BIOL 306 Exam 4 Content

How does reverse transcription of viral RNA occur in a retrovirus?

(I recommend looking at exam 4 powerpoint slides 61 and 62 to help visualize this). -tRNA brought in capsid base pairs with PBS so the 3' end of tRNA can act as a primer. -Reverse transcriptase uses reverse transcriptase function to make a DNA copy of the U5 and R from the 3' primer end of tRNA. -RNase function of reverse transcriptase degrades the U5 and 5' R of the RNA. -The new DNA R moves to 3' end of RNA to base pair with the RNA 3' R. -Reverse transcriptase uses reverse transcriptase function to synthesize the rest of the DNA strand, down to the PBS. -RNase H function of reverse transcriptase degrades all of the remaining RNA template except for the hard-to-degrade polypurine tract and tRNA. -The polypurine tract acts as a primer for the DNA-dependent DNA polymerase function of reverse transcriptase to begin synthesizing the second strand of DNA down to the tRNA. -RNase H function of reverse transcriptase degrades the last of the RNA (polypurine tract and tRNA). -The new DNA (U3, R, U5, PBS) moves so that its 3' PBS base pairs with the 3' PBS on the other strand. -DNA-dependent DNA polymerase function of reverse transcriptase fills in to make the final dsDNA product. --This is a DNA copy of the RNA genome, but it may have some mutations because reverse transcriptase doesn't have proofreading abilities. --Longer than the original viral RNA genome because the Rs have been lengthened to long terminal repeats (LTRs) because a unique RNA sequence was added to each end.

How does Salmonella enterica use a type III secretion system encoded by genomic/pathogenic islands? How do lumen microbiota affect Salmonella proliferation?

(this card scrolls!) -Facultative intracellular pathogen that can exist within host cells. -Many pathogenicity islands!! -Infects M cells (identify friend + foe lumen microbes) in small intestine. Pathogenicity island 1 -Attachment to M cell --> pathogenicity-island-1-encoded TIIISS expressed to inject effectors into cell. -Some effectors change host cytoskeleton to cause membrane ruffling: ruffles surround Salmonella and engulf it into a vacuole. -Some effectors loosen junctions between epithelial cells. -Some effectors increase host cytokine production to elicit inflammation. --Cytokine IL-8 recruits neutrophils from bloodstream through loosened junctions to lumen. --Neutrophils kill microbial competitors, but not Salmonella. --Neutrophils generate reactive oxygen species (ROS) that kill microbial competitors, but not Salmonella. --H2S produced by lumen bacteria is changed into thiosulfate and then tetrathionate by ROS. Only Salmonella can use tetrathionate as a terminal electron acceptor = advantage because doing more-efficient respiration while other bacteria do fermentation (remember that colon is anaerobic). -So, inflammation causes Salmonella to outcompete other bacteria and proliferate, resulting in diarrhea. Pathogenicity island 2 -Salmonella senses it is in vacuole --> pathogenicity-island-2-encoded TIIISS expressed to inject effectors into cell. -Effectors modify vacuole surface so that: --Lysosomes don't identify vacuole to fuse with it to acidify it to kill microbial contents. --Vacuole is trafficked to basal membrane to spill Salmonella into area below mucous. From there, it may reach the bloodstream and spread through the body. Microbiota in lumen... -Hurt Salmonella by... --Taking up niches. --Producing indole via tryptothanase, inhibiting Salmonella gene expression and reducing inflammation. -Help Salmonella by... --Generating H2 gas and H2S.

What is included in the genome of HIV?

(this card scrolls!) More complex than simple retroviruses! *Same as in simple retrovirus:* -90% of time, full length RNA for gag; 10% of time, frameshift for gag-pro. --each is proteolyticallly modified. -Splicing of env. ---cleaved from gp160 to gp41 and gp120 by viral protease. *Because HIV is a lenti virus, its genome contains many other regulatory genes (some of which must be spliced together to be expressed) that are expressed from three different reading frames:* -rev gene mediates transport of unspliced mRNAs out of nucleus. --rev must be spliced together; fully spliced = enters cytoplasm early. --Rev returns to nucleus and binds to any RRE (Rev Response Element; stem looping on unspliced and singly spliced mRNAs) --> nuclear export signal (NES) on Rev binds to CRM1 nuclear export protein to bring these transcripts to cytoplasm for late gene expression. Gag, Pol expressed late because they aren't needed at the time expect for packaging in new viruses. -nef gene downregulates CD4 to prevent superinfection. --Fully spliced = enters cytoplasm early. --Nef reduces binding to new retroviruses; like how E. coli containing F plasmid doesn't bring in another copy of F plasmid. -vpu gene allows release of virus from cell during budding. --Expressed late. --Cleave off host tetherin to free virion from cell.

What are the four major antibiotic mechanisms? How do they work and what are some examples of each?

*1. Target bacterial cell wall synthesis*: -Peptidoglycan is essential to viability of most bacteria. -ex: vancomycin, beta-lactams like penicillin (see semi-comp quizlet). *2. Inhibit protein synthesis*: -Complexity of multiple proteins and rRNAs in ribosome = many targets = diverse antibiotics. -Bacterial and eukaryotic ribosomes are different enough that this can be targeted. -ex: streptomycin binds to 30S subunit, causing ribosome to make mistakes. Resistance bound to happen because it only requires a 2 aa mutation in ribosome. *3. Target nucleic acid replication and synthesis*: -Exploit differences in DNA replication strategies and RNAP of bacteria and eukaryotes. -ex: ciprofloxacin (fluoroquinolone) inhibits DNA gyrase to interfere with DNA replication. -ex: rifampicin inhibits mRNA synthesis by bacterial RNAP. *4. Inhibit essential metabolic pathways*: -Target pathways that exist in bacteria, but not in hosts. -Ex: sulfanilamide inhibits folic acid production.

