Degenerate In Vitro Genetic Selection Reveals Mutations That Diminish Alfalfa Mosaic Virus RNA Replication without Affecting Coat Protein Binding

Abstract

The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5 hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3 RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3 hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity.Alfalfa mosaic virus (AMV) has a number of exceptional features that make it a useful system for studying RNA-protein interactions (reviewed in references 6 and 17). The three positive-sense genomic AMV RNAs (RNAs 1 to 3) and the subgenomic coat protein mRNA (RNA 4) are packaged individually into four bacilliform, aphid-transmitted particles. The viral genomic RNAs are infectious only in the presence of the viral coat protein (7), which binds specifically to the RNA 3Ј termini. Unlike the arginine-rich RNA binding domains of the human immunodeficiency virus Tat and Rev proteins, the AMV coat protein RNA binding domain is lysine rich (3); however, it includes an arginine residue whose position and side chain identity are crucial for specific, high-affinity RNA binding (2). The AMV RNAs lack a poly(A) tail and are further distinguished from closely related plant viruses (e.g., brome mosaic virus) by the absence of a canonical CCA 3Ј terminus and the inability to be charged by aminoacyl synthetases, both of which are defining features of tRNA-like ends (12).The relationship between the formation of the AMV coat protein-RNA complex and functional activity in viral RNA translation, replication, and assembly is poorly understood. Previous biochemical characterization has defined specific binding determinants in both the RNA and coat protein (1,3,14,15,18,(26)(27)(28...

Evaluation of the Conformational Switch Model for Alfalfa Mosaic Virus RNA Replication

“…The A4, G3, C3, and GC3 mutations can be added to a collection of 3Ј nucleotide substitutions that disrupt replication. In general, AMV RNA replication is sensitive to nucleotide substitutions in the 3Ј UTR, and a number of changes outside of the pseudoknot have also been shown to affect viral RNA replication in this system (16,26). The molecular basis for the effects on replication is largely unknown, but others have shown that viral RNA replication can be affected by both cis and trans mutations in positive-strand RNA viruses (17).…”

“…To provide unequivocal evidence of RNA replication by detection of subgenomic RNA4, replication was activated in some experiments by a truncated RNA4 construct (AMV4-trUTR) wherein nucleotides 719 through 842 of the 3Ј untranslated region (Fig. 1B) were deleted (26).…”

“…Following the incubation period, the RNAs were precipitated by the addition of lithium chloride according to the manufacturer's protocol. Constructs encoding infectious clones of AMV RNAs were prepared as described previously (16,26). The RNA4 clone was provided by L. Sue Loesch-Fries (18).…”

“…We assayed the ability of these variant RNAs to activate replication by transfecting wild-type RNA1, -2, and -3 along with variant RNA4, as described previously (16,26). We have shown previously that, using this approach, virus replication levels correlate with CP translation efficiency (26). If the mechanism underlying the absence of replication observed using variant RNA3 constructs (Fig.…”

“…To distinguish between effects the pseudoknot mutations may have on replication and translation, the nucleotide changes were introduced into the full-length 3Ј UTR of RNA4. We assayed the ability of these variant RNAs to activate replication by transfecting wild-type RNA1, -2, and -3 along with variant RNA4, as described previously (16,26). We have shown previously that, using this approach, virus replication levels correlate with CP translation efficiency (26).…”