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Journal of Virology, February 2002, p. 1569-1577, Vol. 76, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.4.1569-1577.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Functional Replacement and Positional Dependence of Homologous and Heterologous L Domains in Equine Infectious Anemia Virus Replication

Feng Li, Chaoping Chen, Bridget A. Puffer,{dagger}, and Ronald C. Montelaro*

Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Received 17 August 2001/ Accepted 13 November 2001


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ABSTRACT
 
We have previously demonstrated by Gag polyprotein budding assays that the Gag p9 protein of equine infectious anemia virus (EIAV) utilizes a unique YPDL motif as a late assembly domain (L domain) to facilitate release of the budding virus particle from the host cell plasma membrane (B. A. Puffer, L. J. Parent, J. W. Wills, and R. C. Montelaro, J. Virol. 71:6541-6546, 1997). To characterize in more detail the role of the YPDL L domain in the EIAV life cycle, we have examined the replication properties of a series of EIAV proviral mutants in which the parental YPDL L domain was replaced by a human immunodeficiency virus type 1 (HIV-1) PTAP or Rous sarcoma virus (RSV) PPPY L domain in the p9 protein or by proviruses in which the parental YPDL or HIV-1 PTAP L domain was inserted in the viral matrix protein. The replication properties of these L-domain variants were examined with respect to Gag protein expression and processing, virus particle production, and virus infectivity. The data from these experiments indicate that (i) the YPDL L domain of p9 is required for replication competence (assembly and infectivity) in equine cell cultures, including the natural target equine macrophages; (ii) all of the functions of the YPDL L domain in the EIAV life cycle can be replaced by replacement of the parental YPDL sequence in p9 with the PTAP L-domain segment of HIV-1 p6 or the PPPY L domain of RSV p2b; and (iii) the assembly, but not infectivity, functions of the EIAV proviral YPDL substitution mutants can be partially rescued by inclusions of YPDL and PTAP L-domain sequences in the C-terminal region of the EIAV MA protein. Taken together, these data demonstrate that the EIAV YPDL L domain mediates distinct functions in viral budding and infectivity and that the HIV-1 PTAP and RSV PPPY L domains can effectively facilitate these dual replication functions in the context of the p9 protein. In light of the fact that YPDL, PTAP, and PPPY domains evidently have distinct characteristic binding specificities, these observations may indicate different portals into common cellular processes that mediate EIAV budding and infectivity, respectively.


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INTRODUCTION
 
General models for the assembly of retroviruses were first proposed over 20 years ago (1), but only within the past several years have there been substantial research and new information detailing the role of viral structural proteins and specific cellular proteins in virion assembly and budding from infected cells and in the infection of target cells by free virions (4, 24). The results of these studies have clearly demonstrated that the retrovirus Gag proteins are sufficient for the assembly of virus particles from transfected cells in the absence of other viral proteins or genomic RNA. Despite the lack of amino acid sequence homology, the Gag proteins of widely diverse retroviruses of different genera appear to be functionally and structurally similar. All retrovirus gag genes encode polyproteins that include matrix (MA), capsid (CA), and nucleocapsid (NC) proteins that provide common essential budding functions of plasma membrane targeting (M domain), Gag polyprotein interaction (I domain), and genomic RNA encapsidation, respectively (6). Thus, these studies have clearly indicated common assembly motifs among apparently diverse retrovirus Gag proteins, with the potential for the elucidation of unique viral functions that may be targeted by antiviral strategies.

In addition to the consensus retrovirus Gag proteins, the gag genes of retroviruses also encode proteins with a late budding function that is required for the efficient release of budding virions from the plasma membrane of infected cells (8, 10, 14, 17, 25, 30). Characterization of the Gag sequences that mediate this late budding function (L domain) has revealed an unexpected diversity in terms of the functional amino acid sequences and their location in specific Gag proteins other than MA, CA, or NC. For example, the L domains of Rous sarcoma virus (RSV) and Moloney murine leukemia virus, both oncoviruses, and Mason-Pfizer monkey virus, a type D retrovirus, all map to a common PPPY motif that is included in diverse protein sequences characteristically located between the viral MA and CA gene sequences, i.e., RSV p2b, Moloney murine leukemia virus p12, and Mason-Pfizer monkey virus p24 (26, 27, 30). Proline-rich L domains have also been identified in lentivirus Gag proteins and localized to a specific PTAP sequence, as exemplified by the p6 proteins of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV) (8, 10, 20, 22, 23). It has been suggested that an MA-associated PTAP sequence found in some isolates of HIV-2 and SIV may also provide L-domain functions, although this has not been demonstrated experimentally. In contrast to these proline-rich L-domain motifs, we discovered that the late budding function of equine infectious anemia virus (EIAV) is mediated by a YPDL sequence in the viral p9 protein that is the C-terminal segment of the EIAV Gag polyprotein (17). Despite the distinct differences in L-domain sequences, it has been demonstrated previously that the YPDL and PTAP or PPPY L domains are functionally interchangeable and positionally independent in Gag polyprotein budding assays (14, 17). These observations suggest that there may be different entry points into a common cellular pathway that is utilized in retrovirus assembly and budding at the plasma membrane.