What are two (hint: really three) examples of AB toxins? How do they work? What diseases do they cause?

*Diptheria toxin (Dtx)* -Encoded by temperate bacteriophage existing as prophage in Corynebacterium diphtheria genome. -Only expressed when bacteria is stressed for iron (will allow lysis of host cell to get iron, overcoming host nutritional immunity). -Unusual for an AB toxin, Dtx is a single protein with a 1:1 A:B ratio. -Dtx binds to receptor --> receptor-mediated endocytosis --> host trypsin cleaves Dtx to activate it --> acidification of endosome (backfires!) --> A exposed to cytoplasm --> Dtx disulfide bridges providing protein structure are reduced --> A escapes to cytoplasm --> A cleaves NAD+ --> ADP-ribosyl portion of NAD+ bound to elongation factor 2 (EF-2) while nicotinamide portion is released --> protein synthesis inhibited. -Causes diptheria: airborne disease, replication in pharynx, overactive immune response forms psuedomembrane that can potentially cause suffocation, toxin may enter bloodstream to harm other tissues (like heart and CNS). *E. coli heat labile toxin (Ltx) and Cholera toxin (Ctx)* -Nearly identical due to horizontal gene transfer, and accordingly act identically = can be discussed interchangeably. -In enterotoxigenic (ETEC) E. coli strains, colonization factor (CF) attaches to small intestine cell via binding of B of Ltx to receptor --> endocytosis --> acidification --> A to cytoplasm --> A cleaves NAD+ --> ADP-ribosyl added to GTP-binding regulatory subunit of adenyl cyclase --> unregulated adenyl cyclase overproduces cAMP --> protein kinase A activated to phosphorylate cystic fibrosis transmembrane regulator (CFTR) --> Cl- constantly pumped out of cell while Na+ is blocked from entering --> osmotic imbalance because solute concentration is too high in lumen --> mass diffusion of water out of cell -Causes diarrhea, facilitating spread of bacteria.

What is an example of an exotoxin that activates second messenger pathways? How does it work?

*E. coli heat stable toxin (ST)* -Produced by 1/3 of enterotoxigenic (ETEC) E. coli strains instead of Ltx (AB toxin). -ST mimics guanylin hormone that regulates intestinal fluid homeostasis. -ST binds to guanylate cyclase (normal receptor for guanylin) --> cGMP accumulates --> protein kinase G --> CFTR phosphorylated --> Cl- pumped out and Na+ kept out --> water diffuses out of cells --> diarrhea. Same result as Ltx and Ctx! -Causes diarrhea primarily in newborns because they have higher expression of guanylate cyclase.

What is an example of a exotoxin that damages cell membranes? How does it work?

*Hemolysin.* -Staph aureus and many Streps secrete hemolysin, which forms pores in blood cells to cause them to lyse. -Although hemolysin does lyse red blood cells, its intended target is white blood cells to defend against immune responses to infection.

How do AB toxins generally work?

-A= active subunit, B= binding subunit. -Typically 1 A subunit and 5 B subunits with A and B encoded as different proteins. -B binds to receptor --> receptor-mediated endocytosis --> acidification --> protein conformation change uncouples A and B --> A released to cytoplasm --> ADP-ribosyltransferase activity. --ADP-ribosyltransferase activity: A cuts NAD+ into nicotinamide (released) and ADP-ribosyl, which will be bound to a host protein (protein target differs for different toxins) to inactivate it, which will result in inhibition of protein synthesis. -AB toxins are enzymes, so you only need one to kill the whole cell!

What are the four mechanisms of bacteria becoming antibiotic resistant? Which can be encoded by plasmids?

*not coded by plasmids*: 1. Mutations alter antibiotic targets so the antibiotic doesn't bind (ex: ribosome or DNA gyrase mutations). *usually coded by plasmids*: 2. Enzymes to break down antibiotics (ex: beta-lactamases in gram neg bac). 3. Antibiotic-altering enzymes (ex: CAT (like HATs) acetylates chloramphenicol so it can't interact with ribosomes). 4. Efflux pumps export the antibiotic (ex: tetracycline resistance genes on transposon; repressor TetR detaches when tetracycline is present so tetA will be expressed to produce efflux pump).

What is the structure of the SARS-CoV-2 genome as it enters the host cell, and what different genes are encoded by it?

+ssRNA that functionally acts as mRNA and can be translated immediately upon entry into the host. -5' UTR and methylguanosine cap. -3' UTR and poly-A tail. Immediate translation will produce nonstructural proteins. Because there is a stop codon at the end of the nonstructural genes, structural and accessory proteins will not be automatically translated. There are four major structural proteins: -Spike protein. -E (envelope) protein: viral glycoprotein. -M (membrane) protein: membrane-embedded glycoprotein. -N (nucleic acid) protein: binds to CoV genome.

What kind of virus is Coronavirus? What is its virion structure?

+ssRNA virus associated with N proteins. -Already 5' capped with UTR and 3' poly-A tail. Virion structure: -Enveloped icosahedral virus. -Trimer spike proteins in envelope.

How do sialic acids differ in birds, humans, and pigs, and what implications does this have for Influenza A?

