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Originally published In Press as doi:10.1074/jbc.M509317200 on October 7, 2005

J. Biol. Chem., Vol. 280, Issue 49, 40474-40480, December 9, 2005
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Functions of Early (AP-2) and Late (AIP1/ALIX) Endocytic Proteins in Equine Infectious Anemia Virus Budding*

Chaoping Chen{ddagger}, Olivier Vincent§1, Jing Jin{ddagger}, Ora A. Weisz¶||, and Ronald C. Montelaro{ddagger}2

From the {ddagger}Molecular Genetics and Biochemistry, Cell Biology and Physiology, and ||Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 and the §Departamento de Microbiologia Molecular, Centro de Investigaciones Biologicas del Consejo Superior de Investigaciones Cientificas, Madrid 28006, Spain

Received for publication, August 24, 2005 , and in revised form, October 6, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The proline-rich L domains of human immunodeficiency virus 1 (HIV-1) and other retroviruses interact with late endocytic proteins during virion assembly and budding. In contrast, the YPDL L domain of equine infectious anemia virus (EIAV) is apparently unique in its reported ability to interact both with the µ2 subunit of the AP-2 adaptor protein complex and with ALG-2-interacting protein 1 (AIP1/Alix) protein factors involved in early and late endosome formation, respectively. To define further the mechanisms by which EIAV adapts vesicle trafficking machinery to facilitate virion production, we have examined the specificity of EIAV p9 binding to endocytic factors and the effects on virion production of alterations in early and late endocytic protein expression. The results of these studies demonstrated that (i) an ~300-residue region of AIP1/Alix-(409-715) was sufficient for binding to the EIAV YPDL motif; (ii) overexpression of AIP1/Alix or AP-2 µ2 subunit specifically inhibited YPDL-mediated EIAV budding; (iii) virion budding from a replication-competent EIAV variant with its L domain replaced by the HIV PTAP sequence was inhibited by wild type or mutant µ2 to a level similar to that observed when a dominant-negative mutant of Tsg101 was expressed; and (iv) overexpression or siRNA silencing of AIP1/Alix and AP-2 revealed additive suppression of YPDL-mediated EIAV budding. Taken together, these results indicated that both early and late endocytic proteins facilitate EIAV production mediated by either YPDL or PTAP L domains, suggesting a comprehensive involvement of endocytic factors in retroviral assembly and budding that can be accessed by distinct L domain specificities.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Equine infectious anemia virus (EIAV)2 is a member of the lentivirus subfamily of retroviruses, which also includes human immunodeficiency virus 1 (HIV-1). The EIAV genome is the simplest among the lentivirus family and encodes three major structural proteins (Gag, Pol, and Env) along with three accessory gene products (Tat, Rev, S2). Like all retroviruses, EIAV Gag protein is synthesized as a polyprotein that upon virion maturation is processed by virus-encoded protease to yield four major structural proteins: matrix, capsid, nucleocapsid, and p9 protein. All four structural proteins of Gag polyprotein play critical and distinctive roles in retrovirus assembly and budding. Matrix proteins direct targeting of Gag polyproteins to cell membranes for viral assembly and budding (1, 2). Both capsid and nucleocapsid have been shown to mediate Gag-Gag interactions during viral assembly, with capsid responsible for the dimerization of homologous Gag polyproteins (3, 4) and nucleocapsid essential for multimerization of Gag molecules (5, 6). The EIAV p9 protein contains a YPDL late (L) domain that is critical for virion release during the late stage of virus budding. Proline-rich L domains such as PTAP and PPPY with similar functions have also been identified from HIV-1, Rous sarcoma virus, and a variety of other enveloped viruses (7).

L domains appear to serve as docking sites to recruit cellular factors, providing essential functions for virion release. The HIV-1 PTAP motif specifically interacts with the N terminus of tumor suppression gene 101 products (Tsg101), an essential component of the ESCRT I complex responsible for multivesicular body (MVB) formation (8, 9). The PPPY motif binds to ubiquitin ligase Nedd4 (10, 11). Interestingly, the EIAV YPDL motif has been shown to interact with two cellular proteins, ALG-2-interacting-protein-1 (AIP1/Alix) (AIP1 is used hereinafter) (12, 13) and the µ2 subunit of the AP-2 adaptor protein complex (14). Although these L domains appear to interact with different cellular proteins, certain functional interchangeability was previously reported. For example, both PTAP and PPPY motifs can substitute for the YPDL domain to support EIAV replication (15). The PTAP function for HIV-1 budding and replication, however, can be replaced by the PPPY but not the YPDL motif (16). These observations indicate that retroviruses, along with other enveloped viruses, may have evolved different L domains to exploit redundant cellular endocytic machineries to achieve virus budding and release.

