Journal of Virology, December 1998, p. 10218-10221, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Equine Infectious Anemia Virus Gag Polyprotein Late
Domain Specifically Recruits Cellular AP-2 Adapter Protein
Complexes during Virion Assembly
Bridget A.
Puffer,1
S. C.
Watkins,2 and
Ronald C.
Montelaro1,*
Department of Molecular Genetics and
Biochemistry1 and
Department of Cell
Biology and Physiology,2 University of
Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
Received 16 June 1998/Accepted 21 August 1998
 |
ABSTRACT |
We have identified an interaction between the equine infectious
anemia virus (EIAV) late assembly domain and the cellular AP-2
clathrin-associated adapter protein complex. A YXXL motif within the
EIAV Gag late assembly domain was previously characterized as a
sequence critical for release of assembling virions. We now show that
this YXXL sequence interacts in vitro with the AP-50 subunit of the
AP-2 complex, while the functionally interchangeable late assembly
domains carried by the Rous sarcoma virus p2b protein and human
immunodeficiency virus type 1 p6 protein, which utilize PPPY and PTAPP
L domains, respectively, do not bind AP-50 in vitro. In addition, EIAV
late domain mutants containing mutations that have previously been
shown to abrogate budding also exhibit marked decreases in AP-50
binding efficiencies. A role for AP-2 complex in viral assembly is
supported by immunofluorescence analysis of EIAV-infected equine dermal
cells demonstrating specific colocalization of the 
adaptin subunit
of AP-2 with the EIAV p9 protein at sites of virus budding on the
plasma membrane. These data provide strong evidence that EIAV utilizes
the cellular AP-2 complex to accomplish virion assembly and release.
| |
TEXT |
The assembly of retroviral
particles is driven by the Gag polyprotein. Gag
polyproteins are synthesized in the cytoplasm from genome-length mRNA and are targeted to the plasma membrane, where approximately 2,000 associate to form immature budding particles (21). Maturation of the virion to an infectious particle
occurs at some point either late in budding or immediately after
release of the virus particle when the Gag polyproteins are
processed by the virus-encoded protease. The equine infectious anemia
virus (EIAV) Gag polyprotein is processed to generate the
matrix (MA; p15), capsid (CA; p26), nucleocapsid (NC; p11), and p9
proteins (8, 11). The p9 sequence at the C terminus of Gag
has been shown to function at a point late in virus assembly and is
critical for release of assembled virus particles (14, 16).
The human immunodeficiency virus type 1 (HIV-1) p6, Rous sarcoma virus
(RSV) p2b, and EIAV p9 proteins were identified to be functional
homologs by construction of chimeric Gag polyproteins and
analysis of particle assembly (14, 16). While HIV-1 p6, RSV
p2b and EIAV p9 are functionally interchangeable, they share
little amino acid sequence similarity. It has been determined
that a PTAP sequence contained in HIV-1 p6 and conserved among all
lentiviruses, with the exception of EIAV, was critical for late domain
function (4, 7, 9). This sequence is suggestive of PXXP
binding motifs which have been shown to interact with the SH3 domain
protein structural module (15). Mutational analysis of RSV
p2b and, recently, pp16 of Mason-Pfizer monkey virus identified a PPPY
sequence, also highly conserved among the oncoviruses, as being
critical for late domain function (22-25). This motif has
been shown to interact with a semiconserved structural module referred
to as a WW domain (3, 19). The interaction was verified in
vitro; however, the "natural partner" in vivo remains to be defined
(6).
Alanine scanning mutagenesis of the EIAV late assembly (L)
domain identified p9 amino acids Y23,
P24, and L26 (YPXL) as critical for a
functional late assembly domain, suggesting a YXXL motif
(16). YXXL sequences in other proteins have been shown
to bind the medium chain subunits of the AP-1 and AP-2
clathrin-associated adapter protein complexes by the yeast two-hybrid
assay, and this interaction has been characterized in vitro (13,
20). Based upon this observation, we decided to examine possible
interactions between retroviral late assembly domains and the medium
chain (AP-50 subunit) of the plasma membrane-localized AP-2
clathrin-associated adapter protein complex. Glutathione
S-transferase (GST) and a GST-p9 fusion protein
(encoding the first 30 residues of p9) were purified and analyzed for
binding to a truncated AP-50 subunit (residues 121 to 435) in a
standard binding assay (13). Figure 1 shows that while GST alone did not bind
to AP-50 (Fig. 1, lane 1), the GST-p9 protein was able to bind
and precipitate AP-50 (Fig. 1, lane 2). We also tested the
PTAPP sequence of HIV-1 (in p6) and the PPPPY sequence of RSV
(in p2b) for binding to AP-50. The GST-p6 and GST-p2b proteins both
failed to bind and precipitate AP-50 (Fig. 1, lanes 3 and 4, respectively). Thus, in vitro the EIAV YXXL sequence critical for
release of budding virions does interact with the AP-50 subunit of
AP-2, and this binding is specific for the EIAV p9 late domain.