-Avian sialic acid is α-2,3-linked to galactose, so avian influenza binds to this kind of sialic acid. -Human sialic acid is α-2,6-linked to galactose, so human influenza binds there. -Pigs have both α-2,3- and α-2,6- sialic acid linkages to galactose, so they can be infected by both avian and human influenzas at once. Virions budding off of pig cells will likely have a mix of avian and human RNA segments. -Viruses don't have a selective means to make sure all 8 different RNA segments are packaged in the virion, so they instead have evolved to grab 11 segments with the hopes that they'll grab all 8 they need in the process. This is very inefficient; many virions won't get all 8 segments and won't be infectious. -However, virions that do successfully get all 8 segments from a new assortment of avian and human RNAs create viruses we don't have immunity to because we've never been exposed to = potential for severe pandemics.

How does SARS-CoV-2 enter host cells?

-CoV-2 spike proteins bind to angiotensin-converting enzyme (ACE2) on host. -Host proteases cleave spike protein to S1 and S2. -Conformational change of remaining protein brings membranes so close together that they fuse. - +ssRNA and associated N proteins spill into cell.

How does CCR5 expression in an individual affect HIV-1 infection?

-Elite non-progressors have two defective copies of the CCR5 gene due to deletions, so they do not express CCR5. Therefore, they are mostly protected from HIV-1. -People that are heterozygous CCR5 have delayed onset; takes longer for them to be infected. -Most people have 2 functioning CCR5 alleles and are infected more quickly. Defective CCR5 alleles are rare, but were likely somewhat selected for due to protection against smallpox.

What is the general structure of a retrovirus?

-Enveloped virus with surface viral glycoproteins in host-derived lipid bilayer. -Viral protein capsid within membrane holds: --Two copies of +ssRNA genome. One tRNA is bound to each +ssRNA copy to serve as a primer for reverse transcriptase. --Preformed enzymes: --> Reverse transcriptase (replicates genome via dsDNA intermediate). --> Integrase (recombines dsDNA genome copies into host). --> Protease (processes membrane envelope proteins to activate them).

What are three classifications of exotoxins?

-Exotoxins that damage cell membranes. -Exotoxins that inhibit protein synthesis. -Exotoxin that mimics another molecule to bind to receptor to activate second messenger pathways.

What are the related functions of hemagglutinin (HA) and neuraminidase (NA) in Influenza A?

-Hemagglutinin (HA) binds to sialic acid on host glycoproteins (widespread on eukaryotic cells and in mucus secretions of tracts) to aid viral entry into a cell. -Neuramindase (NA) cleaves sialic acid off of glycoproteins so HAs don't get stuck on cell during budding.

How does a healthy microbiome protect against Clostridium difficile?

-In a healthy colon microbiome, C. diff is outcompeted. -When people are treated with antibiotics, there is no longer microbiota to outcompete C. diff --> C. diff replicates and causes pseudomembranous colitis, which results in severe damage to the colon lining and may necessitate removal of some of the colon or cause death. --Especially problematic in hospitals, where nosocomial infections are contracted from hyper virulent hospital strains that have increased spore formation and increased secretion of the toxin TcdAB that causes inflammation. Disease progression: -C. diff spores survive passage through stomach. -Bile in small intestine is a signal activating germination of the spores. -C. diff is fully functional vegetative cells by the time they reach their replicative environment of the colon. --In a healthy microbiome, C. diff is outcompeted and their population is low enough that TcdAB doesn't harm the host. --In an unbalanced microbiome without competition, the C. diff population spikes and produces lots of TcdAB that causes hyperinflammation, creating a psuedomembrane that may be lethal. Treatment: -Fecal microbiota transplants reestablish healthy microbiota and introduce bacteriophage to reduce C. diff population.

What are two *possible* reasons why the highly lethal H5N1 avian Influenza A can spread from bird-to-human, but not from human-to-human?

-May be because human proteases can't cleave the H5 (note that many distinct hemagglutinins exist, H1-H16) of H5N1, so Influenza A virions budding off of human cells aren't infectious. -May be because H5 finds more receptors in the lower respiratory tract than typical H1 does, so its harder to spread by coughing and sneezing than if it were in the upper respiratory tract like H1.

How do mRNA SARS-CoV-2 vaccines work?

-Our cells have a large immune response against foreign mRNA, so psuedouridine is substituted for uracil in the mRNAs so they won't trigger this response. -The mRNA is bound to ionizable lipids inside a liposome (double membrane of various lipids). --Once in the alkaline bloodstream, the ionizable lipids will dissociate from the mRNA. -The mRNA will be translatedby the host tom produce the SARS-CoV-2 spike protein, which will elicit a host immune response.

How do PB1-F2 Influenza A strains originate? What is unique about them?

-PB1 mRNA is normally translated from the first AUG. -Translating from a further down AUG produces a short PB1-F2 protein in highly pathogenic avian Influenza A strains. --Spanish flu came from an avian flu with PB1-F2. PB1-F2 strains are more virulent because they cause mitochondrial-induced apoptosis. -Mitochondrial antiviral-signaling protein (MAVS) in mitochondrial outer membrane uses PMF to induce the interferon response when a virus is present to alert other cells that infection is beginning so they can prepare themselves. -PB1-F2 binds to MAVS and dissipates PMF, interfering with this innate defense system.

What are the three distinct enzymatic activities of reverse transcriptase?

-RNA-dependent DNA polymerase (reverse transcriptase). -DNA-dependent DNA polymerase. -RNase H (degrades RNA associated in RNA-DNA hybrid) (H for Hybrid).

What is the general procedure of retroviral replication?

-Reverse transcription (RT) creates a dsDNA copy of the viral +ssRNA genome. The repeats (Rs) of the RNA genome have been lengthened to long terminal repeats (LTRs). -Integration of proviral DNA into host genome. -Transcription of proviral DNA regenerates viral mRNA/genomic ssRNA. -Capsid expressed. -Budding and release of the retrovirus.

