HMPV and RSV viruses are major contributors to respiratory tract infections in infants and young children. In most infants, these viruses cause symptoms resembling those of the common cold. However, in infants born prematurely, children with chronic lung disease, or children with congenital heart disease, these viruses can result in a severe or even life threatening disease. As many as 125,000 hospitalizations occur annually in children less than one year old due to lower respiratory infection or bronchiolitis (47
). Developing new therapeutics to prevent and treat these infections is therefore of considerable importance.
Limiting virus infection requires rapidly mounted defences, which include in large part the release of type I IFN (IFNα/β). Interferon limits viral replication directly and enhances viral clearance by activating adaptive immunity. Understanding how viruses are sensed and how type I IFN is regulated may facilitate the rational design of novel anti-viral therapeutics and/or better vaccine candidates useful in the prevention or treatment of lower respiratory tract infections in children. In this study, we demonstrate that type I IFN production during infection with HMPV viruses involves differential sensing mechanisms, which work in a cell-type specific manner. Sensing of HMPV-A1 virus occurs via
the cytosolic RNA helicase RIG-I in most cell types, with the exception of PDC, where TLR7 mediates these responses. A recent study by Casola and colleagues also implicated RIG-I in the sensing of HMPV in airway epithelial cells (22
). We have confirmed these observations using mice with targeted deletions in the RIG-I pathway and have extended these studies to include an analysis of additional cell types and a comparison of two closely related clinical viral isolates. We have identified 5’- triphosphate RNA as the HMPV viral ligand triggering the RIG-I-IFNβ response. Importantly, we also identified a RIG-I-independent pathway for sensing HMPV-A1 and B1 viruses in epithelial cell lines. HMPV-A1 and B1 viruses activated NFκB and AP-1 dependent reporter genes in a RIG-I independent manner. These data suggest additional mechanism of HMPV sensing. The NLR family member, NOD2 was recently shown to act as a cytosolic sensor for RSV infection (48
). Further studies should delineate if NOD2 also senses HMPV to regulate NFκB and AP-1 signalling described herein.
A major focus of this study was a comparison of the innate response to two closely related strains. While both strains induced type I IFN responses in PDC, the B1 strain failed to elicit a type I IFN response in monocytes and cell lines, despite its ability to infect and replicate as efficiently as the A1 virus and despite the ability of naked viral RNA to trigger RIG-I if delivered by lipofection to the cytoplasm. The fact that the B1 virus could prevent type I IFN induction by the A1 virus, but not that induced by NDV indicates that RIG-I signalling per se is not blocked by the B1 virus. Like the A1 virus, purified B1 viral RNA could trigger IFN and pretreatment of the B1 viral RNA with RNAse or removal of phosphate groups with alkaline phosphatase ablated sensing of the viral RNA by RIG-I. These studies suggest that RNA of both strains are ligands for RIG-I, however, in the context of the virus, the B1 viral RNA is prevented from being sensed by RIG-I. During viral infection, RNA viruses like HMPV fuse with the cell membrane and deliver their ribonucleoprotein (RNP) complex into cells. The RNP complex consists of the viral RNA associated with the viral polymerase L, the nucleoprotein N and the phosphoprotein P. Upon fusion, the viral RNA is protected from free cellular RNases by this protein complex. Proteins within the RNP therefore could prevent the recognition of the vRNA by the RIG-I pathway. In fact, our studies using overexpressed B1 proteins indicated that the B1 virus P protein but not other HMPV proteins could block IFN production by live A1 virus or viral RNA. Although this approach indicated that the B1 P protein was the most likely candidate, we found that if we overexpressed the A1 P protein we could also observe an inhibitory effect. To determine if the B1 P protein was responsible for the inhibitory effect in the context of the virus, we generated recombinant viruses where we replaced the P protein in the A1 virus with that from the B1 virus. This is a more physiologically relevant system to assess its contribution where associations of the B-1 P with other viral proteins could also be accounted for. A recombinant A1 virus encoding the P protein from the B1 strain was generated and its ability to induce RIG-I signaling examined. Unlike the wild type A1 virus recovered from cDNA, the recombinant A1 virus containing the B virus P protein had a substantially reduced ability to induce IFN, suggesting that in the context of the entire virus, B1-P may indeed prevent RIG-I from sensing HMPV virus. Specific inhibition of RIG-I sensing by the B1-P protein could be because of higher levels of expression of the P protein in the B1 virus rather than what is found in the A1 virus. Another possibility is that the B-1 P protein could have higher affinity for the RNA or for other components of the RNP complex (HMPV RNA or N and L), which would preclude the RNA from being sensed. The two P proteins share 86% identity (see ) and therefore unique residues in B1-P could account for these effects.
