Mutation in the RNA-Dependent RNA Polymerase of a Symbiotic Virus Is Associated With the Adaptability of the Viral Host

Abstract

Host adaptation has the potential to cause rapid genetic variation in symbiotic microorganisms in insects. How mutations in symbiotic viruses favor viral fitness in hosts and even influence host adaptability to new environments remains elusive. Here, we explored the role of genetic divergence at one site of a symbiotic virus, Acyrthosiphon pisum virus (APV), in the host aphid's adaptation to unfavorable plants. Based on the transcriptomes of the pea aphid Vicia faba colony and Vicia villosa colony, 46 single nucleotide polymorphism (SNP) sites were found in the APV genomes from the two aphid colonies. One SNP at site 5,990, G5990A, located at the RNA-dependent RNA polymerase (RdRp) domain, demonstrated a predominance from G to A when the host aphids were shifted from V. faba to the low-fitness plants V. villosa or Medicago sativa. This SNP resulted in a substitution from serine (S) to asparagine (N) at site 196 in RdRp. Although S196N was predicted to be located at a random coil far away from conserved functional motifs, the polymerase activity of the N196 type of RdRp was increased by 44.5% compared to that of the S196 type. The promoted enzymatic activity of RdRp was associated with a higher replication level of APV, which was beneficial for aphids as APV suppressed plant's resistance reactions toward aphids. The findings showed a novel case in which mutations selected in a symbiotic virus may confer a favor on the host as the host adapts to new environmental conditions.

Keywords: Acyrthosiphon pisum virus; RdRp; pea aphid; polymerase activity; single nucleotide polymorphism; symbiotic virus.

Figure 1

Sequence logo showing allele frequencies at 46 SNP sites in APV genomes from the pea aphids Vicia faba colony and Vicia villosa colony. The genome position and corresponding codon position for each SNP are shown. Four domains are marked in red rectangles. Five SNPs resulting in nonsynonymous mutations are boxed. Site 5,990 is marked with a triangle. A logo represents each column of the alignment by a stack of letters, with the height of each letter proportional to the observed frequency of the corresponding nucleotide.

Figure 2

Divergence of SNP at site 5,990 of APV in different colonies of pea aphids. (A) Vicia faba colony. (B) Vicia villosa colony. (C) V. villosa colony that was raised on V. faba for 1–5 months. (D) Medicago sativa colony. Twenty individual pea aphids were sequenced in (A–C), and 27 individuals were sequenced in (D).

Figure 3

Location of site 5,990 in the structure of RdRp. (A) Alignment of amino acid sequences among two types of APV RdRp and Kelp fly virus RdRp. Eight conserved motifs (I–VII, X) are boxed. The S or N at amino acid residue 196 is shown in red. Identical or similar amino acid residues among the three sequences are shaded in gray. (B) Three-dimensional structures of APV RdRp predicted in silico by the Robetta and I-TASSER servers. Motifs are shown in assorted colors: I, orange; II, cyan; III, magenta; IV, green; V, blue; VI, yellow; VII, smudge; X, wheat. Site S196N is marked in red.

Figure 4

Effects of SNP at site 5,990 on the enzymatic activity of APV RdRp and viral replication levels. (A) Dot blots showing the biotin-labeled RNA products synthesized by the recombinantly expressed S196-type RdRp-His and N196-type RdRp-His. The 1 and 0.1 mM biotin-NTP were used as positive controls. One hundred micrograms of crude protein from the cells transformed with pET28a vector and 20 mM Tris–HCl (pH 8.0) buffer were used as negative controls. The RdRp-His proteins used in the assays were measured by western blotting with an anti-His monoclonal antibody. Six replicates were prepared. (B) The relative intensity of synthesized RNA products to that of RdRp-His from (A) quantified with ImageJ software. The values represent the means ± SEs. (C) Comparison of the relative RNA levels of APV CP between the V. faba colony and V. villosa colony. Five to six biological replicates were prepared. (D) Comparison of the relative RNA levels of APV CP in the V. villosa colony that was acclimated on V. faba for 3–5 months. Thirteen to nineteen individual aphids were measured. (E) Comparison of the relative RNA levels of APV CP in the aphids 8 days post-infection with the A5990-enriched APV and the G5990-enriched APV. Nine biological replicates were prepared. The relative RNA level of APV CP to the transcript level of L27 is represented as the mean ± SE. Differences were analyzed using Student’s t test. **p < 0.01; ***p < 0.001.