift of H. Göttlinger and were described previously (39).

Generation of viral supernatants and analysis of transduction efficiency. Supernatants containing recombinant viral particles were generated essentially as described earlier (10, 12, 15). FV supernatants were generated by cotransfection of 293T cells with the Gag/Pol-expressing vector pDWP01 and an Env expression plasmid as indicated or by transfection of cells with the proviral expression construct pcHSRV2 and variants thereof. Transductions of recombinant EGFP expressing PFV vector particles were performed by infection of 2 x 104 target cells plated 24 h in advance in 12-well plates for 4 h using 1 ml of viral supernatant or dilutions thereof. The amount of EGFP-positive cells was determined by fluorescence-activated cell sorting analysis 48 h after infection and was in the range of 0.5 to 80% depending on the virus dilution used for infection. All transduction experiments were performed at least three times and in each independent experiment the values obtained with wild-type PFV Env (EM002 or EM015) were arbitrarily set to 100. Viral titers of replication-competent PFV were titrated by endpoint dilution on BHK/LTR(HFV)lacZ cells as described previously (33).

In some experiments intracellular viral particles were artificially released by freeze-thawing of the transfected 293T cells and subsequent centrifugation and filtration of the supernatant through 0.45-µm-pore-size filters to remove cellular debris. The resulting supernatants were then assayed as described above.

Antisera and Western blot expression analysis. Western blot expression analysis of cell- and particle-associated viral proteins was performed essentially as described previously (14) with the exception that blots to be analyzed with some ubiquitin-specific antibodies were autoclaved for 30 min in distilled water using a liquid cycle immediately after blotting to the nitrocellulose to enhance the ubiquitin-specific signal, as suggested previously (18). Subviral particles were isolated similar to FV particles by ultracentrifugation through 20% sucrose. Polyclonal antisera used were specific for PFV Gag (1), the LP of PFV Env, amino acids 1 to 86 (14), the LP and SU subunits, amino acids 87 to 148, or bovine ubiquitin (Sigma U-5379). Furthermore, hybridoma supernatants specific for the HA tag (clone 12CA5), the SU subunit (clone P3E10) of PFV Env (5), or commercially available monoclonal antibodies specific for ubiquitin (Biomol: FK1 and FK2; Covance: P4D1 and P4G7) were employed.

Electron microscopy analysis. Forty-eight hours after transfection, 293T cells were harvested and processed for electron microscopy analysis as described previously (11).


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RESULTS
 
Distinct particle-associated FV Env LP cleavage products arise by differential posttranslational modification. We observed previously that purified FV particles contain different glycoprotein precursor-derived cleavage products recognized by an LP-specific antiserum (14). However, the nature of these distinct forms remained unclear. Basically there are two different potential explanations for their occurrence. The first is that alternative proteolytic cleavage sites are used during FV Env precursor processing. The second is that slower-migrating forms are posttranslationally modified variants of the major cleavage product gp18LP. The recent determination of the LP/SU cleavage site by sequencing of the N terminus of the PFV SU subunit revealed no indication of alternatively processed forms of SU (5).

In order to shed light on the nature of these higher-molecular-weight LP cleavage variants of PFV Env, therefore, we concentrated our efforts on the identification of potential posttranslational modifications of gp18LP. First, we examined if alternative N-glycosylation of gp18LP might account for the observed difference in molecular weight. Therefore PFV particles, purified by ultracentrifugation of cell culture supernatants of 293T cells transfected with the replication-deficient proviral PFV vector pDL01 through a sucrose cushion, were digested with different endoglycosidases or mock incubated (Fig. 2). This analysis revealed the presence of at least three LP cleavage fragments with distinct molecular weights in all samples, although the treatment of PFV particles by either endoglycosidase H (Fig. 2A and B, lane 3) or N-glycosidase F (Fig. 2A and B, lane 2) resulted in a similar increase in mobility of all PFV Env LP cleavage products compared to mock incubated particles (Fig. 2A and B, lane 1). This indicated that the higher-molecular-weight forms of gp18LP are not the result of alternative N-glycosylation. The analysis of the particle-associated composition of LP cleavage products of mutant PFV Env expression constructs (Fig. 1) that had individual potential N-linked glycosylation (N-Gly) sites inactivated further supported this explanation (data not shown).



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  		FIG. 2. Glycosylation analysis of PFV particles. PFV particles generated by transfection of 293T cells with the replication-deficient PFV proviral vector pDL01 (lanes 1 to 3) or empty expression vector (lane 4) were purified by ultracentrifugation through 20% sucrose. Subsequently viral particles were digested with N-glycosidase F (PNGaseF) or endoglycosidase H (EndoH) or mock incubated (mock) followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blot analysis using polyclonal rabbit sera specific for (A) PFV Env LP ({alpha}-LP), (B) PFV Env LP/SU ({alpha}-LP/SU) and PFV Gag ({alpha}-Gag), or (C) ubiquitin ({alpha}-Ubi). Viral proteins are indicated on the right. The different glycosylated and deglycosylated forms of the PFV SU subunit are marked by asterisks. The band of about 25 kDa in all lanes in panel C represents a staining artifact in this specific experiment that was present even in lanes loaded only with sample buffer.

