RNA Sequence Features Are at the Core of Influenza A Virus Genome Packaging

The influenza A virus (IAV), a respiratory pathogen for humans, poses serious medical and economic challenges to global healthcare systems. The IAV genome, consisting of eight single-stranded viral RNA segments, is incorporated into virions by a complex process known as genome packaging. Specific RNA sequences within the viral RNA segments serve as signals that are necessary for genome packaging. Although efficient packaging is a prerequisite for viral infectivity, many of the mechanistic details about this process are still missing. In this review, we discuss the recent advances toward the understanding of IAV genome packaging and focus on the RNA features that play a role in this process.     

Rab11a mediates cell-cell spread and reassortment of influenza A virus genomes via tunneling nanotubes

Influenza A virus [IAV] genomes comprise eight negative strand RNAs packaged into virions in the form of viral ribonucleoproteins [vRNPs]. Rab11a plays a crucial role in the transport of vRNPs from the nucleus to the plasma membrane via microtubules, allowing assembly and virus production. Here, we identify a novel function for Rab11a in the inter-cellular transport of IAV vRNPs using tunneling nanotubes [TNTs]as molecular highways. TNTs are F-Actin rich tubules that link the cytoplasm of nearby cells. In IAV-infected cells, Rab11a was visualized together with vRNPs in these actin-rich intercellular connections. To better examine viral spread via TNTs, we devised an infection system in which conventional, virion-mediated, spread was not possible. Namely, we generated HA-deficient reporter viruses which are unable to produce progeny virions but whose genomes can be replicated and trafficked. In this system, vRNP transfer to neighboring cells was observed and this transfer was found to be dependent on both actin and Rab11a. Generation of infectious virus via TNT transfer was confirmed using donor cells infected with HA-deficient virus and recipient cells stably expressing HA protein. Mixing donor cells infected with genetically distinct IAVs furthermore revealed the potential for Rab11a and TNTs to serve as a conduit for genome mixing and reassortment in IAV infections. These data therefore reveal a novel role for Rab11a in the IAV life cycle, which could have significant implications for within-host spread, genome reassortment and immune evasion.     

Cross-Protective Effect of Antisense Oligonucleotide Developed Against the Common 3′ NCR of Influenza A Virus Genome

The influenza A virus (IAV) has eight segmented single-stranded RNA genome containing a common and evolutionarily conserved non-coding region (NCRs) at 5′ and 3′ ends that are important for the virus replication. In this study, we designed an antisense oligonucleotide against the 3′ NCR of vital segments of the IAV genome to inhibit its replication. The results demonstrated that the co-transfection of Madine Darby Canine Kidney (MDCK) cells with the antisense oligonucleotide and the plasmids encoding the viral genes led to the down-regulation of the viral gene expression. The designed antisense molecules reduced the cytopathic effect caused by A/PR/8/34 (H1N1), A/Udorn/307/72 (H3N2), and A/New Caledonia/20/99 (H1N1) strains of IAV for almost 48 h. Furthermore, the intra-venous delivery of this oligonucleotide significantly reduced the viral titers in the lungs of infected mice and protected the mice from lethal effects of all the strains of influenza virus. The study demonstrated that the antisense oligonucleotide designed against the NCR region inhibits the expression of the viral genome. The decrease of the cytopathic effect in the MDCK cells and increase in survival of mice confirmed the reduction of virus multiplication and pathogenesis in the presence of antisense oligonucleotide. Thus, we demonstrate that a single antisense oligonucleotide is capable of providing protection against more than one strains of the IAV.     