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FIG. 1.
Analysis of AP-50 binding to retroviral late domains in
the context of GST fusion proteins. Truncated AP-50 was synthesized
from the 3M9 cDNA in vitro by using the TNT Quick Coupled Master mix
reticulocyte lysate with [35S]methionine. Binding to 10 µg of an eluted GST fusion protein was initiated in 600 µl of
binding buffer (0.05% Triton X-100, 50 mM HEPES [pH 7.3], 10%
glycerol, 0.1% bovine serum albumin), and protein complexes were
precipitated with glutathione-Sepharose beads, washed twice with
binding buffer, washed once with binding buffer supplemented with 100 mM NaCl, resolved by sodium dodecyl sulfate-12% polyacrylamide gel
electrophoresis, and detected by autoradiography.
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|
To further test our hypothesis that the EIAV L domain interacts with
AP-50 to facilitate virion assembly, we constructed EIAV L domain GST
fusion protein analogs containing late domain alanine scanning
mutations that were shown to be functionally defective in a COS-1
cellular budding assay (16). GST-p9 fusion proteins containing substitution of residue Y23, P24, or
L26 with alanine were tested with the in vitro AP-50
binding assay, and the resultant bands were quantitated by densitometry
and normalized to the value obtained for GST-p9. We observed that
alanine substitution for either Y23, P24, or
L26 resulted in a decrease in the ability to bind AP-50
(Fig. 2, lanes 3, 4, and 5, respectively)
when compared to GST-p9 binding (Fig. 2, lane 2), reflecting the
activity levels obtained in a COS-1 cell budding assay (16).
Quantitation of AP-50 bound by GST-p9 analogs demonstrated an average
75% decrease in AP-50 binding compared to the level observed for the
GST-p9 wild-type sequence (Fig. 2B), in spite of the high
concentrations of protein contained in the reaction mixtures.
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FIG. 2.
Analysis of AP-50 binding by EIAV late assembly domain
wild-type and mutant sequences. (A) Binding activity was determined as
described in the legend to Fig. 1. (B) Quantitation of binding
efficiencies relative to that of the native p9 late domain sequence.
|
|
Having identified an in vitro interaction between AP-50 and EIAV p9, we
next used immunofluorescence analysis to determine whether these
proteins colocalize in EIAV-infected cells. To determine if the AP-2
complex associates with EIAV p9, we obtained a commercially available
monoclonal antibody directed against the adaptin subunit, a
component of adapter protein complexes that is specific for the plasma
membrane-localized complex AP-2 (1, 17, 18). Infected and
uninfected equine dermal (ED) cells were fixed, permeabilized, labeled
with anti- adaptin and anti-EIAV p9, and examined by fluorescence
microscopy. Examination of uninfected ED cells for adaptin
demonstrated a diffuse staining throughout the cell, typical of adaptin staining (Fig. 3E) (1, 17,
18). Visualization of adaptin staining in EIAV-infected cells
(Fig. 3B) revealed normal diffuse staining, in addition to specific
sites of concentrated adaptin staining at the plasma membrane of the
cell and intense apparently intracellular staining corresponding to a
perinuclear localization (Fig. 3B). Visualization of the anti-p9
staining in uninfected cells demonstrated the absence of background
antibody reactivity (Fig. 3D). In marked contrast to uninfected cells, infected ED cells showed intense p9 staining of virions at the plasma
membrane, as well as an apparently perinuclear staining (Fig. 3A). To
identify sites of colocalized adaptin and p9, the Cy3 and fluorescein
isothiocyanate staining patterns were merged (Fig. 3C). Importantly,
the pattern of dual staining (in orange) indicated specific
concentrations of EIAV p9 and adaptin at apparent sites of virus
budding. Examination of cells producing lower levels of viral protein
also indicated colocalization of adaptin and p9 at cellular membranes
(Fig. 3F [see inset]); however, these cells showed more typical
diffuse adaptin staining and did not have intense perinuclear
staining, as observed above. These results demonstrated that adaptin, and thus the AP-2 complex, specifically colocalizes with sites
of virus assembly in ED cells.
