Characterization of provirus clones of simian foamy virus type 1

Abstract

We have cloned proviral DNA of simian foamy virus type 1 (SFV-1) from linear unintegrated DNA (pSFV-1). Transfection of pSFV-1 induces cytopathology in several cell lines with supernatants from the transfected cell culture containing infectious viral particles. Electron microscopy of the transfected cells revealed foamy virus particles. Deletion analysis of pSFV-1 indicated that the transcriptional transactivator (tas) gene located between env and the long terminal repeat is critical for virus replication, whereas the second open reading frame (ORF-2) in this region is dispensable. Although the tas and ORF-2 regions of foamy viruses have significantly diverged, the results presented here suggested that the gene products have similar functions. Recombinant pSFV-1 containing the cat gene was able to transduce the heterologous gene, indicating the utility of SFV-1 as a vector. An infectious clone of SFV-1 which is distantly related to the human foamy virus will provide a means to understand the biology of this unique group of viruses.

FIG. 1

Infectivity of pSFV-1 in the 293, L-929, and COS-7 cell lines. (A) Levels of CPE on infected cells at different time points after pSFV-1 transfection. Levels of cytopathology were scored as follows: −, no CPE; +, 10 to 20% CPE; ++, 30 to 50% CPE; +++, 60 to 70% CPE; ++++, 80 to 90% CPE. Lysis refers to greater than 99% loss of cells in the infected cell culture. The numbers at the top indicate days after DNA transfection or virus infection. (B) RT analysis of supernatants from cell cultures transfected with pSFV-1 at different time intervals. RT was assayed under conditions optimized for foamy virus with Mn²⁺ cation (3). Samples were harvested at the indicated time intervals in triplicate, and RT activity was assayed. Less than 10% variation in replicate samples was observed.

FIG. 2

Transmission EM analysis of virus particles from pSFV-1-transfected (A) and SFV-1-infected (B) L-929 cells. Virus particles are indicated with arrowheads (magnification, ×92,000). For ultrathin sectioning, cells were fixed with 2.5% glutaraldehyde in phosphate buffer at room temperature for 1 h, postfixed with 1% osmium tetroxide for 1 h, dehydrated in a graded series of ethanol, and embedded in Embed 812 (Polysciences, Warrington, Pa.). Ultrathin sections were stained with uranyl acetate and lead citrate. Stained sections were placed on grids and examined with a Hitachi H7000 transmission electron microscope (Hitachi Instruments, San Jose, Calif.).

FIG. 3

Mutational analysis of infectious pSFV-1. (A) Genome organization of pSFV-1 and mutant derivatives. The hatched lines represent the deleted regions. pSFV-1/taorf2 and pSFV-1/enorf2 are proviral clones with natural deletions. (B) Effect of mutations on virus replication. Plus and minus signs represent the presence and the absence of a CPE, respectively. Samples for RT assays were harvested at day 9 posttransfection.

FIG. 4

Assays of CAT reporter transient expression in cells infected with recombinant virus particles containing the cat gene. Recombinant pSFV-1 containing the cat gene (pSFV-1/cat) or the cat gene under control of the SV40 promoter (pSFV-1/svcat) was constructed by replacing the indicated region of the SFV-1 genome. Filtered supernatants from cell cultures transfected with this plasmid were used to infect cells. pSFV-1 LTR/CAT41 and pTM1/CAT are positive controls transfected into cells to monitor the level of CAT activity. p22A2 is a plasmid containing a promoterless cat gene that was used as a negative control. The values shown are from reactions measuring the conversion of ³H-acetyl coenzyme A to ³H-acetylated chloramphenicol. Generally, less than 5% variation in replicate samples was observed. The CAT values in L-929 cells represent basal activity. Relative activity was calculated by dividing the CAT values in L-929-tas cells by the basal levels in L-929 cells. To calculate the relative activity for pSFV-1/svcat-infected cells, the CAT value of pSFV-1/cat in L-929 cells was used as the basal level.