What are some notable human coronaviruses?

-SARS -MERS -SARS-CoV-2 -Russian "flu" in 1890s was actually HCov-OC43. --Epizootic (animal epidemic) in cows that jumped to humans. Immunity and mutations are hypothesized to make this evolve into a mild common cold.

Why has CoV-2 been a harder Coronavirus to control than SARS?

-SARS spillover events can be tracked to eliminate viral sources; can't for Cov-2. -Most SARS transmission occurs in hospitals and spreading can be blocked from there; Cov-2 is widespread in communities. -SARS always shows symptoms quickly, so self-isolation and contact tracing is possible; Cov-2 may be asymptomatic.

What are some of the Coronavirus spillovers caused by bats? Why are they responsible for so many CoV spillovers?

-SARS spillover from bats to civet intermediate, then humans. -MERS (more deadly than SARS) spillover from bats to camel intermediate, then humans. Why so many spillovers? -Bats are the only mammals that can fly and they live longer than many mammals, so they have higher metabolic demands that cause DNA damage and release of self-DNA, changing the ability of bats to react to cytoplasmic DNA. -DNA normally isn't free in cytoplasm. --In *humans*, cGAS binds to DNA in cytoplasm and cyclizes ATP and GTP to cGAMP --> DNA is assumed to be an indicator of a viral infection, so cGAMP starts the interferon response by activating STING --> TBK1 (a kinase) recruited to phosphorylate STING and IRF3 --> IRF3 transported to nucleus for massive interferon response. --In *bats*, one STING codon is changed from coding serine to coding another amino acid (all other mammals have serine at this codon). This results in no phosphorylation of STING, creating a much smaller inflammatory response that means bats can better tolerate viral infections. This viral tolerance means they can be infected by so many viruses and thus cause so many spillovers.

What kind of virus is Influenza A? What family is it in? What is its virion and genomic structure?

-ssRNA virus. -Orthomyxovirus family. Virion structure: -Enveloped icosahedral virus. -Three viral proteins exist in the host-derived lipid bilayer: hemagglutinin (HA) (trimer), neuraminidase (NA) (tetramer), and the matrix 2 ion channel (M2). Genomic structure: -Segmented genome consisting of 8 segments of -ssRNA. -Must enter cell with preformed viral replicase/RNA-dependent RNA polymerase (RDRP) on each -ssRNA segment to make +ssRNA copies for translation. --RDRP consists of the PA, PB1, and PB2 components. -Each segment is wound around nuclear protein (NP) monomers to form ribonucleoprotein (RNP) complexes. --Connections exist between the RNP complexes.

What are the broad overall steps of a SARS-CoV-2 infection, entry to budding?

1. Attachment to receptor via spike protein. 2. Membrane fusion. 3. mRNA spilled into host cell. 4. Ribosomes translate mRNA to pp1a or pp1ab, which will be auto-proteolyzed to nsps. 5. Some of the nsps steal membranes from the ER (causing stress on the cell) to make double membrane vesicles (DMVs) as replication transcription complexes (RTCs). 6. In DMVs, replicase/RDRP replicates or transcribes the viral genome: +gRNA genome --> -gRNA anti-genome --> +gRNA copies. --Replication outcome: +gRNA copies packaged in new viruses. --Transcription outcome: +gRNA transcripts translated to more pp1a and pp1ab. ---> To make transcripts of structural and accessory proteins, RDRP makes a nested set of subgenomic RNAs (sgRNAs) of different lengths so ribosomes will bind right in front of these genes. 7. M, E, and S +sgRNAs will be shuttled to the ER for translation. --Part of the ER will be redirected to endoplasmic reticulum Golgi intermediate complex (ERGIC) (causing stress on cell) that these proteins embed in to await budding. 8. N +sgRNAs translated in cytoplasm and bind to +gRNA. 9. N-bound +gRNAs assemble beneath M, E, and S proteins on ERGIC. 10. Transport to Golgi. 11. Budding via a smooth-walled vesicle.

What are the broad overall steps of an M-tropic HIV infection, entry to budding?

1. HIV binds to CD4 and CCR5 co-receptor. 2. Fusion of viral envelope with host membrane, spilling viral contents into host. 3. Reverse transcriptase makes dsDNA copy of RNA genome. 4. dsDNA copy and integrase (from capsid) brought into nucleus. 5. Integrase randomly inserts viral dsDNA into host genome. 6. Viral DNA transcribed upon activation. --Initial transcription will be unprocessed mRNAs that move to cytoplasm late: --> env mRNAs translated in ER to gp160, glycosylated in Golgi, cleaved by protease to gp120 and gp41 envelope proteins that are trafficked to membrane to wait for budding. While HIV envelope proteins are embedded in host membrane, they may bind to receptors on uninfected cells and cause the two cells to fuse to form a multinucleate syncytium with multiple nuclei, often killing both cells. --> 90% gag proteins and 10% gag-pol due to frameshift, proteolytic processing 7. Reverse transcriptase, integrase, protease, and 2 unprocessed mRNAs are packaged as genome within Gag protein capsid. 8. Budding of virus below envelope proteins. 9. Vpu cleaves host tetherin membrane protein to release virus from host cell.

What are the broad overall steps of an Influenza A infection, entry to budding?

