RNA X-ray footprinting reveals the consequences of an in vivo acquired determinant of viral infectivity

The secondary structures of the bacteriophage MS2 ssRNA genome, frozen in defined states, were determined with minimal perturbation using constraints from X-ray synchrotron footprinting (XRF). The footprints of the gRNA in the virion and as transcript are consistent with single, dominant but distinct conformations, and reveal the presence of multiple Packaging Signals potentially involved in assembly regulation that have not been detected by other techniques. XRF also reveals the dramatic effect of the unique Maturation Protein (MP) on both the capsid lattice, and the gRNA conformation inside the phage compared with a virus-like-particle composed only of coat protein subunits. Aspects of genome organisation in the phage, their impacts on the capsid shell, and the distortion of lattice geometry by MP, are hallmarks of molecular frustration. Phage assembly therefore appears to prepare the particle for the next step of the infectious cycle.

Engineered promoter-switched viruses reveal the role of poxvirus maturation protein A26 as a negative regulator of viral spread

Vaccinia virus produces two types of virions known as single-membraned intracellular mature virus (MV) and double-membraned extracellular enveloped virus (EV). EV production peaks earlier when initial MV are further wrapped and secreted to spread infection within the host. However, late during infection MV accumulate intracellularly and become important for host-to-host transmission. The process that regulates this switch remains elusive and is thought to be influenced by host factors. Here we examined the hypothesis that EV and MV production are regulated by the virus through expression of F13 and the MV-specific protein A26. By switching the promoters and altering the expression kinetics of F13 and A26, we demonstrate that A26 expression downregulates EV production and plaque size, thus limiting viral spread. This process correlates with A26 association with the MV surface protein A27 and exclusion of F13, thus reducing EV titres. Thus, MV maturation is controlled by the abundance of the viral A26 protein, independently of other factors, and is rate-limiting for EV production. The A26 gene is conserved within vertebrate poxviruses, but strikingly lost in poxviruses known to be transmitted exclusively by biting arthropods. A26-mediated virus maturation thus has the appearance to be an ancient evolutionary adaptation to enhance transmission of poxviruses that has subsequently been lost from vector-adapted species, for which it may serve as a genetic signature. The existence of virus-regulated mechanisms to produce virions adapted to fulfil different functions represents a novel level of complexity in mammalian viruses with major impact on evolution, adaptation and transmission. IMPORTANCE Chordopoxviruses are mammalian viruses that uniquely produce a first type of virion adapted to spread within the host and a second type that enhances transmission between hosts, which can take place by multiple ways including direct contact, respiratory droplets, oral/fecal routes, or via vectors. Both virion types are important to balance intra-host dissemination and inter-host transmission, so virus maturation pathways must be tightly controlled. Here we provide evidence that the abundance and kinetics of expression of the viral protein A26 regulates this process by preventing formation of the first form and shifting maturation towards the second form. A26 is expressed late after the initial wave of progeny virions is produced, so sufficient viral dissemination is ensured, and provides virions with enhanced environmental stability. Conservation of A26 in all vertebrate poxviruses but those transmitted exclusively via biting arthropods reveals the importance of A26-controlled virus maturation for transmission routes involving environmental exposure.

cDNA-derived RNA phage assembly reveals critical residues in the maturation protein of the Pseudomonas aeruginosa leviphage, PP7

PP7 is a leviphage with single-stranded RNA genome, which infects Pseudomonas aeruginosa PAO1. A reverse genetic system for PP7 was previously created by using reverse-transcribed cDNA (PP7O) from virion-derived RNA genome. Here, we have found that the PP7O cDNA contained 20 nucleotide differences from the PP7 genome sequence deposited in the database. We created another reverse genetic system exploiting chemically synthesized cDNA (PP7S) based on the database sequence. Unlike PP7O that rendered infectious PP7 virions, PP7S-derived particles were incapable of plaque formation on PAO1 cells, which was restored on the PAO1 cells expressing the maturation protein (MP) from PP7O. Using this reverse genetic system, we revealed two amino acid residues involved in the known roles of MP (i.e. adsorption and genome replication), fortuitously providing a lesson that the viral RNA genome sequencing needs functional verification possibly by a reverse genetic system. IMPORTANCE Biological significance of RNA phages has been largely ignored, ironically because few studies have been focusing on RNA phages. As an initial attempt to properly represent RNA phages in the phageome, we previously created, by using reverse-transcribed cDNA, a reverse genetic system for the small RNA phage, PP7 that infects the opportunistic human pathogen, Pseudomonas aeruginosa. We here report another system by using chemically synthesized cDNA based on the database genome that has 20 nucleotide differences from the previous cDNA. Investigation of those cDNA-derived phage virions unveiled that two amino acids of the maturation protein are crucial for the normal phage lifecycle at different steps. Our study provides an insight into the molecular basis for the RNA phage lifecycle and a lesson that the RNA genome sequencing needs to be carefully validated by cDNA-based phage assembly systems.

