- Research article
- Open Access
Endophilins interact with Moloney murine leukemia virus Gag and modulate virion production
© Wang et al., licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. 2003
Received: 29 April 2003
Accepted: 30 September 2003
Published: 4 December 2003
The retroviral Gag protein is the central player in the process of virion assembly at the plasma membrane, and is sufficient to induce the formation and release of virus-like particles. Recent evidence suggests that Gag may co-opt the host cell's endocytic machinery to facilitate retroviral assembly and release.
A search for novel partners interacting with the Gag protein of the Moloney murine leukemia virus (Mo-MuLV) via the yeast two-hybrid protein-protein interaction assay resulted in the identification of endophilin 2, a component of the machinery involved in clathrin-mediated endocytosis. We demonstrate that endophilin interacts with the matrix or MA domain of the Gag protein of Mo-MuLV, but not of human immunodeficiency virus, HIV. Both exogenously expressed and endogenous endophilin are incorporated into Mo-MuLV viral particles. Titration experiments suggest that the binding sites for inclusion of endophilin into viral particles are limited and saturable. Knock-down of endophilin with small interfering RNA (siRNA) had no effect on virion production, but overexpression of endophilin and, to a lesser extent, of several fragments of the protein, result in inhibition of Mo-MuLV virion production, but not of HIV virion production.
This study shows that endophilins interact with Mo-MuLV Gag and affect virion production. The findings imply that endophilin is another component of the large complex that is hijacked by retroviruses to promote virion production.
The Gag protein of retroviruses plays a critical role in virion assembly (for reviews, see [1–3]). When expressed in the absence of all the other virus-encoded components, this polyprotein alone is sufficient for inducing virus-like particle formation from the cell. Hence, the Gag protein has been referred to as a "particle-making machine" . The Gag proteins of the mammalian gamma-retroviruses, such as the Moloney murine leukemia virus (Mo-MuLV), are translated on free ribosomes in the cytoplasm and myristylated at the amino terminus before being translocated to the plasma membrane . They assemble into enveloped, spherical structures and are then released from the cell. During and after virion assembly, Gag precursors are cleaved by the viral protease into four structural proteins - termed MA, p12, CA, and NC – to form infectious virions.
The MA domain is the major region involved in targeting Gag to the membrane. The precursor Gag proteins are anchored to the plasma membrane by an amino-terminal myristate and by ionic interactions between an amino-terminal cluster of basic residues in the MA domain and the acidic plasma membrane surface [6–8]. Amino-acid substitutions or deletions in the matrix protein of type 1 human immunodeficieny virus (HIV-1) or Mo-MuLV were found to block membrane association [9–12] or to redirect virus assembly to cytoplasmic vacuoles (multivesicular bodies, MVBs). These observations highlight the critical role of MA in intracellular transport of Gag polyproteins to the site of viral assembly. Other mutations in MA have been reported to allow Gag proteins to reach the plasma membrane but to result in Gag accumulation beneath the plasma membrane [13, 14]. Slightly curved, electron-dense patches were formed, and no further capsid assembly was observed. These studies provide evidence of involvement of the MA domain in an early step during viral budding.
L domains (for 'late assembly functions') are required for the late stages of viral budding. These domains are located at different regions of Gag in different retroviruses. In Mo-MuLV, Rous sarcoma virus (RSV) and Mason-Pfizer monkey virus (MPMV), the L domains contain a highly conserved PPPY motif (Pro-Pro-Pro-Tyr) and are located between the matrix and capsid domains [15–17]. In lentiviruses, the L domains have distinct motifs - PTAPP in HIV-1 [18, 19] and YXXL (in the single-letter amino-acid code where X is any amino acid) in equine infectious anemia virus (EIAV)  - and are located at the carboxyl terminus of the Gag protein. Despite the lack of sequence homology, many late domains can be functionally interchanged [21–23].
Recent studies have revealed that a group of cellular proteins of the endocytic/multivesicular pathway are involved in the late stages of viral assembly. Tsg101, a protein involved in vacuolar protein sorting, binds to the PTAPP motif and is required for budding of HIV-1 and Ebola virus [24–27]. Similarly, the WW domains of members of the Nedd4-like family of ubiquitin protein ligases interact with the PPPY motif and play roles in the release of viral particles [28, 29]. EIAV utilizes the YXXL motif within its Gag to recruit AP-2, a component of the endocytic machinery, and possibly other components such as AIP-1/ALIX, to promote virion assembly and release .
To understand further how retroviruses such as Mo-MuLV recruit host cellular factors to promote virion production, we performed a two-hybrid screen of a mouse T-lymphoma cDNA library using a murine viral Gag as 'bait', and identified endophilin 2 as a Gag-interacting partner. Endophilins are involved in the formation of endocytic vesicles from the early onset of budding until fission [30, 31]. In this study, we describe characterization of the Gag-endophilin association and its potential role in virion production.
Identification of endophilin 2 as a Gag-interacting protein
The yeast two-hybrid system was used to search for proteins interacting with murine Gag. The Gag protein of the murine AIDS (MAIDS) defective virus is responsible for its pathogenesis, a hyperplasia and immunodeficiency disease . The gag gene product of one isolate, BM5def Gag, closely resembles Mo-MuLV Gag in the MA, capsid and NC regions but contains a highly divergent p12 region . A mouse T-lymphoma cDNA library was screened in a two-hybrid assay with the full-length BM5def Gag as bait to identify potential cellular binding partners. From 150,000 yeast transformants screened, 31 positive clones were isolated. On the basis of sequence similarity, ten of the cDNAs encode overlapping portions of the mouse homolog of human endophilin 2  (also known as SH3P8 , SH3GL1 , and EEN ).