The L domain-interacting cellular proteins identified to date are endocytic components associated with the formation of either early endosomes (e.g. ubiquitin ligase, AP-2) or late MVBs (e.g. Tsg101, AIP1). Although it is currently assumed that MVBs play a central role in retrovirus budding, it remains uncertain whether and how components of other endosomal vesicles, including early endosomes, contribute to retroviral budding. It is reported that HLTV-1 Gag polyproteins interact first with Nedd4.1 at the plasma membrane and then with Tsg101 in late endosomes/MVB, suggesting a successive assembly and budding process through the endocytic pathway (17, 18). However, neither early nor late endosomes are optimally evolved for viral assembly and release. For example, the formation of early endosomes is topologically opposite that of viral budding, whereas budding into late endosome is topologically correct but imposes subsequent obstacles to virion release from infected cells. Therefore, it remains to be determined whether and how retroviruses combine and/or integrate early and late endosomal machinery to facilitate virion assembly and budding. In the light of previous observations that the EIAV YPDL L domain interacts with both AP-2 and AIP1 in different model systems, the current study was designed to examine the specificity of EIAV p9 binding to early and late endocytic factors and the effects of alterations in early and late endocytic protein expression on virion production. The results of these studies demonstrated a previously unrecognized involvement of both early and late endocytic machinery in EIAV virion assembly and budding, whether accessed by YPDL or PTAP L domains.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and DNA Mutagenesis—Construction of CMVuk proviral EIAV and the CMVuk PTAP derivative was described previously (15). The plasmid encoding LexA-*p9 was constructed by inserting the *p9 fragment of a GST-*p9 construct (14) in the polylinker site of pLexA-(1-202)+PL (19). Full-length AIP1, AIP1372-869, or AIP1409-715 were PCR-amplified and inserted into the Gal4 activation domain expression vector pACTII (Clontech). Plasmids encoding GST-*p2b, GST-*p6, and GST-*p9 or mutant derivatives were described previously (14) and confirmed by DNA sequencing. The gene segment encoding AIP1 residues 409-715 was PCR-amplified and inserted in-frame into pET32c(+) (Novagen, San Diego, CA). Plasmids expressing Tsg1011-204 were obtained from Dr. Eric Freed (NIAID, National Institutes of Health, Frederick, MD) with the permission of Dr. Zijie Sun (Stanford University School of Medicine). Vectors expressing HA-tagged full-length and truncated AIP1/Alix proteins were generated by inserting PCR-amplified fragment into pCMV-HA (Clontech). pCDNA3.1 derivative expressing HA-tagged wild type µ2 was a gift from Dr. Alexander Sorkin (University of Colorado Health Science Center), and the mutant µ2DW was generated using standard molecular cloning protocols. pSilencer plasmid was purchased from Ambion (Austin, TX), and sequences of AIP1 (5'-gctcaagatggtgtgataa-3') or (5'-gatcaagcgcatggcattcat-3') were selected to generate pSilencer constructs that produce small hairpin RNA (shRNA), which was then spontaneously processed in vivo into siRNA.

Cell Culture and Transfection—COS-1 cells were cultured in minimum essential medium as described previously (20). HeLa and 293T cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum. GenePorter II (Gene Therapy Systems, San Diego, CA) or FuGENE 6 (Roche Diagnostics) was used to transfect cells according to the manufacturer's recommendation. SiRNA duplexes were synthesized in vitro and transfected into HeLa cells using GeneEraserTM (Stratagene, La Jolla, CA).

