Previous Article | Next Article 
Journal of Virology, March 1999, p. 2045-2051, Vol. 73, No. 3
Department of Microbiology and Immunology,
Pennsylvania State University College of Medicine, Hershey,
Pennsylvania 17033
Received 22 July 1998/Accepted 2 December 1998
Rous sarcoma virus (RSV) and murine leukemia virus (MLV) are
examples of distantly related retroviruses that normally do not encounter one another in nature. Their Gag proteins direct particle assembly at the plasma membrane but possess very little sequence similarity. As expected, coexpression of these two Gag proteins did not
result in particles that contain both. However, when the N-terminal
membrane-binding domain of each molecule was replaced with that of the
Src oncoprotein, which is also targeted to the cytoplasmic face of the
plasma membrane, efficient copackaging was observed in genetic
complementation and coimmunoprecipitation assays. We hypothesize that
the RSV and MLV Gag proteins normally use distinct locations on the
plasma membrane for particle assembly but otherwise have assembly
domains that are sufficiently similar in function (but not sequence) to
allow heterologous interactions when these proteins are redirected to a
common membrane location.
Many eukaryotic proteins are
synthesized on cytosolic ribosomes and subsequently targeted to the
cytoplasmic faces of very different membranes. Two examples include
NADH cytochrome b5 reductase, which is involved
in lipid biosynthesis on the cytoplasmic face of the endoplasmic
reticulum, and the Src protein, which is involved in signal
transduction events on the inner face of the plasma membrane. Membrane
targeting is thought to involve the N-terminal membrane-binding domains
of such proteins (3, 6, 14, 15, 18-20, 22, 25, 26, 31, 35),
but the sorting mechanism by which specific membranes are recognized is
not understood. Furthermore, little is known about the number of
distinct compartments that exist on the cytoplasmic faces of each of
the various membranes of the eukaryotic cell.
The Gag proteins of retroviruses also exhibit specific membrane
targeting following their synthesis on free ribosomes in the cytosolic
compartments of the cell (36). Most infectious retroviruses, such as human immunodeficiency virus (HIV), murine leukemia virus (MLV), and Rous sarcoma virus (RSV) (an avian retrovirus), express Gag
proteins that mediate budding from the cytoplasmic face of the plasma
membrane; however, certain endogenous viruses bud into the lumen of the
endoplasmic reticulum to produce what are known as intracisternal type
A particles. Specific membrane targeting (and budding) does not require
any virus-encoded product other than the Gag protein. That is, the
glycoproteins (env gene products), the reverse transcriptase
and integrase activities (pol gene products), and the viral
RNA genome are all dispensable for budding from the appropriate
membrane. Although the Env proteins of some retroviruses appear to
direct budding to the basolateral membrane in polarized cells (reviewed
in reference 36), even without Env the Gag proteins of these viruses are still directed to the plasma membrane.
Individual N-terminal membrane-binding domains from Gag proteins are
capable of directing heterologous proteins to the appropriate membrane
(37, 44, 45), but the mechanism(s) by which this happens is
not known. It seems likely that Gag proteins would take advantage of
cellular mechanisms normally utilized to sort host proteins to
different membranes (e.g., those for NADH cytochrome b5 reductase and Src), but, as mentioned above,
these events are poorly understood. Likewise, it is unclear whether Gag
proteins that are targeted to the same membrane (e.g., those of HIV,
MLV, and RSV) assemble into particles at molecularly distinct locations on that membrane.
In the experiments described below, we have examined the possibility
that Gag proteins from two distantly related retroviruses Briefly, assembly domains provide the functions of Gag required for
budding (see Fig. 1A) (reviewed in references 5, 9, and 36). The M (membrane-binding) domain is located
at the N terminus and provides the information needed for
binding/targeting to the plasma membrane. M mutants have very tight
blocks to budding. The I (interaction) domains are located in the NC
sequence and provide the major region of interaction, during which
~1,500 Gag molecules come together to form a particle. I mutants
release low-density particles, if any are produced at all. The L (late) domain is needed just prior to release, as the particle separates from
cell. L can be located at different positions in different Gag proteins
and can function in a positionally independent manner. L mutants
accumulate tethered particles on the membrane, and the few that are
released have normal density.
The ability to exchange assembly domains among distantly related Gag
proteins suggests that their functions may be conserved while their
amino acid sequences are not. Thus, it was difficult to know what to
expect from our attempts to copackage the RSV and MLV Gag proteins. To
our surprise, we found that these two Gag proteins could be copackaged
during budding, but only when the same membrane-binding domain (that of
the Src protein) was placed at their N termini. This provides
unprecedented evidence that distinct membrane-binding sites may exist
for different Gag proteins.
Parental gag genes.
The wild-type RSV
gag gene is from pATV-8, an infectious clone of the RSV
Prague C genome (34). The wild-type MLV gag and pol genes are from pRR88, an infectious clone of the Moloney
murine leukemia virus (MMLV) genome in plasmid pGCcos3neo
(12) (kindly provided by Alan Rein, Frederick Cancer
Research and Development Center). All of the expression plasmids used
in this study have been described previously and are briefly explained
below. They were propagated in Escherichia coli DH-1 by
using medium supplemented with 25 µg of ampicillin per ml.