Taken together, these results are in line with earlier results from our laboratory (5) suggesting that all three products are derived by proteolytic precursor cleavage at amino acid 126 and not at alternative sites. Furthermore, they indicate that the slower-migrating forms of the LP do not arise by differential N-glycosylation of gp18LP.

Ubiquitination of the FV Env gp18LP cleavage product. Another posttranslational modification that leads to a substantial increase in molecular weight and that has recently been associated with retroviral particle release is ubiquitination (41). Therefore, we analyzed PFV particle preparations by Western blot using a ubiquitin-specific polyclonal antiserum. As shown in Fig. 2, digestion of wild-type PFV particles by N-glycosidase F or endoglycosidase H resulted in a mobility shift of all ubiquitin-specific proteins bands (Fig. 2C, lanes 2 and 3) that were identical to the higher-molecular-weight bands detected by the LP-specific antiserum (Fig. 2B, lanes 2 and 3). Similarly, only the minor particle-associated gp28LP and gp38LP fragments but not the predominant gp18LP fragments of the wild-type PFV Env protein (data not shown) and the {Delta}N1 or {Delta}N3 mutant (data not shown) and the faster-migrating bands of the {Delta}N2 mutant (data not shown) were detected by this antiserum. Furthermore, at least two to three additional distinct higher-molecular-weight forms, stained by the ubiquitin-specific antiserum, were detectable (Fig. 2C, lanes 1 to 3; data not shown). However, these were only poorly detectable by the LP- or LP-SU-specific antisera (Fig. 2A and B, lanes 1 to 3; data not shown).

In order to exclude detection of the higher-molecular-weight bands as a result of unspecific binding of the ubiquitin specific antiserum we cotransfected in an additional experiment expression constructs for HA-tagged or unmodified ubiquitin together with different combinations of PFV Env and Gag/Pol vector constructs (Fig. 3). Subsequent probing of particle preparations with the ubiquitin-specific antiserum again stained only slower migrating forms other than gp18LP, regardless of whether HA-tagged ubiquitin, untagged ubiquitin, or empty expression vectors were cotransfected (Fig. 3A). Using an HA-specific monoclonal antibody, these higher-molecular-weight forms and not gp18LP could be detected only in samples where the HA-tagged ubiquitin expression construct had been cotransfected (Fig. 3B, lanes 1 and 2), whereas in samples cotransfected with untagged ubiquitin (Fig. 3B, lanes 4 and 5) or empty expression vector (Fig. 3B, lanes 7 and 8) no HA-specific signal was detectable. Staining with an LP-specific antiserum resulted in detection of gp18LP and at least two other slower-migrating forms of about 28 kDa and 38 kDa in all three transfection groups (Fig. 3C). In addition, four different types of ubiquitin-specific antibodies specifically stained the gp28LP and gp38LP proteins but not the gp18LP protein (data not shown).



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  		FIG. 3. Labeling of particle-associated PFV Env with HA-tagged ubiquitin. Mutant PFV particles were generated by transient cotransfection of 293T cells with the Gag/Pol-expressing PFV vector pDWP01 and the PFV Env expression construct or empty vector as indicated. In addition, HA-tagged ubiquitin (HA-Ubi), untagged ubiquitin (Ubi), or empty expression vector (pUC) was cotransfected as indicated. Subsequently, lysates of viral particles purified by ultracentrifugation through 20% sucrose were analyzed by Western blotting using consecutive incubation with antibodies specific for (A) ubiquitin ({alpha}-Ubi), (B) the HA tag ({alpha}-HA), and (C) PFV Env LP ({alpha}-LP) and PFV Gag ({alpha}-Gag). Viral proteins are indicated on the left and between panels B and C. Cells were transfected with pDWP01 and: lane 1, pczHFVenvEM015 (wt) plus pSG5 HA-Ubi (HA-Ubi); lane 2, pczHFVenvEM077 ({Delta}N2) plus pSG5 HA-Ubi (HA-Ubi); lane 3, pUC19 (pUC) plus pSG5 HA-Ubi (HA-Ubi); lane 4, pczHFVenvEM015 (wt) plus pSG5 Ubi (Ubi); lane 5, pczHFVenvEM077 ({Delta}N2) plus pSG5 Ubi (Ubi); lane 6, pUC19 (pUC) plus pSG5 Ubi (Ubi); lane 7, pczHFVenvEM015 (wt) plus pUC19 (pUC); lane 8, pczHFVenvEM077 ({Delta}N2) plus pUC19 (pUC); lane 9, only pUC19 (pUC).

These results showed that the particle-associated PFV Env LP is strongly ubiquitinated, giving rise to differentially migrating proteins. Furthermore, the detection of similarly sized higher-molecular-weight forms by a simian foamy virus (SFVmac) LP-specific antiserum in PFV particles pseudotyped by SFVmac Env (data not shown) suggested that this posttranslational modification of the glycoprotein is not unique to PFV and might be common to all FV species.

