GPED review

Influenza and Flu Season Lecture Notes

• Lecture Overview The lecture will cover the respiratory system, microorganisms, viruses, influenza, and flu seasons [2].

• Learning Objectives Key goals are to describe viruses, influenza history/reservoirs, and factors causing flu seasons [2].

• Why Influenza? It's medically relevant, well-understood, and oscillates between seasonal and pandemic [2].

• Respiratory System Divided into upper (head, neck) and lower (chest) sections [3]. Lower respiratory infections are more serious [3].

• Micro-organisms Include eukaryotic (algae, protozoa, fungi), prokaryotic (bacteria, archaea), and non-living viruses [4, 5].

• Viruses Non-living infectious particles with varied sizes and shapes [4, 6, 7]. Require nucleic acid and protein [8]. Some have a lipid bilayer envelope [8].

• Influenza Types Includes A, B, C, and D viruses, with varying host ranges [9]. Influenza A has the widest range [9].

• H and N Naming based on hemagglutinin (H) and neuraminidase (N) proteins [10]. Waterfowl are the original host for all influenza A viruses [11].

• Viral Replication Cycle Virus must attach to a host cell, enter, release its genome, create viral parts, assemble, and exit [12].

• Flu Seasonality Respiratory infections peak in winter due to biological, social, and climate-related factors [13, 14]. Transmission occurs through direct contact, fomites, or aerosols [15, 16].

• Viability vs. Climate Higher humidity and temperature reduce virion viability and transmission [17, 18].

• US/Australia Flu data Flu searches peak at different times of the year depending on the hemisphere [13]. The more searches, the more infections [19].

• Trends and Changes The predominant infecting strain is around for a couple of years until another strain takes over [20].

Viral Attachment Lecture Notes

• Viral Attachment Viral attachment is the first step in the viral process [1, 2].

• Lecture Overview The lecture covers basic anatomy, mucins and mucus, host receptors, and viral adhesins [2, 3].

• Key Terms It's important to use the correct term when referring to a virus versus a disease, for example, influenza (virus) and flu (disease) [4].

• Viral Replication Cycle A virus must attach, enter/penetrate, uncoat, replicate, assemble, mature, and release/egress [2]. This is a replication cycle, not a life cycle, because viruses are not living [5].

• Flu Seasonality Several environmental factors affect the flu's seasonality, including humidity, pressure, and temperature [5].

  Definitions

• Epithelium: The thin layer of outermost cells of a tissue [2, 6].

• Mucosa: Internal cell surfaces that interact with the environment [2, 6].

• Lumen: The opening part of a tubular structure [2, 6].

• How Viruses Enter Viruses can enter via direct contact, fomites, droplets, and expelled aerosol particles [7, 8]. After a virus is expelled and picked up by a host, it must attach to a host cell [7].

• Airway Interactions Viruses are most likely to interact with host cells in the upper respiratory system [9].

• Adhesion Hemagglutinin (HA) is the viral adhesion that binds to the host receptor [10].

• Receptor The receptor on the host side is called sialic acid, a sugar that is the terminal sugar on many proteoglycans [10].

• Hemagglutinin The viral adhesion has a stalk (fusion) and a globular head (attachment) [11]. It has three binding sites to increase the opportunity to find the sialic acid receptor [11].

• Two Types of Sialic Acids Two types of sialic acids, 23 and 26, differ based on the carbon that it's bound to [11]. Avian strains of influenza preferentially bind to 23, while human strains preferentially bind to 26 [12].

• Mucus The host creates mucus, secreted proteins with sugars, to hinder viruses [12]. Mucus is made by goblet cells [12]. Mucus is a physical barrier, not part of the immune system, though it contains immune components [13].

• Mucociliary Elevator Ciliated epithelial cells form the mucociliary elevator, which moves mucus up and out of the airway [14].

• Sialic Acid and Mucus Sialic acid in mucus binds influenza [15].

• Viral Adhesions All viruses must have a functional adhesion to infect a host [16]. SARS-CoV-2's adhesion is the spike protein, which binds to the ACE2 receptor [16].

• Tropism Tropism is the cells that a virus can attach to and infect [16].

• Receptors and Tissue ACE2 is found in more tissue throughout the body than sialic acid [17].

• Polio Viability Polio does not have an envelope, but has a robust protein coat that allows it to survive the stomach and intestines all the way through [18, 19].

• HIV Receptors HIV uses GP120 adhesion and must bind to both CD4 and CCR5 receptors on the host cell. A delta 32 mutation makes individuals resistant to HIV infection [18].