RSV Gag mutants.
Several derivatives of the RSV Gag protein
were used (Fig. 1A). The wild-type
protein was expressed by pSV.Myr0 (41, 42). A myristylated
form of this protein containing the first 10 residues of the Src
oncoprotein was expressed by pSV.Myr1 (41, 42) and is here
given the name R.M1. A protease-minus form of this chimera, here named
R.M1.Pr
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Conditions for Copackaging Rous Sarcoma Virus and
Murine Leukemia Virus Gag Proteins during Retroviral Budding
and
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
RSV and
MLVmight interact during particle assembly and budding. Although these viral proteins direct assembly events that are morphogenetically and kinetically similar, they possess little sequence similarity. Only
72 of the 538 residues of MLV Gag (13%) can be scored as identical to
RSV Gag after optimizing the alignment of the sequences (data not
shown). Many of these residues are widely scattered, and most of the
similarity resides in the C-terminal halves of the molecules. That part
of Gag contains the major homology region of the capsid sequence and
the zinc finger motifs
(Cys-X2-Cys-X4-His-X4-Cys) of the
nucleocapsid (NC) sequence (36). However, even in this most
conserved portion of Gag, only 24% of the residues are identical in
the optimized alignment. No sequence similarity is found in the
N-terminal membrane-binding domains of the RSV and MLV Gag proteins.
Indeed, it is well established that MLV Gag is dependent on the
addition of myristate whereas RSV Gag is not myristylated at all
(7, 21, 30). For all of these reasons, it seemed unlikely
that the RSV and MLV Gag proteins would be capable of interacting
during the assembly process. On the other hand, a variety of
experiments have shown that the assembly domains of the RSV Gag protein
(named M, L, and I) can be replaced by those from other distantly
related Gag proteins, such as that of HIV (1, 9, 23, 24).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
, was expressed by pSV.Myr1.D37S (4). An
unmyristylated form of R.M1, the result of changing the second amino
acid from Gly to Glu (2), was produced by pSV.Myr1() and
is here referred to as R.M(). The product of pSV.Bg-Bs (38, 39) is a C-terminally truncated form of R.M1 lacking the I
domains, and the product of pSV.T-10C (23, 40) has an
internal deletion that removes the L domain.
View larger version (29K):
[in a new window]
FIG. 1.
Derivatives of the RSV and MLV Gag proteins. RSV
sequences are represented by open boxes, and those of MLV are
represented by hatched boxes. The restriction sites used to create the
chimeras are noted above each sequence. Myristylation is indicated by a
squiggly line, and M( ) indicates that the glycine encoded by the
second codon is replaced, eliminating the site of myristic acid
addition. Numbers below the Gag proteins refer to amino acids counted
from the N terminus of each Gag protein and mark positions cleaved by
the viral protease. (A) Derivatives of the RSV Gag protein. The
wild-type molecule is illustrated at the top, where the positions of
domains essential for budding are indicated with black bars (M, L, and
I). Gag derivatives in which the first 10 residues are replaced with
those of the Src oncoprotein are also depicted. The location of the
inactivating amino acid substitution in the RSV protease, D37S, is
noted. (B) Derivatives of the MLV Gag protein. The wild-type protein is
illustrated at the top, and versions in which the first 39 amino acids
are replaced with the first 10 residues of the Src protein are also
shown. The site of in-frame suppression ("is") of the stop codon
between the gag and pol genes is indicated. (C)
RSV-MLV Gag chimeras. The RSV BglII site was destroyed,
while the MLV XhoI site was restored during construction of
the chimeric gene.
MLV Gag mutants.
The wild-type MLV Gag protein (Fig. 1B) was
expressed by two different plasmids. One of these, pSV.MLV, also
produces a nearly full-length (lacking the last 168 of the 1,737 residues) protease-plus form of Gag-Pol, whereas the other,
pSV.MLV.Pr, produces a protease-minus form (2). We also
expressed chimeras of MLV Gag in which the first 39 residues are
replaced with the first 10 residues of Src. The Src chimera produced by
pSV.M.M1 also expresses the MLV protease, whereas that produced by
pSV.M.M1.Pr does not (2). A myristate-minus (G2E) form of
the Src-MLV chimera was expressed by pSV.M.M() (2).
Chimeric RSV-MLV gag genes.
pSV.BgM (Fig. 1C)
contains a chimeric gag gene in which the first half of the
myr1 allele of RSV gag is linked to the second half of the wild-type MLV gag gene (within their
capsid-coding sequences) and has been described previously
(2). Plasmids that express a myristate-minus form of this
chimera, pSV.BgM.M(), or an internally deleted chimera,
pSV.T10M.Pr, have also been described previously (2).