The exact mechanisms and cellular processes utilized by the retrovirus L domains in completing viral budding and release remain to be defined. In the case of EIAV, the p9 L domain has been shown previously to specifically recruit and bind to the cellular adapter protein AP-2 at the site of virus budding in infected equine cells (18), suggesting that the virus may adapt cellular endocytosis machinery for virion budding. The oncovirus PPPY L domain has similarly been shown elsewhere by in vitro assays to specifically bind to WW domains from the Yes-kinase-associated proteins and from members of the Nedd4 ubiquitin ligase family (5, 9, 16), but similar specific interactions in infected cells have not been reported. More recently, there have been several reports indicating the possible role of ubiquitin and ubiquitination complexes in retrovirus budding (11, 15, 20, 22, 23). For example, certain proteasome inhibitors that lower cellular ubiquitin content can suppress HIV-1, HIV-2, and RSV replication or budding (15, 20, 22). In addition, recent studies demonstrate specific interactions of the HIV-1 PTAP L domain with Tsg101 (a homologue of ubiquitin-conjugating E2 enzymes lacking the active-site Cys) and of the RSV PPPY L domain with the Nedd4-family-like proteins of ubiquitin ligase E3 (7, 11, 23). While it is tempting to extrapolate these observations to all retroviruses, there is to date no published evidence for the involvement of ubiquitination in EIAV budding. In addition to the role of EIAV p9 in viral budding, we recently demonstrated for the first time that the EIAV p9 is involved in viral infectivity and that the amino acid sequences required for infectivity are distinct from the L domain of p9 (2). These diverse observations emphasize the need for additional studies of the cellular biology of retrovirus budding, in particular comparative studies among diverse retroviruses.

To characterize in more detail the mechanisms of EIAV budding, we have in the present study examined in detail the positional dependence and functional equivalence of PTAP or PPPY and YPDL L domains in the context of EIAV proviral gene expression and replication. Specifically, we addressed the following questions. What is the role of the YPDL L domain in viral budding and infectivity in the context of the EIAV proviral DNA clone? Can the HIV-1 PTAP or RSV PPPY L domain facilitate EIAV budding and replication when substituted for the YPDL motif of the p9 protein? What is the role of MA-associated L domains in EIAV budding and replication? The results of these studies revealed new fundamental information about the basic mechanisms of retrovirus budding and the roles of the L domain in viral replication.


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MATERIALS AND METHODS
 
Construction of mutant plasmid DNA clones. All of the mutant constructs in this study were generated on the basis of two versions of our reference infectious molecular clones, EIAVUK (3) and cmvEIAVUK (2). cmvEIAVUK and its derivatives constructs contain the cytomegalovirus (CMV) immediate-early promoter between the U3 and R regions of the 5" long terminal repeat (LTR) and were used when large amounts of viruses were needed for biochemical analysis. All DNA mutagenesis was carried out using the PCR-ligation-PCR strategy as previously described (12) and other standard molecular cloning approaches. At least two independent clones of each mutant construct were examined in transfection experiments to minimize any possible nonsense mutations generated during PCR and cloning processing.

(i) Construction of YPDL-, PTAP, and PPPY mutants. To study the functions of the EIAV YPDL L domain in virus assembly and replication, we replaced the YPDL L-domain sequence with a sequence of SRSA residues that retained the original amino acid sequence of the overlapping pol reading frame. The YPDL L-domain substitution construct was named EIAVUK(YPDL-). To address functional replacement of homologous and heterologous L domains in virus assembly and replication, we generated the HIV-1 p6 PTAP L-domain substitution by replacing YPDL and flanking sequences (QNLYPDL) in the p9 protein with the HIV-1 p6 L domain and flanking sequences (RPRPTAPP). This substitution construct is designated EIAVUK(PTAP). A similar approach was used to generate the RSV L-domain substitution in which the EIAVUK L domain and flanking sequences (QNLYPDL) were replaced in the same location in the p9 protein with the RSV p2b L domain and flanking sequences (ASAPPPPYVG). This substitution construct is designated EIAVUK(PPPY).