• Influenza Strains and Sialic Acid Birds predominantly have 23, humans predominantly have 26, and pigs express both, making them a mixing vessel for new influenza strains [20].

Central Dogma, Gene Expression, and Molecular Biology Notes

• Central Dogma The central dogma is DNA to RNA to protein [1, 2].

• Gene Expression RNA polymerase transcribes DNA to produce an RNA transcript [1]. Ribosomes translate mRNA to synthesize a polypeptide [1]. Translation follows the genetic code [1].

• DNA Composition DNA has four nucleotides linked in a chain [3]. Nucleotides have deoxyribose sugar, a phosphate group, and a nitrogenous base [3]. Phosphodiester bonds link adjacent nucleotides [3].

• DNA Structure DNA strands are antiparallel with a sugar-phosphate backbone on the outside [4]. Bases pair in the middle, A with T and G with C, held by hydrogen bonds [4].

• Chromatin Structure Inactive genes have promoters hidden in nucleosomes [4]. To activate a gene, transcription factors bind to enhancers and recruit chromatin remodeling proteins [4]. Promoters are exposed by repositioning nucleosomes [4].

• Nucleosome Core The nucleosome core is an octamer of two each of histones H2A, H2B, H3, and H4 [4, 5]. About 160 base pairs of DNA wrap twice around this core [5].

• Histone H1 Histone H1 associates with linker DNA and helps compact the chromosome [5, 6]. Removing H1 loosens the structure [6].

• Histone Tails Histone tails can be modified by methylation or acetylation [5, 7, 8].

• Histone Methylation Histone methyltransferases add methyl groups to histone tails [5]. Methylation favors heterochromatin formation, and the process is reversed by histone demethylases [5].

• Histone Acetylation Histone acetyltransferases add acetyl groups to histone tails, preventing close packing and favoring gene expression in euchromatin [7]. The process is reversed by histone deacetylases [7]. Acetylation opens the structure because it reduces the positive charge of histones, weakening their attraction to negatively charged DNA [9].

• RNA Splicing Exons are expressed regions found in DNA and mature mRNA, while introns are intervening regions found in DNA but not mRNA [10]. Some eukaryotic genes contain many introns [10].

• Transcription RNA polymerase (RNA pol II) and transcription factors are involved in transcription [7, 11]. Basal factors and Pol II lead to low-level transcription, which increases when activators are bound to DNA [12].

• Enhancers Enhancers are DNA elements that help activate transcription.

• Elongation During elongation, the sigma (σ) factor separates, and core RNA polymerase moves along the template strand adding NTPs to the 3' end of the mRNA.

• Infection observations An observation was made regarding viral infection, noting that lower humidity leads to quicker infection and a potentially more robust infection, as the initial inoculum received by exposed guinea pigs is higher [13].

• Plaque Size The size of plaques formed by a virus can indicate its ability to spread; smaller plaques suggest a deficiency in spreading, such as with a neuraminidase-deficient virus [14]. Neuraminidase’s real power comes at the end of the infection, allowing the progeny virions to be released [15].

• Tamiflu Tamiflu is a neuraminidase inhibitor that works by slowing the spread of the virus to neighboring cells [16, 17].

• Histone Conservation Histones are highly conserved across species, suggesting that their structure cannot change much without loss of function [18, 19].

Glycoprotein Synthesis, Transcription, and Sialic Acid: Lecture Notes

Glycoprotein Synthesis and Sialic Acid [1]

• Overview The lecture covers signal sequences, cellular layout, protein trafficking, and the distribution of sialylated glycoproteins [1].

• Learning Objectives Describe the ER and Golgi, signal/recognition sequences, glycosylation, the function of sialyltransferase, and identify the distributions of SA in key influenza species [1].

  Definitions

• Endoplasmic Reticulum (ER) Extension from the nucleus where protein and lipid synthesis occurs [3].

• Golgi Apparatus Near the ER; processes and packages proteins and lipids exiting the ER [3].

• A protein made in the ER, therefore (ergo) it moves to the Golgi [3].

• Signal Recognition Particle A ribonucleoprotein that recognizes a peptide sequence and directs a nascent peptide to the ER [4].

• Signal Peptidase An enzyme that cleaves the nascent peptide downstream of the C-region, releasing the protein into the lumen of the ER [4].

• Glycoproteins All cells in nature have glycoproteins on their exterior [5].

• Sialic Acid A 9-carbon molecule, also called N-acetylneuraminic acid (Neu5Ac). Sialic acid is a generic name for a family of about 40 different compounds, all derived from [5].

