AZT-resistant foamy virus

Abstract

Azidothymidine (AZT) is a reverse transcriptase (RT) inhibitor that efficiently blocks the replication of spumaretroviruses or foamy viruses (FVs). To more precisely elucidate the mechanism of action of the FV RT enzyme, we generated an AZT-resistant FV in cell culture. Biologically resistant virus was obtained for simian foamy virus from macaque (SFVmac), which was insensitive to AZT concentrations of 1 mM, but not for FVs derived from chimpanzees. Nucleotide sequencing revealed four non-silent mutations in the pol gene. Introduction of these mutations into an infectious molecular clone identified all changes to be required for the fully AZT-resistant phenotype of SFVmac. The alteration of individual sites showed that AZT resistance in SFVmac was likely acquired by consecutive acquisition of pol mutations in a defined order, because some alterations on their own did not result in an efficiently replicating virus, neither in the presence nor in the absence of AZT. The introduction of the mutations into the RT of the closely related prototypic FV (PFV) did not yield an AZT-resistant virus, instead they significantly impaired the viral fitness.

Fig. 1

Homology of Pol proteins of different FV and HIV-1. The RT regions of known FVs relevant to this study were aligned using the Genbank accession numbers for SFVmac (X58484 and M33561), PFV (Y07725), the chimpanzee isolate SFVcpz (U04327), orangutan FV (SFVora; AJ544579), African green monkey virus (SFVagm; M74895), FV from felines (FFV: AJ564746), bovines (BFV; U94514) and equines (EFV; AF20190).

Fig. 2

Genome organization of the wild-type and mutant FV Pol open reading frames. The pol gene of SFVmac is shown in the upper panel with the relative location of the RT active center and the four amino acid residues associated with AZT resistance. The SFVmac mutants representing all possible combinations of the four mutations are shown in the lower panel together with the approximate levels of AZT resistance deduced from Table 2. The plasmids pBK04-ITTK and pBK64-ITTK are identical. Plasmid pBK04-ITTK was made by exchanging a pol gene fragment containing the four mutations and amplified from DNA of cell cultures infected with SFVAZTres for a corresponding fragment of pSK29-KISE and pBK64-ITTK was made by in vitro mutagenesis. M108 is a PFV mutant with the changed residues found in SFVAZTres.

Fig. 3

SFVmac protein expression. The expression of Gag (pr71/p68) and Pol (pr127) proteins was analyzed by immunoblot with polyclonal rabbit antisera in lysates from cells transfected with the indicated plasmids.

Fig. 4

Most likely order of mutagenic events resulting in AZT-resistant SFVmac.