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. 2001 Oct;75(20):9762-70.
doi: 10.1128/JVI.75.20.9762-9770.2001.

Functional roles of equine infectious anemia virus Gag p9 in viral budding and infection

C Chen  1 , F Li, R C Montelaro
Affiliations

Functional roles of equine infectious anemia virus Gag p9 in viral budding and infection

C Chen et al. J Virol. 2001 Oct.

Abstract

Previous studies utilizing Gag polyprotein budding assays with transfected cells reveal that the equine infectious anemia virus (EIAV) Gag p9 protein provides a late assembly function mediated by a critical Y(23)P(24)D(25)L(26) motif (L-domain) to release viral particles from the plasma membrane. To elucidate further the role of EIAV p9 in virus assembly and replication, we have examined the replication properties of a defined series of p9 truncation and site-directed mutations in the context of a reference infectious molecular proviral clone, EIAV(uk). Characterization of these p9 proviral mutants revealed new functional properties of p9 in EIAV replication, not previously elucidated by Gag polyprotein budding assays. The results of these studies demonstrated that only the N-terminal 31 amino acids of a total of 51 residues in the complete p9 protein were required to maintain replication competence in transfected equine cells; proviral mutants with p9 C-terminal truncations of 20 or fewer amino acids remained replication competent, while mutants with truncations of 21 or more residues were completely replication defective. The inability of the defective p9 proviral mutations to produce infectious virus could not be attributed to defects in Gag polyprotein expression or processing, in virion RT activity, or in virus budding. While proviral replication competence appeared to be associated with the presence of a K(30)K(31) motif and potential ubiquitination of the EIAV p9 protein, mutations of these lysine residues to methionines produced variant proviruses that replicated as well as the parental EIAV(uk) in transfected ED cells. Thus, these observations reveal for the first time that EIAV p9 is not absolutely required for virus budding in the context of proviral gene expression, suggesting that other EIAV proteins can at least in part mediate late budding functions previously associated with the p9 protein. In addition, the data define a function for EIAV p9 in the infectivity of virus particles, indicating a previously unrecognized role for this Gag protein in EIAV replication.

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Figures

FIG. 1
FIG. 1
Summary of p9 truncations engineered into the reference EIAVuk proviral clone. (A) Diagram of the Gag and Pol genome and amino acid sequences of EIAV p9. The boxed YPDL is the L-domain for EIAV budding and release. (B) Schematic summary of p9 truncations. Each p9 mutant is denoted by a single-letter amino acid followed by a number to represent the residue of p9 that is replaced with a stop codon. For example, the K31 mutant designation indicates that the lysine residue at the 31st position of p9 is replaced with a stop codon (asterisk).
FIG. 2
FIG. 2
Replication properties of EIAV proviral p9 mutants in transfected ED cells as monitored by extracellular RT activity. ED cells were transfected in parallel with the various proviral mutant DNA samples, and supernatants from transfected cells were collected at weekly intervals posttransfection for measurements of RT activity, as described in Materials and Methods. Proviral p9 mutants are under the EIAVuk backbone as described in Fig. 1.
FIG. 3
FIG. 3
Effects of p9 truncations on production of extracellular virions. Cos-1 cells were transfected in parallel with equal amounts of cmvEIAVuk plasmid DNA preparations from the indicated p9 truncation mutants. After 3 days, equal volumes of supernatant medium from each culture were collected and subjected to ultracentrifugation to pellet virus particles. The quantities of virions from different p9 truncations were then analyzed by SDS-PAGE and immunoblotting with a high-titer reference equine immune serum to blot virion Gag proteins. Full-length Gag precursor p55, the intermediates p47 (MA-CA-NC) and p40 (MA-CA), and the mature capsid protein p26 are indicated by arrows. The data presented in this figure are representative of at least three independent transfections. Estimations of relative levels of extracellular virion Gag protein production by each proviral mutant were made using densitometer scans of Gag protein bands in the immunoblot.
FIG. 4
FIG. 4
Effect of p9 truncations on incorporation of RT activity into progeny virions. Cos-1 cells were transfected in parallel with equal quantities of the indicated cmvEIAVuk proviral plasmid DNAs using standard transfection conditions. After 3 days, virus particles were pelleted by centrifugation of equal volumes of cell supernatant medium, and the quantity of EIAV Gag p26 was determined by Western blotting with a reference murine monoclonal antibody, as described in Materials and Methods. Based on p26 content, equal amounts of EIAV virions were then assayed for RT activity. The presented data represent the averages of results from at least three experiments.
FIG. 5
FIG. 5
Effects of p9 truncations on Gag protein expression and proteolytic processing. Equal amounts of plasmid DNA samples containing the respective p9 truncation mutations were used to transfect Cos-1 cells. After 3 days, the transfected cells and supernatant medium were collected for analysis of intracellular and virion-associated EIAV Gag proteins, respectively. (A) Transfected cells were lysed, and intracellular EIAV Gag protein expression was analyzed by SDS-PAGE and Western blotting using the reference (Lady) equine immune serum. (B) Supernatant media from the transfected cells were subjected to ultracentifugation to pellet virus particles that were resuspended and quantified for p26 content using a murine monoclonal antibody. Based on virion p26 content, equal amounts of pelleted virus were then analyzed by SDS-PAGE and immunoblotting with Lady serum for Gag protein profiles. The various p9 mutants are listed on the top of the gel. The full-length Gag precursor p55 is indicated with an arrow. The p9 truncation mutations generated a series of Gag precursors with decreasing molecular weights from right to left, as expected. The major processing intermediates, p47 (MA-CA-NC) and p40 (MA-CA), and mature capsid protein p26 are also indicated by arrows.
FIG. 6
FIG. 6
Role of the K30K31 sequences of EIAV p9 in viral replication. The EIAVuk proviral clone was specifically mutated to substitute methionine residues for both the K31 and K32 residues. The replication properties of the KK/MM mutant were then analyzed by transfection of ED cells and compared to a parallel transfection with an identical amount of the parental EIAVuk proviral plasmid. Viral replication was monitored by extracellular RT activity, as described in the legend for Fig. 2.
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