The 3′-untranslated region of alfalfa mosaic virus RNA 3 contains at least two independent binding sites for viral coat protein

Abstract

The 3'-termini of the three genomic RNAs of alfalfa mosaic virus contain a common sequence of 145 nucleotides (nt) with a specific binding site for coat protein (CP). This sequence consists of several stem/loop structures interspersed with single-stranded AUGC-motifs; in RNA 3 this folding pattern is extended to a region upstream of the homologous sequence. By band-shift assays a minimum of two specific binding sites for CP were identified near the 3'-end of RNA 3. Site 1 consists of the region between nt 11 and 127 from the 3'-end and contains two AUGC-motifs. Site 2 is located between nt 133 and 208 from the 3'-end in a sequence that is largely unique to RNA 3 and contains also two AUGC-motifs. Deletion studies revealed that the two sites could bind CP independently of each other and permitted the identification of sequence elements that are essential for the activity of each site. By site-directed mutagenesis it was shown that the AUGC-motifs are important for binding of CP to both sites. These binding sites may play a role in the phenomenon that each genomic RNA has to be complexed with a few CP molecules to initiate infection. Later in the replication cycle they may act as origins for the assembly of virus particles.

The 3' untranslated region of alfalfa mosaic virus RNA3 contains a core promoter for minus-strand RNA synthesis and an enhancer element.

“…2 (b) shows that RNAs with a mutation in AUGC motif 1, 2, 3, 4 or 5 (lanes 3-7) or a mutation in motifs 3 and 5 (lane 8) had template activity similar to that of the wt transcript (lane 2). Previously, we showed that mutations in motifs 2 and 3 affected CP binding to site I in the 3h UTR of RNA3 in vitro whereas mutations in motifs 4 and 5 affected CP binding to site II (Reusken et al, 1994 ;Reusken & Bol, 1996). In addition, we observed that replacing AUGC motif 2 or 3 by AGGC reduced or abolished accumulation of RNA3 in plants, respectively (van der Vossen et al, 1994).…”

“…The 3h UTRs can be folded into a number of stem-loop structures that are flanked by AUGC motifs and contain specific binding sites for viral CP. Previously, we have shown that the 3h UTR of RNA3 contains a minimum of two independent binding sites for CP, site I being located in the homologous sequence of 145 nt and site II requiring at least part of the region that is unique to the Author for correspondence : John Bol.Fax j31 71 527 4469. e-mail J.BOL!chem.LeidenUniv.nl 3h UTR of RNA3 (Reusken et al, 1994). A CP binding site in the 3h-terminal 39 nt of RNA3 has been analysed in most detail (Houser-Scott et al, 1994Reusken & Bol, 1996).…”

“…1 (a) and Fig. 2 (a), has been described previously (Reusken et al, 1994). Plasmid pBRWT contains an insert consisting of the T7 promoter, a pBR-derived sequence of 783 nt (thin line in Figs 1 a and 2 a) and a sequence corresponding to the 3h-terminal 208 nt of RNA3 (thick line in Figs 1 a and 2 a).…”

“…Fax j31 71 527 4469. e-mail J.BOL!chem.LeidenUniv.nl 3h UTR of RNA3 (Reusken et al, 1994). A CP binding site in the 3h-terminal 39 nt of RNA3 has been analysed in most detail (Houser-Scott et al, 1994Reusken & Bol, 1996).…”

“…3 a). Replacing these AUGC motifs by AGGC had various effects on CP binding in vitro (Reusken et al, 1994) andRNA 1972). RNA was isolated from the particles and an amount of RNA corresponding to 5 mg of fresh leaf material was used per slot.…”