Molecular defects caused by temperature-sensitive mutations in Semliki Forest virus nsP1

Alphavirus replicase protein nsP1 has multiple functions during viral RNA synthesis. It catalyzes methyltransferase and guanylyltransferase activities needed in viral mRNA capping, attaches the viral replication complex to cytoplasmic membranes, and is required for minus-strand RNA synthesis. Two temperature-sensitive (ts) mutations in Semliki Forest virus (SFV) were previously identified within nsP1: ts10 (E529D) and ts14 (D119N). Recombinant viruses containing these individual mutations reproduced the features of the original ts strains. We now find that the capping-associated enzymatic activities of recombinant nsP1, containing ts10 or ts14 lesions, were not ts. The mutant proteins and polyproteins also were membrane bound, mutant nsP1 interacted normally with the other nonstructural proteins, and there was no major defect in nonstructural polyprotein processing in the mutants, although ts14 surprisingly displayed slightly retarded processing. The two mutant viruses were specifically defective in minus-strand RNA synthesis at the restrictive temperature. Integrating data from SFV and Sindbis virus, we discuss the domain structure of nsP1 and the relative positioning of and interactions between the replicase proteins. nsP1 is suggested to contain a specific subdomain involved in minus-strand synthesis and interaction with the polymerase nsP4 and the protease nsP2.     

Antibiotic innovation may contribute to slowing the dissemination of multiresistant Streptococcus pneumoniae: the example of ketolides

Background

Despite increasingly frequent bacterial resistance to antibiotics, antibacterial innovation is rare. Ketolides constitute one of the very few new antibiotic classes active against Streptococcus pneumoniae developed during the last 25 years. Their mechanism of action resembles that of macrolides, but they are unaffected by common resistance mechanisms. However, cross-resistance to ketolides has been observed in some macrolide-resistant strains. We examined how new antibiotic exposure may affect overall pneumococcal resistance patterns in the population. The aims of this study were to assess the potential dissemination of newly emerged resistances and to control the selection of strains already multiresistant to existing antimicrobials.

Methodology/Principal Findings

We developed an age-structured population model for S. pneumoniae transmission in a human community exposed to heptavalent vaccine, and β-lactams, macrolides and ketolides. The dynamics of intra-individual selection of resistant strains under antibiotic exposure and interindividual transmission were simulated, with antibiotic-specific resistance mechanisms defining the path to co-resistances and cross-resistances, and parameters concerning the French situation. Results of this simulation study suggest that new antibiotic consumption could markedly slow the diffusion of multiresistant strains. Wider use was associated with slower progression of multiresistance. When ketolides were prescribed to all ages, resistance to them reached 10% after >15 years, while it took >40 years when they were prescribed only to adults. In the scenario according to which new antibiotics totally replaced former antimicrobials, the β-lactam resistance rate was limited at 70%.

Conclusions

In a context of widespread vaccination and rational use of antibiotics, innovative antibiotic, prescribed to all age groups, may have an added impact on multiresistant-strain dissemination in the population.     

Targeted deletion of RIC8A in mouse neural precursor cells interferes with the development of the brain,eyes, and muscles

Autosomal recessive disorders such as Fukuyama congenital muscular dystrophy, Walker–Warburg syndrome, and the muscle–eye–brain disease are characterized by defects in the development of patient's brain, eyes, and skeletal muscles. These syndromes are accompanied by brain malformations like type II lissencephaly in the cerebral cortex with characteristic overmigrations of neurons through the breaches of the pial basement membrane. The signaling pathways activated by laminin receptors, dystroglycan and integrins, control the integrity of the basement membrane, and their malfunctioning may underlie the pathologies found in the rise of defects reminiscent of these syndromes. Similar defects in corticogenesis and neuromuscular disorders were found in mice when RIC8A was specifically removed from neural precursor cells. RIC8A regulates a subset of G‐protein α subunits and in several model organisms, it has been reported to participate in the control of cell division, signaling, and migration. Here, we studied the role of RIC8A in the development of the brain, muscles, and eyes of the neural precursor‐specific conditional Ric8a knockout mice. The absence of RIC8A severely affected the attachment and positioning of radial glial processes, Cajal‐Retzius’ cells, and the arachnoid trabeculae, and these mice displayed additional defects in the lens, skeletal muscles, and heart development. All the discovered defects might be linked to aberrancies in cell adhesion and migration, suggesting that RIC8A has a crucial role in the regulation of cell–extracellular matrix interactions and that its removal leads to the phenotype characteristic to type II lissencephaly‐associated diseases. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 374–390, 2018     

