CK2beta gene silencing increases cell susceptibility to influenza A virus infection resulting in accelerated virus entry and higher viral protein content

Background: Influenza A virus (IVA) exploits diverse cellular gene products to support its replication in the host. The significance of the regulatory (β) subunit of casein kinase 2 (CK2β) in various cellular mechanisms is well established, but less is known about its potential role in IVA replication. We studied the role of CK2β in IVA-infected A549 human epithelial lung cells. Results: Activation of CK2β was observed in A549 cells during virus binding and internalization but appeared to be constrained as replication began. We used small interfering RNAs (siRNAs) targeting CK2β mRNA to silence CK2β protein expression in A549 cells without affecting expression of the CK2α subunit. CK2β gene silencing led to increased virus titers, consistent with the inhibition of CK2β during IVA replication. Notably, virus titers increased significantly when CK2β siRNA-transfected cells were inoculated at a lower multiplicity of infection. Virus titers also increased in cells treated with a specific CK2 inhibitor but decreased in cells treated with a CK2β stimulator. CK2β absence did not impair nuclear export of viral ribonucleoprotein complexes (6 h and 8 h after inoculation) or viral polymerase activity (analyzed in a minigenome system). The enhancement of virus titers by CK2β siRNA reflects increased cell susceptibility to influenza virus infection resulting in accelerated virus entry and higher viral protein content. Conclusion: This study demonstrates the role of cellular CK2β protein in the viral biology. Our results are the first to demonstrate a functional link between siRNA-mediated inhibition of the CK2β protein and regulation of influenza A virus replication in infected cells. Overall, the data suggest that expression and activation of CK2β inhibits influenza virus replication by regulating the virus entry process and viral protein synthesis. © 2008 Marjuki et al; licensee BioMed Central Ltd.link_to_subscribed_fulltex

Higher polymerase activity of a human influenza virus enhances activation of the hemagglutinin-induced Raf/MEK/ERK signal cascade

Influenza viruses replicate within the nucleus of infected cells. Viral genomic RNA, three polymerase subunits (PB2, PB1, and PA), and the nucleoprotein (NP) form ribonucleoprotein complexes (RNPs) that are exported from the nucleus late during the infectious cycle. The virus-induced Raf/MEK/ERK (MAPK) signal cascade is crucial for efficient virus replication. Blockade of this pathway retards RNP export and reduces virus titers. Hemagglutinin (HA) accumulation and its tight association with lipid rafts activate ERK and enhance localization of cytoplasmic RNPs. We studied the induction of MAPK signal cascade by two seasonal human influenza A viruses A/HK/218449/06 (H3N2) and A/HK/218847/06 (H1N1) that differed substantially in their replication efficiency in tissue culture. Infection with H3N2 virus, which replicates efficiently, resulted in higher HA expression and its accumulation on the cell membrane, leading to substantially increased activation of MAPK signaling compared to that caused by H1N1 subtype. More H3N2-HAs were expressed and accumulated on the cell membrane than did H1N1-HAs. Viral polymerase genes, particularly H3N2-PB1 and H3N2-PB2, were observed to contribute to increased viral polymerase activity. Applying plasmid-based reverse genetics to analyze the role of PB1 protein in activating HA-induced MAPK cascade showed that recombinant H1N1 virus possessing the H3N2-PB1 (rgH1N1/H3N2-PB1) induced greater ERK activation, resulting in increased nuclear export of the viral genome and higr virus titers. We conclude that enhanced viral polymerase activity promotes the replication and transcription of viral RNA leading to increased accumulation of HA on the cell surface and thereby resulting in an upregulation of the MAPK cascade and more efficient nuclear RNP-export as well as virus production

Isolation of Highly Pathogenic Avian Influenza H5N1 Virus from Saker Falcons ( Falco cherrug

