Severe viral respiratory infections in children with IFIH1 loss-of-function mutations.

Viral respiratory infections are usually mild and self-limiting; still they exceptionally result in life-threatening infections in previously healthy children. To investigate a potential genetic cause, we recruited 120 previously healthy children requiring support in intensive care because of a severe illness caused by a respiratory virus. Using exome and transcriptome sequencing, we identified and characterized three rare loss-of-function variants in IFIH1, which encodes an RIG-I-like receptor involved in the sensing of viral RNA. Functional testing of the variants IFIH1 alleles demonstrated that the resulting proteins are unable to induce IFN-ß, are intrinsically less stable than wild-type IFIH1, and lack ATPase activity. In vitro assays showed that IFIH1 effectively restricts replication of human respiratory syncytial virus and rhinoviruses. We conclude that IFIH1 deficiency causes a primary immunodeficiency manifested in extreme susceptibility to common respiratory RNA viruses.

A Short Double-Stapled Peptide Inhibits Respiratory Syncytial Virus Entry and Spreading.

Synthetic peptides derived from the heptad repeat (HR) of fusion (F) proteins can be used as dominant negative inhibitors to inhibit the fusion mechanism of class I viral F proteins. Here, we have performed a stapled-peptide scan across the HR2 domain of the respiratory syncytial virus (RSV) F protein with the aim to identify a minimal domain capable of disrupting the formation of the postfusion six-helix bundle required for viral cell entry. Constraining the peptides with a single staple was not sufficient to inhibit RSV infection. However, the insertion of double staples led to the identification of novel short stapled peptides that display nanomolar potency in HEp-2 cells and are exceptionally robust to proteolytic degradation. By replacing each amino acid of the peptides by an alanine, we found that the substitution of residues 506 to 509, located in a patch of polar contacts between HR2 and HR1, severely affected inhibition. Finally, we show that intranasal delivery of the most potent peptide to BALB/c mice significantly decreased RSV infection in upper and lower respiratory tracts. The discovery of this minimal HR2 sequence as a means for inhibition of RSV infection provides the basis for further medicinal chemistry efforts toward developing RSV fusion antivirals.

Mismatches in the Influenza A Virus RNA Panhandle Prevent Retinoic Acid-Inducible Gene I (RIG-I) Sensing by Impairing RNA/RIG-I Complex Formation.

Influenza virus RNA (vRNA) promoter panhandle structures are believed to be sensed by retinoic acid-inducible gene I (RIG-I). The occurrence of mismatches in this double-stranded RNA structure raises questions about their effect on innate sensing. Our results suggest that mismatches in vRNA promoters decrease binding to RIG-I in vivo, affecting RNA/RIG-I complex formation and preventing RIG-I activation. These results can be inferred to apply to other viruses and suggest that mismatches may represent a general viral strategy to escape RIG-I sensing.

Paramyxovirus ultrastructure and genome packaging: cryo-electron tomography of sendai virus.

Members of the Paramyxoviridae such as measles, mumps, and parainfluenza viruses have pleomorphic, enveloped virions that contain negative-sense unsegmented RNA genomes. This is encapsidated by multiple copies of a viral nucleocapsid protein N to form a helical ribonucleoprotein complex (termed the nucleocapsid), which acts as the template for both transcription and replication. Structure analysis of these viruses has proven challenging, owing to disordered regions in important constituent proteins, conformational flexibility in the nucleocapsid and the pleomorphic nature of virus particles. We conducted a low-resolution ultrastructural analysis of Sendai virus, a prototype paramyxovirus, using cryo-electron tomography. Virions are highly variable in size, ranging approximately from 110 to 540 nm in diameter. Envelope glycoproteins are densely packed on the virion surface, while nucleocapsids are clearly resolved in the virion interior. Subtomogram segmentation and filament tracing allowed us to define the path of many nucleocapsids and in some cases to determine the number of putative genomes within a single virus particle. Our findings indicate that these viruses may contain between one and six copies of their genome per virion and that there is no discernible order to nucleocapsid packaging.

Bacterial cell cycle control by citrate synthase independent of enzymatic activity.

Proliferating cells must coordinate central metabolism with the cell cycle. How central energy metabolism regulates bacterial cell cycle functions is not well understood. Our forward genetic selection unearthed the Krebs cycle enzyme citrate synthase (CitA) as a checkpoint regulator controlling the G1→S transition in the polarized alpha-proteobacterium Caulobacter crescentus, a model for cell cycle regulation and asymmetric cell division. We find that loss of CitA promotes the accumulation of active CtrA, an essential cell cycle transcriptional regulator that maintains cells in G1-phase, provided that the (p)ppGpp alarmone is present. The enzymatic activity of CitA is dispensable for CtrA control, and functional citrate synthase paralogs cannot replace CitA in promoting S-phase entry. Our evidence suggests that CitA was appropriated specifically to function as a moonlighting enzyme to link central energy metabolism with S-phase entry. Control of the G1-phase by a central metabolic enzyme may be a common mechanism of cellular regulation.

Patchwork structure-function analysis of the Sendai virus matrix protein.