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FIG. 3.
Immunofluorescence colocalization of EIAV p9 and AP-2.
EIAV-infected (A, B, C, and F) and uninfected (D and E) ED cells were
grown on glass coverslips, fixed, permeabilized, and stained for EIAV
p9 (A and D) and adaptin (B and E). EIAV p9 and adaptin were
detected by indirect immunofluorescence microscopy with a rabbit
polyclonal p9 antiserum with Cy3-conjugated antirabbit secondary
antibody and monoclonal mouse anti- adaptin with fluorescein
isothiocyanate-conjugated antimouse antibody. Nuclei (blue) were
stained with Hoechst stain (D, E, and F). The p9 (red) and adaptin
(green) colocalization (orange) is shown in panels C and F. Bar, 10 µm.
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|
YXXL sequences in other lentiviral proteins have been identified as
also functioning in replication. The simian immunodeficiency virus
transmembrane protein contains a YXX
sequence critical for the
regulation of surface envelope protein levels (10). Specific
interactions between the HIV-1 and simian immunodeficiency virus
envelope proteins and adapter protein medium chains have also recently
been demonstrated (2, 12). Examination of the EIAV
transmembrane protein and accessory gene sequences fails to identify
YXX motifs; thus, we believe that the demonstrated colocalization of
EIAV p9 and AP-2 is the result of the p9 gene-encoded YXXL sequence.
We report here for the first time the characterization of an
interaction between the late assembly domain of EIAV p9 and the cellular clathrin-associated adapter protein complex AP-2. We used an
in vitro AP-50 binding assay to demonstrate for the first time a
specific interaction between that subunit of the AP-2 complex and
the EIAV late assembly domain YXXL motif. While the EIAV p9 late
assembly domain bound to AP-50, the functionally homologous late
domains of HIV-1 and RSV that utilize PTAPP and PPPPY motifs, respectively, did not bind AP-50 in this in vitro assay. We also demonstrated that proteins containing L domain mutations
previously shown to abrogate function in a COS-1 cell budding
assay were also defective in AP-50 binding. Finally, we demonstrated
colocalization of AP-2 complexes with EIAV p9 at sites of virus
assembly within the cell, suggesting a role for AP-2 complex in virion
assembly and release.
The mechanisms through which retroviral late domains facilitate budding
remain to be determined. The identification here of the involvement of
clathrin-associated adapter proteins indicates that endocytosis
machinery may be necessary for a step in virus assembly, possibly by
recruiting other cellular proteins to the site of assembly to
facilitate a membrane fission event, or by interacting with the Gag
polyprotein and membrane phospholipids to facilitate assembly
of the immature virus particle in a manner similar to the
native function of adapter protein complexes in clathrin-coated pit
formation (5). The cellular partners of the PTAP- and
PPPY-type late domains remain to be identified. We have shown here that
despite the interchangeable nature of the YXXL and PTAP or PPPY late
domains to facilitate Gag budding in vitro, the PTAP and PPPY domains
failed to bind with the AP-50, in contrast to the EIAV YXXL late
domain. This difference indicates that different retroviruses may
utilize different sites in cellular processing pathways to accomplish a
common assembly step in virus replication in their respective host
cells. For example, the various late domains may interact either with
different subunits of a particular complex or with different proteins
involved in the same cellular pathway. Alternatively, the cellular
protein partners of late domains could be involved in entirely
different cellular pathways as dictated by their host cell. While
further experiments are necessary to elucidate the cellular machinery
involved in late assembly and release of retroviral particles, the
results of this study define a cellular process that has been adopted by EIAV and possibly other retroviruses to facilitate a critical step
during virus assembly.
| |
ACKNOWLEDGMENTS |
We thank John Wills and Laurence Garnier for helpful conversations
and assistance in preparing the manuscript and for providing the
wild-type L domain GST fusion proteins. We acknowledge Markus Thali for
assistance with AP-50 binding experiments and Juan Bonifacino for
kindly providing the 3M2 and 3M9 cDNAs for AP-50. Finally, we recognize
John Gibbs, Sean Alber, and Ciprian Almonte for assistance with
immunofluorescence staining.
This work was supported by National Institutes of Health grant
5R01CA49296 (R.C.M.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Genetics and Biochemistry, University of Pittsburgh School of
Medicine, W1144 Biomedical Science Tower, Pittsburgh, PA 15261. Phone:
(412) 648-8869. Fax: (412) 383-8859. E-mail:
rmont{at}pop.pitt.edu.
| |
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Journal of Virology, December 1998, p. 10218-10221, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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