Long terminal repeat regions from exogenous but not endogenous feline leukemia viruses transactivate cellular gene expression

Abstract

We have previously reported that the long terminal repeat (LTR) region of feline leukemia viruses (FeLVs) can enhance expression of certain cellular genes such as the collagenase IV gene and MCP-1 in trans (S. K. Ghosh and D. V. Faller, J. Virol. 73:4931-4940, 1999). Genomic DNA of all healthy feline species also contains LTR-like sequences that are related to exogenous FeLV LTRs. In this study, we evaluated the cellular gene transactivational potential of these endogenous FeLV LTR sequences. Unlike their exogenous FeLV counterparts, neither nearly full-length endogenous FeLV molecular clones (CFE-6 and CFE-16) nor their isolated LTRs were able to activate collagenase IV gene or MCP-1 expression in transient transfection assays. We had also demonstrated previously that production of an RNA transcript from exogenous FeLV LTRs correlates with their transactivational activity. In the present study, we demonstrate that the endogenous FeLV LTRs do not generate LTR-specific RNA transcripts in the feline embryo fibroblast cell line AH927. Furthermore, infection of AH927 cells by an exogenous FeLV subgroup A virus did not induce production of such LTR-specific transcripts from the endogenous proviral genomes, although the LTR-specific transcripts from the exogenous virus were readily detected. Finally, LTR-specific transcripts were not generated in BALB/3T3 cells transiently transfected with isolated CFE-6 LTR, in contrast to transfections with LTRs from exogenous viruses. Our data thus suggest that the inability of endogenous FeLV LTRs in gene transactivation is not due to cell line specificity or presence of any upstream inhibitory cis-acting element. Endogenous, nonleukemogenic FeLV LTRs, therefore, do not transactivate cellular gene expression, and this property appears to be specific to exogenous, leukemogenic FeLVs.

FIG. 1

Transcriptional activation of a collagenase IV gene promoter reporter by endogenous FeLV proviral clones. One microgram of the −517/+62 Coll-CAT reporter plasmid was cotransfected with 10 μg of clone CFE-6 or CFE-16 into BALB/3T3 cells by the DEAE-dextran method. Exogenous full-length FeLV-A clone 61E (10 μg) and its LTR subclone 61E-LTR (7.5 μg) were used as positive controls. Cotransfection with 7.5 μg of backbone vector plasmid pTZ19U was used to determine the constitutive basal expression of the collagenase IV gene promoter reporter vector. Transfections with clones 61E, CFE-6, and CFE-16 were done in duplicate. Efficiency of transfection was monitored by cotransfection of 1 μg of an expression plasmid for green fluorescent protein for each plate. Forty-eight hours after transfection, cells were washed with phosphate-buffered saline and assessed microscopically for green fluorescence under UV light to normalize transfection efficiency. CAT assay was performed on the cell lysates, and products were separated by thin-layer chromatography. These experiments were repeated three times. The thin-layer chromatogram of one representative experiment is shown. Autoradiographs were photographed by AlphaImager 3.4, and quantitative analysis of the percent conversion (fold activation) for each sample was done by densitometric analysis of the image using the AlphaEase program (Alpha Innotech). Ac-Cam, acetylated chloramphenicol; Cam, chloramphenicol.

FIG. 2

Nucleotide sequence alignment of exogenous FeLV-A LTR (61E) with endogenous FeLV LTRs CFE-6 and CFE-16. Sequence information was obtained from GenBank (accession numbers are M18247, M21479, and M21480, respectively) and from reference . Sequence alignment was performed by the MegAlign program available in the sequence analysis program package LASERGENE from DNASTAR, Inc., Madison, Wis. Positions of the RT and PCR primers used in the study are shown in boxes with the primer name above (for 61E) or below (for CFE-6). Complete information on these primers is available in Table 1.

FIG. 3

RT-PCR analysis of the cellular RNA transcripts from uninfected and FeLV-A-infected AH927 cells. Total cellular RNA was isolated from actively growing AH927 cells by guanidine thiocyanate extraction followed by phenol-chloroform extraction as described elsewhere (17). Genomic DNA from AH927 cells was isolated by the sodium dodecyl sulfate–proteinase K digestion method (31). PCR products were separated on 2% agarose gels. PstI-cut lambda DNA was used as molecular weight markers (M). The migration positions and sizes of the amplified products are indicated. To demonstrate specificity of the primers used, simple PCR analysis of genomic or plasmid DNA was also carried out and analyzed in the same gel. (A) Analysis with oligonucleotide P4 as the RT- and 3′-PCR primer. Lanes 1, 3, and 5, no RT; lanes 1 and 2, RNA from FeLV-A 61E-infected AH927 cells; lanes 3 to 6, RNA from uninfected AH927 cells; lane 11, control (Cont) PCR with P4 and P2 primers with no template added. (B) Analysis with oligonucleotide P3 as RT- and 3′-PCR primer. Lanes 1, and 3, no RT; lanes 1 to 4, RNA from uninfected AH927 cells. (C) Analysis of total RNA from FeLV-A 61E-infected AH927 cells. Lanes 1 and 3, no RT; lanes 1 to 6, RNA from FeLV-A 61E-infected AH927 cells. Exogenous FeLV-A plasmid clones 61E-LTR (lanes 7 and 10) and endogenous FeLV plasmid clone CFE6-LTR (lanes 8 and 11) were used as PCR controls. Lane 9 is a control PCR with P3 and P6 primers with no template added. (D) Schematic diagram of the FeLV LTR and locations of primers used for RT-PCR. T, TATA box; A, polyadenylation site.

FIG. 4

Analysis of transactivational activity and LTR-specific RNA transcript production by the endogenous LTR clones. (A) Schematic diagram of the exogenous (Exo) and endogenous (Endo) FeLV LTR clones. Specific LTR fragments were PCR amplified using primer pairs indicated in the text and cloned into the pGEM3 vector. (B) Transactivational activity of the LTR clones. Each LTR-containing plasmid (7.5 μg) was cotransfected with 1 μg of −517/+62 Coll-CAT reporter plasmid into BALB/3T3 cells. CAT activities in these cells were analyzed 48 h later as described for Fig. 1. Cotransfection of pGEM3 and −517/+62 Coll-CAT was used to determine basal expression of the reporter. This assay was performed three times with similar results. One representative chromatogram is shown. (C) RT-PCR analysis of LTR-specific RNA transcript production by the LTR clones. Individual clones were transfected in BALB/3T3 cells as described above; 48 h later, total RNA was extracted from the transfected cells. RNA samples were DNase treated prior to RT-PCR analysis as described for Fig. 2. RT- and 3′-PCR primers were P3 for 61E-LTR and CFE6-LTR, P19 for 61E-H, P20 for CFE6-3, and P4 for 61E. The 5′-PCR primer for LTR clones of exogenous origin (61E, 61E-LTR, and 61E-H) was P2; the 5′-PCR primer for LTR clones of endogenous origin (CFE6-LTR and CFE6-3) was P5. PstI-digested lambda DNA was used as molecular weight markers (M). Sizes of the amplified products are indicated.