Expression and analysis of Gag proteins. COS-1 cells were transfected by the DEAE-dextran-chloroquine method, as described previously (41). The plasmid DNAs were digested with XbaI and ligated at a concentration of 25 µg/ml before transfection. This removes the bacterial plasmid sequence and joins the 3' end of the gag or pol genes with the simian virus 40 late polyadenylation signal (41).
Cells contained in 35-mm plates were labeled at 48 h after transfection for 2.5 h in a manner similar to that described previously (41). Because the MLV Gag protein contains very few methionine residues, labeling was performed with 150 µCi of L-[4,5-3H(N)]leucine. The cells and growth medium from each labeled culture were mixed with standard radioimmunoprecipitation assay lysis buffer (final concentration, 25 mM Tris-HCl [pH 8.0], 0.15 M NaCl, 0.1% sodium dodecyl sulfate [SDS], 1% Triton X-100, 1% deoxycholate) containing protease inhibitors, and the Gag proteins were immunoprecipitated for 1 h on ice with polyclonal rabbit serum against whole RSV (38) or polyclonal goat serum against MLV CA (kindly provided by Alan Rein). Immunoprecipitated proteins were separated by electrophoresis in SDS-10% polyacrylamide gels which were then fixed and dried. The radiolabeled proteins were detected by fluorography with Fluoro-Hance (Research Products International, Inc.) and Kodak X-Omat AR5 film at80°C. Overnight exposures were typically required.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
The overall purpose of our experiments was to determine whether the Gag proteins of RSV and MLV could be copackaged into viral particles when coexpressed in simian (COS-1) cells. The properties of the individual Gag derivatives used in our experiments are described below and are followed by the results of the two types of coexpression experiments used to investigate copackaging.
Expression of RSV Gag derivatives. To study the process of particle assembly mediated by the RSV Gag protein, we have been using a simian virus 40-based vector that expresses Gag in the absence of all other viral proteins (41). The wild-type protein, Pr76 (Fig. 1A), is not myristylated and therefore has been referred to as Myr0 when expressed alone in mammalian cells. Myr0 expression in COS-1 cells results in the rapid release of membrane-enclosed, virus-like particles into the culture medium (Fig. 2, lanes 1). These are similar to authentic RSV particles with regard to their rate of release from the cell, their morphology in electron micrographs, and their density in isopycnic sucrose gradients (16, 38, 41). Moreover, during or immediately after particle release, the viral protease cleaves the Myr0 protein into products that are indistinguishable from the mature products of RSV, with the most prominent protein being CA. Proteolytic processing is not a prerequisite for budding, and many of the experiments described below make use of protease (PR)-minus derivatives.
|
) (Fig. 1A)] blocked particle assembly and
reduced the level of cell-associated processing by the viral PR (Fig.
2, lanes 3). This demonstrates that R.M1 is dependent on the
myristylated Src sequence and demonstrates the importance of the
extreme N terminus of RSV Gag for particle assembly and budding (i.e.,
the M domain has been inactivated by substitution of the Src sequence
[21]). The reduction in cell-associated Gag processing
observed for the myristate-minus derivative is indicative of the
requirement of membrane binding for PR activity and provides an
indirect assay for the targeting of Gag proteins to the plasma membrane
(21, 42).
Expression of MLV Gag derivatives. To investigate MLV assembly in COS-1 cells, we utilized pSV.MLV, which expresses the gag and pol genes of MMLV (Fig. 1B). The products of this vector are the MLV Gag protein (Pr65) and a larger fusion protein (Gag-Pol) that arises from in-frame suppression of a UAG codon at a frequency of 5% (29). Digestion with XbaI before transfection (see Materials and Methods) leaves the PR and reverse transcriptase (RT) coding sequences intact, but only the first 239 codons of integrase (IN) remain. Upon transfection, MLV Gag proteins were synthesized and released into the medium in particles that predominantly contained Gag cleavage products (e.g., CA), but noticeable levels of uncleaved Gag molecules were consistently observed (Fig. 2, lanes 4).
A Src-MLV chimera, M.M1, was constructed by replacing the first 39 amino acids of Gag with the first 10 residues of the Src protein (Fig. 1B). This chimera was detectably smaller than the full-length MLV molecule but was released and processed in a manner identical to the authentic MLV protein (Fig. 2, lanes 5). Because deletions within the first 40 residues of wild-type MLV Gag have been shown to disrupt the budding process (13), it is clear that the heterologous membrane-binding domain from Src can substitute for this M domain in a manner similar to that of the RSV Gag protein. The particles made by the wild type and the Src chimera of MLV Gag appeared to be normal in every way that we measured. Both contained normal levels of RT activity relative to the authentic virus (data not shown). Moreover, sucrose density gradients showed that the derivatives are released into the medium only within particles of a density similar (MLV, 1.18 g/ml) or identical (M.M1, 1.16 g/ml) to authentic retrovirions (data not shown). The significance of the minor shift in density observed with the wild-type MLV construct is unknown, but similar shifts have been reported for particles made by expressing the RSV Gag protein in mammalian cells (42). When the site of myristic acid addition was destroyed by changing Gly to Glu at the second residue of the Src-MLV chimera, the resulting protein [M.M() (Fig. 1B)] was not released into the medium (Fig. 2,
lanes 6). Therefore, the Src chimera of MLV, like its RSV counterpart,
is dependent on the foreign membrane-binding domain for particle
assembly at the plasma membrane. In addition, intracellular processing
was reduced for the myristate-minus chimera, indicating that membrane
binding is required for PR activity in MLV. Indeed, this mutant behaves
identically to the unmyristylated mutant of MMLV Gag originally
described by Rein et al. (30), which was unable to bind to
membranes or assemble virions and demonstrated no proteolytic activity.
