Rubisco is the predominant enzymatic mechanism in the biosphere by which autotrophic bacteria, algae, and terrestrial plants fix CO(2) into organic biomass via the Calvin-Benson-Basham reductive pentose phosphate pathway. Rubisco is not a perfect catalyst, suffering from low turnover rates, a low affinity for its CO(2) substrate, and a competitive inhibition by O(2) as an alternative substrate. As a consequence of changing environmental conditions over the past 3.5 billion years, with decreasing CO(2) and increasing O(2) in the atmosphere, Rubisco has evolved into multiple enzymatic forms with a range of kinetic properties, as well as co-evolving with CO(2)-concentrating mechanisms to cope with the different environmental contexts in which it must operate. The most dramatic evidence of this is the occurrence of multiple forms of Rubisco within autotrophic proteobacteria, where Forms II, IC, IBc, IAc, and IAq can be found either singly or in multiple combinations within a particular bacterial genome. Over the past few years there has been increasing availability of genomic sequence data for bacteria and this has allowed us to gain more extensive insights into the functional significance of this diversification. This paper is focused on summarizing what is known about the diversity of Rubisco forms, their kinetic properties, development of bacterial CO(2)-concentrating mechanisms, and correlations with metabolic flexibility and inorganic carbon environments in which proteobacteria perform various types of obligate and facultative chemo- and photoautotrophic CO(2) fixation.

It has been shown in animal models that ribavirin-resistant poliovirus with a G64S mutation in its 3D polymerase has high replication fidelity coupled with attenuated virulence. Here, we describe the effects of mutagenesis in the human enterovirus 71 (HEV71) 3D polymerase on ribavirin resistance and replication fidelity. Seven substitutions were introduced at amino acid position 3D-G64 of a HEV71 full-length infectious cDNA clone (26M). Viable clone-derived virus populations were rescued from the G64N, G64R, and G64T mutant cDNA clones. The clone-derived G64R and G64T mutant virus populations were resistant to growth inhibition in the presence of 1,600 M ribavirin, whereas the growth of parental 26M and the G64N mutant viruses were inhibited in the presence of 800 M ribavirin. Nucleotide sequencing of the 2C and 3D coding regions revealed that the rate of random mutagenesis after 13 passages in the presence of 400 M ribavirin was nearly 10 times higher in the 26M genome than in the mutant G64R virus genome. Furthermore, random mutations acquired in the 2C coding regions of 26M and G64N conferred resistance to growth inhibition in the presence of 0.5 mM guanidine, whereas the G64R and G64T mutant virus populations remained susceptible to growth inhibition by 0.5 mM guanidine. Interestingly, a S264L mutation identified in the 3D coding region of 26M after ribavirin selection was also associated with both ribavirin-resistant and high replication fidelity phenotypes. These findings are consistent with the hypothesis that the 3D-G64R, 3D-G64T, and 3D-S264L mutations confer resistance upon HEV71 to the antiviral mutagen ribavirin, coupled with a high replication fidelity phenotype during growth in cell culture.

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Human enterovirus 71 (HEV71) is the causative agent of hand, foot, and mouth disease and associated acute neurological disease. At present, little is known about the genetic determinants of HEV71 neurovirulence. Studies of related enteroviruses have indicated that the untranslated regions (UTRs), which control virus-directed translation and replication, also exert significant influence on neurovirulence. We used an infectious cDNA clone of a subgenogroup B3 strain to construct and characterize chimeras with 5=-and 3=-UTR modifications. Replacement of the entire HEV71 5= UTR with that of human rhinovirus 2 (HRV2) resulted in a small reduction in growth efficiency in cells of both nonneuronal (rhabdomyosarcoma) and neuronal (SH-SY5Y) origin due to reduced translational efficiency. However, the introduction of a 17-nucleotide deletion into the proximal region of the 3= UTR significantly decreased the growth of HEV71-HRV2 in SH-SY5Y cells. This observation is similar to that made with stem-loop domain Z (SLD Z)-deleted coxsackievirus B3-HRV2 5=-UTR chimeras reported previously and provides the first evidence of a potentially functional SLD Z in the 3= UTR in human enterovirus A species viruses. We further showed that the cellspecific growth impairment was caused by the synergistic effects of cis-acting UTR control elements on different stages of the virus life cycle. These chimeras will further improve our understanding of the control of HEV71 replication and its relationship to neurovirulence.

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