Ubiquitination sites in the FV Env leader peptide. Ubiquitin is covalently linked to proteins by attachment to the {varepsilon} amino group of lysine residues. Inspection of the N-terminal cytoplasmic domain of the PFV Env LP amino acid sequence revealed five potential ubiquitination sites, K14, K15, K18, K34, and K53 (Fig. 1B). Mutant PFV Env expression constructs having individual lysine residues or combinations thereof changed to arginine were generated to determine the sites of ubiquitination in PFV Env LP (Fig. 1B). Recombinant PFV particles were produced by transient cotransfection of the individual Env expression constructs and a Gag/Pol and EGFP marker gene expressing the PFV vector (pDWP01) into 293T cells. Western blot analysis of transfected cell lysates showed that all mutants were expressed at similar levels (Fig. 4D), although the K14 single mutant ({Delta}Ubi1) and the K14, K15, K18, K34, and K53 quintuple mutant ({Delta}Ubi1-5) showed somewhat reduced or increased LP cleavage, respectively (Fig. 4D, lanes 5 and 10).



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  		FIG. 4. Analysis of PFV Env mutants with inactivated ubiquitination sites. Mutant PFV particles were generated by transient cotransfection of 293T cells with the Gag/Pol-expressing PFV vector pDWP01 and the PFV Env expression construct or empty vector as indicated. Western blot analysis of purified PFV particles using consecutive incubation with antisera specific for (A) PFV Gag ({alpha}-Gag), (B) PFV Env LP/SU ({alpha}-LP/SU), or (C) ubiquitin ({alpha}-Ubi) or cell lysates using antisera specific for (D) PFV Gag ({alpha}-Gag) or PFV Env LP ({alpha}-LP). Viral proteins are indicated on the side of the blots. (E) Relative infectivity of 293T cell supernatants (extracellular) and freeze-thaw cell lysates (intracellular) using the GFP marker gene transfer assay on HT1080 target cells. The values obtained using the wild-type PFV Env expression plasmid (wt) were arbitrarily set to 100%. The mean values and standard deviations of at least three independent experiments are shown. Cells were either transfected with pcDNA3.1+zeo alone (lane 1: pcDNA) or cotransfected with pDWP01 and: lane 2, pcDNA3.1+zeo (pcDNA); lane 3, pczHFVenvEM015 (wt); lane 4, pczHFVenvEM134 ({Delta}Ubi1-3); lane 5, pczHFVenvEM135 ({Delta}Ubi1); lane 6, pczHFVenvEM136 ({Delta}Ubi2); lane 7, pczHFVenvEM137 ({Delta}Ubi3); lane 8, pczHFVenvEM138 ({Delta}Ubi4); lane 9, pczHFVenvEM139 ({Delta}Ubi5); lane 10, pczHFVenvEM140 ({Delta}Ubi1-5).

Examination of particle preparations using a PFV Gag-specific (Fig. 4A) or PFV Env LP/SU-specific (Fig. 4B) antiserum revealed that all mutant Env proteins supported FV particle release. However, probing the same blot with the ubiquitin-specific antiserum showed a strong decrease or complete disappearance of ubiquitination for the K14, K15, and K18 triple mutant {Delta}Ubi1-3 and the quintuple mutant {Delta}Ubi1-5, respectively (Fig. 4C, lanes 4 and 10). Mutants having only individual lysine residues replaced ({Delta}Ubi1 to {Delta}Ubi5) displayed ubiquitination patterns similar to that of the wild-type protein (Fig. 4C, lanes 3 and 5 to 9). Furthermore, no indication of ubiquitination of other PFV Env domains, in particular the TM subunit containing at least additional four lysine residues in its C-terminal cytoplasmic tail, or the 648-amino-acid PFV Gag protein containing only a single lysine residue, was obtained, even when assaying the PFV Env mutant {Delta}Ubi1-5, which has all potential ubiquitination sites in the LP cytoplasmic domain inactivated (Fig. 4C, lane 10, and data not shown). Interestingly, some additional higher-molecular-weight forms, showing a slightly but clearly different mobility than gp28LP and gp38LP, were detectable for all Env proteins analyzed by the PFV LP/SU- (Fig. 4B) or PFV LP-specific antiserum (data not shown) but not by the ubiquitin-specific antiserum (Fig. 4C), although at much weaker intensity. This was most prominent for the {Delta}Ubi1-5 mutant sample, where gp28LP and gp38LP were absent (Fig. 4B, lane 10). The nature of these additional bands is currently unclear.

Single-cycle infectivity analysis of the recombinant mutant particles in a fluorescence-activated cell sorting-based EGFP gene transfer assay revealed wild-type infectivities for cell culture supernatants of most mutants (Fig. 4E). Only supernatants of cells transfected with the {Delta}Ubi1-5 mutant, lacking all potential ubiquitination sites, showed a two- to threefold increase in infectivity (Fig. 4E). However, in the proviral context, no significant difference in viral titers or spread of the mutant virus compared to the wild type was observed (data not shown).