(ii) Construction of MAY, MAP, MY. YPDL-, and MP.YPDL- mutants. To study L-domain positional dependence in virus budding and infectivity, we inserted individually, into the C-terminal MA gene of EIAVUK, without any alteration of the native MA-CA cleavage site, the EIAV YPDL L domain or the HIV-2 (or SIV) MA-associated PTAP L domain for the generation of EIAVUK(MAY) or EIAVUK(MAP), respectively. EIAVUK(MAY) contains two YPDL-based L-domain segments, the parental YPDL sequence in p9 and the QNLYPDL sequence inserted toward the C-terminal end of the MA gene. EIAVUK(MAP) contains an HIV-2-SIV MA-associated PTAP L domain and flanking sequences (TSRPTAPP) inserted into the C-terminal sequences of the MA gene of EIAV in addition to the parental YPDL L domain in p9. Subsequent replacements of the native YPDL L domain in p9 with a segment of SRSA residues were individually performed to generate EIAVUK(MY.YPDL-) in the context of EIAVUK(MAY) and EIAVUK(MP.YPDL-) in the EIAVUK(MAP) backbone.

Cell culture, DNA transfection, and infection. COS-1, fetal equine kidney (FEK), and equine dermal (ED) cells were maintained in Eagle's minimum essential medium (MEM) (Gibco BRL, Grand Island, N.Y.) with 10% fetal bovine serum and antibiotics (100 U of penicillin/ml and 100 µg of streptomycin/ml) and passaged upon confluence. Primary equine blood monocyte-derived-macrophage (MDM) cells were prepared as described previously (19). Briefly, mononuclear cells were isolated from heparinized equine whole blood and prepared by centrifugation through Histopaque (Sigma, St. Louis, Mo.). Following several washes in Ca2+- and Mg2+-free phosphate-buffered saline (PBS), cells were seeded in {alpha}-MEM (Gibco BRL) with 10% heat-inactivated horse serum (Sigma) on each gelatin (Sigma)- and plasma-coated tissue culture dish (150 cm2) (Corning, Corning, N.Y.) overnight at 37°C with 6% CO2. On the following day, the nonadherent and loosely adherent cells were removed by repeated vigorous washing with {alpha}-MEM (Gibco BRL). The adherent cells were detached with 5 mM EDTA in {alpha}-MEM with 10% heat-inactivated horse serum and seeded into 48-well plates (Corning) at a density of 105 cells per well.

FEK and ED cells were transiently transfected with wild-type and mutant EIAVUK proviral DNAs using GenePorter 2 transfection reagent (Gene Therapy Systems Inc.). Briefly, cells were plated in a 60-mm-diameter petri dish (Corning) the day before transfection to reach 60 to 90% confluence on the day of transfection. For each transfection, 8 µg of DNA was diluted into 200 µl of DNA diluent provided, and diluted DNA was then added dropwise into 160 µl of serum- and antibiotic-free medium containing 40 µl of GenePorter 2 reagent for a 15-min incubation at room temperature. The mixture of GenePorter 2-DNA complexes was gently overlaid onto the rinsed cells containing 2.5 ml of serum- and antibiotic-free medium. Following a 5-h incubation, 2.5 ml of Eagle's MEM containing 20% serum was added into the transfected cells. Twenty-four hours posttransfection, medium containing GenePorter 2-DNA complexes was removed, 4 ml of fresh medium was added, and cells were then incubated and maintained for up to 35 days at 37°C in a 5% CO2 incubator. Aliquots of the tissue culture supernatants were taken at periodic intervals and analyzed by using a standard reverse transcriptase (RT) assay as a measure for virus production (13).

