Abstract Transmission of influenza A viruses (IAV) between hosts is subject to numerous physical and biological barriers that impose genetic bottlenecks, constraining viral diversity and adaptation. The bottlenecks within hosts and their potential impacts on evolutionary pathways taken during infection are poorly understood. To address this, we created highly diverse IAV libraries bearing molecular barcodes on two gene segments, enabling high-resolution tracking and quantification of unique virus lineages within hosts. Here we show that IAV infection in lungs is characterized by multiple within-host bottlenecks that result in “islands” of infection in lung lobes, each with genetically distinct populations. We perform site-specific inoculation of barcoded IAV in the upper respiratory tract of ferrets and track viral diversity as infection spreads to the trachea and lungs. We detect extensive compartmentalization of discrete populations within lung lobes. Bottleneck events and localized replication stochastically sample individual viruses from the upper respiratory tract or the trachea that become the dominant genotype in a particular lobe. These populations are shaped strongly by founder effects, with limited evidence for positive selection. The segregated sites of replication highlight the jackpot-style events that contribute to within-host influenza virus evolution and may account for low rates of intrahost adaptation.
INTRODUCTION: The goal of “Biology of Viruses” (MMI/Biochem 575) is to introduce upperlevel undergraduate students to the biology and biochemistry of viruses and virus infection. We will address the fundamentals of virus properties, virus multiplication, disease mechanisms, prevention and intervention of infection, and how viruses pose new threats to human and animal health through emergence and evolution. We will focus primarily on viruses that are pathogenic to animals. This course is intended to cover important concepts and themes in virology. We will discuss carefully selected examples of viruses that impact our world and everyday life. Thus, this course will not be a bug per day style of class. Important prerequisite classes for this course are Biocore 301/302, Zoology 151/152, or MMI one in immunology will be helpful. GRADUATE COURSE ATTRIBUTE: This course is open to upper-level undergraduate students and graduate students. However, graduate students must concurrently enroll in Biochem 910 and report back to Biochem / MMI 575 on 4 topics discussed in Biochem 910 in order to receive a passing grade and have this class count toward the 50% minimum graduate course requirement.
Precise coordination of cellular processes requires prompt specification of protein function in response to various stimuli. This specification includes regulating protein abundance, localization, catalysis, and binding. Post-translational modifications (PTMs) provide cells the plasticity for dynamic and reversible control of protein function. Viral infections provide an exciting lens through which to study PTMs, since PTMs contribute to both cellular responses to infection and viral hijacking of the host. PTMs enhance the already multifunctional nature of viral proteins and offer another level of functional diversity within limited genetic space. Influenza virus protein functions are fine-tuned by diverse types of PTMs, including phosphorylation, ubiquitination, SUMOylation, neddylation, ISGylation, glycosylation, ADP-ribosylation, palmitoylation, and acetylation. All of the major viral proteins are subject to at least one type of PTM. Additionally, as influenza viruses encode no known protein-modifying enzymes, all of these PTMs are mediated by host machinery. Here, we use influenza virus and its proteins as exemplars for how PTMs impact virus replication (Fig 1).
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Fig 1
Post-translation control of key steps during the influenza virus replication cycle.
Simplified diagram of key steps during the influenza virus life cycle highlighting events that are regulated by PTMs to viral or host proteins. The specific modifications, target proteins, and references are listed for each step. Two processes are highlighted in depth; these examples were chosen because the PTMs and causative host enzymes are known and the modifications have discrete effects on replication. In addition, a large number of PTMs have been identified on viral proteins, but no discrete function has yet been assigned [14]. Ac, acetylation; ADPr, ADP-ribosylation; cRNA, plus-sense genomic RNA; dsRNA, double-stranded RNA; Glycos., N-linked glycosylation; ISG15, ISGylation; Nedd8, neddylation; NP, nucleoprotein; NS1, nonstructural protein 1; PO4, phosphorylation; PTM, post-translational modification; RNP, ribonucleoprotein complex; SA, sialic acid; SUMO, SUMOylation; Ub, ubiquitin and ubiquitination; vRNA, minus-sense genomic RNA.
