Viruses of the Paramyxoviridae family, such as the respiratory syncytial virus (RSV), suppress cellular innate immunity represented by type I interferon (IFN) for optimal growth in their hosts. The two unique nonstructural (NS) proteins, NS1 and NS2, of RSV suppress IFN synthesis, as well as IFN function, but their exact targets are still uncharacterized. Here, we investigate if either or both of the NS proteins affect the steady-state levels of key members of the IFN pathway. We found that both NS1 and NS2 decreased the levels of TRAF3, a strategic integrator of multiple IFN-inducing signals, although NS1 was more efficient. Only NS1 reduced IKK, a key protein kinase that specifically phosphorylates and activates IFN regulatory factor 3. Loss of the TRAF3 and IKK proteins appeared to involve a nonproteasomal mechanism. Interestingly, NS2 modestly increased IKK levels. In the IFN response pathway, NS2 decreased the levels of STAT2, the essential transcription factor for IFN-inducible antiviral genes. Preliminary mapping revealed that the C-terminal 10 residues of NS1 were essential for reducing IKK levels and the C-terminal 10 residues of NS2 were essential for increasing and reducing IKK and STAT2, respectively. In contrast, deletion of up to 20 residues of the C termini of NS1 and NS2 did not diminish their TRAF3-reducing activity. Coimmunoprecipitation studies revealed that NS1 and NS2 form a heterodimer. Clearly, the NS proteins of RSV, working individually and together, regulate key signaling molecules of both the IFN activation and response pathways.Respiratory syncytial virus (RSV) is by far the most significant agent of pediatric respiratory disease for which no reliable antiviral or vaccine yet exists (45, 59). The lukewarm success of traditional active-immunization-based strategies has drawn focus to cellular innate immunity, which acts as a broad antiviral defense. A major arm of innate antiviral immunity is the type I IFN family, represented by alpha interferon (IFN-␣) and 46,58). In counterdefense, however, members of the Paramyxoviridae family have evolved accessory gene products that neutralize or inhibit various steps of the IFN pathway, thus permitting robust virus growth (18,27,57,71). It is now appreciated that a better understanding of the viral IFN suppression mechanism(s) is essential for the prudent design of attenuated vaccine strains and better overall therapy.RSV encodes unique anti-IFN genes not found in any other member virus of this family. While other viruses generate IFNsuppressive proteins, mainly the V protein (1,12,18,19,21,22,27,33,50,51,57), from alternative translational reading frames in the P gene through "RNA editing," the P gene of RSV codes for the P protein only. Instead, the RSV genome contains two promoter-proximal genes that code for nonstructural (NS) proteins, NS1 and NS2, which are so named because they are synthesized in RSV-infected cells but are not packaged in the mature virion structure. The predicted primary structures of the NS proteins do not share any sig...

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Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKK, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo-as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKK reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression. Human respiratory syncytial virus (RSV) is a member of thePneumovirus genus in the Paramyxoviridae family (9, 25). RSV replicates primarily in the respiratory epithelium and is a leading cause of respiratory disease and asthma in the very young and the elderly (41, 42). The nonsegmented negative-strand RNA genome of RSV encodes 10 genes, of which the two most promoterproximal and abundantly transcribed genes code for nonstructural proteins 1 and 2 (NS1 and NS2), which are small proteins, 139 and 124 amino acid residues long, respectively (9, 25). The NS proteins circumvent the host innate immune system by preventing the induction of type I interferons (IFNs) as well as IFN-induced antiviral responses (6,12,20,22,23,29,(33)(34)(35)(36)39), thus allowing a more robust replication of the virus, leading to the severe respiratory disease that characterizes RSV infection.The IFN pathways of the cell can be divided into two functional ones, the IFN induction pathway, in which the cells produce IFN, and the IFN response pathway, in which the cells respond to exogenous IFN. One of the most proximal steps in the IFN induction pathway is the activation of the cytoplasmic RNA sensors of the RIG-I family (27,37,43,44). This triggers a cascade of signaling events whereby the CARD sequence of RIG-I interacts with and activates the CARD-like domaincontaining mitochondrial protein, MAVS (7, 18). Activated MAVS then activates TRAF3 (7, 31), which in turn activates two downstream kinases, IKKε and TBK1. The latter are Ser/ Thr kinases that phosphorylate the C-terminal domain of interferon regulatory factor 3 (IRF3) and IRF7, leading to...

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Purpose The activity of the cationic antimicrobial peptide WLBU2 was evaluated against planktonic cells and biofilms of multi-drug resistant (MDR) Acinetobacter baumannii and Klebsiella pneumoniae , alone and in combination with classical antimicrobial agents. Methods Control American Type Culture Collection (ATCC) strains and MDR clinical isolates of A. baumannii and K. pneumoniae were utilized. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of WLBU2 alone and in combination with antimicrobials were determined by classical methods. The Calgary biofilm device was used to determine the minimum biofilm eradication concentration (MBEC). The MTT assay was used to determine the cytotoxicity of agents on eukaryotic cells. The electrophoretic mobility shift assay was used to evaluate the ability of WLBU2 to bind bacterial DNA. Results The WLBU2 MIC and MBC values were identical indicating bactericidal activity. The MIC/MBC values ranged from 1.5625 to 12.5 µM. At these concentrations, Vero cells and human skin fibroblasts were viable. The MBEC of WLBU2 ranged from 25 to 200 µM. A significant loss of eukaryotic cell viability was observed at the MBEC range. The combination of sub-inhibitory concentrations of WLBU2 with amoxicillin-clavulanate or ciprofloxacin for K. pneumoniae , and with tobramycin or imipenem for A. baumannii , demonstrated synergism, leading to a significant decrease in MIC and MBEC values for some isolates and ATCC strains. However, all combinations were associated with considerable loss in eukaryotic cells’ viability. WLBU2 did not demonstrate the ability to bind bacterial plasmid DNA. Conclusion WLBU2 in combination with antimicrobials holds promise in eradication of MDR pathogens.