These data indicate that all lysine residues of the FV LP cytoplasmic domain can potentially be ubiquitinated, although there seems to be a preference for usage of the first three, as the ubiquitin-specific signal decreased significantly for the triple mutant, {Delta}Ubi1-3, having these lysine residues changed to arginine. Furthermore, the complete absence of ubiquitin-specific signals in the quintuple mutant {Delta}Ubi1-5 indicates that these are the only sites of ubiquitination in the PFV Env LP. In addition, the results suggest that FV Env LP ubiquitination is not required for viral particle release and has only a minor effect on viral particle infectivity.

Electron microscopy analysis of mutant particle release. In order to analyze the particle morphogenesis of the ubiquitination-deficient PFV Env mutant in further detail, we performed electron microscopy analysis on 293T cells transfected with either different proviral expression constructs or proviral vector constructs expressing Gag, Pol, and Env. In cells expressing PFV Gag/Pol and the wild-type PFV Env protein, viral particles containing typical FV ring-like capsid structures and glycoprotein spikes were readily detectable (Fig. 5A). In contrast, in addition to regular wild-type PFV particles, significant numbers of subviral particles (SVPs) containing the typical glycoprotein spikes but lacking the ring-like capsid structure were detected in cells expressing the mutant EM140 Env in the context of a proviral expression construct (Fig. 5B to F). In corresponding wild-type samples these SVP structures were not detectable (Fig. 5A, and data not shown).



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  		FIG. 5. Electron microscopy analysis of proviral expression clones. Electron micrographs showing representative thin sections of 293T cells transiently transfected with the wild-type PFV proviral expression vector pczHSRV2EM026 (A) or the mutant proviral vector pczHSRV2EM140 expressing the ubiquitination-deficient PFV Env protein {Delta}Ubi1-5 (B to F). Subviral particles are marked by solid arrows. Magnifications: (A) 89,000x; (B) 70,000x; (C) 110,000x; (D) 90,000x; (E) 95,000x; (F) 127,000x. Bar, 200 nm.

Increased subviral particle release by ubiquitination-deficient PFV Env mutants. Similar to hepadnaviruses, PFV has recently been shown to secrete envelope glycoprotein-containing SVPs, although at much lower levels (36). Therefore, the influence of PFV Env LP ubiquitination on SVP release was analyzed by transfection of 293T cells with the individual mutant glycoprotein expression constructs in the absence of expression of any other PFV protein. Subsequent Western blot analysis of particle preparations using a PFV Env LP-specific antiserum revealed a massive SVP release for the triple mutant {Delta}Ubi1-3 and an even greater SVP release for the quintuple mutant {Delta}Ubi1-5 (Fig. 6B, lanes 1 and 7). In contrast, for wild-type PFV Env as well as for the single mutants ({Delta}Ubi1 to {Delta}Ubi5), displaying a wild-type ubiquitination pattern (Fig. 4C), no SVP release was detectable using the same amount of transfected cell culture supernatant (Fig. 6B, lanes 2 to 6 and 9), although all Env proteins showed similar cellular expression levels (Fig. 6D, lanes 1 to 9).



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  		FIG. 6. Analysis of subviral particle release. Subviral particles were harvested by ultracentrifugation through 20% sucrose from supernatants of 293T cells transfected with the individual Env expression constructs or empty expression vector as indicated (lanes 1 to 9). As a control wild-type PFV particles were generated by cotransfecting the PFV Gag/Pol expression vector pDWP01 and the wild-type PFV Env expression construct pczHFVenvEM015 or empty expression vector (lanes 10 and 11). Western blot analysis of particle preparations using consecutive incubation with (A) a ubiquitin-specific monoclonal antibody ({alpha}-Ubi, P4D1) and (B) a polyclonal antiserum specific for PFV LP ({alpha}-LP). Western blot analysis of the corresponding cell lysates using polyclonal antisera specific for (C) PFV Gag ({alpha}-Gag) and for (D) PFV LP ({alpha}-LP). Cells were transfected with: lane 1, pczHFVenvEM134 ({Delta}Ubi1-3); lane 2, pczHFVenvEM135 ({Delta}Ubi1); lane 3, pczHFVenvEM136 ({Delta}Ubi2); lane 4, pczHFVenvEM137 ({Delta}Ubi3); lane 5, pczHFVenvEM138 ({Delta}Ubi4); lane 6, pczHFVenvEM139 ({Delta}Ubi5); lane 7, pczHFVenvEM140 ({Delta}Ubi1-5); lane 8, pcDNA3.1+zeo (pcDNA); or lane 9, pczHFVenvEM015 (wt); or cotransfected with pDWP01 and pcDNA3.1+zeo (pcDNA, lane 10) or pczHFVenvEM015 (wt, lane 11).