1 Department of Microbiology and Molecular Genetics, Michigan State University, 567 Wilson Road, East Lansing, MI 48824, USA; ctbrown@ucdavis.edu (C.T.B.); zhengyo@msu.edu (Y.-H.Z.); adami@msu.edu (C.A.) 2 BEACON Center for the Study of Evolution in Action, Michigan State University, 567 Wilson Road, East Lansing, MI 48824, USA 3 Department of Computer Science and Engineering, Michigan State University, 428 S. Shaw Lane, East Lansing, MI 48824, USA 4 Department of Physics and Astronomy, Michigan State University, 567 Wilson Road, East Lansing, MI 48824, USA * Correspondence: agupta@msu.edu; Tel.: +1-517-355-8733, Fax: +1-517-353-8957
CD16+ monocytes represent 5–10% of peripheral blood monocytes in normal individuals and are dramatically expanded in several pathological conditions including sepsis, human immunodeficiency virus 1 infection, and cancer. CD16+ monocytes produce high levels of proinflammatory cytokines and may represent dendritic cell precursors in vivo. The mechanisms that mediate the recruitment of CD16+ monocytes into tissues remain unknown. Here we investigate molecular mechanisms of CD16+ monocyte trafficking and show that migration of CD16+ and CD16− monocytes is mediated by distinct combinations of adhesion molecules and chemokine receptors. In contrast to CD16− monocytes, CD16+ monocytes expressed high CX3CR1 and CXCR4 but low CCR2 and CD62L levels and underwent efficient transendo-thelial migration in response to fractalkine (FKN; FKN/CX3CL1) and stromal-derived factor 1α (CXCL12) but not monocyte chemoattractant protein 1 (CCL2). CD16+ monocytes arrested on cell surface–expressed FKN under flow with higher frequency compared with CD16− monocytes. These results demonstrate that FKN preferentially mediates arrest and migration of CD16+ monocytes and suggest that recruitment of this proinflammatory monocyte subset to vessel walls via the CX3CR1-FKN pathway may contribute to vascular and tissue injury during pathological conditions.
Abstract Adaptation of viruses to their host can result in specialization and a restricted host range. Species-specific polymorphisms in the influenza virus polymerase restrict its host range during transmission from birds to mammals. ANP32A was recently been identified as a cellular co-factor impacting polymerase adaption and activity. Avian influenza polymerases require ANP32A containing an insertion resulting from an exon duplication uniquely encoded in birds. Here we find that natural splice variants surrounding this exon create avian ANP32A proteins with distinct effects on polymerase activity. We demonstrate species-independent direct interactions between all ANP32A variants and the PB2 polymerase subunit. This interaction is enhanced in the presence of viral genomic RNA. In contrast, only avian ANP32A restored ribonucleoprotein complex assembly for a restricted polymerase by enhancing RNA synthesis. Our data suggest that ANP32A splicing variation amongst birds differentially impacts viral replication, polymerase adaption, and the potential of avian hosts to be reservoirs.
The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-β expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-β induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-β, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-β mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-β via mitochondrial dsDNA release.
Abstract Viruses must balance their reliance on host cell machinery for replication while avoiding host defense. Influenza A viruses are zoonotic agents that frequently switch hosts, causing localized outbreaks with the potential for larger pandemics. The host range of influenza virus is limited by the need for successful interactions between the virus and cellular partners. Here we used immuno-competitive capture-mass spectrometry to identify cellular proteins that interact with human- and avian-style viral polymerases. We focused on the pro-viral activity of heterogenous nuclear ribonuclear protein U-like 1 (hnRNP UL1) and the anti-viral activity of mitochondrial enoyl CoA-reductase (MECR). MECR is localized to mitochondria where it functions in mitochondrial fatty acid synthesis (mtFAS). While a small fraction of the polymerase subunit PB2 localizes to the mitochondria, PB2 did not interact with full-length MECR. By contrast, a minor splice variant produces cytoplasmic MECR (cMECR) that interacts with PB2. cMECR binds the viral polymerase and suppresses viral replication by blocking assembly of viral ribonucleoprotein complexes (RNPs). MECR ablation through genome editing or drug treatment is detrimental for cell health, creating a generic block to virus replication. Using the yeast homolog Etr1 to supply the metabolic functions of MECR in MECR-null cells, we showed that specific antiviral activity is independent of mtFAS and lies solely within cMECR. Thus, alternative splicing produces a cryptic antiviral protein that is embedded within a key metabolic enzyme.
The emergence of new pandemic influenza A viruses requires overcoming barriers to cross-species transmission as viruses move from animal reservoirs into humans. This complicated process is driven by both individual gene mutations and genome reassortments. The viral polymerase complex, composed of the proteins PB1, PB2, and PA, is a major factor controlling host adaptation, and reassortment events involving polymerase gene segments occurred with past pandemic viruses. Here we investigate the ability of polymerase reassortment to restore the activity of an avian influenza virus polymerase that is normally impaired in human cells. Our data show that the substitution of human-origin PA subunits into an avian influenza virus polymerase alleviates restriction in human cells and increases polymerase activity in vitro. Reassortants with 2009 pandemic H1N1 PA proteins were the most active. Mutational analyses demonstrated that the majority of the enhancing activity in human PA results from a threonine-to-serine change at residue 552. Reassortant viruses with avian polymerases and human PA subunits, or simply the T552S mutation, displayed faster replication kinetics in culture and increased pathogenicity in mice compared to those containing a wholly avian polymerase complex. Thus, the acquisition of a human PA subunit, or the signature T552S mutation, is a potential mechanism to overcome the species-specific restriction of avian polymerases and increase virus replication. Our data suggest that the human, avian, swine, and 2009 H1N1-like viruses that are currently cocirculating in pig populations set the stage for PA reassortments with the potential to generate novel viruses that could possess expanded tropism and enhanced pathogenicity.