Transfections of COS-1 cells with the parental and mutant cmvEIAVUK proviral DNAs were performed as described above. At 72 h posttransfection, medium containing viral particles was collected and clarified by centrifugation at 2,000 rpm for 20 min in a Sorvall RT 6000B centrifuge followed by filtration through a 0.2-µm-pore-size membrane. Virus particle-containing supernatants were concentrated by ultracentrifugation for 50 min at 100,000 x g through a cushion of 20% (wt/vol) glycerol, and pellets were resuspended in PBS for RT assay.

Infection of wild-type EIAVUK and EIAVUKPTAP viruses in natural target equine macrophage cells was performed as previously described (12, 13). Virus stocks were prepared by harvesting the medium from GenePorter 2-transfected FEK cells at 28 days posttransfection, and titers of each virus stock were determined by a cell enzyme-linked immunosorbent assay-based assay (13).

Metabolic labeling and radioimmunoprecipitation. Metabolic labeling and radioimmunoprecipitation of cell- and virion-associated proteins were performed essentially as described previously (17), with minor modifications. Briefly, COS-1 cells in 60-mm-diameter petri dishes (Corning) were transfected with the parental and mutant cmvEIAVUK proviral DNAs using GenePorter 2 transfection reagent. At 48 h posttransfection, transfected COS-1 cells were starved for 45 min in the methionine- and cysteine-free MEM (Sigma) and then labeled with 2 ml of Met- and Cys-free labeling medium containing 200 µCi of [35S]Met-[35S]Cys (7,000 Ci/mmol; NEN, Boston, Mass.) for 6 h. Following the completion of labeling, cells were washed once with cold PBS and lysed in lysis buffer (150 mM Tris-HCl, 5% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], pH 8.0). The cell lysates were cleared of nuclei by centrifugation at 8,000 x g at 4°C for 5 min, and postnuclear supernatants were then preabsorbed with Pansorbin cell beads containing protein A (Calbiochem, San Diego, Calif.). Virions were purified from the clarified supernatant containing virus particles by ultracentrifugation at 4°C for 50 min at 100,000 x g through a cushion of 20% (wt/vol) glycerol, and pellets were resuspended in lysis buffer as described above. Cell- and virion-associated fractions were then immunoprecipitated for 1 h at room temperature with a reference polyclonal immune serum from an EIAV-infected horse. One hundred microliters of Pansorbin cell beads containing protein A (Calbiochem) was then added for 1 h of further incubation at room temperature. Beads were washed three times, and proteins bound to beads were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and quantified by phosphorimager analysis (Molecular Dynamics, Sunnyvale, Calif.).


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RESULTS
 
Construction of EIAV proviral p9 mutants. Previous studies of EIAV p9 L-domain function in viral replication have been predominantly restricted to Gag polyprotein budding assays in COS-1 cells that failed to evaluate potential functions of the p9 protein in viral infectivity (17). Therefore, to examine further the roles of the EIAV p9 protein and its YPDL L domain in viral replication, we engineered a series of EIAV proviral mutations specifically designed to alter the specificity or location of the L domain. Figure 1 summarizes the panel of EIAV p9 variants that were constructed for the examination of the role of EIAV p9 in viral assembly and infectivity. All proviral constructs for characterization of replication competence were constructed in the reference pathogenic infectious molecular clone designated EIAVUK. For the analyses of EIAV protein expression, processing, and assembly in transfected COS-1 cells, the panel of proviral mutants was subcloned into a cmvEIAVUK plasmid to achieve higher levels of gene expression.



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  		FIG. 1. Schematic diagrams of EIAV proviral mutant constructs. (A) The top diagram shows the EIAV Gag polyprotein organization and the partial nucleotide sequences and corresponding amino acid sequences in Gag (p9) and Pol (pro) for the parental and the p9 mutant constructs used in this study. Nucleotides and amino acids that differ from those in the wild-type sequences are in boldface and underlined. (B) The top diagram shows the EIAV Gag polyprotein organization and the partial amino acid sequences of the junction site between MA and CA of the parental and the p9 mutant constructs used in this study. The boldface amino acid sequences in each of the mutant constructs are insertions of the EIAV YPDL or HIV-2 (or SIV) PTAP L domain with its respective flanking sequences at the C-terminal MA region of EIAV Gag. All mutants were constructed as described in Materials and Methods.