1. Hemagglutinin (HA) on Influenza A binds to sialic acid on host glycoprotein. 2. The host cell endocytosizes the virus. 3. The host cell acidifies the endosome (kills most other viruses, but not Influenza!). 4. HA changes conformation. 5. Viral and endosome membranes fuse. 6. Viral -ssRNAs spill into cytoplasm. 7. Viral -ssRNAs trafficked to nucleus to be: --*transcribed* to +ssRNA that will be capped via cap snatching and given poly-A tail--> +ssRNA translated in cytoplasm or ER --> assembly of proteins in/under host membrane. --*replicated* (occurs when enough protein has been produced, indicated by NP and NS in nucleus) to +ssRNA that is uncapped and has no tail --> -ssRNA genomes made from this anti-genome --> transport of NP-coated virus-RDRP complex to viral proteins at host membrane. 8. Virus buds from cell. 9. Neuraminidase (NA) cleaves sialic acid so virus isn't stuck to host. 10. HA activated by host protease to make virus infectious.

How does Influenza A enter host cells?

1. Infleunza A viruses in the respiratory tract use their membrane protein hemagglutinin (HA) to bind to siliac acid on their host's membrane glycoproteins. --Neuraminidase (NA) cleaves sialic acid in mucus secretions of respiratory tract to give the virus access to underlying tissue. --If a virus gets stuck on an erythrocyte (red blood cell), NA cleaves sialic acid to remove the virus from that cell because these cells can't do endocytosis and don't have a nucleus, so they aren't Influenza A hosts. 2. The host eukaryotic cell endocytosizes the virus. 3. The host cell pumps protons into the endosome (kills most other viruses), causing a conformational change in HA pulling the viral and endosome membranes so close together that they fuse, spilling -ssRNA fragments into the cytoplasm. --M2 proton channel pumps protons from endosome into virus to facilitate protein conformational change. --Membrane fusion is common in enveloped viruses; also occurs for HIV retrovirus!

How many proteins are created from the genome of Influenza A?

10 proteins. -Although the genome consists of only 8 -ssRNA segments, two of them are alternatively spliced. --Export to cytoplasm in full-length form or spliced form with a frameshift creating an entirely different protein. --Segment 7 makes M1 (capsid protein) or M2 (ion channel). --Segment 8 makes NS1 or NS2.

Where do most antibiotics come from that aren't beta-lactams?

Actinomycetes soil bacteria group.

How does expression of retroviral mRNA occur? What is the secondary purpose of these mRNAs?

All retroviruses make at least two mRNAs via differential splicing: *Unspliced form* -Enters cytoplasm late because the host cell tries to prevent mRNA with introns from exiting the nucleus. -90% of the time, ribosome in cytoplasm recognizes 5' cap and translates it to gag polyprotein before translation is terminated at stop codon. --Autoproteolysis degrades gag into capsid proteins and protease. -10% of the time, the ribosome ignores the stop codon and produces a longer gag-pol polyprotein. --Psuedoknot formation (mRNA base pairs with itself at loop of stem-loop) causes ribosome to pause and change reading frame, causing it to translate through what once was the stop codon. --Protease breaks off polymerase from gag protein. --Gag protein autoproteolysis. --Polymerase cleaved into reverse transcriptase and integrase. Produced at small concentrations because this only occurs 10% of the time and they are just needed for packaging into capsids, not for use in the current cell. --Hard stop after pol genes prevents env expression from unspliced RNA. *Spliced form* -Host recognizes gag and pol as intron, splicing mRNA. -Spliced transcript transported to cytoplasm earlier than unspliced transcript, translation to env. *Secondary purpose*: vmRNAs have terminal repeats instead of LTRs due to transcription start point location, so unprocessed full-length (unspliced) vmRNAs will also be packaged to serve as genomes in infectious virions.

What are some drugs that have been used to treat Influenza A?

Amantadine -plugs M2 to prevent acidification of virion --> no HA conformation change --> virus doesn't escape endosome. -no longer used because Influenza A has evolved resistance. Neuraminidase inhibitors (ex: Tamiflu) -bind to NA so viruses get stuck on cell's sialic acid during viral release, preventing the virus from spreading to other cells or other hosts.

Why can't we clinically use all antibiotics isolated from bacteria?

Antibiotics can only be used if they have selective toxicity-- hurting bacteria, but not hurting us. -High (wide) therapeutic index (TI) is best-- indicates that you need a high dose to be toxic to patient, but only a low dose is needed for treatment. Safe to use with minimal worry. -Antibiotics with low (narrow) TI can have severe toxicity in patients (hearing loss, renal failure, etc) and their usage must be monitored. --More commonly used in hospitals for monitoring purposes.

What is the type III secretion system?

An exotoxin secretion system for gram negative cells that secretes toxins from cytoplasm directly into target cell. -Needle-shaped machinery with translocation channel that exotoxins move through. --Modified flagellum! Duplication of flagellum --> evolution to secrete proteins through tip rather than synthesize flagellum from tip. -Driven by ATP hydrolysis in cytoplasm.

What are antigenic shift and antigenic drift? How do they occur for Influenza A?

Antigenic shift: rapid alteration of viral genotype and phenotype due to acquisition of a different RNA segment. -Simultaneous infection with two different types of Influenza A in pigs can lead to reassortment creating new major Influenza A strains we aren't immune to. -Ex: H1N1 swine flu has pig, bird, and human Influenza A RNA fragments. Luckily, it didn't get avian PB1-F2, making it less lethal! Antigenic drift: gradual drift in viral amino acid sequences, meaning our immune system won't be able to recognize the virus as well. -Due to Influenza A RDRP's non-fidelitous nature and lack of proofreading systems = high mutation rate. -This is why we need booster flu shots each year.

What is the difference between an antimicrobial and an antibiotic?

Antimicrobials are of natural, semisynthetic, or synthetic origin. Antibiotics are antimicrobials of natural origin. -Produced by microorganism. -May be modified by chemists, but still originally natural.