Endophilins are evolutionarily conserved proteins involved in the formation of endocytic vesicles . All family members contain an amino-terminal coiled-coil domain, a variable region and a carboxy-terminal SH3 domain. Members of the two major subgroups in the endophilin family, A and B, are only about 20% identical to each other. Endophilin A associates with the cytoplasmic surface of membranes , while endophilin B appears to operate at the endoplasmic reticulum and the Golgi complex . Endophilin A has three members in mammals - 1, 2 and 3 - that are about 70% identical to each other at the amino-acid level.
Identifying binding domains in endophilin 2 for Mo-MuLV Gag using the yeast two-hybrid system
To determine the region in endophilin sufficient for binding to Mo-MuLV Gag, amino- or carboxy-terminal fragments of endophilin 2 were fused to the activation domain in Gal4-AD (Figure 1b) and were tested for their interactions with a LexA-Mo-MuLV Gag fusion. Two fragments, ΔSH3 and V+SH3, displayed only weak interaction with Mo-MuLV Gag. Removal of additional portions of endophilin 2 almost completely abrogated the interaction. These results suggest that the intact endophilin, including both amino terminus and SH3 domains, is required for the strongest interaction with Mo-MuLV Gag. No single region could be identified as sufficient for strong binding. Several fragments showed weak binding, significantly above the background level seen with controls.
Interactions between endophilin 2 and other retroviral Gags in the yeast two-hybrid system
Interactions between endophilin 2 and retroviral Gag proteins in the yeast two-hybrid system
Mo-MuLV Pr65 Gag
RSV Pr65 Gag
MPMV Pr76 Gag
SIV Pr57 Gag
HIV-1 Pr55 Gag
Human endophilin 2
Mo-MuLV Gag interacts with another endophilin family member
Mo-MuLV Gag interacts with another endophilin family member in the yeast two-hybrid system
Rat endophilin 1
Rat endophilin 2
Human endophilin 2
Endophilin 2 binds to Mo-MuLV Gag in vitro
Proteins eluted from beads were analyzed by western blotting with an anti-capsid antibody. We found that Mo-MuLV Gag was captured only by GST-endophilin-2 beads, but not by GST, GST-N125, or GST-ΔSH3 beads (Figure 2b; compare lane 5 with lanes 2, 3 and 4). Reprobing the same blot with anti-GST antiserum showed that all the GST fusion proteins were successfully bound to the beads and recovered, and so were available for the interaction (data not shown). Gag was detected only in Mo-MuLV-infected NIH 3T3 cell lysates, and not in uninfected NIH 3T3 cell lysates (Figure 2b; lane 5 versus lane 9), indicating that the Gag antiserum did not cross-react with the GST fusion proteins themselves or with any other proteins on the beads. Although the levels of bound Gag proteins were low, binding to full-length endophilin was readily detectable in repeated experiments. Binding of Gag to any of the fragments was too low for detection. These results demonstrate that endophilin 2 and Mo-MuLV Gag can interact in vitro.
Exogenously expressed endophilin 2 associates with Mo-MuLV virion particles
To monitor the interaction between endophilin and Mo-MuLV Gag in vivo, we investigated whether exogenously expressed endophilin 2 could be incorporated into virion particles. We transfected 293T cells with an expression vector encoding amino-terminal HA-epitope-tagged endophilin 2, either alone or together with a wild-type Mo-MuLV proviral DNA. The culture medium was harvested 48 h post-transfection and virions were purified by sedimentation through 25/45% sucrose step gradients. Virions were collected from the interface of the sucrose gradient, and virion proteins in pellets were solubilized in SDS sample buffer, resolved by gel electrophoresis and analyzed by western blotting. Plasmid DNA encoding an irrelevant HA-tagged protein, annexin II light chain (HA-p11), was used as a negative control. Plasmid DNA encoding an HA-tagged fragment of nucleolin (HA-nuc212), known to be efficiently incorporated into virion particles, served as a positive control .
To obtain an estimate of the proportion of intracellular endophilin 2 that is incorporated into virions, serial dilutions of cell lysates were prepared and compared with virion lysates by analysis on the same western blots. These experiments suggest that virions in the culture medium contain approximately 0.1-0.2% of the HA-endophilin 2 present intracellularly (Figure 3e). This fraction is as much as 100 times lower than the corresponding fraction of a positive control protein, HA-Nuc212, which is very efficiently incorporated into virions. While low, the fraction for endophilin 2 is at least 10 times higher than that for the negative control protein (Figure 3f; less than 0.01%).
Exogenously expressed endophilin 2 is protected from proteases within Mo-MuLV virion particles
The incorporation of exogenously expressed endophilin 2 is saturable
Incorporation of endogenous endophilin 2 and other endocytic proteins into Mo-MuLV virion particles
Two of these virion-associated proteins were tested for their resistance to protease digestion by subtilisin, as was done previously for exogenously expressed HA-endophilin. Both α-adaptin and clathrin present in the virion preparations were fully protected from proteolysis, under conditions in which the external viral Env protein was fully degraded (Figure 7e,f). Thus, these proteins are not simply bound to the outside of the virions, nor released from cells in association with the microvesicles that are known to contaminate virion preparations .