RSV-MLV Gag chimeras.
We also made use of an RSV-MLV chimera
named BgM in which the I domains of RSV are replaced with that of MLV
(Fig. 1C). We have previously shown that this chimera produces
particles at the wild-type rate and density (2). In
contrast, the same N-terminal sequence from RSV without the MLV
sequence (mutant Bg-Bs [Fig. 1A]) is released poorly and the
particles have a very low density (1, 39). The MLV protease
is capable of cleaving BgM to release the chimeric capsid sequence,
p34CA, and this product is not observed when MLV PR is
mutated (2). Moreover, both the uncleaved Gag protein and
p34CA were reactive to RSV and MLV antisera (not shown).
When the site of myristylation was destroyed [BgM() Fig. 1C], the
chimera was unable to produce particles and the level of
cell-associated proteolytic processing was reduced (data not shown), as
was the case for the myristate-minus Src derivatives of the RSV and MLV
Gag proteins.
(Fig. 1C). It lacks
RSV residues 122 to 336, which contain the L domain needed for
efficient release of particles (Fig. 1A). Although this chimera has the
membrane-binding domain from Src, it exhibits a severe block to
budding. The magnitude of this defect is similar to that of RSV mutant
T10C.Pr (23, 40), which contains the very same deletion
(Fig. 3A; compare lanes 3 and 4). A
derivative of T10M that expresses the functional MLV protease was also
unable to produce particles (not shown), as would be expected for a
mutant lacking L domain activity. Because BgM is dependent on the L
domain of RSV, we conclude that the C-terminal sequence from MLV Gag must not possess L domain activity.
|
Genetic evidence for copackaging of heterologous Gag proteins.
We have previously shown that L domain mutants of RSV are associated
with membranes and can be readily rescued into particles when
coexpressed with assembly-competent RSV Gag proteins (40). An example of this is shown in Fig. 3A, where it is clear that T10C.Pr is not released when expressed alone but is easily detected in the medium when coexpressed with R.M1.Pr (compare lanes 3 and 5).
The high efficiency of this complementation experiment allowed us to
investigate the ability of heterologous Gag proteins to rescue L domain
mutants. To our surprise, RSV mutant T10C.Pr was readily released
into the medium when the Src-MLV chimera (M.M1.Pr) was coexpressed
(Fig. 3A, lanes 6). This is remarkable because the only sequence in
common in these two molecules is the small piece of the Src protein
found at their N termini (Fig. 1). Identical results were obtained with
the RSV-MLV chimera T10M.Pr, which is defective for budding on its
own but was rescued by the Src chimeras of both RSV and MLV (Fig. 3A;
compare lanes 4 with lanes 7 and 8).
with M.M1.Pr was
dependent upon the Src N terminus. That is, when the wild-type
membrane-binding domain of RSV was present on T10C.Pr, the authentic
MLV Gag protein was unable to rescue the T10C derivative (data not
shown). Although it seemed unlikely that the very small segment from
Src (10 residues) would be able to provide interactions between Gag
proteins while simultaneously interacting with the membrane, we
addressed this possibility experimentally. Mutant Bg-Bs (Fig. 1A),
which contains the Src sequence but no I domains, was coexpressed with
assembly-competent Src chimeras of RSV and MLV. Neither R.M1.Pr nor
M.M1.Pr were able to enhance the release of Bg-Bs (Fig. 3B, lanes 4 and 5), even though this I domain mutant has been shown to be membrane
associated (42). These experiments suggest that the I
domains located in the C-terminal regions, and not the Src sequences at
the N termini, are responsible for the interactions among heterologous
Gag molecules in the complementation experiments.
Direct interactions between RSV and MLV Gag sequences. Although the genetic evidence for copackaging of RSV and MLV Gag molecules is strong, it does not alone rule out the possibility that budding-competent molecules might indirectly exert an effect on ones that are defective. That is, mutant T10C (and presumably T10M) is blocked at a very late step in budding, just prior to the virus-cell separation step, and it seemed possible that any budding activity by assembly-competent Gag proteins in the vicinity of these arrested particles might be enough to trigger their release into the medium without copackaging. Therefore, it was necessary to determine whether the Src chimeras of RSV and MLV Gag were actually present in the very same particles.