For wild-type samples, SVP-associated Env was only detectable if using 20- to 50-fold higher amounts of transfected cell culture supernatant than for the {Delta}Ubi1-5 mutant (data not shown). Interestingly, whereas for the triple mutant {Delta}Ubi1-3, ubiquitinated LP forms could be still detected in the SVP preparations (Fig. 6A, lane 1), ubiquitinated LP forms were absent from SVP preparations of the quintuple mutant {Delta}Ubi1-5 (Fig. 6A, lane 7). Based on this biochemical analysis, not surprisingly, in HT1080 cells stably expressing the {Delta}Ubi1-5 (EM140) Env (Fig. 7A and B) or 293T cells transiently transfected with a {Delta}Ubi1-5 (EM140) Env expression vector (Fig. 7C and D) SVPs containing the typical PFV Env spike structures were readily detectable by electron microscopy analysis. Similar structures were not detectable in corresponding samples expressing a wild-type PFV Env protein (data not shown).



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  		FIG. 7. Electron microscopy analysis of {Delta}Ubi1-5 PFV Env-expressing cells. Electron micrographs showing representative thin sections of (A and B) HT1080 cells stably expressing the mutant {Delta}Ubi1-5 mutant PFV Env protein or (C and D) 293T cells transiently transfected with the mutant PFV Env expression vector pczHFVenvEM140 ({Delta}Ubi1-5). Magnifications: (A) 72,000x; (B) 180,000x; (C) 57,000x; (D) 138,000x. Bar, 200 nm.

Thus, this indicates that ubiquitination of the PFV Env LP regulates SVP formation and interference with LP ubiquitination, in particular at the first three potential attachment sites, leads to a massive increase in PFV Env-mediated SVP release.


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DISCUSSION
 
The cellular ubiquitination machinery is implicated in the budding processes of different types of viruses (41). For different retroviruses monoubiquitination of Gag subunits and the presence of particle-associated free ubiquitin were reported (21-24, 29). Whether there is a specific function of Gag ubiquitination for viral egress, however, remains unclear. Mutation of target lysine residues in HIV-1 p6 or murine leukemia virus p12 had no effect on virus release or replication and did not result in a loss of incorporation of free ubiquitin (21). However, a recent study demonstrated that wild-type HIV-1 Gag and mutants lacking p6 or having the target lysines in p6 replaced are ubiquitinated at different subunits (9). Therefore, the lack of functional phenotypes for the p6 lysine mutants might be explained by compensatory ubiquitination of other HIV-1 Gag subunits. Furthermore, it is currently unclear whether HIV-1 Gag is ubiquitinated specifically or if Gag ubiquitination is the result of a bystander effect of the nearby presence of an ubiquitin ligase, whose real function is to ubiquitinate a cellular protein at the budding site. In addition, there is no clear correlation in the sensitivity of different viruses to proteasome inhibitors, which is thought to reduce the free pool of ubiquitin within the cells, and the ubiquitination of the viral Gag proteins (23, 24, 34).

We and others have shown recently that PFV, like other viruses, links to the cellular vacuolar protein sorting machinery for particle egress through a PSAP L-domain motif in PFV Gag (25, 38). Interestingly, unlike other retroviral Gag proteins the 648-amino-acid PFV Gag protein contains only a single lysine residue and some primate FV isolates harbor none at all (data not shown). Although we readily detected PFV Env LP ubiquitination in this study, we were unable to detect ubiquitination of the TM subunit harboring additional cytoplasmic lysine residues or of PFV Gag, suggesting that they either are not a substrate for or are not accessible to the cellular ubiquitination machinery associated with viral particle egress.

To our knowledge, this is the first report of viral glycoprotein ubiquitination associated with a function other than protein degradation (2). Ubiquitination of HIV-1 Env at the extracellular domains of the gp41 subunit has been reported previously (2). However, this report suggests that ubiquitination is functionally associated with the probable misfolding and degradation of the majority of the HIV-1 glycoprotein retained within the endoplasmic reticulum.

Unlike the lack of functional consequences of mutation of individual ubiquitination attachment sites in other viral systems, ubiquitination-deficient PFV Env leads to a massive appearance of subviral particles in the subgenomic and proviral context. This might be a consequence of altered intracellular trafficking of ubiquitylation-deficient Env resulting in a less efficient interaction of PFV capsids with the glycoprotein and thereby enhancing the glycoprotein's intrinsic capacity to form subviral particles. In contrast, no significant change in the in vitro infectivity of wild-type and mutant viruses was observed, although subviral and viral particles were generated in similar amounts in the proviral context by the ubiquitination-deficient mutant. However, since there is no good small-animal model for primate foamy viruses, it cannot be easily tested whether such mutant viruses have additional in vivo phenotypes and are able to spread and establish productive infections in animals.

Electron microscopy analysis of cells expressing ubiquitination-deficient PFV Env indicated that SVPs are apparently released at the cell surface but also at intracellular compartments, suggesting that SVP release might take place at different types of cellular membranes. The mechanism underlying the regulation of PFV SVP release by ubiquitination of the glycoprotein leader peptide is unknown at this time. One can envision several different mechanisms. For example, wild-type PFV Env ubiquitinated at the cell surface might be endocytosed at a higher rate than the ubiquitination-deficient mutant protein and thereby SVP release at the plasma membrane is minimized. However, even at intracellular membranes, SVP release was not detectable in samples expressing the wild-type glycoprotein.