EIAVUK proviral L-domain variations were essentially of two types. The first set of proviral constructs was designed to assess the functional role of the EIAV YPDL L domain in viral replication in the context of an infectious proviral clone and the ability of a substitute HIV-1 PTAP or RSV PPPY L domain to provide these replication functions. Thus, the EIAVUK parental proviral clone was modified to contain a specific replacement of the YPDL L-domain sequences of p9 with a sequence of SRSA residues (EIAVUKYPDL-) or to contain a replacement of the YPDL L domain with the PTAP L domain of HIV-1 p6 protein or the PPPY L domain of RSV p2b protein, as depicted in Fig. 1A. In the YPDL- construct, the parental YPDL sequence was replaced with a SRSA substitution that retained the original amino acid sequence of the overlapping pol reading frame. For the HIV-1 L-domain substitution, the EIAVUK L domain and flanking sequences (QNLYPDL) were replaced in the same location in the p9 protein with the HIV-1 p6 L domain and flanking sequences (RPRPTAPP). This substitution construct is designated EIAVUK(PTAP). A similar approach was used to generate the RSV L-domain substitution in which the EIAVUK L domain and flanking sequences (QNLYPDL) were replaced in the same location in the p9 protein with the RSV p2b L domain and flanking sequences (ASAPPPPYVG). This substitution construct is designated EIAVUK(PPPY). Thus, the proviral constructs summarized in Fig. 1A encode EIAVUK p9 with the parental L domain (YPDL), a substituted L domain (YPDL-), a substituted HIV-1 PTAP L domain (PTAP), or a substituted RSV PPPY L domain (PPPY) for functional analyses in directing viral budding and infectivity.

The second set of EIAVUK proviral mutations, depicted in Fig. 1B, was designed to assess the positional dependence of L-domain sequences in viral budding and infectivity. For these studies, YPDL or PTAP sequences were inserted into the MA gene of the EIAVUK provirus containing either the parental p9 sequence or a YPDL- mutation. For example, the MAY construct contained two YPDL-based L-domain segments, the parental YPDL sequence in p9 and the QNLYPDL sequence inserted toward the C-terminal end of the MA gene, but retained the native MA-CA cleavage site (Fig. 1B). In contrast, the MY.YPDL- construct contains only the YPDL L-domain insertion in the MA protein sequence, with the substituted p9 L domain. Similarly, the MAP and MP.YPDL- constructs contained a PTAP L domain and flanking sequences from HIV-2 (or SIV) MA protein inserted into the C-terminal sequences of the MA protein in the proviral genome containing parental p9 or the YPDL- p9, respectively. The proviral constructs summarized in Fig. 1B were then analyzed for their replication properties, as described below.

Replication properties of EIAV L-domain variants. To determine the replication properties of the various EIAVUK proviral L-domain mutants, equal amounts of the respective proviral DNAs were transfected into ED cells, and virus replication was monitored by regular measurements of extracellular RT activity, as described previously (13). The resulting data (Fig. 2) clearly demonstrated a definitive differentiation into replication-competent and -defective phenotypes. Replication-competent phenotypes included the PTAP, PPPY, MAY, and MAP proviral constructs, which all displayed replication kinetics similar to those of the parental EIAVUK provirus in transfected ED cells. In marked contrast, parallel transfections with the YPDL-, MY.YPDL-, and MP.YPDL- proviral DNA constructs produced only background levels of extracellular RT, indicating a lack of viral replication during the 35-day observation period. Similar replication phenotypes were observed in repeated transfections with the ED cell line and in transfections of primary FEK cells (data not shown). A particularly striking result was that both the HIV-1 PTAP and the RSV PPPY L domains were able to effectively substitute for the parental EIAV YPDL L domain of p9 in supporting virus replication (compare EIAVUK and PTAP or PPPY in Fig. 2A), although to a somewhat lower level. In addition, the data revealed that neither the YPDL nor the PTAP L domain sustained replication competence when inserted into the C terminus of the MA protein in the absence of an L domain in the p9 sequences of the proviral construct (compare MY.YPDL- and MP.YPDL- in Fig. 2A). However, it was demonstrated that the insertion of the L domains into the MA did not produce a replication-defective phenotype, as the proviral constructs containing MA-associated L domains and the p9 YPDL L domain displayed the same replication kinetics as those of the parental EIAVUK in transfected ED cells (compare MAY and MAP in Fig. 2A.). Taken together, these observations suggest that a p9-associated L domain is required for replication in transfected ED cells and that the YPDL or a proline-rich L domain can supply the necessary functions during viral budding as well as in infection of target cells. Homologous or heterologous L domains inserted in the MA protein in the absence of a p9 L domain cannot support virus replication, indicating a positional dependence of the L-domain function for replication competence.