Knock-down of endophilin 2 does not inhibit virion production
Inhibition of virion production by overexpression of fragments of endophilin 2
If endophilins are important for virion production, the overexpression of fragments or of wild-type endophilin 2 could exert dominant-negative effects on virion production. A number of such constructs have been shown to affect endocytosis [45–48]. N125 is a fragment of endophilin 2, similar to the equivalent construct of endophilin 1, which binds to lipsomes and tubulates them in vitro; N156 is a fragment with a coiled-coil region; ΔSH3 contains both N125 and N156 regions but lacks the SH3 domain; V+SH3 contains both variable and SH3 domains and has previously been shown to interact with dynamin, synaptojanin and amphiphysin [34, 38, 49]. These fragments were each tagged with an influenza virus HA epitope at the amino terminus. We first examined incorporation of these fragments into virions with low expression in producer cells. We cotransfected 293T cells transiently with a plasmid containing an individual HA-tagged fragment and a proviral DNA at a 1:5 ratio. Culture supernatants were collected 48 h post-transfection and virions were purified through a 25%/45% sucrose step gradient.
To extend these observations, we cotransfected 293T cells with Mo-MuLV proviral construct pNCS and full-length endophilin at increasing DNA ratios (1:0, 1:1, 1:2.5 and 1:5; Figure 10b). We found that endophilin overexpression induced a clear dose-dependent inhibition of Mo-MuLV virion production. At a 1:2.5 ratio, virion production was inhibited 3- to 4-fold; at a 1:5 ratio, more than 10-fold inhibition was observed (Figure 10b; lanes 4 and 5).
We also tested the effect of overexpressing full-length endophilin 2 or fragments of it on HIV-1 virion production. The experiment was carried out the same way as for Mo-MuLV, except an HIV-1 codon-optimized gag-pol DNA construct (pCMVgagpolBNkan) was used to generate HIV-1 virus-like particles. Overexpression of full-length endophilin or fragments of it had only a minor effect, if any, on HIV-1 pr55Gag-based virus-like particle production (Figure 10c). The N125 fragment had the only reproducible effect, and this was a marginal one. Taken together, these results suggest that high-level overexpression of endophilin 2 can induce a significant inhibition of virion production for Mo-MuLV, but not for HIV Gag production in 293T cells. The almost complete lack of effect on HIV-1 virion production suggests that the overexpression of these proteins is not causing broad metabolic toxicity or depletion of plasma membrane as the mechanism of blocking Mo-MuLV virion production.
In the experiments described here, we have presented evidence that endophilins interact with the matrix or MA domain of the Mo-MuLV Gag protein and may contribute to the process of virion production. We detected an interaction between Mo-MuLV Gag and endophilin 2 in the yeast two-hybrid system, and in vitro, and in vivo. The analogous interaction was not detected for all retroviral Gags, but was seen for Mo-MuLV and RSV Gags. Further tests showed that exogenously expressed endophilin 2 is associated with virion particles, and is protected within the viral envelope. Several fragments of endophilin 2 were also incorporated into virions; these experiments did not identify any specific domain of endophilin as essential for the process, and it is possible that more than one domain can direct incorporation. The endophilin fragments may have been targeted to virions by dimerization with endogenous endophilin, or by indirect interactions with other proteins. About 0.1-0.2% of the intracellular levels of endophilin were recovered in virus, even for those fragments which bound poorly to Gag in yeast. Titrating the levels of endophilin expression showed that the binding sites for endophilins during virion formation are limited and the level of incorporated protein is saturable. These observations suggest that the presence of endophilins within virion particles is not simply attributable to mass action, but that the incorporation might be mediated through specific contacts, with only a limited number of sites for Gag-endophilin association. It is not clear whether the incorporation per se is involved in virion production.
If the interaction is crucial for virion production, we thought it possible that overexpression of full-length or fragments of endophilins might interfere with this process by perturbing the correct stoichiometry of the interaction between endophilin and Gag, or other proteins required for this process. Consistent with this notion, overexpression of full-length endophilin 2 did act in a dominant-negative fashion to significantly reduce Mo-MuLV virion production. The inhibition occurred in a dose-responsive manner. Overexpression of endophilin 2 or N156, a fragment that contains a coiled-coil region, had no effect on the level of expression of Gag precursor or a reporter gene within the cells. Moreover, overexpression of endophilin or its fragments had no or little effect on the production of HIV virus-like particles, correlating with our observation that there is no direct interaction between HIV-1 pr55Gag and endophilin. These data further support the notion that the inhibition of Mo-MuLV virion production we observed is not a nonspecific consequence of overexpression on cell viability or physiology. Rather, overexpression could titrate out Mo-MuLV Gag or other interacting proteins that are specifically required for Mo-MuLV and not HIV production.
We used the siRNA method to knock-down endogenous endophilin 2. This technique has been successfully used to document the requirement of TSG101 in virion assembly . In our case, however, the knock-down of endogenous endophilin 2 had no significant effect on virion yield. One possibility is that the levels of endophilins remaining inside cells after knock-down are sufficient to execute the required functions; indeed, the levels required for virion production may be very low, as only a very small proportion of the intracellular protein is incorporated. The other possibility is that other endophilin family members could compensate for the loss of expression. Indeed, this is very likely because endophilin 1 is expressed in 293T cells (data not shown) and we have shown that it can interact with Gag. It is not clear if the various endophilin family members are fully interchangeable for this or even host functions. We do know that the sequence of the siRNA oligonucleotides used in these experiments is specific to endophilin 2, and would not be able to affect the levels of endophilin 1. We were unable to identify a sequence of suitable length that was an identical match between the two mRNAs.
