Identification of the N-Terminal Domain of the Influenza Virus PA Responsible for the Suppression of Host Protein Synthesis

Cellular protein synthesis is suppressed during influenza virus infection, allowing for preferential production of viral proteins. To explore the impact of polymerase subunits on protein synthesis, we coexpressed enhanced green fluorescent protein (eGFP) or luciferase together with each polymerase component or NS1 of A/California/04/2009 (Cal) and found that PA has a significant impact on the expression of eGFP and luciferase. Comparison of the suppressive activity on coexpressed proteins between various strains revealed that avian virus or avian-origin PAs have much stronger activity than human-origin PAs, such as the one from A/WSN/33 (WSN). Protein synthesis data suggested that reduced expression of coexpressed proteins is not due to PA''s reported proteolytic activity. A recombinant WSN containing Cal PA showed enhanced host protein synthesis shutoff and induction of apoptosis. Further characterization of the PA fragment indicated that the N-terminal domain (PANt), which includes the endonuclease active site, is sufficient to suppress cotransfected gene expression. By characterizing various chimeric PANts, we found that multiple regions of PA, mainly the helix α4 and the flexible loop of amino acids 51 to 74, affect the activity. The suppressive effect of PANt cDNA was mainly due to PA-X, which was expressed by ribosomal frameshifting. In both Cal and WSN viruses, PA-X showed a stronger effect than the corresponding PANt, suggesting that the unique C-terminal sequences of PA-X also play a role in suppressing cotransfected gene expression. Our data indicate strain variations in PA gene products, which play a major role in suppression of host protein synthesis.     

Ribavirin-Resistant Mutants of Human Enterovirus 71 Express a High Replication Fidelity Phenotype during Growth in Cell Culture

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 μM ribavirin, whereas the growth of parental 26M and the G64N mutant viruses were inhibited in the presence of 800 μM 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 μM 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.     

What could arsenic bacteria teach us about life?

In this paper, I discuss the recent discovery of alleged arsenic bacteria in Mono Lake, California, and the ensuing debate in the scientific community about the validity and significance of these results. By situating this case in the broader context of projects that search for anomalous life forms, I examine the methodology and upshots of challenging biochemical constraints on living things. I distinguish between a narrower and a broader sense in which we might challenge or change our knowledge of life as the result of such a project, and discuss two different kinds of projects that differ in their potential to overhaul our knowledge of life. I argue that the arsenic bacteria case, while potentially illuminating, is the kind of constraint-challenging project that could not—in spite of what was said when it was presented to the public—change our knowledge of life in the deeper sense.     

CORRELATION BETWEEN ANOLIS LIZARD DEWLAP PHENOTYPE AND ENVIRONMENTAL VARIATION INDICATES ADAPTIVE DIVERGENCE OF A SIGNAL IMPORTANT TO SEXUAL SELECTION AND SPECIES RECOGNITION

Although the importance of signals involved in species recognition and sexual selection to speciation is widely recognized, the processes that underlie signal divergence are still a matter of debate. Several possible processes have been hypothesized, including genetic drift, arbitrary sexual selection, and adaptation to local signaling environments. We use comparative analyses to investigate whether the remarkable geographic variation of dewlap phenotype in a Hispaniolan trunk Anolis lizard (A. distichus) is a result of adaptive signal divergence to heterogeneous environments. We recover a repeated pattern of divergence in A. distichus dewlap color, pattern, and size with environmental variation across Hispaniola. These results are aligned with ecological models of signal divergence and provide strong evidence for dewlap adaptation to local signaling environments. We also find that A. distichus dewlaps vary with the environment in a different manner to other previously studied anoles, thus expanding upon previous predictions on the direction dewlaps will diverge in perceptual color space in response to the environment.     

Sequence and domain arrangements influence mechanical properties of elastin‐like polymeric elastomers

Elastin is the polymeric, extracellular matrix protein that provides properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Elastin assembles by crosslinking through lysine residues of its monomeric precursor, tropoelastin. Tropoelastin, as well as polypeptides based on tropoelastin sequences, undergo a process of self‐assembly that aligns lysine residues for crosslinking. As a result, both the full‐length monomer as well as elastin‐like polypeptides (ELPs) can be made into biomaterials whose properties resemble those of native polymeric elastin. Using both full‐length human tropoelastin (hTE) as well as ELPs, we and others have previously reported on the influence of sequence and domain arrangements on self‐assembly properties. Here we investigate the role of domain sequence and organization on the tensile mechanical properties of crosslinked biomaterials fabricated from ELP variants. In general, substitutions in ELPs involving similiar domain types (hydrophobic or crosslinking) had little effect on mechanical properties. However, modifications altering either the structure or the characteristic sequence style of these domains had significant effects on such properties. In addition, using a series of deletion and replacement constructs for full‐length hTE, we provide new insights into the role of conserved domains of tropoelastin in determining mechanical properties. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 392–407, 2013.     

