Avian influenza A viruses exhibit plasticity in sialylglycoconjugate receptor usage in human lung cells.

It is well known that influenza A viruses (IAV) initiate host cell infection by binding to sialic acid, a sugar molecule present at the ends of various sugar chains called glycoconjugates. These sugar chains can vary in chain length, structure, and composition. However, it remains unknown if IAV strains preferentially bind to sialic acid on specific glycoconjugate type(s) for host cell infection.

Host factor Rab11a is critical for efficient assembly of influenza A virus genomic segments.

It is well documented that influenza A viruses selectively package 8 distinct viral ribonucleoprotein complexes (vRNPs) into each virion; however, the role of host factors in genome assembly is not completely understood. To evaluate the significance of cellular factors in genome assembly, we generated a reporter virus carrying a tetracysteine tag in the NP gene (NP-Tc virus) and assessed the dynamics of vRNP localization with cellular components by fluorescence microscopy. At early time points, vRNP complexes were preferentially exported to the MTOC; subsequently, vRNPs associated on vesicles positive for cellular factor Rab11a and formed distinct vRNP bundles that trafficked to the plasma membrane on microtubule networks.

The p150 Isoform of ADAR1 Blocks Sustained RLR signaling and Apoptosis during Influenza Virus Infection.

Signaling through retinoic acid inducible gene I (RIG-I) like receptors (RLRs) is tightly regulated, with activation occurring upon sensing of viral nucleic acids, and suppression mediated by negative regulators. Under homeostatic conditions aberrant activation of melanoma differentiation-associated protein-5 (MDA5) is prevented through editing of endogenous dsRNA by RNA editing enzyme Adenosine Deaminase Acting on RNA (ADAR1). In addition, ADAR1 is postulated to play pro-viral and antiviral roles during viral infections that are dependent or independent of RNA editing activity.

Structure of avian influenza hemagglutinin in complex with a small molecule entry inhibitor.

HA plays a critical role in influenza infection and, thus HA is a potential target for antivirals. Recently, our laboratories have described a novel fusion inhibitor, termed CBS1117, with EC ∼3 μM against group 1 HA. In this work, we characterize the binding properties of CBS1117 to avian H5 HA by x-ray crystallography, NMR, and mutagenesis.

Identification of entry inhibitors with 4-aminopiperidine scaffold targeting group 1 influenza A virus.

Influenza A viruses (IAVs) cause seasonal flu and occasionally pandemics. The current therapeutics against IAVs target two viral proteins - neuraminidase (NA) and M2 ion-channel protein. However, M2 ion channel inhibitors (amantadine and rimantadine) are no longer recommended by CDC for use due to the emergence of high level of antiviral resistance among the circulating influenza viruses, and resistant strains to NA inhibitors (oseltamivir and zanamivir) have also been reported.

Suppression of Cytotoxic T Cell Functions and Decreased Levels of Tissue-Resident Memory T Cells during H5N1 Infection.

Seasonal influenza virus infections cause mild illness in healthy adults, as timely viral clearance is mediated by the functions of cytotoxic T cells. However, avian H5N1 influenza virus infections can result in prolonged and fatal illness across all age groups, which has been attributed to the overt and uncontrolled activation of host immune responses. Here, we investigate how excessive innate immune responses to H5N1 impair subsequent adaptive T cell responses in the lungs.

Genome-wide CRISPR/Cas9 Screen Identifies Host Factors Essential for Influenza Virus Replication.

The emergence of influenza A viruses (IAVs) from zoonotic reservoirs poses a great threat to human health. As seasonal vaccines are ineffective against zoonotic strains, and newly transmitted viruses can quickly acquire drug resistance, there remains a need for host-directed therapeutics against IAVs. Here, we performed a genome-scale CRISPR/Cas9 knockout screen in human lung epithelial cells with a human isolate of an avian H5N1 strain.

Development of Potential Small Molecule Therapeutics for Treatment of Ebola Virus Disease.

Ebola virus has caused 26 outbreaks in 10 different countries since its identification in 1976, making it one of the deadliest emerging viral pathogens. The most recent outbreak in West Africa from 2014-16 was the deadliest yet and culminated in 11,310 deaths out of 28,616 confirmed cases. Currently, there are no FDA-approved therapeutics or vaccines to treat Ebola virus infections.

Endothelial cell tropism is a determinant of H5N1 pathogenesis in mammalian species.