Yeast Two-hybrid Analysis—The Saccharomyces cerevisiae strain used for two-hybrid studies was CTY10.5d (Mata ade2-101 his3-{Delta}200leu2-{Delta}1 trp1-{Delta}901gal4 gal80 URA3::lexAop-lacZ). Yeast cells were grown in synthetic dextrose medium lacking appropriate supplements to maintain selection for plasmids. {beta}-Galactosidase activity was assayed on permeabilized cells grown to mid-log phase in the selective synthetic dextrose medium and expressed in Miller units. Yeast protein extracts for immunoblot analysis were prepared as described previously (21) and analyzed by immunoblotting with a monoclonal anti-HA antibody (Roche Diagnostics).

GST Pull-down Assays—GST fusion proteins and N-terminally His-tagged AIP1409-715 were expressed from Escherichia coli BL21(DE3)pLysS (Novagen, Madison, WI). Bacterial extracts were prepared in STE buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA) + 1% (v/v) Triton X-100, 5 mM dithiothreitol, and CompleteTM protease inhibitor mixture (Roche Diagnostics), as described previously (22). Extracts containing GST-*p2b, GST-*p6, and GST-*p9 or mutant derivatives were incubated with glutathione-Sepharose 4B beads (Amersham Biosciences) at 4 °C for 1 h. After six washes with 1 ml of buffer STE/1% Triton X-100, beads with bound proteins were incubated with bacterial extract (500 µl) containing His-tagged AIP1409-715 at 4 °C for 1 h. After six washes with STE/1% Triton X-100, beads were boiled in sample buffer, and proteins were separated by 10% polyacrylamide SDS-PAGE. Bound His-tagged AIP1409-715 was detected by Western analysis with an anti-His tag antibody and enhanced chemiluminescence with reagents from Amersham Biosciences.

Reverse Transcriptase (RT) and Western Blotting Assays—To measure virion production, culture medium collected from 24 to 48 h after transfection was clarified of cell debris by low speed centrifugation and then centrifuged at 20,800 x g for 3 h at 4°C to pellet virions. Virion pellets were resuspended in 50 µl of phosphate-buffered saline, and 10-µl samples were assayed in RT assays as described previously (20).

To examine EIAV-specific protein content in transfected cells and pelleted virions, total cell lysates prepared as described previously (20) and pelleted virions were resolved by electrophoresis through 4-15% gradient gels (Bio-Rad) and immunoblotted using a reference immune serum from a field-infected horse (20). Horseradish peroxidase-conjugated goat anti-horse IgG F(ab')2 (Jackson ImmunoResearch, West Grove, PA) was used as secondary antibody. The immunoblots were developed by incubation with SuperSignal West Pico chemiluminescent substrate (Pierce). Horseradish peroxidase-conjugated high affinity anti-HA antibody (Roche Applied Science) was used to specifically detect HA-tagged proteins.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Interaction between AIP1 and EIAV p9 Proteins—Interaction between AIP1 and EIAV p9 was previously demonstrated using yeast two-hybrid and GST pull-down assays (12, 13, 23). To serve as a starting point for a comprehensive comparison, we initially examined the p9-AIP1 interaction using similar approaches. We previously demonstrated interaction between the µ2 subunit of the AP-2 adaptor protein complex and EIAV p9 using GST pull-down assay (24). In that study, a recombinant protein containing only the N-terminal 30 amino acids of EIAV p9 was fused to GST, in contrast to the full-length p9 protein (51 residues) used in studies of p9-AIP1 binding (12, 13, 23). Therefore, we performed p9-AIP1 binding assays using the N terminus of p9 (designated as *p9) in this study. Fig. 1 summarizes the yeast two-hybrid results using *p9 as bait to examine binding activities of full-length and two truncated AIP1 proteins. Full-length AIP1 bound to *p9 as expected (Fig. 1A), and interaction of two truncated fragments (AIP1372-869 and AIP1409-715) with *p9 was also detected. The stronger interaction of full-length AIP1 was not due to higher expression of the fusion protein as shown by Western blot analysis (Fig. 1B). The observed binding properties are consistent with previous studies showing that both the N terminus (1-175) and the C-terminal proline-rich domain (717-869) of AIP1 are not required for this interaction (12, 13, 23). Additionally, the current studies further defined the p9 binding domain of AIP1 to residues 409-715. It is interesting to note that the EIAV p9 binding domain of AIP1 (residues 409-715) does not contain the PSAP motif (AIP1717-720) involved in interaction with Tsg101 (13).