To test for direct interactions among coexpressed RSV and MLV Gag proteins, a series of coimmunoprecipitation experiments was used (Fig. 4A). Cells expressing the Src-Gag chimerasR.M1.Pr, M.M1.Pr, or bothwere radiolabeled, and the
particles released into the medium were collected by
ultracentrifugation. As an important control, R.M1.Pr particles and
M.M1.Pr particles that had been made separately were mixed together.
The pelleted particles were disrupted with lysis buffer, and the
released proteins were immunoprecipitated with either anti-MLV or
anti-RSV serum. R.M1.Pr and M.M1.Pr produced particles when
expressed alone, as expected (Fig. 4B, lanes 1 and 4). When these
separately made particles were mixed prior to disruption and
immunoprecipitation, only the M.M1.Pr protein was detected by the
anti-MLV serum and only the R.M1.Pr protein was detected with the
anti-RSV serum, indicating that they could not interact once the
barrier of the viral membrane was removed (Fig. 4B, lanes 2 and 5). In
contrast, when the Src chimeras of RSV and MLV were coexpressed, both
Gag proteins were immunoprecipitated with either antiserum, indicating
that direct interactions had taken place between them (Fig. 4B, lanes 3 and 6). The amount of MLV protein collected with anti-MLV serum was much greater than that obtained with anti-RSV serum, but the amount of
RSV protein was about the same with either serum. This is the expected
result given that the level of expression of RSV Gag was less than MLV
Gag in this particular experiment. Thus, it appears that all of the RSV
protein may have been copackaged, leaving the excess MLV protein to
produce particles alone. While it is not possible to make more
quantitative conclusions, these data clearly demonstrate that the Src
chimeras of RSV and MLV can interact during assembly and become
packaged together within the same particle.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
Evidence for particles that contain Gag proteins from different retroviruses has never been reported; indeed, our experiments show that the wild-type Gag proteins of RSV and MLV cannot be copackaged even when expressed at high levels in the same cells. From this observation, we hypothesized that the mutually exclusive properties of these two Gag proteins might result from (i) the use of different transport pathways to, or different sites of assembly on, the plasma membrane; (ii) an inability of the molecules to interact even though present at the same membrane location; or (iii) both. Our studies of the Src chimeras of RSV and MLV in budding assays support the first possibility and show that the barrier to copackaging can be removed by placing a common membrane targeting/binding signal on the N termini of these two Gag molecules. The evidence presented here comes from complementation and coimmunoprecipitation assays. A third line of evidence consistent with copackaging has been obtained by coexpressing the protease-active form of BgM with protease-minus forms of the two Src chimeras. In this case, particles that contain unique cleavage products as a result of trans-processing among the copackaged Gag molecules (2) are released into the medium.
We interpret our results to mean that the wild-type RSV and MLV Gag proteins are normally directed to different (as yet undefined) locations on the plasma membrane by their unique N-terminal M domains (Fig. 5A). When each of these domains are replaced with the membrane-binding domain of the Src protein, the chimeras arrive at the same membrane location, allowing them to interact and assemble particles containing both Gag molecules (Fig. 5B). The mechanisms of specific and localized membrane targeting are completely unknown, not only for Gag proteins but also for the many cellular proteins that are made in the cytosolic compartments of the cell and then transported to the cytoplasmic faces of the endoplasmic reticulum, Golgi, or plasma membranes (see the introduction). Thus, it appears to us that an unrecognized set of protein-sorting mechanisms remains to be discovered.
|
The idea that retroviruses utilize unique sites on the plasma membrane as they exit the cell, much as they utilize distinct sites as they enter, is further supported by the emerging properties of L domains. In particular, it appears that L domains participate in specific interactions with host proteins, recruiting them to the site of budding to mediate the membrane fusion event needed for the virus to separate from the cell (reviewed in reference 9). The L domain of RSV (P-P-P-P-Y) has been shown to interact in vitro with proteins containing WW domains (11, 40, 43), and the L domain of equine infectious anemia virus (Y-X-X-L) has been shown to interact both in vitro and in vivo with the plasma membrane-localized, clathrin-associated adapter protein complex (AP-2), a component of the endocytosis machinery (27, 28). Clearly, it is important to learn more about the local molecular environment in and around the sites of budding.