There is accumulating evidence that birds of prey are susceptible to fatal infection with highly pathogenic avian influenza (HPAI) virus. We studied the antigenic, molecular, phylogenetic, and pathogenic properties of 2 HPAI H5N1 viruses isolated from dead falcons in Saudi Arabia and Kuwait in 2005 and 2007, respectively. Phylogenetic and antigenic analyses grouped both isolates in clade 2.2 (Qinghai-like viruses). However, the viruses appeared to have spread westward via different flyways. It remains unknown how these viruses spread so rapidly from Qinghai after the 2005 outbreak and how they were introduced into falcons in these two countries. The H5N1 outbreaks in the Middle East are believed by some to be mediated by wild migratory birds. However, sporting falcons may be at additional risk from the illegal import of live quail to feed them

Genomic Diversity and Antimicrobial Susceptibility of Invasive Neisseria meningitidis in South Africa, 2016–2021

Background: Invasive meningococcal isolates in South Africa have in previous years (<2008) been characterized by serogroup B, C, W, and Y lineages over time, with penicillin intermediate resistance (peni) at 6%. We describe the population structure and genomic markers of peni among invasive meningococcal isolates in South Africa, 2016–2021. Methods: Meningococcal isolates were collected through national, laboratory-based invasive meningococcal disease (IMD) surveillance. Phenotypic antimicrobial susceptibility testing and whole-genome sequencing were performed, and the mechanism of reduced penicillin susceptibility was assessed in silico. Results: Of 585 IMD cases reported during the study period, culture and PCR-based capsular group was determined for 477/585 (82%); and 241/477 (51%) were sequenced. Predominant serogroups included NmB (210/477; 44%), NmW (116/477; 24%), NmY (96/477; 20%), and NmC (48/477; 10%). Predominant clonal complexes (CC) were CC41/44 in NmB (27/113; 24%), CC11 in NmW (46/56; 82%), CC167 in NmY (23/44; 53%), and CC865 in NmC (9/24; 38%). Peni was detected in 16% (42/262) of isolates, and was due to the presence of a penA mosaic, with the majority harboring penA7, penA9, or penA14. Conclusions: IMD lineages circulating in South Africa were consistent with those circulating prior to 2008; however, peni was higher than previously reported, and occurred in a variety of lineages

Meningococcal disease in North America: Updates from the Global Meningococcal Initiative

This review summarizes the recent Global Meningococcal Initiative (GMI) regional meeting, which explored meningococcal disease in North America. Invasive meningococcal disease (IMD) cases are documented through both passive and active surveillance networks. IMD appears to be decreasing in many areas, such as the Dominican Republic (2016: 18 cases; 2021: 2 cases) and Panama (2008: 1 case/100,000; 2021: <0.1 cases/100,000); however, there is notable regional and temporal variation. Outbreaks persist in at-risk subpopulations, such as people experiencing homelessness in the US and migrants in Mexico. The recent emergence of β-lactamase-positive and ciprofloxacin-resistant meningococci in the US is a major concern. While vaccination practices vary across North America, vaccine uptake remains relatively high. Monovalent and multivalent conjugate vaccines (which many countries in North America primarily use) can provide herd protection. However, there is no evidence that group B vaccines reduce meningococcal carriage. The coronavirus pandemic illustrates that following public health crises, enhanced surveillance of disease epidemiology and catch-up vaccine schedules is key. Whole genome sequencing is a key epidemiological tool for identifying IMD strain emergence and the evaluation of vaccine strain coverage. The Global Roadmap on Defeating Meningitis by 2030 remains a focus of the GMI.Medical writing support for the development of this manuscript, under the direction of the authors, was provided Matthew Gunther of Ashfield MedComms, an Inizio company. Medical writing support was funded by Sanofi Pasteur. All authors discussed and agreed to the objectives of this manuscript and con- tributed throughout its production. All authors read and approved the final manuscript.S

Beta gene silencing increases cell susceptibility to influenza A virus infection resulting in accelerated virus entry and higher viral protein content-0