To compare the binding properties of AIP1 proteins and the µ2 subunit in parallel, we performed GST pull-down assays using GST-*p9 and the AIP1409-715 construct. Our data revealed that similar levels of GST fusion protein were produced from each construct (Fig. 2A) and that *p9 evidently bound to the AIP1409-715. Single alanine substitutions in the EIAV L domain at residues Tyr-23, Pro-24, or Leu-26 abolished detectable AIP1409-715 binding, suggesting their critical role for AIP1 interaction, whereas mutation of D25A reduced AIP1409-715 binding by about 70% when compared with *p9. The D25A substitution in p9 has previously been reported to have no effect on EIAV viral budding (24). Our data confirmed that the YPDL sequence is a critical determinant of AIP1-p9 interaction, consistent with other reports (12, 13, 23). Interestingly, the YPDL sequence specificity for AIP1 binding reported here is similar to that for p9 binding to µ2 that we described previously (24). The *p6 protein (SRPEPTAPPEES) did not bind to AIP1409-715 as expected due to the absence of the p6 LYP motif responsible for AIP1 interaction in this construct. The observation that RSV *p2b failed to bind to AIP1409-715 further confirmed the specificity of AIP1 interaction. Taken together, these data demonstrated that interaction of EIAV p9 and AIP1 is dependent on the p9 YPDL sequence and on a small region (residues 409-715) of the AIP1 protein. Thus, it appeared that in vitro, both AIP1 and µ2 equally interact with EIAV p9 protein via the YPDL sequence, suggesting possible interactions with early and late endocytic proteins in infected cells.



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  		FIGURE 1.
Characterization of EIAV p9 interaction with AIP1 using the yeast two-hybrid system. The yeast strain CTY10-5d was used and fusion proteins were expressed from pLexA-(1-202)+PL and pACTII derivatives. A, GAD-AIP1 fusions contain the indicated AIP1 residues. Interaction of p9 with AIP1 and its truncated forms was reflected in {beta}-galactosidase activity (average of four transformants with standard deviation). B, Western analysis of protein extracts from transformants expressing LexA-p9 and the indicated GAD-AIP1 protein fusions, which were detected with anti-HA antibodies. The positions of the different protein fusion proteins are indicated by arrows.

 



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  		FIGURE 2.
In vitro binding of AIP1409-715 to EIAV p9 and mutant derivatives. A, GST fusions proteins bound to glutathione-Sepharose beads (one-half of the total) were separated by 10% SDS-PAGE and stained with Coomassie Blue. B, the remaining beads, loaded with GST-*p2b (SAPPPPYVGS), GST-*p6 (SRPEPTAPPEES), and GST-*p9 (SVPKNGKQGAQGRPQKQTFPIQQKSQHNKSVVQETPQTQNLYPDLSEIK) or mutant derivatives, were incubated with a bacterial extract containing His-tagged AIP1409-715. After washing the beads, bound proteins were boiled in sample buffer, separated by SDS-PAGE, and analyzed by immunoblotting with anti-His tag antibody. The lane marked Input contains 2% of the bacterial extract used for binding experiments. Protein markers are in kDa.

 
Effects of AIP1 Proteins on EIAV Budding—Previous studies of AIP1 interaction with the YPDL sequence of EIAV p9 protein were performed using Gag expression vectors and not with full-length EIAV proviral constructs. To examine the functional role of AIP1-p9 interactions in virion production in the context of complete proviral expression, we co-transfected COS-1 cells with an EIAV proviral construct and AIP1 expression vectors. As a specificity control, we also included an expression vector encoding the N-terminal moiety of Tsg1011-204 that contains the HIV-1 PTAP binding domain (25). As summarized in Fig. 3, EIAV production was reduced by 50-70% from cells expressing AIP1 proteins when compared with cells transfected with vector DNA (Fig. 3A). Various AIP1 proteins differed slightly in their inhibitory effect on EIAV budding, with the full-length AIP1 apparently the most potent inhibitor despite the relatively low levels of full-length AIP1 detected in transfected cells. Another interesting observation is that co-expression of Tsg1011-204 reproducibly enhanced EIAV production by an average of 30-40% when compared with the vector control. Overexpression of AIP1 proteins and Tsg1011-204 seemed not to significantly affect the steady state levels of EIAV-specific protein as shown in Fig. 3B (upper panel). Thus, these data demonstrated that EIAV budding mediated by the YPDL domain is specifically inhibited by AIP1 protein overexpression.