Role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure

Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Since the IVA genome does not have the processing protease for the viral hemagglutinin (HA) envelope glycoprotein precursors, entry of this virus into cells and infectious organ tropism of IAV are primarily determined by host cellular trypsin-type HA processing proteases. Several secretion-type HA processing proteases for seasonal IAV in the airway, and ubiquitously expressed furin and pro-protein convertases for highly pathogenic avian influenza (HPAI) virus, have been reported. Recently, other HA-processing proteases for seasonal IAV and HPAI have been identified in the membrane fraction. These proteases proteolytically activate viral multiplication at the time of viral entry and budding. In addition to the role of host cellular proteases in IAV pathogenicity, IAV infection results in marked upregulation of cellular trypsins and matrix metalloproteinase-9 in various organs and cells, particularly endothelial cells, through induced pro-inflammatory cytokines. These host cellular factors interact with each other as the influenza virus-cytokine-protease cycle, which is the major mechanism that induces vascular hyperpermeability and multiorgan failure in severe influenza. This mini-review discusses the roles of cellular proteases in the pathogenesis of IAV and highlights the molecular mechanisms of upregulation of trypsins as effective targets for the control of IAV infection. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Maintenance and dissemination of avian-origin influenza A virus within the northern Atlantic Flyway of North America

Wild waterbirds, the natural reservoirs for avian influenza viruses, undergo migratory movements each year, connecting breeding and wintering grounds within broad corridors known as flyways. In a continental or global view, the study of virus movements within and across flyways is important to understanding virus diversity, evolution, and movement. From 2015 to 2017, we sampled waterfowl from breeding (Maine) and wintering (Maryland) areas within the Atlantic Flyway (AF) along the east coast of North America to investigate the spatio-temporal trends in persistence and spread of influenza A viruses (IAV). We isolated 109 IAVs from 1,821 cloacal / oropharyngeal samples targeting mallards (Anas platyrhynchos) and American black ducks (Anas rubripes), two species having ecological and conservation importance in the flyway that are also host reservoirs of IAV. Isolates with >99% nucleotide similarity at all gene segments were found between eight pairs of birds in the northern site across years, indicating some degree of stability among genome constellations and the possibility of environmental persistence. No movement of whole genome constellations were identified between the two parts of the flyway, however, virus gene flow between the northern and southern study locations was evident. Examination of banding records indicate direct migratory waterfowl movements between the two locations within an annual season, providing a mechanism for the inferred viral gene flow. Bayesian phylogenetic analyses provided evidence for virus dissemination from other North American wild birds to AF dabbling ducks (Anatinae), shorebirds (Charidriformes), and poultry (Galliformes). Evidence was found for virus dissemination from shorebirds to gulls (Laridae), and dabbling ducks to shorebirds and poultry. The findings from this study contribute to the understanding of IAV ecology in waterfowl within the AF.     

Influenza A virus interactions with macrophages: Lessons from epithelial cells

Influenza viruses are an important cause of respiratory infection worldwide. In humans, infection with seasonal influenza A virus (IAV) is generally restricted to the respiratory tract where productive infection of airway epithelial cells promotes viral amplification, dissemination, and disease. Alveolar macrophages (MΦ) are also among the first cells to detect and respond to IAV, where they play a pivotal role in mounting effective innate immune responses. In contrast to epithelial cells, IAV infection of MΦ is a “dead end” for most seasonal strains, where replication is abortive and newly synthesised virions are not released. Although the key replicative stages leading to productive IAV infection in epithelial cells are defined, there is limited knowledge about the abortive IAV life cycle in MΦ. In this review, we will explore host factors and viral elements that support the early stages (entry) through to the late stages (viral egress) of IAV replication in epithelial cells. Similarities, differences, and unknowns for each key stage of the IAV replicative cycle in MΦ will then be highlighted. Herein, we provide mechanistic insights into MΦ‐specific control of seasonal IAV replication through abortive infection, which may in turn, contribute to effective host defence.     