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  		FIG. 2. Replication kinetics of the parental EIAVUK and mutant p9 viruses in transfected ED cells (A) and infected equine MDM cells (B). (A) The ED cells were transfected with 8 µg of the parental EIAVUK and other p9 mutant proviral DNA constructs, and transfected cells were then cultured for 5 weeks. Viral replication was monitored by regular assays of supernatant RT activity. (B) Primary MDM cells were infected with either the parental EIAVUK or the indicated mutant virus stock at an MOI of 0.1. Virus production following infection of equine MDM cells was monitored at various times postinfection by measuring RT activity in the culture supernatant. The data presented in this figure are representative of at least three independent transfections or infections.

In evaluating the role of various EIAV genes in viral replication, we have previously observed differences in replication properties in fibroblastic cells (e.g., ED and FEK) compared to the natural target equine macrophages (13). Similar cell type dependence phenotypes have been reported elsewhere for HIV-1 p6 mutations (21, 28). To confirm the relevance of the replication properties observed in transfections of fibroblastic cells, we next examined the replication kinetics of EIAVUK and EIAVUK(PTAP) by infection of MDMs, the natural target cells for EIAV in vivo. The data in Fig. 2B demonstrated similar replication kinetics and levels for both the parental EIAVUK and the variant EIAVUK(PTAP) virus in MDM cells infected with an identical multiplicity of infection (MOI) of 0.1. The pattern of replication shown in Fig. 2B was reproducible in repeated infection experiments and at different MOI values (data not shown). Thus, these data demonstrate that the PTAP L domain can substitute for the YPDL L domain in supporting EIAV replication in primary macrophages, the natural in vivo target cells.

Effects of p9 mutations on viral particle production. As the retrovirus L domain has been closely associated with viral assembly and budding from the plasma membrane, we next sought to determine whether the replication phenotypes observed with the panel of L-domain variants correlated with virion budding and release from the cellular plasma membrane. To obtain a higher level of proviral gene expression for these experiments, we utilized a cmvEIAVUK expression plasmid containing the panel of L-domain variants summarized in Fig. 1. Thus, COS-1 cells were transfected in parallel with DNA from the parental and variant cmvEIAVUK proviral constructs, and virion production was measured at 72 h posttransfection by assays of RT in viral pellets from cell supernatants (Fig. 3). The results of these experiments demonstrated that all of the proviral transfections yielded extracellular virion-associated RT indicative of virion production but that the apparent levels of virion budding differed by a range of approximately 10-fold. The highest levels of extracellular virion-associated RT production were observed in the transfections with the proviral constructs (MAY and MAP) containing L domains in both the viral p9 and MA genes, while the lowest RT levels were observed with the proviral construct (YPDL-) lacking any L domain in the gag gene. However, there was no definitive correlation between replication competence and virion production, assuming that the transfected COS-1 cells utilized in the experiment represented in Fig. 3 accurately reflect viral replication in the ED cells used for Fig. 2A. For example, the replication-defective cmvEIAVUK(MY.YPDL-) provirus yielded RT levels that were only about 30% lower than those of the replication-competent cmvEIAVUK(PTAP) proviral construct. Therefore, the observed variations in virus particle production do not appear to adequately explain the absolute replication phenotypes. Rather, the data indicate that the viral particles produced by the replication-defective L-domain mutants were noninfectious.



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  		FIG. 3. Analysis of viral budding from COS-1 cells transfected with the parental cmvEIAVUK and mutant L-domain proviral DNA constructs. COS-1 cells were transfected with 8 µg of cmvEIAVUK or mutant p9 proviral DNA constructs as indicated. Virions were pelleted from the transfected culture supernatant and were analyzed for RT activity. The data represent an average of at least three independent experiments, with variation indicated by standard deviation bars.

Effects of L-domain variations on EIAV Gag expression, processing, and budding. Based on the observation that replication-defective L-domain variants were able to produce virus particles that were evidently noninfectious, we next sought to examine the expression and processing of Gag proteins by the various L-domain proviral variants. For these studies, COS-1 cells were transfected in parallel with parental cmvEIAVUK or L-domain variant proviral DNA for 48 h and then radiolabeled with [35S]methionine-[35S]cysteine for 6 h. Cell supernatant medium was then collected and pelleted for analysis of virion-associated proteins, and the labeled cells were solubilized for analysis of intracellular protein expression and processing. For these experiments, the virion proteins and intracellular viral proteins were immunoprecipitated with a reference polyclonal immune serum from an EIAV-infected horse, resolved by SDS-PAGE, and quantified by phosphorimager analysis.