ssRNA phage penetration triggers detachment of the F-pilus

Although the F-specific ssRNA phage MS2 has long had paradigm status, little is known about penetration of the genomic RNA (gRNA) into the cell. The phage initially binds to the F-pilus using its maturation protein (Mat), and then the Mat-bound gRNA is released from the viral capsid and somehow crosses the bacterial envelope into the cytoplasm. To address the mechanics of this process, we fluorescently labeled the ssRNA phage MS2 to track F-pilus dynamics during infection. We discovered that ssRNA phage infection triggers the release of F-pili from host cells, and that higher multiplicity of infection (MOI) correlates with detachment of longer F-pili. We also report that entry of gRNA into the host cytoplasm requires the F-plasmid–encoded coupling protein, TraD, which is located at the cytoplasmic entrance of the F-encoded type IV secretion system (T4SS). However, TraD is not essential for pilus detachment, indicating that detachment is triggered by an early step of MS2 engagement with the F-pilus or T4SS. We propose a multistep model in which the ssRNA phage binds to the F-pilus and through pilus retraction engages with the distal end of the T4SS channel at the cell surface. Continued pilus retraction pulls the Mat-gRNA complex out of the virion into the T4SS channel, causing a torsional stress that breaks the mature F-pilus at the cell surface. We propose that phage-induced disruptions of F-pilus dynamics provides a selective advantage for infecting phages and thus may be prevalent among the phages specific for retractile pili.

Increased Expression of B Lymphocyte Induced Maturation Protein 1 (BLIMP1) in Patients with Common Variable Immunodeficiency (CVID)

Common variable immunodeficiency (CVID) is a primary immune deficiency disorder characterized by a failure in B cell differentiation, impaired immunoglobulin production, and defect in response to vaccines. As a result of defective B cell maturation and differentiation in CVID, the affected patients commonly present with reduced numbers of memory B cell and antibody-secreting plasma cells. B-cell lymphoma 6 protein (BCL6) and B lymphocyte induced maturation protein 1 (BLIMP1) molecules are two important transcription factors that have key roles in the maturation of B cells to plasma cells. Hence, in the current survey, we aimed to evaluate the mRNA and protein expression levels of BCL6 and BLIMP1 in B lymphocytes isolated from peripheral blood in CVID patients. We collected blood samples from 12 CVID patients and 12 healthy controls. We isolated peripheral blood mononuclear cells (PBMCs) using Ficoll density gradient separation. Then, CD19+ B cells were purified using MACS. The protein expression and transcriptional level of BCL6 and BLIMP1 were respectively measured using flow cytometry and real-time PCR. Our results showed that the BLIMP1 mRNA expression, as well as BLIMP1 protein expression, were significantly higher in CVID patients compared to control subjects (p=0.009 and p=0.007, respectively). However, we found no significant difference in mRNA and protein expression of BCL6 between patients and healthy controls. According to our findings, increased mRNA and protein expression levels of BLIMP1 could be involved in defective maturation of B cells in patients with CVID and elucidate mechanistic insights into the pathogenesis of this disorder.