Retrovirus budding is the topological reverse of endocytosis , and membrane curvature changes dramatically from the relatively planar plasma membranes during formation of 100 nm virions. So far it has not been documented whether proteins that modify membrane curvature contribute to retroviral budding. In this study, we have shown that overexpression of N125, a fragment of endophilin that binds liposomes and induces tubules of diameter 20-100 nm , or of full-length endophilin, a protein that presumably promotes both positive and negative membrane curvature changes during the formation of endocytic vesicles [48, 51], causes a significant reduction of virion production. These observations raise the possibility that our overexpression disrupts normal endophilin functions, and that the binding of lipids and subsequent promotion of changes in plasma membrane curvature could be one of the functions normally exerted by endophilin to assist Mo-MuLV virion formation. It is noteworthy that clathrin, like Gag, was long thought to be sufficient to drive membrane budding by itself. More recently it has become evident that groups of proteins have to work together to bend a biological membrane . Endophilin is not the only protein that can induce membrane curvature. At least three other proteins (dynamin, amphiphysin and epsin) that are involved in clathrin-medicated endocytosis can independently trigger membrane tubulation in vitro [53–55]. These proteins might function at multiple stages during vesicle formation. Morphological analyses indicate that endophilin A is required for clathrin-mediated synaptic vesicle endocytosis at multiple stages, including the following: the early stage of endocytosis, the formation of shallow pits; late stages, with the formation of deeply invaginated, elongated pits; and fission [45, 46]. Proteins such as endophilin, which can help generate membrane curvature, might be involved in virion production at several stages, from formation of slightly curved membrane structures to stalk-like structures until viral fission.
Endophilin may act as part of a large complex, and may associate with many other proteins. The SH3 domain of endophilin has been shown to bind to the proline-rich domain of dynamin , amphiphysin  and synaptojanin, a phosphatidylinositol 5'-phosphatase implicated in synaptic-vesicle uncoating . We found that AP-2 and clathrin, but not dynamin 2, are significantly incorporated into virion particles. The incorporation of any one of the various proteins involved in vesicular trafficking may depend on the behavior of the particular complex in which it resides; one explanation for the lack of incorporation of dynamin 2 is that it may interact with a distinct pool of endophilins, one that does not interact with Gag.
Among the proteins associated with Gag are several other proteins that are implicated in retrovirus budding. Tsg101 is known to interact with the PTAP motif of the L domain of HIV-1 and many other retroviruses, and is required for efficient virion release. Members of the Nedd4 family, involved in endocytosis and recycling of membrane proteins, interact with the PPPY motif of the L domain of many other retroviruses and play a similar role. Dominant-negative fragments of Nedd4-like family members inhibit the release of viral particles much as we have seen for endophilin [28, 29]. These observations suggest that a completely functioning vesicular trafficking pathway is required for retroviruses budding [50, 56, 57]. Interactions with vesicles may also be involved in earlier stages of trafficking of genomic RNA, Env and Gag to the cell surface ; possibly the binding of endophilins to Gag can promote their association with endosomal vesicles.
Very recently, HIV-1 Gag has been shown to associate with the endocytic protein AIP-1/Alix through specific contacts with the Gag p6 domain [59, 60]; Alix is known to interact with endophilins . This observation, along with our results, suggests that endophilin could be another component that is hijacked by retroviruses to promote virion production.
Materials and methods
Yeast two-hybrid system
Yeast reporter strains CTY10-5d and GGY::171  were generously provided by R. Sternglanz. The yeast expression vector pSH2-1  encodes an amino-terminal LexA DNA-binding domain (LexADB); pGADNOT encodes a carboxy-terminal Gal4 activation domain (Gal4AD) . Plasmids containing Mo-MuLV, RSV, HIV-1, SIV-1 and MPMV Gag were described previously [40–42]. Plasmids Δ6 and Δ8 are identical to 3' Δ2355 and 5' Δ1304, respectively, as described previously . DNAs encoding Mo-MuLV MA, p12, p12-CA and CA were amplified from pNCA  by PCR and cloned into plasmid pSH2-1 to generate yeast two-hybrid plasmids.
Yeast two-hybrid library screen
BM5def Gag was cloned into pGBT9 DNA-binding domain vector (Clontech, Palo Alto, USA) and used to screen a V13 T-lymphoma cDNA library .
The full-length cDNA encoding human endophilin 2 was obtained from Chi Wai So (University of Hong Kong) [67–69]. DNA encoding human endophilin 2 residues 1-125 (N125), 1-306 (ΔSH3) or full length (1-368) was cloned into pGEX2TKPL, a derivative of pGEX-2TK (Pharmacia, Piscataway, USA). GST-fusion proteins were produced in bacteria cells as described previously .
Mammalian plasmid DNAs
Mo-MuLV was expressed either from plasmid pNCA  or from pNCS, a derivative carrying an SV40 origin of replication in the plasmid backbone. The plasmid pCMVgagpolBNkan (provided by George Pavlakis) contains a Rev-independent, codon-optimized HIV-1 Gag-Pol gene driven by a cytomegalovirus (CMV) promoter. The plasmids encoding hemagglutinin (HA)-tagged nuc212 or p11 were as described . DNA encoding human endophilin 2 residues 1-125 (N125), 125-306 (N156), 1-306 (ΔSH3), 34-368 (Δ34), 268-368 (V+SH3), or full-length 1-368 (enph 2) was cloned into a pcDNA3.1 vector encoding an amino-terminal HA-epitope tag.
Transfection and siRNA
Four 21-nucleotide single-strand RNAs with symmetric 2 nucleotide 3' (2'-deoxy) thymidine overhangs were ordered from Dharmacon (Lafayette, USA). The sequence of siRNA 1 is: sense, 5'-CACGGUGUCCAAGAUCCGTT-3'; antisense, 5'-ACGGAUCUUGGACACCGUGTT-3'. The sequence of siRNA 2 is: sense, 5'-GUUCGAGGAGUCCAAGGAGTT-3'; antisense, 5'-CUCCUUGGACUCCUCGAACTT-3'. These single-strand RNAs were annealed to produce duplexes.