To further verify the specificity of the interactions observed above, we next examined the presence of HA-tagged AIP1 proteins in pelleted EIAV virions. Our data showed that all three AIP1 proteins as well as Tsg101 were detected in pelleted virions (Fig. 3B, lower panel). To exclude the possibility that overexpression of these endocytic proteins results in their appearance in the pelletable fraction of the culture medium, we transfected COS-1 cells with the various endocytic protein expression vectors in the absence of EIAV proviral DNA and centrifuged the culture medium under conditions used to pellet EIAV virions. No AIP1 or Tsg101 could be detected by immunoblotting in these control pelleted preparations in the absence of virion production (data not shown). Therefore, our results further confirmed specific incorporation of AIP1 into EIAV virions. The incorporation of Tsg101 into EIAV virions that we observed also suggested its involvement in EIAV budding, perhaps through indirect interactions mediated by AIP1-Tsg101 interaction (13).



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  		FIGURE 3.
Effect of overexpressed AIP1/Alix proteins on EIAV budding. COS-1 cells grown on 6-well plates were co-transfected with CMVuk proviral DNA and plasmids encoding for the indicated proteins at a 1:3 mass ratio with GenePorterII. Culture medium from 24 to 48 h after transfection was collected and centrifuged to pellet virions. A, RT activity of 10 µl of resuspended virions was assayed to quantify virion production. Data represent means ± ranges for duplicate samples from one of two independent experiments. B, at 48 h after transfection, cell lysates were prepared to examine cell-associated EIAV-specific viral proteins (upper panel) and expression of HA-tagged Tsg101 or AIP1 proteins (lower panel). C, resuspended virions were examined for content of the major viral structural protein p26 (upper) and of HA-tagged proteins (lower).

 
Effects of AIP1 and µ2 Expression on EIAV Budding—Results from the preceding experiments and previous reports have confirmed that both AIP1 and µ2 bind to the YPDL sequence of EIAV p9 protein in GST pull-down assays and that overexpression of AIP1 inhibits EIAV budding from transfected COS-1 cells. We next compared the roles of AIP1 and µ2 in EIAV budding using wild type EIAV (CMVukYPDL) and a mutant EIAV (CMVukPTAP) with its YPDL motif replaced by the HIV-1-derived PTAP motif. AIP1 and µ2 inhibited EIAV budding by 60 and 50%, respectively, as measured by pelleted virion-associated RT activity (Fig. 4A). The specificity of µ2 inhibition was further confirmed by the observation that overexpression of a mutant µ2(µ2DW) that is unable to bind to EIAV p9 protein had no significant effect on EIAV production (Fig. 4A). Thus, both AIP1 and µ2 contributed to EIAV budding.

Overexpression of Tsg1011-204 enhanced CMVuk(YPDL) budding by about 30% (Fig. 4A) as observed in Fig. 3A, and budding of CMVuk(PTAP) was inhibited about 55% by overexpressed Tsg1011-204 as expected (Fig. 4B). These two proviral constructs only differ from each other in the specificity of the L domain sequences, and both are previously demonstrated to be replication-competent (15). Our data indicated that Tsg101 is involved in both PTAP- and YPDL-mediated retrovirus budding but probably through different interaction mechanisms. Interestingly, expression of both wild type and mutant µ2 reduced CMVuk(PTAP) budding by about 80% (Fig. 4B), indicating that the AP-2 adaptor protein complex might also participate in PTAP-mediated budding, although direct interaction with the PTAP motif may not be required.

The involvement of both early and late endocytic proteins in retrovirus budding is also supported by the observation that these proteins could be co-pelleted with the EIAV virion (Fig. 4, C and D). It appeared that there was more Tsg1011-204 and AIP1409-715 incorporated into pelleted EIAV virions when compared with the levels of µ2 subunit. There are several possible explanations for the observed differences. Although all proteins were detected via their HA epitope tags, the positions of these tags differ (N-terminal for Tsg101 and AIP1, internal for µ2), possibly affecting the level of antibody reactivity of each protein to the anti-HA conjugate used in our immunoblots. If equal reactivity is assumed, different incorporation of these proteins might be the result of varied levels of these proteins expressed in the transfected cells (cell lysate data not shown). In particular, AP-2 expression is under such a tight regulation that loss of one subunit leads to complete depletion of AP-2 (26), and robust expression of AP-2 complex is difficult to achieve by overexpressing only one subunit of the complex. Taken together, our results demonstrated that both AIP1 and the µ2 subunit of AP-2 contribute to YPDL-mediated EIAV budding and that AP-2 also participates in PTAP-mediated budding.

