Ontogeny of Carbon Monoxide-Related Gene Expression in a Deep-Diving Marine Mammal.

Marine mammals such as northern elephant seals (NES) routinely experience hypoxemia and ischemia-reperfusion events to many tissues during deep dives with no apparent adverse effects. Adaptations to diving include increased antioxidants and elevated oxygen storage capacity associated with high hemoprotein content in blood and muscle. The natural turnover of heme by heme oxygenase enzymes (encoded by HMOX1 and HMOX2) produces endogenous carbon monoxide (CO), which is present at high levels in NES blood and has been shown to have cytoprotective effects in laboratory systems exposed to hypoxia. To understand how pathways associated with endogenous CO production and signaling change across ontogeny in diving mammals, we measured muscle CO and baseline expression of 17 CO-related genes in skeletal muscle and whole blood of three age classes of NES. Muscle CO levels approached those of animals exposed to high exogenous CO, increased with age, and were significantly correlated with gene expression levels. Muscle expression of genes associated with CO production and antioxidant defenses (HMOX1, BVR, GPX3, PRDX1) increased with age and was highest in adult females, while that of genes associated with protection from lipid peroxidation (GPX4, PRDX6, PRDX1, SIRT1) was highest in adult males. In contrast, muscle expression of mitochondrial biogenesis regulators (PGC1A, ESRRA, ESRRG) was highest in pups, while genes associated with inflammation (HMOX2, NRF2, IL1B) did not vary with age or sex. Blood expression of genes involved in regulation of inflammation (IL1B, NRF2, BVR, IL10) was highest in pups, while HMOX1, HMOX2 and pro-inflammatory markers (TLR4, CCL4, PRDX1, TNFA) did not vary with age. We propose that ontogenetic upregulation of baseline HMOX1 expression in skeletal muscle of NES may, in part, underlie increases in CO levels and expression of genes encoding antioxidant enzymes. HMOX2, in turn, may play a role in regulating inflammation related to ischemia and reperfusion in muscle and circulating immune cells. Our data suggest putative ontogenetic mechanisms that may enable phocid pups to transition to a deep-diving lifestyle, including high baseline expression of genes associated with mitochondrial biogenesis and immune system activation during postnatal development and increased expression of genes associated with protection from lipid peroxidation in adulthood.

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

Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3Dpol) 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 3Dpol 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) A24 Cruzeiro strain in BHK cells, and both high- and low-fidelity variants retained fitness during coinfection with the wild-type virus. The higher-fidelity W237F (W237FHF) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237ILF) and W237LLF 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 3Dpol mutations that affect polymerase fidelity. Recombinant FMDVs containing substitutions at 3Dpol 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 W237FHF substitution or W237ILF and W237LLF mutations were highly attenuated in animals. Our study shows that obtaining 3Dpol 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.