On the Species Specificity of Acceptor RNA and Attachment Enzymes

One of the steps in protein biosynthesis appears to be the attachment of each amino acid to a specific acceptor (SRNA) molecule. According to the adaptor hypothesis, each SRNA molecule would then fit to a specific complementary base sequence on a linear RNA template, specifying the sequence of amino acids in the resultant protein [1,2]. An adaptor molecule thus could have two specificities: one recognizing the correct amino acid and activating enzyme; the other, the proper position on the template. The correctness of the amino-acid sequence therefore would depend upon the precision and constancy of the adaptors. However, the structures of the enzymes and adaptors are presumably under the genetic control of the organism and might be subject to heritable modifications. It is therefore conceivable that one or both ends of an adaptor might change sufficiently to cause occasional errors and, in the long run, an alteration of the genetic code might evolve. This notion, prompted by genetic observations [3] which suggested that mutation of a bacterium might modify its translation of genetic information, lead to the present comparison of the specificities of the acceptor RNA and activating enzymes of different organisms. Several differences in specificity have been reported previously. Berg et al. [4] demonstrated that SRNA from Escherichia coli contains two distinguishable acceptors for methionine. An enzyme prepared from yeast could attach methionine to one of these, while the enzyme from E. coli could attach to both. Webster found, in pig liver, a difference between the nuclear and cytoplasmic attachment enzymes for alanine. Rendi and Ochoa [6] noted that, for leucine, the enzymes in yeast and in E. coli could attach only to their homologous SRNA. Furthermore, in the case of leucine, rat liver enzyme and SRNA were interchangeable with those from E. coli. The observations presented below show that whether the enzymes and/or acceptors from two organisms are interchangeable depends upon not only the organisms in question but also the particular amino aci

West Nile virus methyltransferase domain interacts with protein kinase G

Background: The flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent protein kinase, has previously been shown to phosphorylate dengue virus NS5. Methods: We used mass spectrometry to specifically identify NS5 phosphosites. Co-immunoprecipitation assays were used to study protein-protein interactions. Effects on viral replication were measured via replicon system and plaque assay titering. Results: We identified multiple sites in West Nile virus (WNV) NS5 that are phosphorylated during a WNV infection, and showed that the N-terminal methyltransferase domain of WNV NS5 can be specifically phosphorylated by PKG in vitro. Expressing PKG in cell culture led to an enhancement of WNV viral production. We hypothesized this effect on replication could be caused by factors beyond the specific phosphorylations of NS5. Here we show for the first time that PKG is also able to stably interact with a viral substrate, WNV NS5, in cell culture and in vitro. While the mosquito-borne WNV NS5 interacted with PKG, tick-borne Langat virus NS5 did not. The methyltransferase domain of NS5 is able to mediate the interaction between NS5 and PKG, and mutating positive residues in the αE region of the methyltransferase interrupts the interaction. These same mutations completely inhibited WNV replication. Conclusions: PKG is not required for WNV replication, but does make a stable interaction with NS5. While the consequence of the NS5:PKG interaction when it occurs is unclear, mutational data demonstrates that this interaction occurs in a region of NS5 that is otherwise necessary for replication. Overall, the results identify an interaction between virus and a cellular kinase and suggest a role for a host kinase in enhancing flaviviral replication

The Fourth International Neonatal and Maternal Immunization Symposium (INMIS 2017): Toward Integrating Maternal and Infant Immunization Programs.

