Basic residues in the foamy virus Gag protein

Abstract

Foamy virus (FV) capsid proteins have few lysines. Basic residues are almost exclusively represented by arginines indicating positive selective pressure. To analyze the possible functions of this peculiarity, we mutated an infectious molecular clone of the prototypic FV (PFV) to harbor lysines in the Gag protein at arginine-specifying positions and analyzed various aspects of the FV replication cycle. The majority of mutants replicated equally as well in permanent cell cultures as the original wild-type (wt) virus and were genetically stable in gag upon 10 cell-free passages. With respect to the features of late reverse transcription, nucleic acid content, and infectiousness of the virion DNA genome, the majority of mutants behaved like the wt. Several mutants of PFV were ubiquitinated in Gag but unable to generate virus-like particles (VLPs) or to undergo pseudotyping by a heterologous envelope. Using primary cells, however, a replicative disadvantage of the majority of mutants was disclosed. This disadvantage was enhanced upon interferon (IFN) treatment. We found no evidence that the lysine-bearing gag mutants showed more restriction than the wt virus by tetherin (CD317) or Trim5α. A single lysine in PFV Gag was found to be nonessential for transient replication in permanent cell culture if replaced by an arginine residue. Upon replication in primary cells, even without IFN treatment, this mutant was severely impaired, indicating the importance of specifying at least this lysine residue in PFV Gag. The paucity of lysines in FV Gag proteins may be a consequence of preventing proteasomal Gag degradation.

Fig. 1.

PFV Gag sequences and mutants analyzed in this study. The protein sequence of PFV Gag is shown, and the generated mutants are indicated.

Fig. 2.

Semiquantitative analysis of the infectiousness of virion DNA from PFV. DNAs were extracted from virions, and indicator cells were transfected with 1 μl of virion DNA corresponding to 0.8 ml of supernatant or 10-fold dilutions thereof, as indicated. Controls included the transfection of cells with the infectious plasmid pcPFV2, with pcPFV2 treated with DNase I, and with pcDNA3.1. After the transfection of cells, 1 ml of the cell-free supernatant was transferred to fresh indicator cells, which were subsequently stained for blue foci, scored as follows: +++, approximately 10%; ++, approximately 5%; +, up to 0.1%; −, no blue cell foci; n.d., not determined. A representative example of two individual experiments yielding virtually similar results is shown.

Fig. 3.

Ubiquitination and analysis of pseudotyping and VLP formation by PFV Gag. (A) The ubiquitination of wt and mutated PFV was analyzed by transfecting HEK 293T cells with the respective proviral constructs together with the HA-tagged ubiquitin-encoding plasmid pBJ5 HA-Ubi. Cellular lysates and extracellular viral particles were prepared and probed with antisera against GAPDH and PFV Gag (cellular lysate) or the HA-tag and Gag (viral particles). Oligo-ubiquitination is readily visible for the Env-LP and for the lysine mutants (as indicated). (B) A potential pseudotyping or VLP formation of altered PFV capsids was analyzed by transfecting the cells with the vector pTW01 and subgenomic constructs specifying wt or mutant PFV gag, wt pol, wt env, the heterologous glycoprotein VSV-G, or no glycoprotein (VLP formation), as indicated. The immunoblot was developed using antisera against Gag, VSV-G, and GAPDH. All mutants still required authentic Env for particle export. In contrast, when inserted into the proviral plasmid backbone, M144 led to premature cleavage or expression of a truncated Gag when inserted into the subgenomic expression plasmid. α, anti.