Delayed lysis confers resistance to the nucleoside analogue 5-fluorouracil and alleviates mutation accumulation in the single-stranded DNA bacteriophage ϕX174

Abstract

Rates of spontaneous mutation determine viral fitness and adaptability. In RNA viruses, treatment with mutagenic nucleoside analogues selects for polymerase variants with increased fidelity, showing that viral mutation rates can be adjusted in response to imposed selective pressures. However, this type of resistance is not possible in viruses that do not encode their own polymerases, such as single-stranded DNA viruses. We previously showed that serial passaging of bacteriophage ϕX174 in the presence of the nucleoside analogue 5-fluorouracil (5-FU) favored substitutions in the lysis protein E (P. Domingo-Calap, M. Pereira-Gomez, and R. Sanjuán, J. Virol. 86:: 9640-9646, 2012, doi:10.1128/JVI.00613-12). Here, we found that approximately half (6/12) of the amino acid replacements in the N-terminal region of this protein led to delayed lysis, and two of these changes (V2A and D8A) also conferred partial resistance to 5-FU. By delaying lysis, the V2A and D8A substitutions allowed the virus to increase the burst size per cell in the presence of 5-FU. Furthermore, these substitutions tended to alleviate drug-induced mutagenesis by reducing the number of rounds of copying required for population growth, revealing a new mechanism of resistance. This form of mutation rate regulation may also be utilized by other viruses whose replication mode is similar to that of bacteriophage ϕX174.

Importance: Many viruses display high rates of spontaneous mutations due to defects in proofreading or postreplicative repair, allowing them to rapidly adapt to changing environments. Viral mutation rates may have been optimized to achieve high adaptability without incurring an excessive genetic load. Supporting this, RNA viruses subjected to chemical mutagenesis treatments have been shown to evolve higher-fidelity polymerases. However, many viruses cannot modulate replication fidelity because they do not encode their own polymerase. Here, we show a new mechanism for regulating viral mutation rates. We found that, under mutagenic conditions, the single-stranded bacteriophage ϕX174 evolved delayed lysis, and that this allowed the virus to increase the amount of progeny produced per cell. As a result, the viral population was amplified in fewer infection cycles, reducing the chances for mutation appearance.

FIG 1

Single-amino-acid replacements introduced in the N-terminal region of the lysis protein. (A) Scheme of the cytoplasmic, transmembrane, and periplasmic domains of the lysis protein E. All substitutions were introduced in the periplasmic domain. (B) Nucleotide sequence of the region studied, showing the two overlapping reading frames (top, gene D; bottom, gene E) and the amino acid changes introduced.

FIG 2

Effect of the V2A and D8A substitutions on lytic and growth properties of the virus. Standard growth curves (A and B), lysis assays (C and D), and intracellular growth assays (E and F) are shown for the WT (black circles), V2A (white squares), and D8A (white triangles) viruses in the absence (A, C, and E) and in the presence (B, D, and F) of 10 ng/μl 5-FU. Error bars in panels A to D indicate the standard errors of the means (SEM) and are not shown in panels E and F for clarity.

FIG 3

Expected effect of changes in lysis time and burst size on viral growth rate. The growth rates of the WT (black circles), V2A (white squares), and D8A (white triangles) viruses were predicted based on their experimentally determined lysis times and burst sizes (x axis) and compared to measured growth rates (y axis) in the presence and in the absence of 10 ng/μl 5-FU.

FIG 4

Predicted number of viral generations required to infect a given number of host cells for the WT (black circles), V2A (white squares), and D8A (white triangles) viruses with and without 5-FU. For an infection starting with 1 PFU, generations were calculated as g = 1 + lnH/lnB, where H is the number of infected cells and B is the burst size.