What are the ESKAPE pathogens?

Bacteria of serious concern due to multi-drug resistant strains.

What is the difference between bacteriostatic and bactericidal antibiotics? When is each used?

Bacteriostatic antibiotics inhibit bacterial growth. -Used when the host is assumed to be healthy enough that their immune system can kill the bacteria on its own; this just buys them some time. Bactericidal antibiotics kill bacteria. -Used when the host immune system isn't very robust.

How may reverse transcription be targeted by drugs?

Because reverse transcription is so complex, it's a great target for drugs! -Nucelotide and nucleoside analog drugs stop reverse transcription when they are integrated into newly synthesized DNA. -Commonly used for PrEP (Pre-Exposure Prophylaxis), which is drug treatment to prevent or reduce the risk of HIV infection.

What are beta-lactams? What are two classes of them?

Beta-lactams are antibiotics derived from fungi that have a beta-lactam ring mimicking D-Ala-D-Ala on peptide portion of peptidoglycan. So, beta-lactams will bind to transpeptidase to kill it and prevent crosslinking. -Varying R-groups on beta-lactams change properties. Classes, each derived from different fungi: -Penicillins. -Cephalosporins. --Good example of antibiotic with multiple generations. More complex side groups on each generation to increase effectiveness against gram positive *and* gram negative bacteria and against beta-lactamases.

What are the normal functions of CCR5 and CXCR4? Can they be drug targets? If so, how?

CCR5 receptor on macrophages normally binds to chemokines released by infected cells. -R5 is a safe drug target that may be antagonized without harm to the patient (recall elite non-progressors don't have functional CCR5). -Maravirioc binds to CCR5 to prevent viral infection. CXCR4 receptor on T-cells normally binds to stromal derived factor 1 (SDF-1), which is important for stem cell homeostasis. -Therefore, it cannot be safely antagonized as a drug target.

What is cap-snatching, how does it occur, and what is its role in Influenza A?

Cap-snatching is Influenza A's random decapitation of newly synthesized host mRNAs. In the nucleus, PB2 of RDRP binds to random host mRNA 5' methylguanosine cap and PA decapitates it. -No cap on eukaryotic mRNAs = host protein synthesis halted. --So, no antiviral response will be mounted. --So, resources available for viral replication and translation. -Capped host mRNA fragment base pairs to -ssRNA segment to serve as primer for RDRP to transcribe +ssRNA. --Once RDRP hits a poly adenylation signal, a poly-A tail is added, so transcription is stopped before the full length +ssRNA is created. This +ssRNA is destined for translation; it isn't an anti-genome! --Product is chimeric viral mRNA: host cap, bit of host mRNA, viral +ssRNA, poly-A tail. Cap allows recruitment of host ribosomes for translation. Cap-snatching is potential drug target!

What are notable parts of the SARS-CoV-2 spike protein?

Consists of S1 and S2 subunits in one large polyprotein later cleaved by host. -Receptor-binding domain interacts with ACE2: --A few nucleotides make direct contact with ACE2. --Slightly different between different CoVs. -Junction of cleavage to S1 and S2: --Human SARS-CoV-2 has 4 additional amino acids not found in any closely related CoVs. ---> Causes glycosylation of some amino acids in the junction. ---> Form polybasic cleavage site, making human SARS-CoV-2 more virulent because it can be processed by more proteases. *Original route:* CoV binds to ACE2 receptors --> put in endosome --> acidification causes cathepsin (protease) to cleave spike protein to release viral contents in cell. *New route:* Additional proteases that don't require endocytosis for membrane fusion to occur; may also cleave spike proteins as virus buds off so it is ready to infect next cell immediately upon arrival.

What is the difference between an endotoxin and an exotoxin? What is the normal cause of disease symptoms?

Endotoxin: part of body of bacteria (ex: LPS). Exotoxin: truly secreted proteins. Most diseases rely on host inflammatory responses to bacteria, but some are largely mediated by bacterial exotoxins.

What is sulfanilamide?

First antimicrobial! -Analog of para-aminobenzoic acid (PABA), a precursor of folic acid (vitamin B). -Inhibits conversion of PABA to folic acid. --Bacteria do this conversion but humans don't (get folic acid from diet), so this hurts bacteria but not us :)

What family does HIV-1 belong to? What is the cellular host for HIV-1? What proteins are important for viral entry and how do they accomplish this?

HIV-1 is in the Lenti family of retroviruses. -Infections take years to develop = more complicated than simple retroviruses. Host cell: immune system cells; macrophages and T-cells. gp160 cleaved to gp120 and gp41 (encoded by ENV, gp = glycoprotein). -gp120 is noncovalently associated with gp41, which protrudes from the membrane. -gp120 portion binds to: --CD4 (on all immune system cells). --coreceptor: CCR5 (R5; on macrophages) or CXCR4 (X4; on T-cells). gp120 binds to CD4 --> if CCR5 is also present, gp120 binds to that --> conformational change --> gp120 falls off, gp41 changes shape to pull viral and host membranes close together --> membrane fusion --> nucleocapsid spills into cytoplasm --> nuclear targeting protein on one of the capsid proteins interacts with nuclear pore to bring virus into nucleus.

How does transcription of the proviral DNA of a retrovirus within the host genome occur?

Important that Rs have been lengthened to LTRs because U3 is needed on both ends for transcription! -U3 contains the promoter and enhancer that host transcription factors and RNA polymerase bind to. -Transcription begins at the beginning of the first R and the second R will be the last transcribed region. -5' methylguanosine cap and poly-A tail added, recreating the original viral mRNA with Rs instead of LTRs. 5' cap, R, U5, genes, U3, R, poly-A tail.