The 293T cells were cultured in six-well plates in 2 ml of DMEM medium with 10% FBS and transfected with siRNAs at 40% confluence by lipofectamine 2000 (Invitrogen, Carlsbad, USA). The transfection was carried out twice in a row (one transfection per day). A final concentration of 50 nM siRNA duplexes was used in each transfection experiment.
Transfection, virion purification, western analyses, and subtilisin treatment
The 293T cells were transfected with viral DNAs using calcium phosphate . Cells were lysed and supernatants were harvested 48 h post-transfection unless stated otherwise in the text. Centrifugation of virion particles and subtilisin digestion of virions were performed as previously described . Briefly, 293T cells were transfected with 10 μg of proviral DNA together with 2 μg of plasmid expressing HA-endophilin. The culture medium harvested from 293T cells 48 h post-transfection was filtered through a 0.45 μm filter and virions were purified on 25%/45% sucrose step gradient, followed by a second purification by sedimentation through a 25% sucrose cushion. For quantification of exogenously expressed endophilin and of the control protein HA-p11, in cytosolic and virion fractions, serial dilutions of cytosolic cell lysates and viral particles were prepared in SDS-PAGE sample buffer. Samples were loaded on the same SDS-acrylamide gel for electrophoresis and analyzed by western blotting with anti-HA antibody. To estimate the fraction of the cytosolic protein present in the virions, signal intensities in the lanes were compared and the dilution in the cytosolic series with the correspondingly closest signal to that seen in the virions was identified. The proportion in the virions was then calculated from the dilution giving comparable signals.
For subtilisin treatment, purified virion pellets were resuspended in subtilisin buffer (40 mM Tris pH 8, 2 mM CaCl2) and incubated with various amounts of subtilisin (Boehringer Mannheim, Indianapolis, USA) for digestion overnight at room temperature. Reactions were stopped by adding PMSF and aprotinin to final concentrations of 2 mM and 1 μg/ml, respectively. Particles were then purified through a 25% sucrose cushion. For subtilisin digestion in the presence of detergent, NP-40 was added to a final concentration of 0.2%.
Exogenous reverse transcriptase assay
Exogenous reverse transcriptase assays were performed as described previously . The amount of DNA synthesized was quantitated by PhosphorImager analysis of the radioactivity of the reverse-transcribed product.
Anti-mouse endophilin 2 polyclonal antibody was previously described . We found it also cross-reacted with both the endogenous and the exogenously expressed human endophilin 2, but not with endophilin 1 (data not shown). Anti-dynamin 2 polyclonal antibody was from Santa Cruz Biotech (Santa Cruz, USA). Anti-HA monoclonal antibody was purchased from BabCO (Berkeley, USA). Anti-clathrin and AP-2 monoclonal antibodies were from Pharmingen (San Diego, USA). Goat polyclonal anti-CA antiserum (79S-804) was obtained from the National Cancer Institute (Bethesda, USA).
We thank Jeremy Luban, Gregg Gundersen, Dag Helland, Masha Orlova, Gilda Tachedjian, Mojgan Naghavi, Theodora Hatziioannou, Irene Nunes, Subarna Bhattacharyya and Yong Cang for helpful discussions, and the Kenia de los Santos for lab assistance. This work was supported by PHS grant CA 30488 from the National Cancer Institute. S.P.G. is an Investigator of the Howard Hughes Medical Institute.
- Wills JW, Craven RC: Form, function, and use of retroviral gag proteins. AIDS. 1991, 5: 639-654.PubMedView ArticleGoogle Scholar
- Swanstrom R, Wills JW: Synthesis, assembly, and processing of viral proteins. In Retroviruses. Edited by: Coffin JM, Hughes SH, Varmus HE. 1997, Cold Spring Harbor: Cold Spring Harbor Laboratory PressGoogle Scholar
- Goff SP: Retroviridae: the retroviruses and their replication. In Fields Virology. Edited by: Knipe DM, Howley PM, Griffin D. 2001, Philadelphia: Lippincott Williams & WilkinsGoogle Scholar
- Dickson C, Eisenman R, Fan H, Hunter E, Teich N: Protein biosynthesis and assembly. In RNA tumour viruses. Edited by: Weiss R, Teich N, Varmus HE, Coffin JM. 1984, Cold Spring Harbor: Cold Spring Harbor Laboratory, 2ndGoogle Scholar
- Rein A, McClure MR, Rice NR, Luftig RB, Schultz AM: Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus. Proc Natl Acad Sci USA. 1986, 83: 7246-7250.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhou W, Parent LJ, Wills JW, Resh MD: Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids. J Virol. 1994, 68: 2556-2569.PubMedPubMed CentralGoogle Scholar
- Spearman P, Wang JJ, Vander Heyden N, Ratner L: Identification of human immunodeficiency virus type 1 Gag protein domains essential to membrane binding and particle assembly. J Virol. 1994, 68: 3232-3242.PubMedPubMed CentralGoogle Scholar
