Antisense transcription in gammaretroviruses as a mechanism of insertional activation of host genes

Abstract

Transcription of retroviruses is initiated at the U3-R region boundary in the integrated provirus and continues unidirectionally to produce genomic and mRNA products of positive polarity. Several studies have recently demonstrated the existence of naturally occurring protein-encoding transcripts of negative polarity in complex retroviruses. We report here on the identification of transcripts of negative polarity in simple murine leukemia virus (MLV). In T-cell and B-cell lymphomas induced by SL3-3 and Akv MLV, antisense transcripts initiated in the U3 region of the proviral 5' long terminal repeat (LTR) and continued into the cellular proto-oncogenes Jdp2 and Bach2 to create chimeric transcripts consisting of viral and host sequence. The phenomenon was validated in vivo using a knock-in mouse model homozygous for a single LTR at a position known to activate Nras in B-cell lymphomas. A 5' rapid amplification of cDNA ends (RACE) analysis indicated a broad spectrum of initiation sites within the U3 region of the 5' LTR. Our data show for the first time transcriptional activity of negative polarity initiating in the U3 region of simple retroviruses and suggest a novel mechanism of insertional activation of host genes. Elucidation of the nature and potential regulatory role of 5' LTR antisense transcription will be relevant to the design of therapeutic vectors and may contribute to the increasing recognition of pervasive eukaryotic transcription.

FIG. 1.

Detection of antisense transcripts in lymphomas of SL3-3 and Akv MLV-infected mice. (A) Exons and splicing structure of canonical Jdp2 and the alternative 1f-3-4 mRNAs are depicted with coding sequence in black. Positions and the predominant proviral orientations in integration clusters B/C and D are indicated with arrows. Position and orientation of Jdp2 primers 51 and 57 is also shown. (B) The number of individual 5′ RACE transcription start site (TSS) tags on the negative strand of the 5′LTR U3 of a cluster D integrated provirus. (C) RT-PCR on RNA from eight tumor samples using primers that are amplicon specific to asU3-generated Jdp2 chimeras (140+57), canonical Jdp2 using primers in exon 2 and 4 (51+57), and β-Actin (Actb) as the control. In lane 9, no reverse transcriptase enzyme was added in the cDNA preparation (-enz). A schematic representation of the most frequently found asU3-Jdp2 structure was generated using the splice donor site in exon 1f. The transcriptional orientation of the proviral LTR is indicated with gray triangles. (D) RT-PCR of eight cluster B/C tumors using the indicated primers. Legend as for panel B. (E) RT-PCR on RNA from eight B-cell tumors induced by Akv MLV. To the right, a schematic of two of the three identified asU3-Bach2 chimeric transcripts, showing the usage of a cryptic splice donor site (SD′) and the PCR amplicons.

FIG. 2.

Antisense U3-1f chimeric transcript levels are comparable to those initiated in the cellular promoter of exon 1f. (A) Strategy for comparison of the levels of 1f transcripts initiated using asU3 transcription or using the cellular promoter. Structures of the Jdp2 gene and generated mRNAs are shown together with the positions and orientations of the primers used. (B) RT-PCR on RNA from four selected tumor samples at two concentrations (×5 and ×1). PCR amplifications of 10-fold dilutions (from one million to one thousand copies) of a plasmid with an asU3-1f-3-4 insert are loaded to the left (pDNA). The amplicon 164+57 represents total 1f transcript, whereas the amplicon 140+57 measures the contribution from antisense-generated 1f transcript. RT-PCR for total Jdp2 (amplicon 47+57) and canonical Jdp2 (amplicon 51+57) and Actb is also shown. The 140+57 amplicon size (295 bp) is given for the pDNA amplification product. In lane 14, no reverse transcriptase enzyme was added in the cDNA preparation (-enz). Lane numbers are indicated below. (C) Quantitative real-time PCR assay of tumors predominantly activating Jdp2 exon 1f. Tumors 1 to 3 are the same as in panel B. The contribution of asU3-mediated transcription to total 1f mRNA levels is shown with black bars (left axis), while the relative levels of total Jdp2 (amplicon 47+57) is shown with white bars (right axis). In tumor 6, the signal from the asU3 amplicon was below the meaningful detection level (n.d.).

FIG. 3.

Antisense transcription is dependent only on the LTR. (A) Schematic structure of the Nras gene with coding sequence in black. The transcriptional orientation of the neo-LTR cassettes are indicated with triangles. Also shown are the structures of three asU3-Nras transcripts and the primers used. The relative positions of primers 140 and 2620 on U3 are indicated. (B) RT-PCR on RNA from spleens from two mice with an LTR knock-in transcriptionally identical to Nras (lanes 1 and 2) and two mice with an LTR knock-in transcriptionally opposite to Nras (lanes 3 and 4). The identities of the identified structures are shown to the right, and RT-PCR amplicons are shown to the left. (C) Number of 5′ RACE TSS tags on the negative strand of the 5′LTR U3 isolated from a mouse spleen with a LTR in transcriptionally opposite orientation compared to Nras. The mRNA structures (SD-130, SD-73, and SD+34/R.T.) associated with the TSS regions are indicated on the top. (D) The experiments in panel B were repeated with mice in which the neomycin cassette had been removed. R.T., read-through transcript.

FIG. 4.

Antisense transcription is not detectable in chronically infected cell cultures. Northern blot analyses of total RNA from uninfected (NIH), or SL3-3 MLV (S)- and Akv1-99 (A)-infected NIH 3T3 cells using an oligonucleotide probe specific for antisense or sense LTR transcription as described in Materials and Methods.