What mutations are included in the Omicron variant of SARS-CoV-2, and how do they affect disease outcomes?

Mutation upon Delta; has those mutations plus more, including near 30 mutations in the spike protein alone. -Unknown effects on transmissibility and immune evasion.

How does the nature of an HIV-1 infection change over time?

Initially, an HIV-1 infection is M-tropic (macrophage-tropic) and will bind to CD4 and CCR5 (R5), but not CXCR4 (X4). -This is because macrophages are found at the mucosal surfaces where sexual contact occurs -Initial spike in viral load, then falls to low levels. -CD4 count decreases very gradually due to slow depletion of macrophages. Over time, novel HIV-1 strains in the patient evolve to bind R5 or X4, then lose the ability to bind to R5 and only can bind to X4, becoming T-tropic (T-cell-tropic). -HIV-1 infects most T-cells --> T-cell depletion --> immune system dysfunction --> AIDS diagnosed and opportunistic infections possible. -Viral load spikes again. -CD4 count decreases drastically due to rapid depletion of T-cells. -Once AIDS has been diagnosed, HIV-1 is less likely to spread to a new host because T-cells are in the plasma rather than mucosal surfaces involved in sexual contact.

How is the DNA copy of a retrovirus integrated into the host genome?

Integrase randomly inserts the LTRs of the DNA copy into the host genome, integrating the proviral DNA.

Why must vancomycin be used as little as possible?

Last-resort antibiotic with growing resistance that spreads promiscuously via conjugation of resistance transposon on F plasmid. -If used too much, resistant strains will be selected for. -If used less, there will be no selection and resistant strains will hopefully die naturally.

How does HIV-1 promote its own translation?

Like other viruses, HIV-1 shuts off host gene expression to save energy and resources for its own translation. -Interacts with host translational machinery by cleaving proteins, etc. -Doesn't harm viral translation.

What mutations are included in the Delta variant of SARS-CoV-2, and how do they affect disease outcomes?

Mutations throughout genome, including: -In spike protein --At least two in receptor-binding domain, possibly causing increased affinity for binding, which may make the Delta variant more transmissible. --One near polybasic cleavage site, which may impact processing.

Why do new Influenza pandemics begin? What viruses are included in Influenza vaccines?

New strains of Influenza A have new combinations of HA, NA, etc, so no one has immunity to them. This has the potential to create pandemics. -New pandemic strains *usually* replace the previous one. Influenza vaccines include two influenza A strains and two Influenza B strains based on the strains that are predicted to be most prevalent/have the most pandemic potential that year.

What is a genomic island? What are some bacteria that contain these and use them for pathogenicity?

New stretches of DNA acquired via horizontal gene transfer (by conjugation, transformation, or transduction). -Typically flanked by genes with homology to phage or plasmids and look like they were once in transposons (integrase, insertion sequences, direct repeats, etc). -Contain lower G-C content and more A-T content, which makes them easier to express in foreign cells. -Enteropathogenic (EPEC) E. coli strains and Salmonella enterica use pathogenicity islands to encode TIIISS.

How does enteropathogenic (EPEC) E. coli use a type III secretion system encoded by genomic/pathogenic islands?

Non-intimate attachment of pili to small intestine cell --> TIIISS produced --> pore transferred to host cell --> translocated interim receptor (Tir) transported through pore to host and is embedded in host membrane (EPEC E. coli has no natural receptor, so it brings its own!) --> host mistakes Tir for regular host machinery and phosphorylates it --> EPEC can now bind to Tir --> cytoskeletal filaments are produced in host, generating a pedestal lifting up the EPEC (may provide protection from inflammatory cells, but this is largely unknown).

How are nonstructural proteins (nsp) expressed in SARS-CoV-2?

Nonstructural gene encodes massive polyprotein that does auto-proteolysis to produce nsps. -Most of the time, the ribosome hits a stop codon to produce pp1a, which will be auto-proteolyzed to nsp 1-11. --These nsps are needed earlier in viral replication and in larger quantities. --nsp1 shuts off host gene expression by blocking ribosome complex assembly to halt cell cycle progression. -Less frequently, a ribosome will encounter a psuedoknot and will change its reading frame (normally, this would result in the ribosome breaking down the psuedoknot) to translate the whole gene and produce pp1ab, which will be auto-proteolyzed to nsp 1-10 and 12-16. --nsp 11 lost by frameshift. --nsp 12-16 include RDRP and ExoN.

What are non-structural (NS) proteins in Influenza A?

Not part of the structure of the virion. -Ex: NS1 inhibits host mRNA export from nucleus by binding to host nucleoporin proteins = host antiviral response prevented.

How does a healthy microbiome protect against caries?

Plaque formation: -Saliva protein deposits form salivary pellicle on enamel. -Pioneer species of healthy microflora (many Strep species; have high affinity for pellicle) bind to salivary pellicle proteins to create healthy biofilm (plaque). There isn't much room in this healthy mouth for low-pellicle-affinity early colonizer cariogenic bacteria. -If sugar consumption is high and healthy plaque isn't regularly removed by brushing, then red complex (cariogenic bacteria) late colonizers bind to receptors on early colonizers (healthy bacteria). After meals, pH briefly drops to around 5, but saliva and healthy microbes quickly bring the pH back up to about neutral (~6.7). -Frequent sugar snacking creates acidic conditions killing healthy flora, allowing acid-tolerant low-pellicle-affinity cariogenic bacteria like early colonizer Strep mutans to proliferate and produce acid keeping the pH low, degrading enamel to produce caries.

What kinds of tissue will SARS-CoV-2 infect?