Combined Effect of AIP1 and µ2 Overexpression on EIAV Budding—As both AIP1409-715 and µ2 overexpression inhibited EIAV budding, we sought to determine whether they function independently or in cooperation. For this purpose, we individually titrated the minimum amounts of AIP1409-715 or µ2 expression vector for detectable inhibitory effect (data not shown). Based on these assays, we cotransfected 293T cells using 0.8µg of µ2 and 0.2 µg of AIP1409-715, individually or in combination, whereas keeping the total amount of DNA constant with blank vector plus EIAV proviral DNA. Virions released into the culture medium during the period from 24 to 48 h after transfection were pelleted, and RT activity associated with the resuspended pellet was measured to quantify EIAV budding (Fig. 5). Co-expression of µ2 and AIP1409-715 inhibited EIAV budding more than either protein expressed individually (80% versus 40 and 50% inhibition, respectively), indicating an additive effect of AIP1 and µ2 and further supporting the concept that both AIP1 andµ2 contribute to EIAV budding. To test whether the combined effect was cell type-dependent, we also carried out the same experiment in COS-1 cells and found similar results (Fig. 5B). Incorporation of AIP1409-715 and µ2 was also detected in pelleted EIAV virions (Fig. 5C).



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  		FIGURE 4.
Effect of individually overexpressed endocytic proteins on EIAV budding. COS-1 cells grown on 6-well plates were transfected with CMVuk(YPDL) (A) or CMVuk PTAP proviral DNA (B) along with plasmids encoding for the indicated proteins at a 1:2 mass ratio using FuGENE 6. Culture medium from 24 to 48 h after transfection was collected. RT activity of pelleted virions was assayed to represent virion production. Data represent means ± ranges for duplicate samples from one of two independent experiments. Approximately equal amounts of resuspended virions normalized by RT activity were also subjected to Western blot to examine the content of HA-tagged endocytic proteins associated with the pelleted virions produced from CMVuk(YPDL) (C) or CMVuk(PTAP) (D) transfection.

 



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  		FIGURE 5.
Additive effect of overexpressed µ2 and AIP1409-712 proteins on EIAV budding. 293T (A) and COS-1 (B) cells grown on 6-well plates were co-transfected with 1 µgof CMVuk proviral DNA and a total of 1 µg of indicated plasmids using FuGENE 6. Culture medium from 24 to 48 h after transfection was collected to pellet virions. RT activity of 10 µlof resuspended virions was measured to represent virion production. Data represent means ± ranges for duplicate samples from one of three independent experiments. C, approximately equal amounts of resuspended virions produced from the transfected COS-1 cells normalized by RT activity were also examined for the content of HA-tagged proteins.

 
siRNA and shRNA Silencing of µ2 and AIP1 on EIAV Budding—It has been cautioned that overexpression of certain proteins might nonspecifically affect global cellular functions rather than specifically disturbing the precise cellular pathway under investigation. To complement our overexpression study on the roles of µ2 and AIP1 in EIAV budding, we examined the effects of µ2 and AIP1 down-regulation on EIAV budding. Fraile-Ramos et al. (26) previously tested specific siRNA sequences against the µ2 subunit of AP-2 and demonstrated an average 50% reduction in µ2 expression in HeLa cells at 48 h after transfection. Based on the same siRNA sequence, we generated a pSilencer-derived DNA construct that produces small hairpin RNA (shRNAµ2) upon transfection, which is spontaneously processed into siRNA in the transfected cells. The silencing effect of this construct on COS-1 cells cotransfected with HA-tagged µ2 was examined (Fig. 6A). Consistent with the results of Fraile-Ramos et al. (26), shRNAµ2 reduced µ2 expression by about 50% at 48 h after transfection. To knock down AIP1 expression, we cloned several sequences targeting various regions of AIP1 into the pSilencer vector and identified a specific sequence (5'-gctcaagatggtgtgataa-3') to achieve efficient silencing (Fig. 6B). At 48 h after co-transfection, expression of full-length AIP1 was reduced by about 80% and expression of AIP1409-715 was reduced by about 70%, respectively.

