Capsid protein mediated evasion of IRAK1-dependent signalling is essential to Sindbis virus neuroinvasion and virulence in mice.

ABSTRACTAlphaviruses are arthropod-borne, single-stranded positive-sense RNA viruses that are recognized as rapidly emerging pathogens. Despite being exquisitely sensitive to the effects of the innate immune response alphaviruses can readily replicate, disseminate, and induce pathogenesis in immunologically competent hosts. Nonetheless, how alphaviruses evade the induction of an innate immune response prior to viral gene expression, or in non-permissive infections, is unknown. Previously we reported the identification of a novel host/pathogen interaction between the viral Capsid (CP) protein and the host IRAK1 protein. The CP/IRAK1 interaction was determined to negatively impact IRAK1-dependent PAMP detection in vitro, however, the precise importance of the CP/IRAK1 interaction to alphaviral infection remained unknown. Here we detail the identification of the CP/IRAK1 interaction determinants of the Sindbis virus (SINV) CP protein and examine the importance of the interaction to alphaviral infection and pathogenesis in vivo using an interaction deficient mutant of the model neurotropic strain of SINV. Importantly, these interaction determinants are highly conserved across multiple Old-World alphaviruses, including Ross River virus (RRV), Mayaro virus (MAYV), Chikungunya virus (CHIKV), and Semliki Forest virus (SFV). In the absence of a functional CP/IRAK1 interaction, SINV replication is significantly restricted and fails to disseminate from the primary site of inoculation due to the induction of a robust type-I Interferon response. Altogether these data indicate that the evasion of IRAK1-dependent signalling is critical to overcoming the host innate immune response and the in vivo data presented here demonstrate the importance of the CP/IRAK1 interaction to neurovirulence and pathogenesis.

Dancing with the Devil: A Review of the Importance of Host RNA-Binding Proteins to Alphaviral RNAs during Infection.

Alphaviruses are arthropod-borne, single-stranded positive sense RNA viruses that rely on the engagement of host RNA-binding proteins to efficiently complete the viral lifecycle. Because of this reliance on host proteins, the identification of host/pathogen interactions and the subsequent characterization of their importance to viral infection has been an intensive area of study for several decades. Many of these host protein interaction studies have evaluated the Protein:Protein interactions of viral proteins during infection and a significant number of host proteins identified by these discovery efforts have been RNA Binding Proteins (RBPs). Considering this recognition, the field has shifted towards discovery efforts involving the direct identification of host factors that engage viral RNAs during infection using innovative discovery approaches. Collectively, these efforts have led to significant advancements in the understanding of alphaviral molecular biology; however, the precise extent and means by which many RBPs influence viral infection is unclear as their specific contributions to infection, as per any RNA:Protein interaction, have often been overlooked. The purpose of this review is to summarize the discovery of host/pathogen interactions during alphaviral infection with a specific emphasis on RBPs, to use new ontological analyses to reveal potential functional commonalities across alphaviral RBP interactants, and to identify host RBPs that have, and have yet to be, evaluated in their native context as RNA:Protein interactors.

Evaluating Metabolic Pathways and Biofilm Formation in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia has recently arisen as a prominent nosocomial pathogen because of its high antimicrobial resistance and ability to cause chronic respiratory infections. Often the infections are worsened by biofilm formation which enhances antibiotic tolerance. We have previously found that mutation of the gpmA gene, encoding the glycolytic enzyme phosphoglycerate mutase, impacts the formation of this biofilm on biotic and abiotic surfaces at early time points. This finding, indicating an association between carbon source and biofilm formation, led us to hypothesize that metabolism would influence S. maltophilia biofilm formation and planktonic growth. In the present study, we tested the impact of various growth substrates on biofilm levels and growth kinetics to determine metabolic requirements for these processes. We found that S. maltophilia wild type preferred amino acids versus glucose for planktonic and biofilm growth and that gpmA deletion inhibited growth in amino acids. Furthermore, supplementation of the ΔgpmA strain by glucose or ribose phenotypically complemented growth defects. These results suggest that S. maltophilia shuttles amino acid carbon through gluconeogenesis to an undefined metabolic pathway supporting planktonic and biofilm growth. Further evaluation of these metabolic pathways might reveal novel metabolic activities of this pathogen. IMPORTANCE Stenotrophomonas maltophilia is a prominent opportunistic pathogen that often forms biofilms during infection. However, the molecular mechanisms of virulence and biofilm formation are poorly understood. The glycolytic enzyme phosphoglycerate mutase appears to play a role in biofilm formation, and we used a mutant in its gene (gpmA) to probe the metabolic circuitry potentially involved in biofilm development. The results of our study indicate that S. maltophilia displays unique metabolic activities, which could be exploited for inhibiting growth and biofilm formation of this pathogen.

Cyanuric chloride as the basis for compositionally diverse lipids.

Cyanuric chloride has been utilized in the development of new synthetic lipid compounds using two differing schemes. The resulting lipids, presented in this manuscript, were characterized and evaluated for their ability to form nanoparticles and subsequently tested for their utility in various biological applications, including gene delivery and immunization. Of the 12 lipids synthesized, 8 formed nanoparticles that remained stable, based on dynamic light scattering, for at least one month. The compounds were then assessed for their toxicity, and subsequently tested for their ability to encapsulate drugs, genes and peptides. While the compounds did not seem to encapsulate carboxyfluorescein, we demonstrate that these lipids are capable of plasmid delivery in vitro, and inducing antibody profiles similar to other hydrophobic anchors in liposomal peptide vaccines. This strategy for accessing diverse lipid compounds offers a way to easily optimize lipid-based therapeutics for research in an expedited manner.

Autoantibody Responses to Apolipoprotein A-I Are Not Diet- or Sex-Linked in C57BL/6 Mice.

Atherosclerosis is responsible for a large percentage of all-cause mortality worldwide, but it is only now beginning to be understood as a complex disease process involving metabolic insult, chronic inflammation, and multiple immune mechanisms. Abs targeting apolipoprotein A-I (ApoA-I) have been found in patients with cardiovascular disease, autoimmune conditions, as well as those with no documented history of either. However, relatively little is known about how these Abs are generated and their relationship to diet and sex. In the current study, we modeled this aspect of autoimmunity using anti-ApoA-I immunization of male and female C57BL/6 mice. Unexpectedly, we found that autoantibodies directed against a single, previously unknown, epitope within the ApoA-I protein developed irrespective of immunization status or dyslipidemia in mice. When total IgG subclasses were analyzed over the course of time, we observed that rather than driving an increase in inflammatory IgG subclasses, consumption of Western diet suppressed age-dependent increases in IgG2b and IgG2c in male mice only. The lack of change observed in female mice suggested that diet and sex might play a combined role in Th1/Th2 balance and, ultimately, in immunity to pathogen challenge. This report demonstrates the need for inclusion of both sexes in studies pertaining to diet and aging and suggests that further study of immunogenic epitopes present in ApoA-I is warranted.