Alternatively, a differential intracellular localization of ubiquitinated and nonubiquitinated glycoproteins, and therefore differential access to the cellular machinery associated with viral budding, might account for the different levels of SVP egress observed. Another mechanism might be a selective binding of ubiquitinated glycoprotein to cellular proteins specifically inhibiting SVP release or an increased affinity of ubiquitinated Env for PFV Gag resulting in more efficient incorporation into viral particles. Otherwise, sequence motifs within the PFV Env cytoplasmic domain required for interaction with cellular proteins during SVP budding and release may be masked by ubiquitination, thereby preventing efficient SVP egress. Whether one of these potential mechanisms or a combination of them are involved in the regulation of FV SVP egress by ubiquitination of the glycoprotein LP requires further analysis. Furthermore it would be interesting to examine other viral glycoproteins, in particular retroviral Env proteins (i.e., HIV-1 Env), for potential posttranslational modification by ubiquitination to see whether it serves a functional role in viral or subviral particle egress.

Recently it has been reported that intracellular release of another retrovirus with a B/D-type assembly strategy, MPMV, potentially involves a pericentriolar recycling endosomal compartment and is enhanced by coexpression of the cognate glycoprotein (35). Furthermore, this report demonstrated that intracellular trafficking of the MPMV Env protein through this compartment is important for initiating export of MPMV capsids from the intracellular site of assembly (35). Although a tyrosine-based endocytosis signal motif in the cytoplasmic domain of the MPMV Env TM subunit is implicated in intracellular trafficking of the MPMV glycoprotein (37) it is tempting to speculate that perhaps the MPMV Env protein is also ubiquitinated and its intracellular trafficking and localization are modulated through this type of posttranslational modification.

FV particle egress is unique among retroviruses because coexpression of the cognate glycoprotein is required for budding and particle release (reviewed in references 13 and 16). Unlike other retroviruses harboring all the structural information essential for these processes within the Gag polyprotein, FVs apparently have split these functions between Gag and Env proteins. However, both may contain redundant structural information, since addition of a membrane-targeting signal to FV Gag enables FV Env independent particle release (6) and FV Env expression by itself results in the generation of SVPs (36). With the demonstration of particle-associated, ubiquitinated forms of the PFV Env LP subunit and regulation of the extent of PFV SVP egress by this posttranslational modification of the glycoprotein in this report, we add another unique feature to the already unusual biosynthesis and function of this viral glycoprotein.


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ACKNOWLEDGMENTS
 
We thank Heinrich Göttlinger for helpful discussions and providing expression constructs, B. Hub for excellent technical assistance, and Axel Rethwilm and J. Bodem for critically reading the manuscript.

This work was supported by grants from the DFG (LI621/3-1 and LI621/4-1) and the BMBF (01ZZ0102) to D.L.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Virologie, Medizinische Fakultät "Carl Gustav Carus," Technische Universität Dresden, Fetscherstr. 74, 01307 Dresden, Germany. Phone: 49 351 458 6210. Fax: 49 351 458 6314. E-mail: dirk.lindemann{at}mailbox.tu-dresden.de. Back


