Nucleotide sequence and structural determinants of specific binding of coat protein or coat protein peptides to the 3' untranslated region of alfalfa mosaic virus RNA 4

Abstract

The specific binding of alfalfa mosaic virus coat protein to viral RNA requires determinants in the 3' untranslated region (UTR). Coat protein and peptide binding sites in the 3' UTR of alfalfa mosaic virus RNA 4 have been analyzed by hydroxyl radical footprinting, deletion mapping, and site-directed mutagenesis experiments. The 3' UTR has several stable hairpins that are flanked by single-stranded (A/U)UGC sequences. Hydroxyl radical footprinting data show that five sites in the 3' UTR of alfalfa mosaic virus RNA 4 are protected by coat protein, and four of the five protected regions contain AUGC or UUGC. Electrophoretic mobility band shift results suggest four coat protein binding sites in the 3' UTR. A 3'-terminal 39-nucleotide RNA fragment containing four AUGC repeats bound coat protein and coat protein peptides with high affinity; however, coat protein bound poorly to antisense 3' UTR transcripts and poly(AUGC)10. Site-directed mutagenesis of AUGC865-868 resulted in a loss of coat protein binding and peptide binding by the RNA fragment. Alignment of alfalfa mosaic RNA sequences with those from several closely related ilarviruses demonstrates that AUGC865-868 is perfectly conserved; moreover, the RNAs are predicted to form similar 3'-terminal secondary structures. The data strongly suggest that alfalfa mosaic virus coat protein and ilavirus coat proteins recognize invariant AUGC sequences in the context of conserved structural elements.

RNA conformational changes in the life cycles of RNA viruses, viroids, and virus-associated RNAs

“…In the presence of viral coat protein, the AUGC regions were not cleaved by T1 ribonuclease (G-specificity), suggesting either that the AUGCs were protected by bound protein, or that the RNA conformation was altered [120]. A wide range of biochemical methods was applied toward mapping the coat protein binding sites and determining the RNA sequence and structural features that are recognized by the coat protein [121][122][123][124][125][126]. However, it was not until the structure of AMV 3′ terminal RNA bound to the N-terminal arm of the viral coat protein was solved [127] that the extent of conformational changes in both the protein and the RNA became clear.…”

Alfalfa mosaic virus coat protein bridges RNA and RNA-dependent RNA polymerase in vitro

“…The 3' terminus of the AMV RNAs is characterized by hairpin structures that are separated by (A/U)UGC tetranucleotide repeats, forming multiple coat protein binding sites Houser-Scott et al, 1994;Reusken, Neeleman, and Bol, 1994). A schematic representation of the AMV RNA 3 3' untranslated region shows the positions of the AUGC or UUGC sequences separating proposed hairpin structures ( Figure 6A).…”

“…To assess potential differential functions correlated with coat protein occupancy at the binding sites, individual AUGC or UUGC coat protein binding sites were converted to AAAA in viral genomic RNA 3. The AUGC to AAAA substitutions have been shown previously to disrupt coat protein binding Houser-Scott et al, 1994). Coat protein binding to the 3' UTR RNA was analyzed by electrophoretic mobility shift assay and viral RNA replication from the mutated RNA template was assessed by coat protein accumulation resulting from viral RNA replication in transfected tobacco protoplasts.…”

In vitro genetic selection analysis of alfalfa mosaic virus coat protein binding to 3'-terminal AUGC repeats in the viral RNAs

“…In this context, the 3Ј terminus of AMV and the related ilarvirus RNAs is structurally distinct because it is nonpolyadenylated, lacks pseudoknots, and cannot be aminoacylated (14). The 3Ј termini of AMV and the ilarvirus RNAs do, however, contain high-affinity coat pro-tein binding sites (15,50,51) (Fig. 1), and tetranucleotide AUGC repeats are a conspicuous feature of the 3Ј-terminal nucleotide sequences.…”