• Sialyltransferases A family of 20 genes, conserved from mouse to human [5]. Each one is specific for a target carbohydrate and linkage type [5]. Two transferases create alpha2-6 linkages (ST6Gal I and II), and six create alpha2-3 linkages (ST3Gal I–VI) [5].

• H5N1 Discovered in birds in 1959, with an outbreak in Hong Kong in 1997 [6]. It binds both 2-6 & 2-3 sialic acid and is more pathogenic and deadly in some species [6].

• Viral Infection A virus can infect a particular host if the receptor the virus utilizes is expressed in that host and in a set of tissue/s that particular virus can reach [7].

Transcription and Gene Expression [2]

• Basal Transcription Normal level of transcription requiring transcription factors and polymerase [2].

• Enhancers DNA sequences that can be upstream or downstream of the gene [2]. Enhancers can enhance activation or enhance repression [8].

• Cis vs. Trans Action Enhancers act in cis, meaning they must be on the same DNA strand and close to the gene [9]. Transcription factors act in trans, affecting genes far away or on different chromosomes [9].

• Exons and Introns Exons are expressed regions, while introns are intervening regions that are removed from the primary transcript [10]. The mature RNA will have the introns removed [10].

• Untranslated Regions Mature mRNA has 5' and 3' untranslated regions (UTRs) that are not translated into protein [11].

• Alternative Splicing Exons can be skipped or mutually exclusive, leading to different protein isoforms from the same gene [11].

• mRNA Processing A methyl cap is added to the 5' end of the RNA, and a poly-A tail is added to the 3' end by poly-A polymerase [12]. These modifications are necessary for proper processing and export from the nucleus [12].

• mRNA Stability RNA-binding proteins stabilize and circularize the RNA for export [12].

Peptide Synthesis and Protein Structure [13]

• Translation tRNA brings amino acids to the ribosome, adding them to the growing peptide chain [13].

• Ribosome Sites The ribosome has three sites: E (ejecting), P (polymerizing), and A (adding) [13].

• Amino Acids You do not need to memorize the amino acids [13].

• Protein StructurePrimary: Amino acid sequence [13].

• Secondary: Alpha helices and beta sheets [13].

• Tertiary: Peripheral interactions [13].

• Quaternary: Subunits come together [13].

• It is important to remember the different orders of protein structure and how they come together to properly answer a question [14].

Glycosylation [14]

• ER and Golgi Proteins typically move from the ER to the Golgi [14].

• Signal Sequences Nothing happens without a signal sequence [15]. Viruses hijack these sequences [15].

• ER signals If we are going to the ER, we need a specific sequence; if we are going to the Golgi after that, the vesicle needs a sequence [16].

• ER energy The ER is one of the largest energy sinks in the cell [17].

• Glycosylation Golgi finishes the job [17]. Carbohydrates are added post-translationally during protein trafficking [4].

• Sialic Acid The receptor that influenza looks for is not a specific protein but sialic acid [18]. It can be on any glycoprotein on the surface of a host cell [18].

• Sialyltransferases Transferases add sialic acid to the end of a sugar [19]. There are 20 of these, fairly highly conserved [19]. Two do 2-6 linkages, and six do 2-3 linkages [19].

• Differential Expression Different expression gives different linkages [20].

• Tissue Distribution2-6 SA is in green, and 2-3 SA is in red [21].

• In the lower respiratory tract, there is more red (2-3) [21].

Influenza and Sialic Acid Linkages [22]

• Avian vs. Human In the upper respiratory tract, it's harder for avian influenza to bind, whereas human influenza will [22].

• H5N1 H5N1 binds to both 2-6 and 2-3 sialic acid and is more pathogenic [23].

• Pigs Pigs have 2-6 all over, but they have 2-3 all over [24]. The pig is the mixing vessel because of this and the proximity to humans [25].

• Transmission Transmissions are coming from up high to up high, not down low to up high [25].

Viral Entry and Uncoating: Mechanisms of Influenza A Virus

Viral Entry and Uncoating Lecture Notes

• Viral Infectious Cycle The cycle includes attachment, entry/penetration, uncoating, replication, assembly, maturation, and release/egress [1].

• Lecture Focus This lecture focuses on viral entry into a host cell, endosomal acidification, viral/endosomal membrane fusion, breakdown of the viral matrix, and release of viral contents into the cytoplasm [1].

Definitions

• Susceptible Cell A cell capable of being entered by a virion [2, 3].

• Permissive Cell A cell capable of supporting viral replication [2, 3].