- Göttlinger HG, Sodroski JG, Haseltine WA: Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. Proc Natl Acad Sci USA. 1989, 86: 5781-5785.PubMedPubMed CentralView ArticleGoogle Scholar
- Cannon PM, Matthews S, Clark N, Byles ED, Iourin O, Hockley DJ, Kingsman SM, Kingsman AJ: Structure-function studies of the human immunodeficiency virus type 1 matrix protein, p17. J Virol. 1997, 71: 3474-3483.PubMedPubMed CentralGoogle Scholar
- Facke M, Janetzko A, Shoeman RL, Krausslich HG: A large deletion in the matrix domain of the human immunodeficiency virus gag gene redirects virus particle assembly from the plasma membrane to the endoplasmic reticulum. J Virol. 1993, 67: 4972-4980.PubMedPubMed CentralGoogle Scholar
- Freed EO, Orenstein JM, Buckler-White AJ, Martin MA: Single amino acid changes in the human immunodeficiency virus type 1 matrix protein block virus particle production. J Virol. 1994, 68: 5311-5320.PubMedPubMed CentralGoogle Scholar
- Soneoka Y, Kingsman SM, Kingsman AJ: Mutagenesis analysis of the murine leukemia virus matrix protein: identification of regions important for membrane localization and intracellular transport. J Virol. 1997, 71: 5549-5559.PubMedPubMed CentralGoogle Scholar
- Choi G, Park S, Choi B, Hong S, Lee J, Hunter E, Rhee SS: Identification of a cytoplasmic targeting/retention signal in a retroviral Gag polyprotein. J Virol. 1999, 73: 5431-5437.PubMedPubMed CentralGoogle Scholar
- Manrique ML, Celma CC, Gonzalez SA, Affranchino JL: Mutational analysis of the feline immunodeficiency virus matrix protein. Virus Res. 2001, 76: 103-113. 10.1016/S0168-1702(01)00249-0.PubMedView ArticleGoogle Scholar
- Yuan B, Li X, Goff SP: Mutations altering the moloney murine leukemia virus p12 Gag protein affect virion production and early events of the virus life cycle. EMBO J. 1999, 18: 4700-4710. 10.1093/emboj/18.17.4700.PubMedPubMed CentralView ArticleGoogle Scholar
- Wills JW, Cameron CE, Wilson CB, Xiang Y, Bennett RP, Leis J: An assembly domain of the Rous sarcoma virus Gag protein required late in budding. J Virol. 1994, 68: 6605-6618.PubMedPubMed CentralGoogle Scholar
- Yasuda J, Hunter E: A proline-rich motif (PPPY) in the Gag polyprotein of Mason-Pfizer monkey virus plays a maturation-independent role in virion release. J Virol. 1998, 72: 4095-4103.PubMedPubMed CentralGoogle Scholar
- Göttlinger HG, Dorfman T, Sodroski JG, Haseltine WA: Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release. Proc Natl Acad Sci USA. 1991, 88: 3195-3199.PubMedPubMed CentralView ArticleGoogle Scholar
- Huang M, Orenstein JM, Martin MA, Freed EO: p6Gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease. J Virol. 1995, 69: 6810-6818.PubMedPubMed CentralGoogle Scholar
- Puffer BA, Watkins SC, Montelaro RC: Equine infectious anemia virus Gag polyprotein late domain specifically recruits cellular AP-2 adapter protein complexes during virion assembly. J Virol. 1998, 72: 10218-10221.PubMedPubMed CentralGoogle Scholar
- Yuan B, Campbell S, Bacharach E, Rein A, Goff SP: Infectivity of Moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses. J Virol. 2000, 74: 7250-7260. 10.1128/JVI.74.16.7250-7260.2000.PubMedPubMed CentralView ArticleGoogle Scholar
- Parent LJ, Bennett RP, Craven RC, Nelle TD, Krishna NK, Bowzard JB, Wilson CB, Puffer BA, Montelaro RC, Wills JW: Positionally independent and exchangeable late budding functions of the Rous sarcoma virus and human immunodeficiency virus Gag proteins. J Virol. 1995, 69: 5455-5460.PubMedPubMed CentralGoogle Scholar
- Xiang Y, Cameron CE, Wills JW, Leis J: Fine mapping and characterization of the Rous sarcoma virus Pr76gag late assembly domain. J Virol. 1996, 70: 5695-5700.PubMedPubMed CentralGoogle Scholar
- VerPlank L, Bouamr F, LaGrassa TJ, Agresta B, Kikonyogo A, Leis J, Carter CA: Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag). Proc Natl Acad Sci USA. 2001, 98: 7724-7729. 10.1073/pnas.131059198.PubMedPubMed CentralView ArticleGoogle Scholar
- Garrus JE, von Schwedler UK, Pornillos OW, Morham SG, Zavitz KH, Wang HE, Wettstein DA, Stray KM, Cote M, Rich RL, et al: Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell. 2001, 107: 55-65.PubMedView ArticleGoogle Scholar
- Demirov DG, Ono A, Orenstein JM, Freed EO: Overexpression of the N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late domain function. Proc Natl Acad Sci USA. 2002, 99: 955-960. 10.1073/pnas.032511899.PubMedPubMed CentralView ArticleGoogle Scholar
- Martin-Serrano J, Zang T, Bieniasz PD: HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Nat Med. 2001, 7: 1313-1319. 10.1038/nm1201-1313.PubMedView ArticleGoogle Scholar
- Kikonyogo A, Bouamr F, Vana ML, Xiang Y, Aiyar A, Carter C, Leis J: Proteins related to the Nedd4 family of ubiquitin protein ligases interact with the L domain of Rous sarcoma virus and are required for gag budding from cells. Proc Natl Acad Sci USA. 2001, 98: 11199-11204. 10.1073/pnas.201268998.PubMedPubMed CentralView ArticleGoogle Scholar