Prevention of serious infections in pregnant mothers, newborns, and young infants through immunization during pregnancy and in early life has the potential to further reduce maternal and neonatal morbidity and mortality worldwide. In the past decade, research in this field has advanced substantially, from the understanding of the biology and immunology of pregnancy and early life, to the active development of several candidate vaccines, for which challenges and opportunities for global implementation are under consideration. Experts from academia, industry, regulatory and funding agencies, public health, and international organizations met in Brussels (Belgium) from 10 to 12 September 2017, at the 4th International Neonatal and Maternal Immunization Symposium (INMIS), to review the most relevant advances in maternal and neonatal immunization. The overarching focus of the conference was to identify the path forward to achieve integration of maternal and early life immunization strategies for the successful implementation of vaccines in antenatal care and pediatric programs for reduction of maternal and infant mortality worldwide.IMPORTANCE This report provides an overview of the proceedings of the 4th International Maternal and Neonatal Immunization Symposium, where presentations focused on the state-of-the-art research on the development and implementation of vaccines given during pregnancy for the protection of mothers and infants

West Nile Virus Methyltransferase Domain Interacts with Protein Kinase

Background The flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent protein kinase, has previously been shown to phosphorylate dengue virus NS5. Methods We used mass spectrometry to specifically identify NS5 phosphosites. Co-immunoprecipitation assays were used to study protein-protein interactions. Effects on viral replication were measured via replicon system and plaque assay titering. Results We identified multiple sites in West Nile virus (WNV) NS5 that are phosphorylated during a WNV infection, and showed that the N-terminal methyltransferase domain of WNV NS5 can be specifically phosphorylated by PKG in vitro. Expressing PKG in cell culture led to an enhancement of WNV viral production. We hypothesized this effect on replication could be caused by factors beyond the specific phosphorylations of NS5. Here we show for the first time that PKG is also able to stably interact with a viral substrate, WNV NS5, in cell culture and in vitro. While the mosquito-borne WNV NS5 interacted with PKG, tick-borne Langat virus NS5 did not. The methyltransferase domain of NS5 is able to mediate the interaction between NS5 and PKG, and mutating positive residues in the αE region of the methyltransferase interrupts the interaction. These same mutations completely inhibited WNV replication. Conclusions PKG is not required for WNV replication, but does make a stable interaction with NS5. While the consequence of the NS5:PKG interaction when it occurs is unclear, mutational data demonstrates that this interaction occurs in a region of NS5 that is otherwise necessary for replication. Overall, the results identify an interaction between virus and a cellular kinase and suggest a role for a host kinase in enhancing flaviviral replication

Persistence of Resistant Staphylococcus epidermidis after Single Course of Clarithromycin

Short course of antimicrobial therapy can select resistant bacteria that persist for 4 years or longer

Mutational escape from the polyclonal antibody response to SARS-CoV-2 infection is largely shaped by a single class of antibodies

Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasma, including plasma from individuals from whom some of the antibodies were isolated. The plasma-escape maps most closely resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is dominated by a single class of antibodies targeting an epitope that is already undergoing rapid evolution

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal IgGs and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1^A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4Å cryo-EM structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses and that characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies

Effects of the antimicrobial tylosin on the microbial community structure of an anaerobic sequencing batch reactor

The effects of the antimicrobial tylosin on a methanogenic microbial community were studied in a glucose-fed laboratory-scale anaerobic sequencing batch reactor (ASBR) exposed to stepwise increases of tylosin (0, 1.67, and 167 mg/L). The microbial community structure was determined using quantitative fluorescence in situ hybridization (FISH) and phylogenetic analyses of bacterial 16S ribosomal RNA (rRNA) gene clone libraries of biomass samples. During the periods without tylosin addition and with an influent tylosin concentration of 1.67 mg/L, 16S rRNA gene sequences related to Syntrophobacter were detected and the relative abundance of Methanosaeta species was high. During the highest tylosin dose of 167 mg/L, 16S rRNA gene sequences related to Syntrophobacter species were not detected and the relative abundance of Methanosaeta decreased considerably. Throughout the experimental period, Propionibacteriaceae and high GC Gram-positive bacteria were present, based on 16S rRNA gene sequences and FISH analyses, respectively. The accumulation of propionate and subsequent reactor failure after long-term exposure to tylosin are attributed to the direct inhibition of propionate-oxidizing syntrophic bacteria closely related to Syntrophobacter and the indirect inhibition of Methanosaeta by high propionate concentrations and low pH. Biotechnol. Bioeng. 2011;108: 296–305. © 2010 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78493/1/22934_ftp.pd