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-β, 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. V iral respiratory tract infections are the most common childhood infections worldwide, with close to 100% of children being infected during the first years of life. Whereas the vast majority of viral respiratory infections are mild and self-limiting, more severe disease leads to the hospitalization of about 3% of individuals in each birth cohort (1). In-hospital mortality rates are limited to <1% with intensive care support; still these infections account for 21% of childhood mortality worldwide (2, 3). The main viral pathogens causing lower respiratory tract infections are human respiratory syncytial virus (RSV), enteroviruses [including human rhinoviruses (HRV)], adenoviruses, human metapneumovirus, coronavirus, influenza, and parainfluenza viruses, with RSV being responsible for the majority of the hospitalized pediatric cases (4, 5).A number of risk factors including socioeconomic and environmental influences, preterm birth, chronic diseases, and immunosuppression are associated with more severe clinical presentation (6). However, ∼1 out of 1,000 children without any known risk factor will require intensive care support due to life-threatening manifestations of common viral respiratory infections. In the absence of established differences in pathogen virulence, we hypothesized that human genetic variation contributes to unusual susceptibility to severe disease due to common viruses. Supporting evidence is provided by a recent study, which showed that rare variants in IRF7 resulted in life-threatening influenza in an otherwise healthy child (7).We combined exome sequencing, transcriptomic analysis, and in vitro functional testing to identify and characterize potentially causal genetic variants in a prospective cohort of previously healthy children requiring intensive care support for common respiratory viral infections. We report the identification of a pathogen-restricted immunodeficiency due to loss-of-function variants in IFIH1, which result in defective innate recognition of RNA viruses, preventing the activation of an efficie...

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.The Paramyxoviridae include many medically important viruses such as measles, mumps, and parainfluenza viruses and the emerging zoonotic agents nipah and hendra viruses. There are also numerous animal pathogens within the Paramyxoviridae including Newcastle disease, rinderpest, peste-des-petitsruminants, and canine distemper viruses. The Paramyxoviridae are divided into two subfamilies, the Paramyxovirinae and the Pneumovirinae. The Paramyxovirinae includes the above-mentioned animal and human pathogens and is divided into five genera: Avulavirus, Henipavirus, Morbillivirus, Respirovirus, and Rubulavirus. The Pneumovirinae comprise human and bovine respiratory syncytial viruses, pneumonia virus of mice, and the metapneumoviruses. Paramyxoviruses have a negative-sense nonsegmented RNA genome that is encapsidated by multiple copies of a virally encoded nucleocapsid protein (N) to form a helical ribonucleoprotein complex known as the nucleocapsid. This is associated with the virally encoded RNA-dependent RNA polymerase, which is made up of the large protein (L) and the phosphoprotein (P). During virion morphogenesis, the nucleocapsid/RNA-dependent RNA polymerase complex is packaged within a lipid envelope at the host-cell plasma membrane. The envelope is studded with glycoproteins necessary for virus egress, attachment, and entry and is lined internally by the matrix protein (M). A number of species-specific accessory proteins are also found within the virion (13).Structural investigation of paramyxovirus biology has proven difficult, owing to disordered regions in some of their proteins, conformational flexibility in the nucleocapsid, and the pleomorphic nature of the paramyxovirus virion. For example, measles virus N has be...

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.

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.Enveloped viruses derive their envelope from cellular membranes after the viral components have assembled at the lipid bilayer. The assembly process brings together the glycoproteins spanning the lipid bilayer with the inner core of the virus particle. The inner layer of the membrane generally contains a viral protein that bridges the glycoproteins and the inner core, dubbed the matrix or M protein. M is generally considered an essential protein, without which the production of virus particle production is highly impaired if not impossible.The M protein of Sendai virus (SeV-M), a member of the Paramyxovirinae subfamily, Paramyxoviridae family, is no exception to the rule. It is synthesized in the cytoplasm and self-associates to form a leaflet at the inner face of the plasma membrane (for a recent review, see reference 45). In the virus particle, it similarly carpets the inner part of the viral envelope, interacting with the two surface glycoproteins, HN and F, on the one hand and with the viral ribonucleoprotein complex (N protein plus viral RNA) associated with the L and P proteins on the other hand (for a review, see reference 29). In addition to its role in virus particle formation, paramyxovirus M has been reported to participate in the regulation of RNA synthesis (19,27,38,40,44). Such a role for M in viral transcription control has been described for other negative-stranded RNA viruses, such as vesicular stomatitis virus (VSV) and rabies virus (both members of the Rhabdoviridae family) (9,11,26,31,49) as well as for the influenza viruses (Orthomyxovirus family) (32, 48). In addition, VSV-M has been implicated in the shutoff of cellular transcription (3, 4), and rabies virus-M has been implicated in the stimulation of viral replication in vivo (14, 15).Our laboratory has long been interested in the SeV-M protein and, in particular, in its function in virus particle formation (13,35,36,43). To perform a structure-function analysis, one would ideally like to silence expression of the resident SeV-M gene and replace it with M mutants in a search for residues or domains that can modulate its functions. One approach would con...

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.