Exome Sequencing Identifies Mutations in CCDC114 as a Cause of Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 14 genes, but they collectively account for only ∼60% of all PCD. To identify mutations that cause PCD, we performed exome sequencing on six unrelated probands with ciliary outer dynein arm (ODA) defects. Mutations in CCDC114, an ortholog of the Chlamydomonas reinhardtii motility gene DCC2, were identified in a family with two affected siblings. Sanger sequencing of 67 additional individuals with PCD with ODA defects from 58 families revealed CCDC114 mutations in 4 individuals in 3 families. All 6 individuals with CCDC114 mutations had characteristic oto-sino-pulmonary disease, but none had situs abnormalities. In the remaining 5 individuals with PCD who underwent exome sequencing, we identified mutations in two genes (DNAI2, DNAH5) known to cause PCD, including an Ashkenazi Jewish founder mutation in DNAI2. These results revealed that mutations in CCDC114 are a cause of ciliary dysmotility and PCD and further demonstrate the utility of exome sequencing to identify genetic causes in heterogeneous recessive disorders.     

Comparative histomorphology of intrinsic vibrissa musculature among primates: implications for the evolution of sensory ecology and “face touch”

Macrovibrissae are specialized tactile sensory hairs present in most mammalian orders, used in maxillary mechanoreception or “face touch.” Some mammals have highly organized vibrissae and are able to “whisk” them. Movement of vibrissae is influenced by intrinsic vibrissa musculature, striated muscle bands that attach directly to the vibrissa capsule. It is unclear if primates have organized vibrissae or intrinsic vibrissa musculature and it is uncertain if they can move their vibrissae. The present study used histomorphological techniques to compare vibrissae among 19 primates and seven non‐primate mammalian taxa. Upper lips of these mammals were sectioned and processed for histochemical analysis. While controlling for phylogenetic effects the following hypotheses were tested: 1) mammals with well‐organized vibrissae possess intrinsic vibrissa musculature and 2) intrinsic vibrissa musculature is best developed in nocturnal, arboreal taxa. Our qualitative analyses show that only arboreal, nocturnal prosimians possess intrinsic musculature. Not all taxa that possessed organized vibrissae had intrinsic vibrissa musculature. Phylogenetic comparative analyses revealed a 70% probability that stem mammals, primates, and haplorhines possessed intrinsic vibrissa musculature and well‐organized vibrissae. These two traits most likely coevolved according to a discrete phylogenetic analysis. These results indicate that nocturnal, arboreal primates have the potential to more actively use their vibrissae in spatial recognition and navigation tasks than diurnal, more terrestrial species, but there is a clear phylogenetic signal involved in the evolution of primate vibrissae and “face touch.” Am J Phys Anthropol, 2013. © 2012 Wiley Periodicals, Inc.     

Whole-Exome Sequencing Identifies Mutated C12orf57 in Recessive Corpus Callosum Hypoplasia

The corpus callosum is the principal cerebral commissure connecting the right and left hemispheres. The development of the corpus callosum is under tight genetic control, as demonstrated by abnormalities in its development in more than 1,000 genetic syndromes. We recruited more than 25 families in which members affected with corpus callosum hypoplasia (CCH) lacked syndromic features and had consanguineous parents, suggesting recessive causes. Exome sequence analysis identified C12orf57 mutations at the initiator methionine codon in four different families. C12orf57 is ubiquitously expressed and encodes a poorly annotated 126 amino acid protein of unknown function. This protein is without significant paralogs but has been tightly conserved across evolution. Our data suggest that this conserved gene is required for development of the human corpus callosum.     

Germline Mutations in NFKB2 Implicate the Noncanonical NF-κB Pathway in the Pathogenesis of Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by antibody deficiency, poor humoral response to antigens, and recurrent infections. To investigate the molecular cause of CVID, we carried out exome sequence analysis of a family diagnosed with CVID and identified a heterozygous frameshift mutation, c.2564delA (p.Lys855Serfs^∗7), in NFKB2 affecting the C terminus of NF-κB2 (also known as p100/p52 or p100/p49). Subsequent screening of NFKB2 in 33 unrelated CVID-affected individuals uncovered a second heterozygous nonsense mutation, c.2557C>T (p.Arg853^∗), in one simplex case. Affected individuals in both families presented with an unusual combination of childhood-onset hypogammaglobulinemia with recurrent infections, autoimmune features, and adrenal insufficiency. NF-κB2 is the principal protein involved in the noncanonical NF-κB pathway, is evolutionarily conserved, and functions in peripheral lymphoid organ development, B cell development, and antibody production. In addition, Nfkb2 mouse models demonstrate a CVID-like phenotype with hypogammaglobulinemia and poor humoral response to antigens. Immunoblot analysis and immunofluorescence microscopy of transformed B cells from affected individuals show that the NFKB2 mutations affect phosphorylation and proteasomal processing of p100 and, ultimately, p52 nuclear translocation. These findings describe germline mutations in NFKB2 and establish the noncanonical NF-κB signaling pathway as a genetic etiology for this primary immunodeficiency syndrome.