Most Influenza A Virions Fail To Express at Least One Essential Viral Protein

Segmentation of the influenza A virus (IAV) genome enables rapid gene reassortment at the cost of complicating the task of assembling the full viral genome. By simultaneously probing for the expression of multiple viral proteins in MDCK cells infected at a low multiplicity with IAV, we observe that the majority of infected cells lack detectable expression of one or more essential viral proteins. Consistent with this observation, up to 90% of IAV-infected cells fail to release infectious progeny, indicating that many IAV virions scored as noninfectious by traditional infectivity assays are capable of single-round infection. This fraction was not significantly affected by target or producer cell type but varied widely between different IAV strains. These data indicate that IAV exists primarily as a swarm of complementation-dependent semi-infectious virions, and thus traditional, propagation-dependent assays of infectivity may drastically misrepresent the true infectious potential of a virus population.     

Influenza A virus neuraminidase protein interacts with Hsp90, to stabilize itself and enhance cell survival

Neuraminidase protein (NA) of influenza A virus (IAV) is popularly known for its sialidase function to assist in the release of progeny virus. However, involvement of NA in other stages of the IAV life cycle also indicates its multifunctional nature and necessity to interact with other host proteins. Here, we report a host protein—heat shock protein 90 (Hsp90), as a novel interacting partner of IAV NA. A classical yeast two-hybrid screen was conducted to identify a new host interacting partner for NA and the interaction was further validated by coimmunoprecipitation from cells, transiently expressing both proteins and also from IAV-infected cells. Confocal imaging showed that both proteins colocalized in the cytoplasm in transfected host cells. Interestingly, increased levels of NA in the presence of Hsp90 was observed, which tends to decrease if adenosine triphosphatase activity of Hsp90 is inhibited using 17-N-allylamino-17-demethoxygeldanamycin (17AAG). This establishes viral NA as a client protein of host chaperone Hsp90 contributing toward NA's stability via the NA-Hsp90 interaction. This is the first report showing the interaction of NA with Hsp90 and its role in stabilizing viral NA thus preventing it from degradation. Enhanced cell survival in the presence of this interaction was also observed, thus suggesting the requirement of stable viral NA, post-IAV infection, for efficient virus production in infected mammalian cells.     

Tannins from Hamamelis virginiana Bark Extract: Characterization and Improvement of the Antiviral Efficacy against Influenza A Virus and Human Papillomavirus

Antiviral activity has been demonstrated for different tannin-rich plant extracts. Since tannins of different classes and molecular weights are often found together in plant extracts and may differ in their antiviral activity, we have compared the effect against influenza A virus (IAV) of Hamamelis virginiana L. bark extract, fractions enriched in tannins of different molecular weights and individual tannins of defined structures, including pseudotannins. We demonstrate antiviral activity of the bark extract against different IAV strains, including the recently emerged H7N9, and show for the first time that a tannin-rich extract inhibits human papillomavirus (HPV) type 16 infection. As the best performing antiviral candidate, we identified a highly potent fraction against both IAV and HPV, enriched in high molecular weight condensed tannins by ultrafiltration, a simple, reproducible and easily upscalable method. This ultrafiltration concentrate and the bark extract inhibited early and, to a minor extent, later steps in the IAV life cycle and tannin-dependently inhibited HPV attachment. We observed interesting mechanistic differences between tannin structures: High molecular weight tannin containing extracts and tannic acid (1702 g/mol) inhibited both IAV receptor binding and neuraminidase activity. In contrast, low molecular weight compounds (<500 g/mol) such as gallic acid, epigallocatechin gallate or hamamelitannin inhibited neuraminidase but not hemagglutination. Average molecular weight of the compounds seemed to positively correlate with receptor binding (but not neuraminidase) inhibition. In general, neuraminidase inhibition seemed to contribute little to the antiviral activity. Importantly, antiviral use of the ultrafiltration fraction enriched in high molecular weight condensed tannins and, to a lesser extent, the unfractionated bark extract was preferable over individual isolated compounds. These results are of interest for developing and improving plant-based antivirals.     