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REFERENCES
 
    1
  1. Baunach, G., B. Maurer, H. Hahn, M. Kranz, and A. Rethwilm. 1993. Functional analysis of human foamy virus accessory reading frames. J. Virol. 67:5411-5418.[Abstract/Free Full Text]
    2. 2
  2. Bültmann, A., J. Eberle, and J. Haas. 2000. Ubiquitination of the human immunodeficiency virus type 1 Env glycoprotein. J. Virol. 74:5373-5376.[Abstract/Free Full Text]
    3. 3
  3. Cosson, P. 1996. Direct interaction between the envelope and matrix proteins of HIV-1. EMBO J. 15:5783-5788.[Medline]
    4. 4
  4. Du Bridge, R. B., P. Tang, H. C. Hsia, P. M. Leong, J. H. Miller, and M. P. Calos. 1987. Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol. Cell. Biol. 7:379-387.[Abstract/Free Full Text]
    5. 5
  5. Duda, A., A. Stange, D. Lüftenegger, N. Stanke, D. Westphal, T. Pietschmann, S. W. Eastman, M. L. Linial, A. Rethwilm, and D. Lindemann. 2004. Prototype foamy virus envelope glycoprotein leader peptide processing is mediated by a furin-like cellular protease, but cleavage is not essential for viral infectivity. J. Virol. 78:13865-13870.[Abstract/Free Full Text]
    6. 6
  6. Eastman, S. W., and M. L. Linial. 2001. Identification of a conserved residue of foamy virus Gag required for intracellular capsid assembly. J. Virol. 75:6857-6864.[Abstract/Free Full Text]
    7. 7
  7. Geiselhart, V., P. Bastone, T. Kempf, M. Schnolzer, and M. Löchelt. 2004. Furin-mediated cleavage of the feline foamy virus Env leader protein. J. Virol. 78:13573-13581.[Abstract/Free Full Text]
    8. 8
  8. Geiselhart, V., A. Schwantes, P. Bastone, M. Frech, and M. Löchelt. 2003. Features of the Env leader protein and the N-terminal Gag domain of feline foamy virus important for virus morphogenesis. Virology 310:235-244.[CrossRef][Medline]
    9. 9
  9. Gottwein, E., and H. G. Kräusslich. 2005. Analysis of human immunodeficiency virus type 1 Gag ubiquitination. J. Virol. 79:9134-9144.[Abstract/Free Full Text]
    10. 10
  10. Heinkelein, M., C. Leurs, M. Rammling, K. Peters, H. Hanenberg, and A. Rethwilm. 2002. Pregenomic RNA is required for efficient incorporation of Pol polyprotein into foamy virus capsids. J. Virol. 76:10069-10073.[Abstract/Free Full Text]
    11. 11
  11. Kräusslich, H. G., M. Fäcke, A. M. Heuser, J. Konvalinka, and H. Zentgraf. 1995. The spacer peptide between human immunodeficiency virus capsid and nucleocapsid proteins is essential for ordered assembly and viral infectivity. J. Virol. 69:3407-3419.[Abstract]
    12. 12
  12. Lindemann, D., M. Bock, M. Schweizer, and A. Rethwilm. 1997. Efficient pseudotyping of murine leukemia virus particles with chimeric human foamy virus envelope proteins. J. Virol. 71:4815-4820.[Abstract]
    13. 13
  13. Lindemann, D., and P. A. Goepfert. 2003. The foamy virus envelope glycoproteins. Curr. Top. Microbiol. Immunol. 277:111-129.[Medline]
    14. 14
  14. Lindemann, D., T. Pietschmann, M. Picard-Maureau, A. Berg, M. Heinkelein, J. Thurow, P. Knaus, H. Zentgraf, and A. Rethwilm. 2001. A particle-associated glycoprotein signal peptide essential for virus maturation and infectivity. J. Virol. 75:5762-5771.[Abstract/Free Full Text]
    15. 15
  15. Lindemann, D., and A. Rethwilm. 1998. Characterization of a human foamy virus 170-kilodalton Env-Bet fusion protein generated by alternative splicing. J. Virol. 72:4088-4094.[Abstract/Free Full Text]
    16. 16
  16. Linial, M. L., and S. W. Eastman. 2003. Particle assembly and genome packaging. Curr. Top. Microbiol. Immunol. 277:89-110.[Medline]
    17. 17
  17. Lüftenegger, D., M. Picard-Maureau, N. Stanke, A. Rethwilm, and D. Lindemann. 2005. Analysis and function of prototype foamy virus envelope N glycosylation. J. Virol. 79:7664-7672.[Abstract/Free Full Text]
    18. 18
  18. Mimnaugh, E. G., P. Bonvini, and L. Neckers. 1999. The measurement of ubiquitin and ubiquitinated proteins. Electrophoresis 20:418-428.[CrossRef][Medline]
    19. 19
  19. Moebes, A., J. Enssle, P. D. Bieniasz, M. Heinkelein, D. Lindemann, M. Bock, M. O. McClure, and A. Rethwilm. 1997. Human foamy virus reverse transcription that occurs late in the viral replication cycle. J. Virol. 71:7305-7311.[Abstract]
    20. 20
  20. Murakami, T., and E. O. Freed. 2000. Genetic evidence for an interaction between human immunodeficiency virus type 1 matrix and alpha-helix 2 of the gp41 cytoplasmic tail. J. Virol. 74:3548-3554.[Abstract/Free Full Text]
    21. 21
  21. Ott, D. E., L. V. Coren, E. N. Chertova, T. D. Gagliardi, and U. Schubert. 2000. Ubiquitination of HIV-1 and MuLV Gag. Virology 278:111-121.[CrossRef][Medline]
    22. 22
  22. Ott, D. E., L. V. Coren, T. D. Copeland, B. P. Kane, D. G. Johnson, R. C. Sowder, 2nd, Y. Yoshinaka, S. Oroszlan, L. O. Arthur, and L. E. Henderson. 1998. Ubiquitin is covalently attached to the p6Gag proteins of human immunodeficiency virus type 1 and simian immunodeficiency virus and to the p12Gag protein of Moloney murine leukemia virus. J. Virol. 72:2962-2968.[Abstract/Free Full Text]
    23. 23
