RNA Silencing May Play a Role in but Is Not the Only Determinant of the Multiplicity of Infection

Abstract

The multiplicity of infection (MOI), i.e., the number of viral genomes that infect a cell, is an important parameter in virus evolution, which for each virus and environment may have an optimum value that maximizes virus fitness. Thus, the MOI might be controlled by virus functions, an underexplored hypothesis in eukaryote-infecting viruses. To analyze if the MOI is controlled by virus functions, we estimated the MOI in plants coinfected by two genetic variants of Tomato bushy stunt virus (TBSV); by TBSV and a TBSV-derived defective interfering RNA (DI-RNA); or by TBSV and a second tombusvirus, Cymbidium ringspot virus (CymRSV). The MOI was significantly larger in TBSV-CymRSV coinfections (~4.0) than in TBSV-TBSV or TBSV-DI-RNA coinfections (~1.7 to 2.2). Coinfections by CymRSV or TBSV with chimeras in which an open reading frame (ORF) of one virus species was replaced by that of the other identified a role of viral proteins in determining the MOI, which ranged from 1.6 to 3.9 depending on the coinfecting genotypes. However, no virus-encoded protein or genomic region was the sole MOI determinant. Coinfections by CymRSV and TBSV mutants in which the expression of the gene-silencing suppressor protein p19 was abolished also showed a possible role of gene silencing in MOI determination. Taken together, these results demonstrate that the MOI is a quantitative trait showing continuous variation and that as such it has a complex determination involving different virus-encoded functions.

Importance: The number of viral genomes infecting a cell, or the multiplicity of infection (MOI), is an important parameter in virus evolution affecting recombination rates, selection intensity on viral genes, evolution of multipartite genomes, or hyperparasitism by satellites or defective interfering particles. For each virus and environment, the MOI may have an optimum value that maximizes virus fitness, but little is known about MOI control in eukaryote-infecting viruses. We show here that in plants coinfected by two genotypes of Tomato bushy stunt virus (TBSV), the MOI was lower than in plants coinfected by TBSV and Cymbidium ringspot virus (CymRSV). Coinfections by CymRSV or TBSV with TBSV-CymRSV chimeras showed a role of viral proteins in MOI determination. Coinfections by CymRSV and TBSV mutants not expressing the gene-silencing suppressor protein also showed a role of gene silencing in MOI determination. The results demonstrate that the MOI is a quantitative trait with a complex determination involving different viral functions.

FIG 1

Schematic representation of the genomes of TBSV-C/P, DI-72, and CymRSV. The organization of the coding regions and UTRs of TBSV-C/P (white boxes) and CymRSV (black boxes) is shown. The four regions of DI-72 (I to IV) derived from TBSV are outlined with boxes.

FIG 2

Schematic representation of chimeras between TBSV-P and CymRSV and the NCG in various coinfections. (A) Chimeras coinoculated with CymRSV or with TBSV-P are shown, with ORFs derived from TBSV-P indicated as white boxes and ORFs derived from CymRSV indicated as black boxes. (B) Frequencies of total infected cells, doubly infected cells, and the NCG for each coinfection. The values are means ± standard errors of the results of three replicate experiments.

FIG 3

Correlation between sequence identity of the genomes of coinfecting viruses and the estimated MOI. Correlation analysis between the percentage of sequence identity and the estimated MOI or NCG is shown as an R² statistic (df = 10). Tendency is represented by a dashed line. Standard error bars are represented.