Structural characterization of genomic RNA-coat protein contacts in single-stranded RNA viruses by high-resolution cryo-EM

Recent developments in cryo-electron microscopy (cryo-EM) hardware along with continuously evolving software tools have led to the discovery of many novel structures that it was not possible to solve until now, resulting in what is termed “the resolution revolution”. In structural virology, it has also led to a re-evaluation of known structures. Most virion structures solved by X-ray crystallography or cryo-EM are focused on the capsid protein (CP) as a result of the application of icosahedral symmetry averaging to “improve” the electron density maps. However, this has the consequence that the intrinsic asymmetry of important components of virions, such as the viral genome and structural proteins lacking such symmetry, are masked. Single-stranded (ss), positive-sense RNA viruses are major pathogens in all kingdoms of life. Asymmetric cryo-EM structure determination of a major model virus in this class, bacteriophage MS2, reveals the limitations of a symmetrized view. As well as the presence and interactions made by the unique Maturation Protein, it also reveals multiple gRNA-CP dimer contacts corresponding to our previous prediction that dispersed, sequence-degenerate RNA motifs (Packaging Signals, PSs) play important roles during the virion assembly. Here, we describe how relaxing symmetry during structure determination can image such gRNA-PS contacts in a range of ssRNA viruses including the picornavirus Bovine Enterovirus-1, the alphaviruses Sindbis and Semliki Forest Viruses, as well as the plant virus Turnip Crinkle Virus. The revelation of these functionally important gRNA-CP contacts changes our fundamental understanding of assembly in these pathogens and may have further translational importance.

Proteomic Analysis of Lipid Droplets in Sesamum indicum

We attempted to identify the total proteome in sesame lipid droplets. Results from two-dimensional electrophoresis showed 139 protein spots in lipid droplet samples. Each spot was isolated, digested with trypsin, and applied to liquid chromatography–tandem mass spectrometry (Q-Tof Premier). As a result, 103 spots were identified. Although oleosin, caleosin, and steroleosin are known major components of the lipid droplet, many other proteins were also found in the lipid droplet. In addition to the three major proteins, TAG factor protein, glyceraldehyde-3-phosphate dehydrogenase, F1 ATPase, 70-kDa heat shock protein, seed maturation protein PM24, and 11S globulin precursor isoforms 3 and 4 were found in the lipid droplet. Three types of oleosins, 15-, 15.5-, and 17-kDa were present in the sesame lipid droplet, and the 15.5-kDa oleosin had high homology with oleosin from Coffea canephora. It has been shown by acid phosphatase treatment that oleosin proteins contain phosphate groups. Protein disulfide-isomerase 2 precursor, calreticulin-1, and BiP, which are known as marker proteins of the endoplasmic reticulum, were found as the components of the lipid droplet. Immunoconfocal microscopy was used to show that 11S globulin precursor isoform 3 and 4 were indeed localized in the lipid droplet. The presence of 11S globulin in the lipid droplets suggested a new mechanism for the lipid droplet formation.

Analysis of Blimp-1 and PD-1/PD-L1 Immune Checkpoint in an Autoimmune Thyroiditis Animal Model

Objective. B lymphocyte-induced maturation protein 1 (Blimp-1) and programmed cell death protein 1 (PD-1) have opposing roles in the development of T cells; however, the mechanism of autoimmune thyroiditis- (AIT-) associated abortion is unclear. The present study investigated the expression of Blimp-1 and PD-1/PD-ligand 1 (PD-L1) in AIT-associated pregnancy loss and elucidated the related signaling pathway involving in the inflammatory response. Methods. An experimental fetal loss model with autoimmune thyroiditis was established after murine thyroglobulin- (mTg-) immunized CBA/J female mice mating with Balb/c males. ELISA was employed to investigate the TgAb level in the serum of CBA/J female mice. The expression of Blimp-1, PD-1/PD-L1, mammalian target protein rapamycin (mTOR), and Foxp3 proteins in the placenta and spleen was detected through immunofluorescence staining and western blotting. Results. ELISA indicated that the serum TgAb level in the mTg group was higher than that in the control group (P<0.001). Fetal resorption rates increased in the mTg group compared with those in the control group (45.63% vs. 3.1%, P<0.05). Blimp-1 levels in the placenta and spleen were higher in the AIT-related miscarriage group than in the control group. However, the expression of PD-1/PD-L1 and Foxp3 was significantly decreased in the placenta and spleen in the AIT-related miscarriage group. Conclusion. Blimp-1 participates in the pathogenesis of autoimmune thyroid disease-associated pregnancy loss through the inflammatory immune response, which is potentially mediated through the PD-1/PD-L1 signaling pathway.