Attenuation of Foot-and-Mouth Disease Virus by Engineered Viral Polymerase Fidelity

Abstract

Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3D pol ) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allow FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3D pol in combination with previously reported results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon. Using biochemical and genetic tools, we show that the replacement of tryptophan 237 with phenylalanine imparts higher fidelity, but replacements with isoleucine and leucine resulted in lower-fidelity phenotypes. Viruses containing these W237 substitutions show in vitro growth kinetics and plaque morphologies similar to those of the wild-type (WT) A 24 Cruzeiro strain in BHK cells, and both high-and low-fidelity variants retained fitness during coinfection with the wildtype virus. The higher-fidelity W237F (W237F HF ) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237I LF ) and W237L LF mutant viruses exhibited lower ribavirin resistance. Interestingly, the variant viruses showed heterogeneous and slightly delayed growth kinetics in primary porcine kidney cells, and they were significantly attenuated in mouse infection experiments. These data demonstrate, for a single virus, that either increased or decreased RdRp fidelity attenuates virus growth in animals, which is a desirable feature for the development of safer and genetically more stable vaccine candidates.IMPORTANCE Foot-and-mouth disease (FMD) is the most devastating disease affecting livestock worldwide. Here, using structural and biochemical analyses, we have identified FMDV 3D pol mutations that affect polymerase fidelity. Recombinant FMDVs containing substitutions at 3D pol tryptophan residue 237 were genetically stable and displayed plaque phenotypes and growth kinetics similar to those of the wild-type virus in cell culture. We further demonstrate that viruses harboring either a W237F HF substitution or W237I LF and W237L LF mutations were highly attenuated in animals. Our study shows that obtaining 3D pol fidelity variants by protein engineering based on polymerase structure and function could be exploited for the development of attenuated FMDV vaccine candidates that are safer and more stable than strains obtained by selective pressure via mutagenic nucleotides or adaptation approaches.

Foot-and-mouth disease virus type O specific mutations determine RNA-dependent RNA polymerase fidelity and virus attenuation

“…Many laboratories have therefore focused their research on discovering the virulence factors that are associated with its causative agent, FMDV. For example, the well-established role of the leader proteinase in the virulence in of swine and cattle (Kleina and Grubman, 1992; Roberts and Belsham, 1995; Skern et al, 1998), the role of RGD sequence in the VP1 G-H loop (Leippert et al, 1997; Mason et al, 1994), and the importance of virus nonstructural proteins (Gladue et al, 2014; Pacheco et al, 2013, 2003; Rai et al, 2017), the 3′ NTR (Rodriguez Pulido et al, 2009) and the codon usage bias (Diaz-San Segundo et al, 2015). We have previously shown that increased 3D pol fidelity of the type Asia1 FMDV strain could reduce its fitness in vitro and attenuate virulence in vivo (Zeng et al, 2014).…”

“…The researchers in this example demonstrated that the mutator strain had decreased fidelity in vivo, which resulted in attenuation in young, aged, and immunocompromised mouse models of human SARS (Graham et al, 2012). With regard to FMDV, it has been shown that either increased or decreased RdRp fidelity attenuates virus growth in animals (Rai et al, 2017; Zeng et al, 2014). Therefore, manipulation of polymerase fidelity is clearly a promising approach to engineer attenuated virus vaccines.…”

“…This increases mutation frequency beyond the natural state of a virus, which also leads to an attenuated phenotype in vivo (Gnadig et al, 2012; Korboukh et al, 2014; Xie et al, 2014). These results have demonstrated that a reduction in replication fidelity is also an available approach to modulate the virulence of an RNA virus population (Gnadig et al, 2012; Liu et al, 2013; Pfeiffer and Kirkegaard, 2005; Rai et al, 2017; Xie et al, 2014). …”

Contribution of a Multifunctional Polymerase Region of Foot-and-Mouth Disease Virus to Lethal Mutagenesis

“…D5N at 3D was selected upon passage of FMDV Asia 1 in BHK-21c2 cells in the presence of ribavirin, and the mutant virus exhibited high polymerase fidelity according to the mutation frequency of the population (55). Modeled on the structural elements of the polymerase of FMDV of serotype C (36), at least 18 different amino acid replacements at 9 separate structural elements in 3D of FMDV serotype C, A, or Asia 1 have been involved in significant alterations of copying fidelity: D5N, K18E, and K20E (at the N-terminal region of the enzyme, with K20 within ␤1); A38V (immediately following ␤2); P44S (loop ␤2-␣2); G62S (loop ␣2-␣3); R84H (within ␣3); D165E (loop ␤5-␤6); P169S (within ␤6), K172R, and V173I (within ␣6) (the three substitutions in motif F); M194I (within ␣7); M296V and M296I (loop ␤9-␣11); W237F, W237I, and W237L (within ␤8, in motif A); G361S (loop ␣12-␤12) (29)(30)(31)(32)(33)(55)(56)(57)(58) (Fig. 6).…”

Trends in antiviral strategies

“…Trends in antiviral strategies genome heterogeneity within a suitable range: too low a diversity impairs adaptability, and too high a diversity may approach the population to an extinction threshold Smith and Denison, 2013;Zeng et al, 2014). Several studies have demonstrated an attenuation phenotype associated with low-or high-fidelity viral mutants (Borderia et al, 2016;Rai et al, 2017). In a clinical setting, maintaining mutant spectra of viruses within a suitable amplitude range is one of the predictors of viral survival and progression toward disease (Section 8.8 in Chapter 8).…”