• Receptor-mediated Endocytosis A process where a cell-surface receptor captures a target molecule and brings it into the cell [2, 3].

• Viral Structural Protein A protein that is part of the mature virion, such as IAV's M1 [2, 4].

• Viral Membrane Protein A protein anchored in the viral envelope, such as IAV's HA, NA, and M2 [2].

IAV (Influenza A Virus) Entry

• Entry Facilitation Hemagglutinin (HA) facilitates entry [5].

• Receptor-Mediated Endocytosis IAV enters host cells through receptor-mediated endocytosis [3].

• HA Structure HA has HA1 and HA2 domains, a transmembrane domain, a fusion peptide (FP), and an activating cleavage site [6-8]. The whole structure before cleavage is HA0 [8, 9].

• HA Cleavage HA FP must be cleaved by a host enzyme for activation before endocytosis [10-12]. Cleavage is essential for viability; it allows HA1 to disassociate from HA2 [12].

• Endosomal Acidification Acidification is required for the influenza infectious cycle.

• Acid-Catalyzed Fusion Lowering the pH in the endosome drives membrane fusion.

• M2 Ion Channel The M2 ion channel is involved in the acidification process [14, 15].

• M1 Matrix Protein The M1 matrix protein provides structure to the virus [16, 17]. M1 breakdown releases vRNPs [17].

Fusion Process

• Membrane Fusion Viral and endosomal membranes must fuse for viral contents to be released [1, 18].

• Hemifusion State An intermediate state where only half of the bilayers have merged [10, 13, 19].

• Fusion Pore Formation of a fusion pore relieves stress and allows viral contents to enter the cytoplasm [10, 19].

• Energy Requirement Overcoming the energy barrier is necessary to proceed from hemifusion to full fusion [19].

• Histidine's Role Histidines in HA become protonated, leading to charge changes and repulsion that facilitate membrane fusion [10, 13, 20].

M2 Ion Channel and M1 Matrix

• M2 Function At low pH, M2 channel activation occurs via protonation of histidine residues, allowing hydration of the channel pore and proton conductance [16, 17, 21].

• M1 Function M1 provides structure and is affected by acidification, leading to the release of vRNPs [16, 17].

vRNP Nuclear Translocation: An Overview of Viral Replication

vRNP Nuclear Translocation: Key Points

Lecture Overview:

• Covers the IAV genome, virion contents, vRNP transport into the nucleus, vRNA transcription, and v-mRNA translation [1].

  Learning Objectives:

• To understand the roles of IAV RdRp components, the makeup of vRNPs, the role of Nuclear Localization Signals, and the mechanism of nuclear import [1].

  Definitions:

• IAV RdRp: Includes PA, PB1, and PB2 [1].

• PA cleaves host RNA [1].

• PB1 is the polymerase subunit [1].

• PB2 binds the host 5’ cap [1].

• (+) sense RNA: Similar to mRNA (e.g., CoV-2) [1].

• (-) sense RNA: Does not directly code for protein (e.g., IAV) [1].

  vRNP Components

• Consist of viral RNA and nucleoprotein (NP) [2].

• Also includes the RNA-dependent RNA polymerase PB1/PB2/PA [2].

  Nuclear Import:

• Essential because influenza replicates in the nucleus [3].

• vRNPs must be transported into the nucleus [1].

• M1 proteins are excluded from the nucleus [4].

  Viral Polymerase:

• Influenza must bring its own RNA polymerase (RDRP) [5].

  RNA Types:

• (+) sense RNA can be directly translated [6].

• (-) sense RNA requires an extra step [6].

  Nuclear Localization Signals (NLS):

• Required for vRNP entry into the nucleus [1].

• Multiple types of NLS exist [7].

• Many NLS sequences are rich in lysine and arginine [8].

  NP Nuclear Localization Signals:

• Two potential NLS regions were identified in NP [9].

• The N-terminal sequence (3-13) is crucial for nuclear import [10].

• For NLS to function it must be accessible and recognizable [11].

  Importin:

• A host protein that binds to the NLS on NP [12, 13].

• Facilitates vRNP import into the nucleus [12, 13].

  Ran GTP:

• Binds to importin in the nucleus [14].

• Causes importin to release the vRNP cargo [14].

  Uncoating:

• The M2 ion channel allows protons into the virus, leading to M1 matrix protein breakdown and vRNP release [15].

• This process, along with membrane fusion, is required to release vRNPs into the cytoplasm [7].