Usurping IFN induction pathways is a common tactic employed by viruses to enable their replication within host cells. Viral IFN antagonists strike at just about every level of the IFN regulatory network, but by far the best-studied strategies relate to the ability of viral proteins to counteract RNA sensing and signaling events. Examples include Influenza virus NS1, which inactivates RIG-I (43
), Hepatitis C Virus, NS3/4A, which cleaves and inactivates MAVS (18
) and the phosphoprotein of Borna disease virus and Rabies virus, which target the IRF3 kinase, TBK1 (49
). In the case of RSV, the genome encodes two non-structural (NS)-1 and NS2 proteins known to inactivate the IFN response (51
). A recent study from Casola and colleagues implicated the G protein from another strain of HMPV in evasion of the RIG-I signaling pathway (52
). A recombinant hMPV lacking the G protein (rhMPV-ΔG) was developed as a potential vaccine candidate and shown to be attenuated in the respiratory tract of a rodent model of infection. Casola and colleagues found that rhMPV-ΔG-infected airway epithelial cells produced higher levels of chemokines and type I interferon compared to cells infected with rhMPV-WT. They showed that RIG-I was the target of G protein inhibitory activity. Indeed the G protein associated with RIG-I and inhibited RIG-I-dependent gene transcription. Our data with the HMPV-B1 virus however do not support a role for the B1-virus G protein but rather indicate that the B1 phosphoprotein can prevent RIG-I from sensing the viral RNA.
The inhibitory effect of the B1 virus P protein was restricted to the RIG-I pathway, since the B1 virus did not prevent the induction of IFNα/β in PDC. IFN production in PDC was sensitive to bafilomycin A1 and chloroquine and was dependent on TLR7. The current model of anti-viral sensing in PDC suggests that TLR-mediated recognition of viruses occurs without direct infection and that the presence of viral genomic nucleic acids within the endosomal/lysosomal compartment is sufficient to trigger TLRs. Iwasaki and colleagues demonstrated recently that RNA viruses such as VSV which do not enter cells via the endosomal compartment but replicate in the cytosolic compartment where cytosolic viral replication intermediates are then delivered into the lysosomal compartment by the process of autophagy to trigger TLRs (53
Generally cell entry of paramyxoviruses requires two glycoproteins: the attachment (G, H or HN) and fusion (F) proteins. In the case of HMPV viruses, however, analysis of recombinant viruses lacking the G protein has suggested that attachment and fusion is mainly dependent on the F protein (54
). The F protein is a type I glycoprotein, synthesized as an inactive precursor, F0 and subsequently converted into its biologically active form, the heterodimer F1/F2. The majority of Paramyxoviridae
F proteins are cleaved intracellularly by host cellular proteases, most notably furin. Cleavage of the F-protein from HMPV however, requires secretory proteases, which restrict HMPV viruses to the lumen of the respiratory and enteric tract for replication in vivo. In vitro
the addition of trypsin to process the F0 protein into its mature form allows efficient propagation of the virus (25
). In contrast to most other Paramyxoviridae
F proteins that require neutral pH for membrane fusion, cleavage of the HMPV F protein might therefore require low pH conditions (55
). These findings might indicate a requirement for low-pH compartments such as the endosome for entry of HMPV viruses in PDC. Receptor-mediated endocytosis at low pH was indeed recently shown in Vero cells for HMPV-A2 strain (56
). Since PDC do not need to be infected to induce IFN, the ability of PDC to respond may be a result of uptake of viral particles to the endosome directly.
Altogether, our data unveil different mechanisms for sensing of HMPV viruses in different cell types. Such cell type specific involvement of the RIG-I versus TLR pathways in induction of antiviral responses is not unique to HMPV viruses, as this differential sensing has previously been reported in the case of sensing of NDV (14
). Understanding how viruses are detected and how viruses exploit innate sensing and signalling pathways is essential for the development of vaccines to harness the power of the innate immune system for the benefit of the host.