Assessment of bovine leukemia virus transcripts in vivo

Abstract

Reverse transcriptase PCR (RT-PCR) consistently detected bovine leukemia virus transcripts in fresh cells, and competitive RT-PCR enumerated these transcripts. The detection of transcripts in limited numbers of tumor cells indicated that expression occurs in a minority of cells. The data suggest that individual cells contain hundreds of copies of the tax/rex transcript in vivo.

FIG. 1

Representation of the BLV genome, open reading frames, transcripts, and the products of translation of spliced transcripts. The locations of primers are indicated on the genomic RNA, and their identifiers are underlined. Vertical dashed lines align common termini of transcripts, exons, and reading frames. Splice donor and acceptor sites are numbered according to the proviral sequence described by Sagata et al. (42). Names and splice sites of alternate transcripts and their predicted protein products are from references (alt), (tof/rof), and (RIII, 305/7018, GIV, and 502/7157).

FIG. 2

Amplification of X region transcripts. Fifty-nanogram quantities of DNase I-treated, poly(A)⁺ RNA preparations were amplified by RT-PCR with BLV primers 5′B1 and 3′B2 (top panel) or with actin primers 5′ACT1 and 3′ACT2 (middle panel). The bottom panel shows the results of hybridization of BLV amplicons with X probe. Samples include PBMC from a BLV-seronegative animal (BLV−), PBMC from seropositive, non-PL animals (BLV+), PBMC from seropositive, PL animals (PL), and ML cells. Five-nanogram quantities of control poly(A)⁺ RNA preparations were used (BL3, BL3*, NBC-13, and NBC-13 plus PMA). The numbers over the bottom panel indicate samples which were subsequently subjected to QC RT-PCR. The molecular weight (MW) lane contained a 50-bp ladder. The amplified products of tax/rex and alt transcripts are 414 and 191 bp, respectively.

FIG. 3

Quantitation of tax/rex transcript in NBC-13 cells. BglI- or HindIII-digested products of QC RT-PCR were subjected to electrophoresis and Southern analysis with the X probe. The input of cellular RNA, in nanograms of poly(A)⁺ RNA, and competitor RNA, in femtograms of competitor, and the presence of RT in reaction mixtures are shown above the autoradiographs. An arrow indicates the relative equivalence point. The molecular weight (mw) lane contains a 50-bp ladder with an intense band at 350 bp. BglI digestion yields a 211-bp fragment from tax/rex transcript amplicons, and HindIII digestion yields a 206-bp band from tax/rex competitor RNA amplicons.

FIG. 4

RT-PCR of limited numbers of BLV-infected cells. Twofold serial dilutions of cells were lysed and subjected to RT-PCR for BLV or β-actin transcripts. The actual numbers of cells in dilutions under 50 were confirmed visually and varied by no more than three cells. (A) Five samples at each dilution of ML cells were tested for tax/rex transcripts, two without and three with RT enzyme. The products of nested primers, 5′TR1 and 3′TR2, are 241 bp. (B) Samples were the same as in panel A but were amplified with nested β-actin primers 5′ACTN1 and 3′ACTN2, which yield a 165-bp product. (C) RT-PCR of lysates from serial twofold dilutions of NBC-13 cells with tax/rex-specific primers 5′TR1 and 3′TR2 (241 bp) or alt specific primers 5′ALT and 3′TR2 (89 bp). (D) Amplification of two BL3* cells as described for panel C. Southern blots were hybridized with X probe or actin probe (Table 1).

FIG. 5

Indirect immunofluorescence staining of NBC-13 cells. (A) Photomicrograph of a rare positive cell, stained with a monoclonal antibody for BLV p24 and goat anti-mouse immunoglobulin G-fluorescein isothiocyanate, prior to PMA treatment (magnification, ×600; Evans blue counterstain). (B) NBC-13 cells 24 h after treatment with 10 nM PMA (magnification, ×600). (C) Flow cytometric analysis of NBC-13 cell preparations as described for panels A and B stained with a control monoclonal antibody (Ab) or with anti-p24.