- Strack B, Calistri A, Accola MA, Palu G, Göttlinger HG: A role for ubiquitin ligase recruitment in retrovirus release. Proc Natl Acad Sci USA. 2000, 97: 13063-13068. 10.1073/pnas.97.24.13063.PubMedPubMed CentralView ArticleGoogle Scholar
- Reutens AT, Begley CG: Endophilin-1: a multifunctional protein. Int J Biochem Cell Biol. 2002, 34: 1173-1177. 10.1016/S1357-2725(02)00063-8.PubMedView ArticleGoogle Scholar
- Huttner WB, Schmidt A: Lipids, lipid modification and lipid-protein interaction in membrane budding and fission -insights from the roles of endophilin A1 and synaptophysin in synaptic vesicle endocytosis. Curr Opin Neurobiol. 2000, 10: 543-551. 10.1016/S0959-4388(00)00126-4.PubMedView ArticleGoogle Scholar
- Aziz DC, Hanna Z, Jolicoeur P: Severe immunodeficiency disease induced by a defective murine leukaemia virus. Nature. 1989, 338: 505-508. 10.1038/338505a0.PubMedView ArticleGoogle Scholar
- Chattopadhyay SK, Sengupta DN, Fredrickson TN, Morse HC, Hartley JW: Characteristics and contributions of defective, ecotropic, and mink cell focus-inducing viruses involved in a retrovirus-induced immunodeficiency syndrome of mice. J Virol. 1991, 65: 4232-4241.PubMedPubMed CentralGoogle Scholar
- Micheva KD, Ramjaun AR, Kay BK, McPherson PS: SH3 domain-dependent interactions of endophilin with amphiphysin. FEBS Lett. 1997, 414: 308-312. 10.1016/S0014-5793(97)01016-8.PubMedView ArticleGoogle Scholar
- Sparks AB, Hoffman NG, McConnell SJ, Fowlkes DM, Kay BK: Cloning of ligand targets: systematic isolation of SH3 domain-containing proteins. Nat Biotechnol. 1996, 14: 741-744.PubMedView ArticleGoogle Scholar
- Giachino C, Lantelme E, Lanzetti L, Saccone S, Bella Valle G, Migone N: A novel SH3-containing human gene family preferentially expressed in the central nervous system. Genomics. 1997, 41: 427-434. 10.1006/geno.1997.4645.PubMedView ArticleGoogle Scholar
- So CW, Caldas C, Liu MM, Chen SJ, Huang QH, Gu LJ, Sham MH, Wiedemann LM, Chan LC: EEN encodes for a member of a new family of proteins containing an Src homology 3 domain and is the third gene located on chromosome 19p13 that fuses to MLL in human leukemia. Proc Natl Acad Sci USA. 1997, 94: 2563-2568. 10.1073/pnas.94.6.2563.PubMedPubMed CentralView ArticleGoogle Scholar
- Ringstad N, Nemoto Y, De Camilli P: The SH3p4/Sh3p8/ SH3p13 protein family: binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain. Proc Natl Acad Sci USA. 1997, 94: 8569-8574. 10.1073/pnas.94.16.8569.PubMedPubMed CentralView ArticleGoogle Scholar
- Farsad K, Ringstad N, Takei K, Floyd SR, Rose K, De Camilli P: Generation of high curvature membranes mediated by direct endophilin bilayer interactions. J Cell Biol. 2001, 155: 193-200. 10.1083/jcb.200107075.PubMedPubMed CentralView ArticleGoogle Scholar
- Alin K, Goff SP: Mutational analysis of interactions between the Gag precursor proteins of murine leukemia viruses. Virology. 1996, 216: 418-424. 10.1006/viro.1996.0078.PubMedView ArticleGoogle Scholar
- Luban J, Alin KB, Bossolt KL, Humaran T, Goff SP: Genetic assay for multimerization of retroviral gag polyproteins. J Virol. 1992, 66: 5157-5160.PubMedPubMed CentralGoogle Scholar
- Li X, Yuan B, Goff SP: Genetic analysis of interactions between Gag proteins of Rous sarcoma virus. J Virol. 1997, 71: 5624-5630.PubMedPubMed CentralGoogle Scholar
- Bacharach E, Gonsky J, Alin K, Orlova M, Goff SP: The carboxy-terminal fragment of nucleolin interacts with the nucleocapsid domain of retroviral gag proteins and inhibits virion assembly. J Virol. 2000, 74: 11027-11039. 10.1128/JVI.74.23.11027-11039.2000.PubMedPubMed CentralView ArticleGoogle Scholar
- Ott DE, Coren LV, Kane BP, Busch LK, Johnson DG, Sowder RC, Chertova EN, Arthur LO, Henderson LE: Cytoskeletal proteins inside human immunodeficiency virus type 1 virions. J Virol. 1996, 70: 7734-7743.PubMedPubMed CentralGoogle Scholar
- Ringstad N, Gad H, Low P, Di Paolo G, Brodin L, Shupliakov O, De Camilli P: Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis. Neuron. 1999, 24: 143-154.PubMedView ArticleGoogle Scholar
- Gad H, Ringstad N, Low P, Kjaerulff O, Gustafsson J, Wenk M, Di Paolo G, Nemoto Y, Crun J, Ellisman MH, et al: Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin. Neuron. 2000, 27: 301-312.PubMedView ArticleGoogle Scholar
- Simpson F, Hussain NK, Qualmann B, Kelly RB, Kay BK, McPherson PS, Schmid SL: SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation. Nat Cell Biol. 1999, 1: 119-124.PubMedView ArticleGoogle Scholar
- Schmidt A, Wolde M, Thiele C, Fest W, Kratzin H, Podtelejnikov AV, Witke W, Huttner WB, Soling HD: Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid. Nature. 1999, 401: 133-141. 10.1038/43613.PubMedView ArticleGoogle Scholar
