Duplicated sequence motif in the long terminal repeat of maedi-visna virus extends cell tropism and is associated with neurovirulence

Abstract

Maedi-visna virus (MVV) is a lentivirus of sheep causing chronic inflammatory disease of the lungs (maedi) and the nervous system (visna). We have previously shown that a duplicated sequence in the long terminal repeat (LTR) of MVV is a determinant of cell tropism. Here, we demonstrate that deletion of a CAAAT sequence from either one of the repeats resulted in poor virus growth in sheep choroid plexus cells. A duplication in the LTR encompassing the CAAAT sequence was found in four neurological field cases that were sequenced, but no duplication was present in the LTRs from seven maedi cases; one maedi isolate was mixed. These results indicate that the duplication in the LTR is associated with neurovirulence.

FIG. 1.

Portion of the U3 region of the LTR of the MVV recombinant clones VA3 and VA4 comprising the duplications studied in this work. The repeated sequence is marked with vertical lines. The potential transcription factor binding sites where the sequence is identical to the consensus sequence are indicated by a solid line under the sequence; dotted lines below the sequence indicate sites that deviate from the consensus sequence by one nucleotide. Nucleotide deletions are indicated by dashes. The ability of each virus strain to replicate (+) or not (−) in SCP cells is indicated on the right.

FIG. 2.

Growth curves of the various LTR deletion variants in SCP cells (A) and macrophages (B) as measured by RT assays. Virus strains with one or two API sites (−10 and −20 respectively), the AML site (AMLa), and CAAAT (CAAATa) deleted from the first copy of the repeat in the LTR and CAAAT deleted from the second copy of the repeat (CAAATb) are shown. kcpm, counts per minute (in thousands).

FIG. 3.

Relative luciferase activity of the LTRs with and without a repeat (VA4 and VA3, respectively) and with deletions of the CAAAT sequence and the AML site from VA3 in SCP cells. pBudCE4.1/lacZ/CAT was cotransfected for normalization of transfection efficiency. The experiments were repeated three times, and the mean values plus standard deviations (error bars) are shown in comparison to VA3 values.

FIG. 4.

Sequences of the regions in the LTRs comprising the duplications in the molecular clones KV1772, VA3, and VA4 and in field cases of visna and maedi. Duplicated sequences are shown only once, but are shown with a thick black line above the sequence. Identical nucleotides (dots) and deletions (dashes) are indicated. The CAAAT sequence is underlined.