  23. Ott, D. E., L. V. Coren, R. C. Sowder, 2nd, J. Adams, K. Nagashima, and U. Schubert. 2002. Equine infectious anemia virus and the ubiquitin-proteasome system. J. Virol. 76:3038-3044.[Abstract/Free Full Text]
    24. 24
  24. Ott, D. E., L. V. Coren, R. C. Sowder, 2nd, J. Adams, and U. Schubert. 2003. Retroviruses have differing requirements for proteasome function in the budding process. J. Virol. 77:3384-3393.[Abstract/Free Full Text]
    25. 25
  25. Patton, G. S., S. A. Morris, W. Chung, P. D. Bieniasz, and M. O. McClure. 2005. Identification of domains in gag important for prototypic foamy virus egress. J. Virol. 79:6392-6399.[Abstract/Free Full Text]
    26. 26
  26. Pelchen-Matthews, A., G. Raposo, and M. Marsh. 2004. Endosomes, exosomes and Trojan viruses. Trends Microbiol. 12:310-316.[CrossRef][Medline]
    27. 27
  27. Pietschmann, T., M. Heinkelein, M. Heldmann, H. Zentgraf, A. Rethwilm, and D. Lindemann. 1999. Foamy virus capsids require the cognate envelope protein for particle export. J. Virol. 73:2613-2621.[Abstract/Free Full Text]
    28. 28
  28. Pietschmann, T., H. Zentgraf, A. Rethwilm, and D. Lindemann. 2000. An evolutionarily conserved positively charged amino acid in the putative membrane-spanning domain of the foamy virus envelope protein controls fusion activity. J. Virol. 74:4474-4482.[Abstract/Free Full Text]
    29. 29
  29. Putterman, D., R. B. Pepinsky, and V. M. Vogt. 1990. Ubiquitin in avian leukosis virus particles. Virology 176:633-637.[CrossRef][Medline]
    30. 30
  30. Rasheed, S., W. A. Nelson-Rees, E. M. Toth, P. Arnstein, and M. B. Gardner. 1974. Characterization of a newly derived human sarcoma cell line (HT-1080). Cancer 33:1027-1033.[CrossRef][Medline]
    31. 31
  31. Resh, M. D. 1999. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim. Biophys. Acta 1451:1-16.[Medline]
    32. 32
  32. Rethwilm, A. 2003. The replication strategy of foamy viruses. Curr. Top. Microbiol. Immunol. 277:1-26.[Medline]
    33. 33
  33. Schmidt, M., and A. Rethwilm. 1995. Replicating foamy virus-based vectors directing high level expression of foreign genes. Virology 210:167-178.[CrossRef][Medline]
    34. 34
  34. Schubert, U., D. E. Ott, E. N. Chertova, R. Welker, U. Tessmer, M. F. Princiotta, J. R. Bennink, H. G. Kräusslich, and J. W. Yewdell. 2000. Proteasome inhibition interferes with gag polyprotein processing, release, and maturation of HIV-1 and HIV-2. Proc. Natl. Acad. Sci. USA 97:13057-13062.[Abstract/Free Full Text]
    35. 35
  35. Sfakianos, J. N., and E. Hunter. 2003. M-PMV capsid transport is mediated by Env/Gag interactions at the pericentriolar recycling endosome. Traffic 4:671-680.[CrossRef][Medline]
    36. 36
  36. Shaw, K. L., D. Lindemann, M. J. Mulligan, and P. A. Goepfert. 2003. Foamy virus envelope glycoprotein is sufficient for particle budding and release. J. Virol. 77:2338-2348.[Abstract/Free Full Text]
    37. 37
  37. Song, C., S. R. Dubay, and E. Hunter. 2003. A tyrosine motif in the cytoplasmic domain of Mason-Pfizer monkey virus is essential for the incorporation of glycoprotein into virions. J. Virol. 77:5192-5200.[Abstract/Free Full Text]
    38. 38
  38. Stange, A., I. Mannigel, K. Peters, M. Heinkelein, N. Stanke, M. Cartellieri, H. Göttlinger, A. Rethwilm, H. Zentgraf, and D. Lindemann. 2005. Characterization of prototype foamy virus gag late assembly domain motifs and their role in particle egress and infectivity. J. Virol. 79:5466-5476.[Abstract/Free Full Text]
    39. 39
  39. Strack, B., A. Calistri, and H. G. Göttlinger. 2002. Late assembly domain function can exhibit context dependence and involves ubiquitin residues implicated in endocytosis. J. Virol. 76:5472-5479.[Abstract/Free Full Text]
    40. 40
  40. Swanstrom, R., and J. W. Wills. 1997. Synthesis, assembly, and processing of viral proteins, p. 263-334. In J. M. Coffin, S. H. Hughes, and H. E. Varmus (ed.), Retroviruses. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
    41. 41
  41. Vogt, V. M. 2000. Ubiquitin in retrovirus assembly: actor or bystander? Proc. Natl. Acad. Sci. USA 97:12945-12947.[Free Full Text]
    42. 42
  42. Wilk, T., F. de Haas, A. Wagner, T. Rutten, S. Fuller, R. M. Flügel, and M. Löchelt. 2000. The intact retroviral Env glycoprotein of human foamy virus is a trimer. J. Virol. 74:2885-2887.[Abstract/Free Full Text]
    43. 43
  43. Wilk, T., V. Geiselhart, M. Frech, S. D. Fuller, R. M. Flügel, and M. Löchelt. 2001. Specific interaction of a novel foamy virus env leader protein with the N-terminal gag domain. J. Virol. 75:7995-8007.[Abstract/Free Full Text]
    44. 44
  44. Wyma, D. J., A. Kotov, and C. Aiken. 2000. Evidence for a stable interaction of gp41 with Pr55Gag in immature human immunodeficiency virus type 1 particles. J. Virol. 74:9381-9387.[Abstract/Free Full Text]

Journal of Virology, December 2005, p. 15074-15083, Vol. 79, No. 24
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.24.15074-15083.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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