Persistent and Compartmentalised Disruption of Dendritic Cell Subpopulations in the Lung following Influenza A Virus Infection

Immunological homeostasis in the respiratory tract is thought to require balanced interactions between networks of dendritic cell (DC) subsets in lung microenvironments in order to regulate tolerance or immunity to inhaled antigens and pathogens. Influenza A virus (IAV) poses a serious threat of long-term disruption to this balance through its potent pro-inflammatory activities. In this study, we have used a BALB/c mouse model of A/PR8/34 H1N1 Influenza Type A Virus infection to examine the effects of IAV on respiratory tissue DC subsets during the recovery phase following clearance of the virus. In adult mice, we found differences in the kinetics and activation states of DC residing in the airway mucosa (AMDC) compared to those in the parenchymal lung (PLDC) compartments. A significant depletion in the percentage of AMDC was observed at day 4 post-infection that was associated with a change in steady-state CD11b⁺ and CD11b⁻ AMDC subset frequencies and significantly elevated CD40 and CD80 expression and that returned to baseline by day 14 post-infection. In contrast, percentages and total numbers of PLDC were significantly elevated at day 14 and remained so until day 21 post-infection. Accompanying this was a change in CD11b⁺and CD11b⁻ PLDC subset frequencies and significant increase in CD40 and CD80 expression at these time points. Furthermore, mice infected with IAV at 4 weeks of age showed a significant increase in total numbers of PLDC, and increased CD40 expression on both AMDC and PLDC, when analysed as adults 35 days later. These data suggest that the rate of recovery of DC populations following IAV infection differs in the mucosal and parenchymal compartments of the lung and that DC populations can remain disrupted and activated for a prolonged period following viral clearance, into adulthood if infection occurred early in life.     

Target Membrane Cholesterol Modulates Single Influenza Virus Membrane Fusion Efficiency but Not Rate

Host lipid composition influences many stages of the influenza A virus (IAV) entry process, including initial binding of IAV to sialylated glycans, fusion between the viral envelope and the host membrane, and the formation of a fusion pore through which the viral genome is transferred into a target cell. In particular, target membrane cholesterol has been shown to preferentially associate with virus receptors and alter physical properties of the membrane like fluidity and curvature. These properties affect both IAV binding and fusion, which makes it difficult to isolate the role of cholesterol in IAV fusion from receptor binding effects. Here, we develop a fusion assay that uses synthetic DNA-lipid conjugates as surrogate viral receptors to tether virions to target vesicles. To avoid the possibly perturbative effect of adding a self-quenched concentration of dye-labeled lipids to the viral membrane, we tether virions to lipid-labeled target vesicles and use fluorescence microscopy to detect individual, pH-triggered IAV membrane fusion events. Through this approach, we find that cholesterol in the target membrane enhances the efficiency of single-particle IAV lipid mixing, whereas the rate of lipid mixing is independent of cholesterol composition. We also find that the single-particle kinetics of influenza lipid mixing to target membranes with different cholesterol compositions is independent of receptor binding, suggesting that cholesterol-mediated spatial clustering of viral receptors within the target membrane does not significantly affect IAV hemifusion. These results are consistent with the hypothesis that target membrane cholesterol increases lipid mixing efficiency by altering host membrane curvature.     

A20 (Tnfaip3) Deficiency in Myeloid Cells Protects against Influenza A Virus Infection