Paramyxoviruses contain a bi-lipidic envelope decorated by two transmembrane glycoproteins and carpeted on the inner surface with a layer of matrix proteins (M), thought to bridge the glycoproteins with the viral nucleocapsids. To characterize M structure-function features, a set of M domains were mutated or deleted. The genes encoding these modified M were incorporated into recombinant Sendai viruses and expressed as supplemental proteins. Using a method of integrated suppression complementation system (ISCS), the functions of these M mutants were analyzed in the context of the infection. Cellular membrane association, localization at the cell periphery, nucleocapsid binding, cellular protein interactions and promotion of viral particle formation were characterized in relation with the mutations. At the end, lack of nucleocapsid binding go together with lack of cell surface localization and both features definitely correlate with loss of M global function estimated by viral particle production.

Sendai virus induced cytoplasmic actin remodeling correlates with efficient virus particle production.

Cytoplasmic actins have been found interacting with viral proteins and identified in virus particles. We analyzed by confocal microscopy the cytoplasmic ß- and γ-actin patterns during the course of Sendai virus infections in polarized cells. We observed a spectacular remodeling of the ß-cytoplasmic actin which correlated with productive viral multiplication. Conversely, suppression of M during the course of a productive infection resulted in the decrease of particle production and the absence of ß-actin remodeling. As concomitant suppression of ß- and γ-actins resulted as well in reduction of virus particle production, we propose that Sendai virus specifically induces actin remodeling in order to promote efficient virion production. Beta- and γ-cytoplasmic actin recruitment could substitute for that of the endosomal sorting complex required for transport (ESCRT) mobilized by other enveloped viruses but apparently not used by Sendai virus.

Sendai virus particle production: basic requirements and role of the SYWST motif present in HN cytoplasmic tail.

Sendai virus (SeV) HN protein is dispensable for virus particle production. HN incorporation into virions strictly depends on a cytoplasmic domain SYWST motif. HNAFYKD, with SYWST replaced with the analogous sequence of measles virus (MeV) H (AFYKD), is not incorporated in virus particles produced by LLCMK2 cells, although it is normally expressed at the plasma membrane. Unlike HNSYWST, HNAFYKD is not internalized to late endosomes, raising the possibility that HN internalization is required for uptake into virus particles. Various mosaic MeV-H containing increasing amounts of the SeV-HN all failed to be taken up in SeV virions. However, when co-expressed with HNAFYKD these MeV-H chimera induced HNAFYKD uptake into virions showing that internalization is not a prerequisite for HN uptake into particles. We propose that HN incorporation in virus particles requires first neutralization by HN of a putative inhibitor of infectious particle formation.

Suppression of the Sendai virus M protein through a novel short interfering RNA approach inhibits viral particle production but does not affect viral RNA synthesis.

Short RNA interference is more and more widely recognized as an effective method to specifically suppress viral functions in eukaryotic cells. Here, we used an experimental system that allows suppression of the Sendai virus (SeV) M protein by using a target sequence, derived from the green fluorescent protein gene, that was introduced in the 3' untranslated region of the M protein mRNA. Silencing of the M protein gene was eventually achieved by a small interfering RNA (siRNA) directed against this target sequence. This siRNA was constitutively expressed in a cell line constructed by transduction with an appropriate lentivirus vector. Suppression of the M protein was sufficient to diminish virus production by 50- to 100-fold. This level of suppression had no apparent effect on viral replication and transcription, supporting the lack of M involvement in SeV transcription or replication control.

From assembly to virus particle budding: pertinence of the detergent resistant membranes.

Detergent resistant membranes (DRMs) are the site of assembly for a variety of viruses. Here, we make use of Sendai virus mutant proteins that are not packaged into virus particles to determine the involvement of this assembly for the virus particle production. We found that, in the context of an infection, (1) all the Sendai virus proteins associated in part with DRMs, (2) mutant HN and M proteins not packaged into virus particles were similarly part of this association, (3) after M protein suppression resulting in a significant reduction of virus production, the floatation profile of the other viral proteins was not altered and finally (4) cellular cholesterol depletion did not decrease the virus particle production, although it somehow reduced their virus infectivity. These results led us to conclude that the assembly complex found in DRM fractions does not constitute a direct precursor of virus particle budding.

Nature of a paramyxovirus replication promoter influences a nearby transcription signal.

The genomic and antigenomic 3' ends of the Sendai virus replication promoters are bi-partite in nature. They are symmetrically composed of leader or trailer sequences, a gene start (gs) or gene end (ge) site, respectively, and a simple hexameric repeat. Studies of how mRNA synthesis initiates from the first gene start site (gs1) have been hampered by the fact that gs1 is located between two essential elements of the replication promoter. Transcription initiation, then, is separated from the replication initiation site by only 56 nt on the genome, so that transcription and replication may sterically interfere with each other. In order to study the initiation of Sendai virus mRNAs without this possible interference, Sendai virus mini-genomes were prepared having tandem promoters in which replication takes place from the external one, whereas mRNA synthesis occurs from the internal one. Transcription now initiates at position 146 rather than position 56 relative to the genome 3' end. Under these conditions, it was found that the frequency with which mRNA synthesis initiates depends, in an inverse fashion, on the strength of the external replication promoter. It was also found that the sequences essential for replication are not required for basic mRNA synthesis as long as there is an external replication promoter at which viral RNA polymerase can enter the nucleocapsid template. The manner in which transcription and replication initiations influence each other is discussed.