We have not tested any other Gag proteins for the ability to interact
in coexpression experiments, so there is little that we can say at this
point about which combinations will work. Those wishing to pursue these
sorts of experiments need to keep four things in mind. First, RSV and
MLV are both oncoviruses. It has become clear from numerous studies
that the Gag proteins of oncoviruses and lentiviruses have many
important differences, including different arrangements of their
assembly domains (M-L-I versus M-I-L, respectively [23]) and the locations of their particle size
determinants (10, 16). Second, RSV and MLV have similar,
rapid kinetics of budding (half-life, 
30 min). It is difficult to
predict whether Gag molecules from retroviruses with lower rates of
budding would be able to be copackaged with RSV. Similarly, Gag
proteins from type B and D retroviruses, which assemble in the
cytoplasm prior to transport to the membrane, might not be compatible
with Gag molecules that assemble only on the membrane. On the other
hand, a mutant of Mason-Pfizer monkey virus that results in the
assembly of this type D virus only on the membrane has been identified, and it might well be capable of copackaging (32). Third, the MLV Gag protein is very different from that of RSV in that it cannot be
rescued into particles when its membrane-binding domain is absent
(33). This and other recently described data suggest that
MLV Gag proteins do not initiate interactions among themselves until
they arrive at, and are concentrated on, the plasma membrane (2). This property might be essential for obtaining
definitive results in copackaging experiments. For example, if two Gag
molecules that are normally targeted to different sites on the plasma
membrane are capable of interacting in the cytoplasm to make complexes that can go to either site, then it would be impossible to discern the
existence of the two unique sites of budding. MLV Gag would also be
useful in those cases where cytoplasmic interactions among heterologous
molecules produce complexes that are incapable of being transported to
any membrane. Fourth, the yeast two-hybrid system has been used to look
for direct interactions among Gag proteins from different retroviruses,
and positive results have been obtained only with the most closely
related molecules (8, 17). RSV and MLV Gag have not been
tested. Whether negative results from this artifical assay are
predictive of what can happen during budding remains to be seen.
| |
ACKNOWLEDGMENTS |
|---|
We thank all of the members of the Wills laboratory (past and present) for helpful suggestions with experiments and comments regarding the manuscript. In particular, we thank Brad Bowzard for critical review of the manuscript. We thank Alan Rein for providing the MLV genome and MLV-specific antiserum. We also thank those who have known about our results and have waited so patiently for us to get them on paper.
This work was supported by grants awarded to J.W.W. from the National Institutes of Health (CA47482) and the American Cancer Society (FRA-427).
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Dr., P. O. Box 850, Hershey, PA 17033. Phone: (717) 531-3528. Fax: (717) 531-6522. E-mail: jwills{at}psu.edu.
Present address: Invitrogen Corporation, Carlsbad, CA 92008.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
| 1. |
Bennett, R. P.,
T. D. Nelle, and J. W. Wills.
1993.
Functional chimeras of the Rous sarcoma virus and human immunodeficiency virus Gag proteins.
J. Virol.
67:6487-6498 |
| 2. |
Bowzard, J. B.,
R. P. Bennett,
N. K. Krishna,
S. M. Ernst,
A. Rein, and J. W. Wills.
1998.
Importance of basic residues in the nucleocapsid sequence for retrovirus Gag assembly and complementation rescue.
J. Virol.
72:9034-9044 |
| 3. | Buser, C. A., C. T. Sigal, M. D. Resh, and S. McLaughlin. 1994. Membrane binding of myristylated peptides corresponding to the NH2 terminus of Src. Biochemistry 33:13093-13101[Medline]. |
| 4. |
Craven, R. C.,
R. P. Bennett, and J. W. Wills.
1991.
Role of the avian retroviral protease in the activation of reverse transcriptase during virion assembly.
J. Virol.
65:6205-6217 |
| 5. | Craven, R. C., and L. J. Parent. 1996. Dynamic interactions of the Gag polyprotein. Curr. Top. Microbiol. Immunol. 214:65-94[Medline]. |
| 6. |
Cross, F. R.,
E. A. Garber,
D. Pellman, and H. Hanafusa.
1984.
A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation.
Mol. Cell. Biol.
4:1834-1842 |
| 7. |
Erdie, C. R., and J. W. Wills.
1990.
Myristylation of Rous sarcoma virus Gag protein does not prevent replication in avian cells.
J. Virol.
64:5204-5208 |
| 8. |
Franke, E. K.,
H. E. H. Yuan,
K. L. Bossolt,
S. P. Goff, and J. Luban.
1994.
Specificity and sequence requirements for interactions between various retroviral Gag proteins.
J. Virol.
68:5300-5305 |
| 9. | Garnier, L., J. B. Bowzard, and J. W. Wills. 1998. Recent advances and remaining problems in HIV assembly. AIDS 12(Suppl. A):S5-S16. |
| 10. |
Garnier, L.,
L. Ratner,
B. Rovinski,
S. X. Cao, and J. W. Wills.
1998.
Particle size determinants in the human immunodeficiency virus type 1 Gag protein.
J. Virol.
72:4667-4677 |
| 11. | Garnier, L., J. W. Wills, M. F. Verderame, and M. Sudol. 1996. WW domains and retrovirus budding. Nature 381:744-745[Medline]. |
| 12. |
Gorelick, R. J.,
L. E. Henderson,
J. P. Hanser, and A. Rein.
1988.
Point mutations of Moloney murine leukemia virus that fail to package viral RNA: evidence for specific RNA recognition by a "zinc finger-like" protein sequence.
Proc. Natl. Acad. Sci. USA
85:8420-8424 |
| 13. |
Jørgensen, E. C. B.,
F. S. Pedersen, and P. Jørgensen.
1992.
Matrix protein of Akv murine leukemia virus: genetic mapping of regions essential for particle formation.
J. Virol.
66:4479-4487 |
| 14. |
Kensil, C. R., and P. Strittmatter.
1986.