The cellular and molecular mechanisms underpinning the unusually high virulence of highly pathogenic avian influenza H5N1 viruses in mammalian species remains unknown. Here, we investigated if the cell tropism of H5N1 virus is a determinant of enhanced virulence in mammalian species. We engineered H5N1 viruses with restricted cell tropism through the exploitation of cell type-specific microRNA expression by incorporating microRNA target sites into the viral genome.

RIG-I Signaling Is Critical for Efficient Polyfunctional T Cell Responses during Influenza Virus Infection.

Retinoic acid inducible gene-I (RIG-I) is an innate RNA sensor that recognizes the influenza A virus (IAV) RNA genome and activates antiviral host responses. Here, we demonstrate that RIG-I signaling plays a crucial role in restricting IAV tropism and regulating host immune responses. Mice deficient in the RIG-I-MAVS pathway show defects in migratory dendritic cell (DC) activation, viral antigen presentation, and priming of CD8+ and CD4+ T cell responses during IAV infection.

Insertion of a GFP reporter gene in influenza virus.

The incorporation of a fluorescent reporter gene into a replication-competent influenza A virus (IAV) has made it possible to trace IAV infection in vivo. This protocol describes the process of inserting a green fluorescent protein (GFP) reporter into the IAV genome using the established reverse genetics system. The strategy begins with the reorganization of segment eight of the IAV genome, during which the open reading frames of nonstructural protein 1 (NS1) and the nuclear export protein (NEP) are separated to allow for GFP fusion to the NS1 protein.

Influenza A virus utilizes suboptimal splicing to coordinate the timing of infection.

Influenza A virus is unique as an RNA virus in that it replicates in the nucleus and undergoes splicing. With only ten major proteins, the virus must gain nuclear access, replicate, assemble progeny virions in the cytoplasm, and then egress. In an effort to elucidate the coordination of these events, we manipulated the transcript levels from the bicistronic nonstructural segment that encodes the spliced virus product responsible for genomic nuclear export.

A small-RNA enhancer of viral polymerase activity.

Influenza A virus (IAV) is an unremitting virus that results in significant morbidity and mortality worldwide. Key to the viral life cycle is the RNA-dependent RNA polymerase (RdRp), a heterotrimeric complex responsible for both transcription and replication of the segmented genome. Here, we demonstrate that the viral polymerase utilizes a small RNA enhancer to regulate enzymatic activity and maintain stoichiometric balance of the viral genome.

Engineered RNA viral synthesis of microRNAs.

MicroRNAs (miRNAs) are short noncoding RNAs that exert posttranscriptional gene silencing and regulate gene expression. In addition to the hundreds of conserved cellular miRNAs that have been identified, miRNAs of viral origin have been isolated and found to modulate both the viral life cycle and the cellular transcriptome. Thus far, detection of virus-derived miRNAs has been largely limited to DNA viruses, suggesting that RNA viruses may be unable to exploit this aspect of transcriptional regulation.

Influenza A virus-generated small RNAs regulate the switch from transcription to replication.

The discovery of regulatory small RNAs continues to reshape paradigms in both molecular biology and virology. Here we describe examples of influenza A virus-derived small viral RNAs (svRNAs). svRNAs are 22-27 nt in length and correspond to the 5' end of each of the viral genomic RNA (vRNA) segments.

MicroRNA-mediated species-specific attenuation of influenza A virus.

Influenza A virus leads to yearly epidemics and sporadic pandemics. Present prophylactic strategies focus on egg-grown, live, attenuated influenza vaccines (LAIVs), in which attenuation is generated by conferring temperature sensitivity onto the virus. Here we describe an alternative approach to attenuating influenza A virus based on microRNA-mediated gene silencing.

Foxp3 transcription-factor-dependent and -independent regulation of the regulatory T cell transcriptional signature.

The CD4(+)CD25(+) lineage of regulatory T (Treg) cells plays a key role in controlling immune and autoimmune responses and is characterized by a unique transcriptional signature. The transcription factor Foxp3 had been thought to determine the Treg cell lineage, a hypothesis challenged by recent observations. We have performed a cross-sectional analysis of the Treg cell signature in Treg-like cells generated under a number of conditions, with or without Foxp3, to delineate the elements that can be ascribed to T cell activation, interleukin-2, transforming growth factor-beta (TGF-beta) signaling, or Foxp3 itself.