Ubated with virus-containing medium (MOI = 5) at 37°C for 1 h, medium was replaced, and cells were further incubated at 37°C for the times as indicated. (c) A549 cells were incubated with virus-containing medium (MOI = 5) at 4°C for 20 min and then at 37°C for the times as indicated. CK2β activation was assessed at the indicated time points by Western blot with polyclonal rabbit anti-phosphorylated CK2β (P-CK2β). Bands in three independent experiments were quantified, and relative CK2β activation (black bars) was calculated and normalized to the loading control (anti-CK2β mAb). White bars represent uninoculated cells.<p><b>Copyright information:</b></p><p>Taken from "beta gene silencing increases cell susceptibility to influenza A virus infection resulting in accelerated virus entry and higher viral protein content"</p><p>http://www.jmolecularsignaling.com/content/3/1/13</p><p>Journal of Molecular Signaling 2008;3():13-13.</p><p>Published online 23 Jul 2008</p><p>PMCID:PMC2494991.</p><p></p

Synergistic Adaptive Mutations in the Hemagglutinin and Polymerase Acidic Protein Lead to Increased Virulence of Pandemic 2009 H1N1 Influenza A Virus in Mice

Influenza impressively reflects the paradigm of a viral disease in which continued evolution of the virus is of paramount importance for annual epidemics and occasional pandemics in humans. Because of the continuous threat of novel influenza outbreaks, it is essential to gather further knowledge about viral pathogenicity determinants. Here, we explored the adaptive potential of the influenza A virus subtype H1N1 variant isolate A/Hamburg/04/09 (HH/04) by sequential passaging in mice lungs. Three passages in mice lungs were sufficient to dramatically enhance pathogenicity of HH/04. Sequence analysis identified 4 nonsynonymous mutations in the third passage virus. Using reverse genetics, 3 synergistically acting mutations were defined as pathogenicity determinants, comprising 2 mutations in the hemagglutinin (HA[D222G] and HA[K163E]), whereby the HA(D222G) mutation was shown to determine receptor binding specificity and the polymerase acidic (PA) protein F35L mutation increasing polymerase activity. In conclusion, synergistic action of all 3 mutations results in a mice lethal pandemic H1N1 virus

Beta gene silencing increases cell susceptibility to influenza A virus infection resulting in accelerated virus entry and higher viral protein content-6

Rol or CK2β siRNA for 48 h, then co-transfected with plasmids containing the PB2, PB1, PA, and NP genes plus a luciferase reporter plasmid. Cells not transfected with the PB1 plasmid were used as negative controls. After 24 h, polymerase (luciferase) activity was assayed in cell extracts. Results represent the mean ± SE of 3 independent experiments.<p><b>Copyright information:</b></p><p>Taken from "beta gene silencing increases cell susceptibility to influenza A virus infection resulting in accelerated virus entry and higher viral protein content"</p><p>http://www.jmolecularsignaling.com/content/3/1/13</p><p>Journal of Molecular Signaling 2008;3():13-13.</p><p>Published online 23 Jul 2008</p><p>PMCID:PMC2494991.</p><p></p

Beta gene silencing increases cell susceptibility to influenza A virus infection resulting in accelerated virus entry and higher viral protein content-1

.t.) by Western blot analysis with a mAb specific for the β-subunit of CK2. Loading was controlled with a mAb against β-actin. (b) Total CK2α protein detected 24 h, 48 h, and 72 h after transfection with CK2β siRNA.<p><b>Copyright information:</b></p><p>Taken from "beta gene silencing increases cell susceptibility to influenza A virus infection resulting in accelerated virus entry and higher viral protein content"</p><p>http://www.jmolecularsignaling.com/content/3/1/13</p><p>Journal of Molecular Signaling 2008;3():13-13.</p><p>Published online 23 Jul 2008</p><p>PMCID:PMC2494991.</p><p></p