IAV: Genomic Transcription and Replication Lecture Notes

Genomic Transcription & Replication Lecture Notes:

Lecture 7 Overview: How IAV (Influenza A Virus) gets more done with less, focusing on v-mRNA transcription and the shift from transcription to replication [1].

Key Definitions:

• Primary Transcription: mRNA production from viral genomic RNA (vRNA) [2, 3].

• Replication: vRNA is used to create cRNA (+ sense), which then creates new vRNA (- sense) [2].

• mRNA and cRNA are both + sense [2, 4].

• Central Dogma (IAV): The central dogma is negative RNA to positive RNA to protein [5, 6].

• Location: This process occurs in the nucleus [3].

• More Proteins, Fewer Segments: IAV gets more proteins than genomic segments by [5]:

• Using host splicing machinery [5].

• Alternative splicing [1, 5].

• Alternative start codons [1, 7].

• Ribosomal frameshifts [7].

• IAV vRNAs Must Be Transcribed: Transcription and replication are not strictly replication but a step in the replication process [2, 3].

• Transcription SpecificsMaking mRNA from viral RNA [2, 3].

• mRNA has a 5’ cap and poly A tail; vRNA does not [8].

• The host cell is fine and doesn't think it has any issues [9].

• RNA polymerase 2 makes mRNA [9].

• Cap Snatching: Because influenza viruses can't make their own caps, they have to steal them [10].

• The virus gets close to the host's RNA polymerase II [10].

• The viral polymerase is made of PB1, PB2, and PA [10].

• PB2 grabs the cap [10, 11]. PB2 is the thief [12].

• PA cuts the host RNA [10-12]. PA has endonuclease activity and its A looks like scissors [11, 12].

• PB1 is catalytic for RNA polymerization [10, 12]. PB1 adds one nucleotide at a time [12].

• Genomic Segments: There are 8 segments in the genome, named and numbered by size [13-15].

• If directly translated, they would only yield 8 proteins, but more are needed [13, 15].

• PB2 strand only encodes PB2 [15].

• PB1 can encode PB1, PB1-40, and PB1F2 [6, 15].

• M segments can create M1 mRNA and M2 mRNA [15].

• NS strand produces NS1 and NS2 [15].

• Alternative Splicing: The host cell machinery splices some of the influenza segments. When spliced differently, there is an additional protein [5, 16, 17].

• More M1 mRNA is created than M2 mRNA [5, 18].

• More NS1 is created earlier than NS2 or NS3 [5, 18, 19].

• Not doing this with introns [5, 18].

• Alternative Start Codons: Used by PB1 strand to create multiple proteins [7, 19].

• Kozak sequences upstream of the start codon increase the chances of ribosome binding [19, 20].

• Shifting reading frames drastically changes the protein composition [20, 21].

• ATG is a start codon [7, 21].

• COV2 vs. Influenza:

• COV2 doesn't enter the nucleus [14].

• COV2 RNA is immediately translated [14].

• COV2 doesn't undergo nuclear import or cap-snatching [14].

• COV2 is (+) RNA [14].

Viral Assembly and Budding: Lecture Notes

Assembly & Budding Lecture Notes:

• Infectious Cycle: Attachment, entry, uncoating, replication, assembly, maturation [1].

• Topics: Focus on viral genome replication, stem-loops, segment packaging, and neuraminidase's role in budding [1].

• Learning: Aim to understand how the correct 8 segments assemble, interact, and arrange [1].

• Replication Definitions:

• Resident RdRp: Holds original strand ends [2].

• Non-Resident RdRp 1: Synthesizes new vRNA [2].

• Non-Resident RdRp 2: Stabilizes vRNA for NP association [2].

• 5’ and 3’ Complementarity: vRNA and cRNA ends form stem-loops for RdRp association [3].

• M1 and NEP Role in Export [4]:

• M1 covers up the NLS on MPs [4].

• NEP/NS1 covers the NLS on M1, exposing an NES [4].

• Exportin recognizes the NES on NEP and facilitates nuclear export [4].

• HA and NA Traffic to Plasma Membrane: HA, NA, and M2 go to the plasma membrane [5].

• M1 Initiates Budding: M1 associates with transmembrane proteins (HA, NA, M2) to start budding [6].

• Specific Segment Packaging:

• Each virion gets 8 segments, not random [6].

• Specific RNA sequences ensure proper association [7].

• Mutating sequences near the 3' and 5' ends can disrupt segment interactions [8].

• Neuraminidase: Neuraminidase cleaves Sialic Acid (SA) and maintains balance with hemagglutinin (HA) [9, 10].