The data in Fig. 4A demonstrated similar levels of intracellular Gag protein expression in cells transfected with the parental cmvEIAVUK and the various L-domain variants, indicating that the L-domain substitutions and insertions did not substantially alter overall intracellular expression of Gag proteins. The data in Fig. 4B, however, revealed a marked difference in the levels of extracellular virions produced by the various cell transfections during the 6-h labeling experiment. The replication-competent MAY and MAP L-domain variants produced levels of extracellular viral particles that were similar to those of the parental EIAVUK transfection, while particle production by the replication-defective L-domain variants lacking the p9 YPDL L domain was apparently reduced by at least 10-fold. For example, particle production in transfections by the replication-defective MY.YPDL- or MP.YPDL-, each containing only an MA-associated L domain, was reproducibly about 10% of the particle production observed with the parental cmvEIAVUK under similar conditions. Transfections with the YPDL- construct, which contained no L domain, were reduced even more, being only about 1% of the particle production from the parental cmvEIAVUK. These results suggested that the observed defect in replication might at least in part be due to reductions in particle production caused by the absence of a parental YPDL-based L domain in the p9 protein. Once again, however, the correlation of replication phenotypes with virion production was not absolute, as transfection with the infectious PTAP variant produced levels of extracellular viral particles that were similar to those for the replication-defective L-domain variants and about 10-fold less than those for the replication-competent constructs (compare EIAVUK, MAY, and MAP in Fig. 4B.) This observation is consistent with the hypothesis that replication-defective L-domain variants produce reduced levels of virus particles and that these particles are noninfectious.



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  		FIG. 4. Analysis of Gag polyprotein expression and processing in COS-1 cells transfected with cmvEIAVUK and mutant L-domain proviral constructs. COS-1 cells transfected with 8 µg of cmvEIAVUK or indicated mutant p9 proviral DNA constructs were metabolically labeled with [35S]Met-[35S]Cys for 6 h. Viral proteins were immunoprecipitated from the cell lysates (A) and pelleted virions (B), separated by SDS-PAGE, and quantified by phosphorimager analysis. Positions of viral proteins are indicated by arrows.

The data presented in Fig. 3 and 4B utilized two different assays of viral budding in COS-1 cells based on measurement of either virion-associated RT or antibody-precipitated virion-associated radiolabeled Gag protein, both in pellets of extracellular virus. While the two different assays indicated similar relative levels of viral budding among the different L-domain mutants compared to wild-type virus, there were evident differences in the apparent level of reduction between certain mutants and wild-type virus as calculated from the two different assay methods. For example, viral budding by the YPDL- mutant appeared to be about 10% of that for the parental EIAV as measured by RT levels but only about 1% of that for the wild type as measured by immunoprecipitation. We attribute these apparent discrepancies to the fundamental differences in the two assay techniques. For measurements of RT levels, extracellular virus is pelleted from transfected cells after 3 days. In contrast, the immunoprecipitation assays are performed on virus pelleted from transfected-cell supernatants after only 6 h of labeling with [35S]methionine-[35S]cysteine. Thus, the latter technique provides only a short-term perspective on viral budding levels, while the RT assays average viral budding over several days. Based on these differences and the semiquantitative nature of immunoprecipitations, we consider the data in Fig. 3 a more reliable measure of virion budding efficiencies among the EIAV L-domain mutants, while Fig. 4 addresses the issue of protein processing among the mutants.


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DISCUSSION
 
Previous studies from our laboratory have demonstrated that the Gag p9 protein of EIAV is necessary for viral budding and for viral infectivity and that these distinct functions map to different p9 sequences (2). The present set of experiments demonstrated for the first time that the late budding and replication competence associated with the YPDL L domain of EIAV p9 can be replaced by the heterologous PTAP L-domain sequence from HIV-1 or the heterologous PPPY from RSV inserted in the p9 protein of an EIAV proviral construct. Thus, these results extend previous observations from our lab and others indicating that the EIAV YPDL L domain is functionally interchangeable with the heterologous RSV PPPY L domain and HIV-1 PTAP in Gag polyprotein budding assays (14, 17). While other investigators have demonstrated the functional interchangeability of proline-rich lentiviral PTAP and oncoviral PPPY L domains in viral budding and replication (29), the present study appears to be the first to reveal the functional interchangeability of heterologous YPDL and PTAP or PPPY L domains in viral replication. Thus, these EIAV data support the hypothesis that the diverse L-domain motifs may utilize different entry points to common cellular machinery for viral budding from the plasma membrane.

