Further characterization of equine foamy virus reveals unusual features among the foamy viruses

Abstract

Foamy viruses (FVs) are nonpathogenic, widely spread complex retroviruses which have been isolated in nonhuman primates, cattle, cats, and more recently in horses. The equine foamy virus (EFV) was isolated from healthy horses and was characterized by molecular cloning and nucleotide sequence analysis. Here, to further characterize this new FV isolate, the location of the transcriptional cap and poly(A) addition sites as well as the main splice donor and acceptor sites were determined, demonstrating the existence of the specific subgenomic pol mRNA, one specific feature of FVs. Moreover, similar to what has been described for the human foamy virus (HFV), the prototype of FVs, a replication-defective EFV genome was identified during persistent infection. At the protein level, the use of specific antibodies allowed us to determine the size and the subcellular localization of EFV Gag, Env, and Tas, the viral transactivators. While EFV Gag was detected in both the cytoplasm and the nucleus, EFV Env mainly localized in the Golgi complex, in contrast to HFV Env, which is sequestered in the endoplasmic reticulum. In addition, electron microscopy analysis demonstrated that EFV budding occurs at the plasma membrane and not intracellularly, as is the case for primate FVs. Interestingly, EFV Tas was detected both in the nucleus and the cytoplasm of Tas-transfected cells, in contrast to the strict nuclear localization of other FV Tas but similar to the equine infectious anemia virus Tat gene product. Taken together, our results reveal that this new FV isolate exhibits remarkable features among FVs, bringing new insights into the biology of these unconventional retroviruses.

FIG. 1.

Characterization of the transcriptional cap and poly(A) addition sites of EFV. (A) Primer extension performed on total RNA from EFV-infected ED cells revealed that the LTR cap site (1) is located at position 1127 (left panel), whereas the IP cap site (2) is located at position 9256 (right panel). These sites are represented on a schematic diagram (B). The polyadenylation signal has been located at position 11860 on the basis of sequence analysis and the poly(A) addition site at position 11890 on the basis of RT-PCR results. (C) Sequence alignment reveals that the EFV IP cap site is more distant from the TATA box than other described FV cap sites (the TATA box is in bold lettering, and cap sites are underlined).

FIG. 2.

EFV splicing pattern. (A) The different viral mRNA species from EFV-infected ED cells are detected by Northern blot with a specific radiolabeled U3-R probe. N.I., not infected. (B) RT-PCR assays performed on total RNA from EFV-infected cells with distinct sets of primers (black arrowheads) reveal a complex pattern of viral mRNA expression. The splice donor (SD) and splice acceptor (SA) sites are indicated for each viral mRNA species. Note the presence of a second SD site at the R region of the 5′ LTR (position 1257). A putative ORF2-encoding mRNA was also isolated. These two mRNA species were found in a single clone (*). (C) Alignment of the pol mRNA splice junctions of HFV, BFV, FFV, and EFV. SD and SA are indicated in bold lettering. nt, nucleotides.

FIG. 3.

Characterization of a Tas-defective EFV DNA genome. Genomic DNA from BHK21 cells acutely or persistently infected with EFV was analyzed by Southern blot using a probe encompassing the bel region. Plasmids coding for EFV Tas (pSVEFV-Tas) or EFV Bet (pSGEFV-Bet, harboring a 297-bp deletion in the tas gene) were used as size controls. A Tas-defective DNA genome is detected in cells persistently infected with EFV.

FIG. 4.

Characterization of the EFV proteins. (A) Proteins extracted from EFV-infected ED cells were immunoprecipitated with a rabbit anti-EFV antiserum or mouse anti-Gag or anti-Env antibodies. The EFV Gag doublet is detected at 66/62 kDa, whereas the Env precursor is detected at 130 kDa and the TM and SU subunits are detected near 46 and 76 to 80 kDa, respectively. EFV Bet is detected at 56 kDa. A 160-kDa protein which is detected with both anti-EFV and anti-Env antisera likely represents an Env-Bet fusion glycoprotein. Immunoprecipitation of nuclear (N) and cytoplasmic (C) fractions of Env- or Gag-expressing BHK21 cells revealed that, while Env is mainly detected in the cytoplasm, Gag is distributed in both the cytoplasm and the nucleus. The efficiency of the subcellular fractionation is assessed by Western blot by using a monoclonal antibody directed against the LDH. (B) Confocal analysis of Gag-transfected BHK21 cells performed 20 h posttransfection (top) or 48 h posttransfection (bottom). Whereas Gag is detected at the vicinity of the centrosome (detected with an anti-centriole antibody, CTR910) early after transfection, its localization is mainly nuclear 48 h posttransfection. Nuclei were revealed with DAPI.

FIG. 5.

Subcellular distribution of the EFV glycoprotein. (A) Confocal microscopy of pSGEFVEnv- or pSGHFVEnv-transfected Cos6 cells reveals that EFV Env mainly localized in the cis-Golgi complex stained with anti-GM130 antibodies. Nuclei are stained with DAPI in merge images. (B) Electron micrographs of ultrathin sections of EFV- or HFV-infected BHK21 cells. Images represent budding of viral particles at the plasma membrane for EFV and at internal membranes for HFV. The bars correspond to 1 μm.

FIG. 6.

Subcellular distribution and transactivation properties of EFV Tas. (A) Western blot analysis of nuclear and cytoplasmic fractions of pSVEFV- and pSVHFV-Tas-transfected Cos6 cells. EFV Tas is distributed both in the nucleus and the cytoplasm, whereas HFV Tas is mainly nuclear. (B) Confocal microscopy of cells expressing EFV Tas or HFV Tas confirms the biochemical data and reveals that EFV Tas is mainly perinuclear. Nuclei are stained with DAPI.