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. 2002 Feb;76(4):1569-77.
doi: 10.1128/jvi.76.4.1569-1577.2002.

Functional replacement and positional dependence of homologous and heterologous L domains in equine infectious anemia virus replication

Feng Li  1 , Chaoping Chen, Bridget A Puffer, Ronald C Montelaro
Affiliations

Functional replacement and positional dependence of homologous and heterologous L domains in equine infectious anemia virus replication

Feng Li et al. J Virol. 2002 Feb.

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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Figures

FIG. 1.
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.
FIG. 2.
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.
FIG. 3.
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.
FIG. 4.
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.
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