Predominantly infects respiratory tissue, but ACE2 is widespread in the body, so the virus can also infect other tissues such as endothelial, heart, kidneys, gut, etc. -*May* (speculative) be due to tissue tropism to secondary locations via viremia, like what occurs with polio.

What is the consequence of Coronavirus' unusually large genome (relative to other viruses)?

RNA viruses (including polio, HIV, and Influenza) usually don't have proofreading activities, so they frequently have mutations. Coronavirus' large genome means that they need to have exonuclease (ExoN) to do proofreading because they would otherwise die from too many mutations.

What are some potential targets for antibiotics against SARS-CoV-2? What do these antibiotics do?

Take place of nucleotides in viral replication to halt replication (ExoN can't fix this). -Remdesivir takes the place of adenosine. -Molnupiravir takes the place of cytosine. -Effectiveness is lower than hoped. Inhibit proteases to prevent viral processing for entry. -Bromohexine is a potential inhibitor of TMPRSS2, one of the protease's on SARS-CoV-2. It also cleaves hemagglutinin on Influenza A. -Paxlovid binds to proteases. -Generally more effective.

What is streptomycin?

The first anti-TB drug! -Antibiotic isolated from soil bacteria. -Inhibits bacterial protein synthesis by inhibiting 30S ribosomal subunit.

What is the general makeup of the genome of a retrovirus?

Three protein-encoding genes: -GAG: group antigens (capsid proteins). -POL: polymerase (reverse transcriptase, integrase, and protease). -ENV: envelope (envelope glycoproteins, which facilitate infection). Oncoviruses (retroviruses causing cancer) have a fourth protein-encoding gene: SRC (inserted into host to cause uncontrolled replication = host cellular transformation to cancer cell). Non-protein coding regions: -U5 (unique 5' sequence) and U3 (unique 3' sequence). -Repeats (R) flank U5 and U3. -Primer-binding sequence (PBS) Overall genome, 5' to 3': R, U5, PBS, GAG, POL, ENV, <SRC if oncovirus>, U3, R

How are structural and accessory proteins expressed in SARS-CoV-2?

Through the creation of subgenomic RNA (sgRNA) by RDRP. RDRP reads the gRNA (genomic) 3' to 5' to make the anti-genome. -For replication, the full-length anti-genome (-gRNA) is produced and used as a template to make +gRNA copies. -For transcription, smaller sgRNAs may be produced. --Identical transcription-regulating sequences (TRS) repeating through the gRNA can base pair , looping out the portion of the genome between them and causing RDRP to synthesize smaller -sgRNAs that will be transcribed to +sgRNAs (mRNAs) --The +sgRNAs will always have the leader sequence needed for translation, and the gene immediately downstream of the leader will be the only one expressed because all stop codons are hard stops . Not very efficient because only the first gene on each +sgRNA will be expressed, but it is necessary to allow structural and accessory protein expression. -Compare to HIV, which addresses the inability of eukaryotic hosts to translate polycistronic mRNA by splicing mRNAs in the nucleus.

What was unusual about the Spanish flu Influenza A pandemic?

Unusual W-shaped mortality curve: -infants and the elderly have high mortality (normal for flu). -abnormal: unusually high mortality for people int heir 20s and 30s.

How does a healthy microbiome protect against sexually transmitted diseases (STDs)? How do oral contraceptives affect this?

Vaginal microbiota differs at different stages of a woman's life due to hormones. -One set of microbiota is dominant during pre-pubescent and post-menopausal years. -During reproductive years, estrogen causes vaginal mucosal cells to produce large amounts of glycogen, shifting microbiota to be dominated by Lactobacillus acidophilus and other species that use glycogen and produce acid, decreasing the vaginal pH. --During the reproductive years when women are most at risk of getting an STI, this acidity protects them from pathogenic bacteria and yeast infections; awesome evolution! Oral contraceptives alter the hormonal balance, causing less glycogen production, resulting in less acidity, meaning that STDs like Gonorrhea see increased cases.

How does influenza A replicate after the viral genome has entered the cytoplasm?

Viral -ssRNAs are trafficked to the nucleus (odd- RNA viruses usually don't use nucleus). Viral RNA in the nucleus has one of two fates: *1. -ssRNA is transcribed.* -Cap-snatching, transcription to create +ssRNA, and poly-adenylation. - +ssRNA is then transported to the cytoplasm for translation. --mRNA for envelope proteins (HA, NA, M2) is translated in the endoplasmic reticulum --> Golgi --> proteins embedded in membrane that will later bud off as virus. --mRNA for non-envelope proteins (M1, NP, NS) is translated in the cytoplasm. M1 will assemble under the membrane where viral proteins have embedded. NP and NS will move back into the nucleus. -When NP and NS concentrations in the nucleus are high, this serves as a signal that enough viral proteins have been produced, so the virus will switch from transcription of -ssRNA to replication of -ssRNA and cap-snatching will stop. *2. -ssRNA is replicated.* - -ssRNA will be copied to +ssRNA, but it won't be capped or poly-adenylated; instead, a full-length +ssRNA anti-genome/copy RNA (cRNA) is produced to serve as a template for making -ssRNAs to package in new viruses. -NP will coat new -ssRNA strands. -RDRP is attached to the -ssRNA (so virus will be able to replicate in next host) --> virus-RDRP complex transported towards M1-lined region of membrane below viral membrane proteins --> virus buds off cell. --NA cleaves sialic acid off host receptors so the HAs of the budding virus don't make it stick to the previous host. --Host protease cleaves HA0 (originally synthesized hemagglutinin) to HA1 and HA2 held together by disulfide bond, allowing conformational change at low pH = HA is now active and virus is now infectious.