The innate immune response provides the first line of defense against viruses and other pathogens by responding to specific microbial molecules. Influenza A virus (IAV) produces double-stranded RNA as an intermediate during the replication life cycle, which activates the intracellular pathogen recognition receptor RIG-I and induces the production of proinflammatory cytokines and antiviral interferon. Understanding the mechanisms that regulate innate immune responses to IAV and other viruses is of key importance to develop novel therapeutic strategies. Here we used myeloid cell specific A20 knockout mice to examine the role of the ubiquitin-editing protein A20 in the response of myeloid cells to IAV infection. A20 deficient macrophages were hyperresponsive to double stranded RNA and IAV infection, as illustrated by enhanced NF-κB and IRF3 activation, concomitant with increased production of proinflammatory cytokines, chemokines and type I interferon. In vivo this was associated with an increased number of alveolar macrophages and neutrophils in the lungs of IAV infected mice. Surprisingly, myeloid cell specific A20 knockout mice are protected against lethal IAV infection. These results challenge the general belief that an excessive host proinflammatory response is associated with IAV-induced lethality, and suggest that under certain conditions inhibition of A20 might be of interest in the management of IAV infections.     

Influenza Virus Reassortment Is Enhanced by Semi-infectious Particles but Can Be Suppressed by Defective Interfering Particles

A high particle to infectivity ratio is a feature common to many RNA viruses, with ~90–99% of particles unable to initiate a productive infection under low multiplicity conditions. A recent publication by Brooke et al. revealed that, for influenza A virus (IAV), a proportion of these seemingly non-infectious particles are in fact semi-infectious. Semi-infectious (SI) particles deliver an incomplete set of viral genes to the cell, and therefore cannot support a full cycle of replication unless complemented through co-infection. In addition to SI particles, IAV populations often contain defective-interfering (DI) particles, which actively interfere with production of infectious progeny. With the aim of understanding the significance to viral evolution of these incomplete particles, we tested the hypothesis that SI and DI particles promote diversification through reassortment. Our approach combined computational simulations with experimental determination of infection, co-infection and reassortment levels following co-inoculation of cultured cells with two distinct influenza A/Panama/2007/99 (H3N2)-based viruses. Computational results predicted enhanced reassortment at a given % infection or multiplicity of infection with increasing semi-infectious particle content. Comparison of experimental data to the model indicated that the likelihood that a given segment is missing varies among the segments and that most particles fail to deliver ≥1 segment. To verify the prediction that SI particles augment reassortment, we performed co-infections using viruses exposed to low dose UV. As expected, the introduction of semi-infectious particles with UV-induced lesions enhanced reassortment. In contrast to SI particles, inclusion of DI particles in modeled virus populations could not account for observed reassortment outcomes. DI particles were furthermore found experimentally to suppress detectable reassortment, relative to that seen with standard virus stocks, most likely by interfering with production of infectious progeny from co-infected cells. These data indicate that semi-infectious particles increase the rate of reassortment and may therefore accelerate adaptive evolution of IAV.     

IL-21R Signaling Suppresses IL-17+ Gamma Delta T Cell Responses and Production of IL-17 Related Cytokines in the Lung at Steady State and After Influenza A Virus Infection

Influenza A virus (IAV) infection of the respiratory tract elicits a robust immune response, which is required for efficient virus clearance but at the same time can contribute to lung damage and enhanced morbidity. IL-21 is a member of the type I cytokine family and has many different immune-modulatory functions during acute and chronic virus infections, although its role in IAV infection has not been fully evaluated. In this report we evaluated the contributions of IL-21/IL-21 receptor (IL-21R) signaling to host defense in a mouse model of primary IAV infection using IL-21R knock out (KO) mice. We found that lack of IL-21R signaling had no significant impact on virus clearance, adaptive T cell responses, or myeloid cell accumulations in the respiratory tract. However, a subset of inflammatory cytokines were elevated in the bronchoalveolar lavage fluid of IL-21R KO mice, including IL-17. Although there was only a small increase in Th17 cells in the lungs of IL-21R KO mice, we observed a dramatic increase in gamma delta (γδ) T cells capable of producing IL-17 both after IAV infection and at steady state in the respiratory tract. Finally, we found that IL-21R signaling suppressed the accumulation of IL-17⁺ γδ T cells in the respiratory tract intrinsically. Thus, our study reveals a previously unrecognized role of IL-21R signaling in regulating IL-17 production by γδ T cells.