Binding and fluorescence properties of the membrane domain of NADH-cytochrome-b5 reductase.
J. Biol. Chem.
261:7316-7321 |
| 15. | Kim, J., M. Mosior, L. A. Chung, H. Wu, and S. McLaughlin. 1991. Binding of peptides with basic residues to membranes containing acidic phospholipids. Biophys. J. 60:135-148[Medline]. |
| 16. |
Krishna, N. K.,
S. Campbell,
V. M. Vogt, and J. W. Wills.
1998.
Genetic determinants of Rous sarcoma virus particle size.
J. Virol.
72:564-577 |
| 17. |
Luban, J.,
K. B. Alin,
K. L. Bossolt,
T. Humaran, and S. P. Goff.
1992.
Genetic assay for multimerization of retroviral Gag polyproteins.
J. Virol.
66:5157-5160 |
| 18. | McLaughlin, S., and A. Aderam. 1995. The myristoyl-electrostatic switch: a modulator of reversible protein-membrane interactions. Trends Biochem. Sci. 20:272-276[Medline]. |
| 19. | Mosior, M., and S. McLaughlin. 1992. Binding of basic peptides to acidic lipids in membranes: effects of inserting alanine(s) between the basic residues. Biochemistry 31:1767-1773[Medline]. |
| 20. |
Murakami, K.,
T. Yubisui,
M. Takeshita, and T. Miyata.
1989.
The NH2-terminal structures of human and rat liver microsomal NADH-cytochrome b5 reductases.
J. Biochem.
105:312-317 |
| 21. | Nelle, T. D., and J. W. Wills. 1996. A large region within the Rous sarcoma virus matrix protein is dispensable for budding and infectivity. J. Virol. 70:2269-2276[Abstract]. |
| 22. |
Ozols, J.,
S. A. Carr, and P. Strittmatter.
1984.
Identification of the NH2-terminal blocking group of NADH-cytochrome b5 reductase as myristic acid and the complete amino acid sequence of the membrane-binding domain.
J. Biol. Chem.
259:13349-13354 |
| 23. | Parent, L. J., R. P. Bennett, R. C. Craven, T. D. Nelle, N. K. Krishna, J. B. Bowzard, C. B. Wilson, B. A. Puffer, R. C. Montelaro, and J. W. Wills. 1995. Positionally independent and exchangeable late budding functions of the Rous sarcoma virus and human immunodeficiency virus Gag proteins. J. Virol. 69:5455-5460[Abstract]. |
| 24. | Parent, L. J., C. B. Wilson, M. D. Resh, and J. W. Wills. 1996. Evidence for a second function of the MA sequence in the Rous sarcoma virus Gag protein. J. Virol. 70:1016-1026[Abstract]. |
| 25. | Peitzsch, R. M., and S. McLaughlin. 1993. Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins. Biochemistry 32:10436-10443[Medline]. |
| 26. | Pellman, D., E. A. Garber, F. R. Cross, and H. Hanafusa. 1985. An N-terminal peptide from p60src can direct myristylation and plasma membrane localization when fused to heterologous proteins. Nature 314:374-377[Medline]. |
| 27. | Puffer, B. A., L. J. Parent, J. W. Wills, and R. C. Montelaro. 1997. Equine infectious anemia virus utilizes a YXXL motif within the late assembly domain of the Gag p9 protein. J. Virol. 71:6541-6546[Abstract]. |
| 28. |
Puffer, B. A.,
S. C. Watkins, and R. C. Montelaro.
1998.
Equine infectious anemia virus Gag polyprotein late domain specifically recruits cellular AP-2 adapter protein complexes during virion assembly.
J. Virol.
72:10218-10221 |
| 29. | Rein, A., and J. G. Levin. 1992. Readthrough suppression in the mammalian type C retroviruses and what it has taught us. New Biol. 4:283-289[Medline]. |
| 30. |
Rein, A.,
M. R. McClure,
N. R. Rice,
R. B. Luftig, and A. M. Schultz.
1986.
Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus.
Proc. Natl. Acad. Sci. USA
83:7246-7250 |
| 31. | Resh, M. D. 1994. Myristylation and palmitylation of Src family members: the fats of the matter. Cell 76:411-413. |
| 32. | Rhee, S. S., and E. Hunter. 1990. A single amino acid substitution within the matrix protein of a type D retrovirus converts its morphogenesis to that of a type C retrovirus. Cell 63:77-86[Medline]. |
| 33. |
Schultz, A. M., and A. Rein.
1989.
Unmyristylated Moloney murine leukemia virus Pr65gag is excluded from virus assembly and maturation events.
J. Virol.
63:2370-2373 |
| 34. | Schwartz, D. E., R. Tizard, and W. Gilbert. 1983. Nucleotide sequence of Rous sarcoma virus. Cell 32:853-869[Medline]. |
| 35. |
Sigal, C. T.,
W.-J. Zhou,
C. A. Buser,
S. McLaughlin, and M. D. Resh.
1994.
The amino-terminal basic residues of Src mediate membrane binding through electrostatic interactions with acidic phospholipids.