While the present data indicate a functional homology between the HIV-1 PTAP or RSV PPPY and EIAV YPDL L domains, they also reveal an important positional dependence that was not evident from earlier Gag polyprotein budding assays (14, 17). In this regard, replication competence of the EIAV proviral constructs required the presence of any known L domain in the p9 protein, while replacement of the p9 L domain by either the YPDL or PTAP motif at the C terminus of the MA protein produced replication-defective proviruses. It is important to note that the insertion of YPDL or PTAP sequences into the EIAV MA protein did not cause the defective phenotype, as the EIAV proviral constructs containing both MA and p9 L domains were replication competent. Therefore, these data suggest that the full functionality of the EIAV L domain in viral replication is dependent on its location in the context of the Gag p9 protein, suggesting an influence of other p9 sequences on L-domain functions.

The correlation of replication-competent and -defective phenotypes with the efficiency of viral Gag polyprotein expression, processing, and budding revealed several interesting aspects of L-domain functions in the context of proviral gene expression. First, there was not a simple correlation between the efficiency of virion budding from transfected cells and viral replication competence, as measured by the production of extracellular virion-associated RT (Fig. 3). For example, virion production by the replication-competent PTAP provirus was about fivefold less than that of the parental EIAVUK and less than twofold higher than that of the replication-defective MY.YPDL- or MP.YPDL- provirus. Thus, these data demonstrated substantial production of progeny virions by the replication-defective proviruses but suggested that the progeny viral particles were noninfectious. This lack of infectivity could not be attributed to a defect in proviral Gag polyprotein expression or processing, as replication-competent and -defective L-domain proviral mutants revealed virtually identical intracellular Gag polyprotein profiles in transfected cells. Taken together, these observations indicate that the EIAV p9 L domain is required for viral infectivity and suggest that L-domain interactions with cellular proteins used to achieve virion budding may also be utilized in the entry of the virion core into cells after fusion of the viral envelope with the cellular plasma membrane. While the budding function of the YPDL L domain can be achieved in either a p9 or an MA location, the infectivity function of the YPDL motif is retained only in the context of the p9 protein.

The indicated role of the EIAV L domain in viral infectivity appears to continue a theme established by previous studies in our lab revealing a critical role for the viral p9 in virion infection as well as virion budding. In this regard, we recently reported that only the N-terminal 31 amino acids of EIAV p9, which include the YPDL motif, are required for proviral replication competence (2). Removal of the p9 residues between E32 and YPDL yielded proviruses that produced noninfectious particles, and further truncations that removed the L domain were defective in virion production. These data indicated a synergistic interaction between the YPDL L domain and adjacent sequences in viral infectivity, with the L domain being sufficient for virion budding. The present study evidently demonstrates that the heterologous PTAP or PPPY L domain is able to effectively interact with these critical neighboring residues in the EIAV p9 to achieve the necessary functions for viral infectivity.

The precise mechanisms employed by retrovirus L domains and neighboring p9 sequences in virion budding as well as in infection of target cells remain to be determined, and these future studies will depend heavily on a more thorough interfacing of cellular biology and retrovirology. The functional homology between PTAP or PPPY and YPDL L-domain motifs implies that diverse retroviruses likely interface with similar cellular proteins and that lessons learned from one retrovirus system may indeed be applicable to others. In this regard, studies with avian and murine oncoviruses and animal lentiviruses can contribute to studies with HIV-1 to identify specific viral-host cell interactions that may serve as novel targets for antiviral drugs.


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ACKNOWLEDGMENTS
 
This work was supported by National Institutes of Health grant 5RO1 CA49296 from the National Cancer Institute.

We acknowledge the assistance of the DNA Sequencing Facility of the Biomedical Research Support Facilities at the University of Pittsburgh and Laryssa Howe for viral titration assays.


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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}pitt.edu. Back

{dagger} Present address: Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104 Back


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Journal of Virology, February 2002, p. 1569-1577, Vol. 76, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.4.1569-1577.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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