Functional Cross-talk between Distant Domains of Chikungunya Virus Non-structural Protein 2 Is Decisive for Its RNA-modulating Activity

Chikungunya virus (CHIKV) non-structural protein 2 (nsP2) is a multifunctional protein that is considered a master regulator of the viral life cycle and a main viral factor responsible for cytopathic effects and subversion of antiviral defense. The C-terminal part of nsP2 possesses protease activity, whereas the N-terminal part exhibits NTPase and RNA triphosphatase activity and is proposed to have helicase activity. Bioinformatics analysis classified CHIKV nsP2 into helicase superfamily 1. However, the biochemical significance of a coexistence of two functionally unrelated modules in this single protein remains unknown. In this study, recombinant nsP2 demonstrated unwinding of double-stranded RNA in a 5′–3′ directionally biased manner and RNA strand annealing activity. Comparative analysis of NTPase and helicase activities of wild type nsP2 with enzymatic capabilities of different truncated or N-terminally extended variants of nsP2 revealed that the C-terminal part of the protein is indispensable for helicase functionality and presumably provides a platform for RNA binding, whereas the N-terminal-most region is apparently involved in obtaining a conformation of nsP2 that allows for its maximal enzymatic activities. The establishment of the protocols for the production of biochemically active CHIKV nsP2 and optimization of the parameters for helicase and NTPase assays are expected to provide the starting point for a further search of possibilities for therapeutic interventions to suppress alphaviral infections.     

Detailed Contact Data and the Dissemination of Staphylococcus aureus in Hospitals

Close proximity interactions (CPIs) measured by wireless electronic devices are increasingly used in epidemiological models. However, no evidence supports that electronically collected CPIs inform on the contacts leading to transmission. Here, we analyzed Staphylococcus aureus carriage and CPIs recorded simultaneously in a long-term care facility for 4 months in 329 patients and 261 healthcare workers to test this hypothesis. In the broad diversity of isolated S. aureus strains, 173 transmission events were observed between participants. The joint analysis of carriage and CPIs showed that CPI paths linking incident cases to other individuals carrying the same strain (i.e. possible infectors) had fewer intermediaries than predicted by chance (P < 0.001), a feature that simulations showed to be the signature of transmission along CPIs. Additional analyses revealed a higher dissemination risk between patients via healthcare workers than via other patients. In conclusion, S. aureus transmission was consistent with contacts defined by electronically collected CPIs, illustrating their potential as a tool to control hospital-acquired infections and help direct surveillance.     

Macromolecular assembly-driven processing of the 2/3 cleavage site in the alphavirus replicase polyprotein

Semliki Forest virus (SFV) is a member of the Alphavirus genus, which produces its replicase proteins in the form of a nonstructural (ns) polyprotein precursor P1234. The maturation of the replicase occurs in a temporally controlled manner by protease activity of nsP2. The template preference and enzymatic capabilities of the alphaviral replication complex have a very important connection with its composition, which is irreversibly altered by proteolysis. The final cleavage of the 2/3 site in the ns polyprotein apparently leads to significant rearrangements within the replication complex and thus denotes the "point of no return" for viral replication progression. Numerous studies have devised rules for when and how ns protease acts, but how the alphaviral 2/3 site is recognized remained largely unexplained. In contrast to the other two cleavage sites within the ns polyprotein, the 2/3 site evidently lacks primary sequence elements in the vicinity of the scissile bond sufficient for specific protease recognition. In this study, we sought to investigate the molecular details of the regulation of the 2/3 site processing in the SFV ns polyprotein. We present evidence that correct macromolecular assembly, presumably strengthened by exosite interactions rather than the functionality of the individual nsP2 protease, is the driving force for specific substrate targeting. We conclude that structural elements within the macrodomain of nsP3 are used for precise positioning of a substrate recognition sequence at the catalytic center of the protease and that this process is coordinated by the exact N-terminal end of nsP2, thus representing a unique regulation mechanism used by alphaviruses.