- Cremona O, Di Paolo G, Wenk MR, Luthi A, Kim WT, Takei K, Daniell L, Nemoto Y, Shears SB, Flavell RA, et al: Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell. 1999, 99: 179-188.PubMedView ArticleGoogle Scholar
- Patnaik A, Chau V, Wills JW: Ubiquitin is part of the retrovirus budding machinery. Proc Natl Acad Sci USA. 2000, 97: 13069-13074. 10.1073/pnas.97.24.13069.PubMedPubMed CentralView ArticleGoogle Scholar
- Scales SJ, Scheller RH: Lipid membranes shape up. Nature. 1999, 401: 123-124. 10.1038/43582.PubMedView ArticleGoogle Scholar
- Schmidt AA: Membrane transport: the making of a vesicle. Nature. 2002, 419: 347-349. 10.1038/419347a.PubMedGoogle Scholar
- Ford MG, Mills IG, Peter BJ, Vallis Y, Praefcke GJ, Evans PR, McMahon HT: Curvature of clathrin-coated pits driven by epsin. Nature. 2002, 419: 361-366. 10.1038/nature01020.PubMedView ArticleGoogle Scholar
- Sweitzer SM, Hinshaw JE: Dynamin undergoes a GTP-dependent conformational change causing vesiculation. Cell. 1998, 93: 1021-1029.PubMedView ArticleGoogle Scholar
- Takei K, Slepnev VI, Haucke V, De Camilli P: Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis. Nat Cell Biol. 1999, 1: 33-39.PubMedView ArticleGoogle Scholar
- Carter CA: Tsg101: HIV-1's ticket to ride. Trends Microbiol. 2002, 10: 203-205. 10.1016/S0966-842X(02)02350-8.PubMedView ArticleGoogle Scholar
- Luban J: HIV-1 and Ebola virus: the getaway driver nabbed. Nat Med. 2001, 7: 1278-1280. 10.1038/nm1201-1278.PubMedView ArticleGoogle Scholar
- Basyuk E, Galli T, Mougel M, Blanchard JM, Sitbon M, Bertrand E: Retroviral genomic RNAs are transported to the plasma membrane by endosomal vesicles. Dev Cell. 2003, 5: 161-174.PubMedView ArticleGoogle Scholar
- Strack B, Calistri A, Popova E, Göttlinger HG: AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell. 2003, 114: 689-699.PubMedView ArticleGoogle Scholar
- von Schwedler UK, Stuchell M, Muller B, Ward DM, Chung HY, Morita E, Wang HE, Davis T, He GP, Cimbora DM, et al: The protein network of HIV budding. Cell. 2003, 114: 701-713.PubMedView ArticleGoogle Scholar
- Chatellard-Causse C, Blot B, Cristina N, Torch S, Missotten M, Sadoul R: Alix (ALG-2-interacting protein X), a protein involved in apoptosis, binds to endophilins and induces cytoplasmic vacuolization. J Biol Chem. 2002, 277: 29108-29115. 10.1074/jbc.M204019200.PubMedView ArticleGoogle Scholar
- Kalpana GV, Goff SP: Genetic analysis of homomeric interactions of human immunodeficiency virus type 1 integrase using the yeast two-hybrid system. Proc Natl Acad Sci USA. 1993, 90: 10593-10597.PubMedPubMed CentralView ArticleGoogle Scholar
- Hanes SD, Brent R: DNA specificity of the bicoid activator protein is determined by homeodomain recognition helix residue 9. Cell. 1989, 57: 1275-1283.PubMedView ArticleGoogle Scholar
- Luban J, Bossolt KL, Franke EK, Kalpana GV, Goff SP: Human immunodeficiency virus type 1 Gag protein binds to cyclophilins A and B. Cell. 1993, 73: 1067-1078.PubMedView ArticleGoogle Scholar
- Colicelli J, Goff SP: Sequence and spacing requirements of a retrovirus integration site. J Mol Biol. 1988, 199: 47-59.PubMedView ArticleGoogle Scholar
- Kim W, Tang Y, Okada Y, Torrey TA, Chattopadhyay SK, Pfleiderer M, Falkner FG, Dorner F, Choi W, Hirokawa N, Morse HC: Binding of murine leukemia virus Gag polyproteins to KIF4, a microtubule-based motor protein. J Virol. 1998, 72: 6898-6901.PubMedPubMed CentralGoogle Scholar
- So CW, Caldas C, Liu MM, Chen SJ, Huang QH, Gu LJ, Sham MH, Wiedemann LM, Chan LC: EEN encodes for a member of a new family of proteins containing an Src homology 3 domain and is the third gene located on chromosome 19p13 that fuses to MLL in human leukemia. Proc Natl Acad Sci USA. 1997, 94: 2563-2568. 10.1073/pnas.94.6.2563.PubMedPubMed CentralView ArticleGoogle Scholar
- So CW, Sham MH, Chew SL, Cheung N, So CK, Chung SK, Caldas C, Wiedemann LM, Chan LC: Expression and protein-binding studies of the EEN gene family, new interacting partners for dynamin, synaptojanin and huntingtin proteins. Biochem J. 2000, 348: 447-458. 10.1042/0264-6021:3480447.PubMedPubMed CentralView ArticleGoogle Scholar
- So CW, So CK, Cheung N, Chew SL, Sham MH, Chan LC: The interaction between EEN and Abi-1, two MLL fusion partners, and synaptojanin and dynamin: implications for leukaemogenesis. Leukemia. 2000, 14: 594-601. 10.1038/sj.leu.2401692.PubMedView ArticleGoogle Scholar
- Pear WS, Nolan GP, Scott ML, Baltimore D: Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci USA. 1993, 90: 8392-8396.PubMedPubMed CentralView ArticleGoogle Scholar
- Tachedjian G, Aronson HE, de los Santos M, Seehra J, McCoy JM, Goff SP: Role of residues in the tryptophan repeat motif for HIV-1 reverse transcriptase dimerization. J Mol Biol. 2003, 326: 381-396. 10.1016/S0022-2836(02)01433-X.PubMedView ArticleGoogle Scholar