Our results revealed that 70-80% of AIP1 silencing could be achieved by shRNAAIP1, but only about 50% of µ2 silencing was observed using either siRNA or shRNA at 48 h after transfection. The long half-life of µ2 subunit (~24 h) (27) might partially account for the relative inefficiency of knockdown procedures. Therefore, to more rigorously suppress AP-2 expression, assays at later time points (72-96 h after transfection) or two consecutive transfections were used to achieve higher levels of µ2 silencing (28). We therefore designed our experiments to first transfect 293T cells with siRNA duplex at day 0, and at day 2, cells were split, reseeded, and incubated for ~12 h prior to transfection using the EIAV proviral DNA with or without pSilencer vectors encoding the specific shRNA. RT activity in culture medium collected from 24 to 48 h after the second transfection was quantitated as a measure of EIAV budding. In Fig. 6C, we first transfected 293T cells with increasing concentrations of µ2 siRNA and used only EIAV proviral DNA during the second transfection. Under these conditions, EIAV budding was inhibited by µ2 siRNA in a dose-dependent manner (Fig. 6C), i.e. no inhibition at 0 nM, 40% inhibition at 10 nM, and 60% inhibition at 25 nM µ2 siRNA. To assay the effect of the specific shRNA encoded by the pSilencers, we mock-transfected the 293T cells with the GeneEraserTM. Following splitting and reseeding of the cells, we transfected them with EIAV proviral DNA along with the shRNA constructs. Our results in Fig. 6D showed that EIAV budding was reduced by about 15-20% by shRNAµ2 and shRNAAIP1, respectively, indicating that the shRNAs we generated affect EIAV budding as expected. In another experiment (Fig. 6E), 293T cells were first transfected with 10 nM µ2 siRNA and then transfected with EIAV proviral DNA and pSilencer constructs at 60 h after the first transfection. Expression of shRNAµ2 further reduced EIAV production by about 50% and shRNAAIP1 by about 60%, respectively. The inhibitory effect was cumulative for µ2 and AIP1 down-regulation, further confirming that both µ2 and AIP1 are involved in EIAV budding.



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  		FIGURE 6.
Effect of siRNA and shRNA targeting µ2 and AIP1409-715 on EIAV budding. An siRNA sequence known to down-regulate µ2 expression (26) was used to generate pSilencer plasmid, denoted as shRNAµ2 here. A sequence (5'-gctcaagatggtgtgataa-3') targeting to 1765-1783 nt downstream of AIP1 start codon was selected to make shRNAAIP1 pSilencer constructs. Down-regulation of µ2(A) and AIP1 (B) was verified in COS-1 cells transfected with the indicated plasmids. Cell lysates were prepared at 48 h after transfection. Approximately equal amounts of cell lysates (normalized to {beta}-actin) were immunoblotted with anti-HA antibody to detect µ2 and AIP1 proteins (arrows), respectively. C, the effect of µ2 siRNA on EIAV budding. 293T cells were transfected with siRNA targeting µ2 at the indicated concentrations. At 48 h after siRNA transfection, cells were split and reseeded and then incubated for another 12 h before they were transfected with EIAV proviral DNA. Culture medium from 24 to 48 h after the second transfection was collected to pellet virions, and RT activity of 10 µl of resuspended virions was measured to represent virion production. D, to evaluate the effect of shRNA alone on EIAV budding, 293T cells were transfected with the indicated constructs along with the EIAV proviral DNA. Virion production was measured as in C to quantify budding efficiency. E, the effects of siRNA and shRNA on EIAV budding. The same protocol used in C was followed except that 293T cells were first transfected with 10 nM siRNA targeting µ2, and EIAV proviral DNA along with the indicated shRNA constructs were included during the second transfection. Data represent means ± ranges for duplicate samples from one of two independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It has become increasingly appreciated that retroviruses have evolved to adapt various host cellular machinery for their replication, and the number of identified cellular factors that interact with viral proteins is rapidly increasing (7, 29-31). Recently, cellular proteins that specifically bind to retrovirus L domains have been extensively characterized (9, 10, 23, 24), providing strong evidence to link retrovirus budding with vesicular trafficking. However, the detailed molecular and cellular mechanisms that underlie the adaptation of endocytic proteins by retroviruses to facilitate budding remain to be illustrated.