Proc. Natl. Acad. Sci. USA
91:12253-12257 |
| 36. | Swanstrom, R., and J. W. Wills. 1997. Retroviral gene expression. II. Synthesis, processing, and assembly of viral proteins, p. 263-334. In R. Weiss, N. Teich, H. Varmus, and J. M. Coffin (ed.), Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 37. | Verderame, M. F., T. D. Nelle, and J. W. Wills. 1996. The membrane-binding domain of the Rous sarcoma virus Gag protein. J. Virol. 70:2664-2668[Abstract]. |
| 38. |
Weldon, R. A., Jr.,
C. R. Erdie,
M. G. Oliver, and J. W. Wills.
1990.
Incorporation of chimeric Gag protein into retroviral particles.
J. Virol.
64:4169-4179 |
| 39. |
Weldon, R. A., Jr., and J. W. Wills.
1993.
Characterization of a small (25-kilodalton) derivative of the Rous sarcoma virus Gag protein competent for particle release.
J. Virol.
67:5550-5561 |
| 40. |
Wills, J. W.,
C. E. Cameron,
C. B. Wilson,
Y. Xiang,
R. P. Bennett, and J. Leis.
1994.
An assembly domain of the Rous sarcoma virus Gag protein required late in budding.
J. Virol.
68:6605-6618 |
| 41. |
Wills, J. W.,
R. C. Craven, and J. A. Achacoso.
1989.
Creation and expression of myristylated forms of Rous sarcoma virus Gag protein in mammalian cells.
J. Virol.
63:4331-4343 |
| 42. |
Wills, J. W.,
R. C. Craven,
R. A. Weldon, Jr.,
T. D. Nelle, and C. R. Erdie.
1991.
Suppression of retroviral MA deletions by the amino-terminal membrane-binding domain of p60src.
J. Virol.
65:3804-3812 |
| 43. |
Xiang, Y.,
C. E. Cameron,
J. W. Wills, and J. Leis.
1996.
Fine mapping and characterization of Rous sarcoma virus pr76gag late assembly domain.
J. Virol.
70:5695-5700 |
| 44. |
Zhou, W.,
L. J. Parent,
J. W. Wills, and M. D. Resh.
1994.
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.
68:2556-2569 |
| 45. | Zhou, W., and M. D. Resh. 1996. Differential membrane binding of the human immunodeficiency virus type 1 matrix protein. J. Virol. 70:8540-8548[Abstract]. |
Journal of Virology, March 1999, p. 2045-2051, Vol. 73, No. 3
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Kenney, S. P., Lochmann, T. L., Schmid, C. L., Parent, L. J.
(2008). Intermolecular Interactions between Retroviral Gag Proteins in the Nucleus. J. Virol.
82: 683-691
[Abstract] [Full Text] -
Ako-Adjei, D., Johnson, M. C., Vogt, V. M.
(2005). The Retroviral Capsid Domain Dictates Virion Size, Morphology, and Coassembly of Gag into Virus-Like Particles. J. Virol.
79: 13463-13472
[Abstract] [Full Text] -
Spidel, J. L., Craven, R. C., Wilson, C. B., Patnaik, A., Wang, H., Mansky, L. M., Wills, J. W.
(2004). Lysines Close to the Rous Sarcoma Virus Late Domain Critical for Budding. J. Virol.
78: 10606-10616
[Abstract] [Full Text] -
Andrawiss, M., Takeuchi, Y., Hewlett, L., Collins, M.
(2003). Murine Leukemia Virus Particle Assembly Quantitated by Fluorescence Microscopy: Role of Gag-Gag Interactions and Membrane Association. J. Virol.
77: 11651-11660
[Abstract] [Full Text] -
Patnaik, A., Wills, J. W.
(2002). In Vivo Interference of Rous Sarcoma Virus Budding by cis Expression of a WW Domain. J. Virol.
76: 2789-2795
[Abstract] [Full Text] -
Krishna, N. K., Wills, J. W.
(2001). Insertion of Capsid Proteins from Nonenveloped Viruses into the Retroviral Budding Pathway. J. Virol.
75: 6527-6536
[Abstract] [Full Text] -
Patnaik, A., Chau, V., Wills, J. W.
(2000). Ubiquitin is part of the retrovirus budding machinery. Proc. Natl. Acad. Sci. USA
97: 13069-13074
[Abstract] [Full Text] -
Bowzard, J. B., Visalli, R. J., Wilson, C. B., Loomis, J. S., Callahan, E. M., Courtney, R. J., Wills, J. W.
(2000). Membrane Targeting Properties of a Herpesvirus Tegument Protein-Retrovirus Gag Chimera. J. Virol.
74: 8692-8699
[Abstract] [Full Text] -
Parent, L. J., Cairns, T. M., Albert, J. A., Wilson, C. B., Wills, J. W., Craven, R. C.
(2000). RNA Dimerization Defect in a Rous Sarcoma Virus Matrix Mutant. J. Virol.
74: 164-172
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»