In the current study, we compared the functions of the µ2 subunit of AP-2 and AIP1 in EIAV budding using complementary approaches. Our results revealed that disruption of µ2 or AIP1 expression, either up-regulation by transient transfection or down-regulation by siRNA silencing, reduced EIAV budding from cells cotransfected with an EIAV proviral construct. Furthermore, the effects of µ2 and AIP1 were cumulative, if not synergistic, as overexpression or knockdown of both resulted in more potent inhibition than that observed for either individual protein. Interestingly, our data demonstrated that HIV-1 PTAP L domain-mediated budding was also inhibited when µ2 subunit was overexpressed in the same cells. Therefore, the current report has provided evidence for a contribution of the AP-2 adaptor protein complex in both PTAP- and YPDL-mediated budding, in addition to the previously reported role of AIP1 in this process.

The µ2 subunit of AP-2 is a critical component for the formation of early endosomes, and identification of its interaction with the EIAV L domain suggested the involvement of the early endocytic pathway in retrovirus budding (24). However, the precise role of early endocytic proteins in viral budding has been controversial as the topology of endocytosis is opposite to that of retrovirus budding. The past few years have seen a collection of evidence that AIP1, a cellular component involved in late endosome/MVB formation, interacts with the EIAV YPDL L domain and an LYP motif located downstream of the PTAP motif in HIV p6 (12, 13, 23). Along with the identification of Tsg101 (a component of the ESCRT I complex essential for MVB formation) as the binding partner of HIV PTAP motif, these results suggest that retroviruses can recruit late endocytic machinery for budding as both processes share the same topology. However, it remains unclear whether and how retroviruses bud into late endosomes before they exit host cells. The current results that both µ2 and AIP1 contribute to YPDL- and PTAP-mediated budding strongly indicated that retrovirus might actually combine both early and late endosomal machinery to facilitate budding. Consistent with this model, MLV Gag polyproteins have been shown to traffic between the plasma membrane and the late endosomes, although the functional significance of this trafficking remains to be defined (17). Here we have reported that PTAP-mediated budding is also sensitive to µ2 overexpression and that both wild type and mutant µ2 had similar effects on budding. These data indicated that the AP-2 adaptor protein complex might contribute to retrovirus budding via a general mechanism that remains to be defined. It is also worth noting that when the µ2 subunit was depleted using high concentrations of siRNA oligonucleotides (25 nM), no further reduction in EIAV budding was observed upon introduction of a second shRNA (shRNAµ2 or shRNAAIP1) by transfection (data not shown). These observations indicated that adaptation of µ2 and AIP1 might be sequential such that complete blockade of one interaction abolishes the entire chain. Further characterizations of the apparently complex interactions between the Gag proteins of different retroviruses and endocytic proteins involved in assembly and budding can provide further insights into the exact role of the endocytic machinery in Gag trafficking, assembly, and budding. In addition, the characterization of these critical Gag-endocytic protein interactions can provide new targets for the development of antiviral drugs.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant 2RO1 CA49296 (to R. C. M.), National Institutes of Health postdoctoral training Grant T32 AI49820 (to C. C.), and Comision Interministerial de Ciencia y Tecnología Grant BIO2002-00803 (to O. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by the Ramón y Cajal Program (Ministerio de Ciencia y Tecnología, Spain). Back

2 To whom correspondence should be addressed: Dept. of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, W1144 Biomedical Science Tower, Pittsburgh, PA 15261. Tel.: 412-648-8869; Fax: 412-383-8859; E-mail: rmont{at}pitt.edu.

2 The abbreviations used are: EIAV, equine infectious anemia virus; HIV-1, human immunodeficiency virus 1; AP-2, activator protein-2; AIP1, ALG-2-interacting-protein 1; Alix, ALG-2-interacting-protein x; MVB, multivesicular body; GST, glutathione S-transferase; HA, hemagglutinin; siRNA, small interfering RNA; shRNA, small hairpin RNA.; RT, reverse transcription Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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