Chapter 13 exam 3 notes microbio PDF

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Dr. Becker...

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★ Viruses: genetic information (DNA or RNA) contained in a protective coat ○ Viruses are obligate Intracellular Parasites ○ Viruses are inert particles: they don't have metabolism, don't replicate, no motility ○ The genome hijacks the host cell's machinery to replicate itself ○ Inert outside of cells, but inside cells they direct the activity of that cell ○ They are infectious agents, NOT microorganisms ○

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Bacteriophages: infect prokaryotes ■ Most viruses infect only specific types of cells in one host ■ Host range is determined by interactions between viral and host cellular surface molecules ■ FDA approved using species-specific bacteriophages to control foodcontaminating bacteria ● May provide alternative to antibiotics ● Study at Yale, they used bacteriophages that attack and kill P. aeruginosa to avoid a lung transplant and she survived. ● Phage treatments from bacteria vs antibiotics Most viruses notable for small size ■ Viruses are 100 to 1000 times smaller than the cells they infect. Virion: viral particle that consists of nucleic acid and protein coat ■ The protein coat is called a capsid: it protects the viral nucleic acids ● The capsid is made out of protein subunits called capsomeres ● Capsid + nucleic acid = nucleocapsid ■ Enveloped viruses: have a lipid bilayer (envelope) that is obtained when leaving the host cell ● Enveloped viruses are more susceptible to disinfectants and hand sanitizer (the lipid bilayer can get damaged, the envelope has spikes that allows it to attach and attack and it can not attach without the envelope) ● There is matrix protein between nucleocapsid and envelope ■ Non-enveloped (naked) viruses lack the envelope ■ Cloaked viruses???? ● In the environments is it naked ● Inside the host, it cloaks itself in the membrane of the host ● Hepatitis A virus naked in the environments, not in the host ■ Viral genome: either DNA or RNA, but never both ● Useful for classification ● Genome is linear or circular ● Double or single stranded ■ Viruses have protein components for attachment ● Phages have tail fibers ● Many eukaryotic viruses have spikes ● Allow viron to attach to specific receptor sites on the host cell ■ Three main shapes of viruses

● Icosahedral: animal and plant ● Helical: animal and plant ● Complex: most phages ■ International committee on Viral Taxonomy (ICVT) publishes classification of viruses. ● Viruses are assigned to families based on sequence of genome ● Virus families end in the suffix -viridae ○ Some names indicate appearance ○ Other named for the geographic area it is from ● Virus Genus ends in -virus ● Species/subtype ○ Often the name of the disease ○ Ex. poliovirus causes polio ○ Unlike bacteria which are characterized by the genus and species name, viruses commonly are referred to only by the species name ● Enteric viruses ○ Transmitted via fecal-oral route (enteric refers to intestine) ● Respiratory viruses ○ Inhaled via infected respiratory droplets ● Zoonotic viruses ○ Transmitted from animal to human via vector (arbovirus) or direct contact ● Sexually transmitted viruses ○ Spread by sexual contact ★ Bacteriophages infect bacterial cells ○ Three general types characterized based on relationship with host ■ Lytic phages ■ Temperate phages ■ Filamentous phages ■

Lytic phages: also called virulent phages replicate using host machinery and then the newly replicated viral particles exit host by lysing the cell ● Lytic phages yield productive infections ○ Production of virions occurs immediately vs latent infection ● T4 phage (dsDNA): entire five step process takes about 30 minutes ○ 1.) Attachment ■ Phage collides with the host cell (T4=E. coli) by chance ■ Viral tail fiber binds to host cell receptor, which is specific for each virus ● Host cell receptor is usually a pilus or other cell surface structure (T4=E. coli LPS)

■ Any cell that lack the receptor are resistant 2.) Genome entry ■ T4 lysozyme (located in tip of the tail) degrades peptidoglycan, the bond between NAG and NAM ■ The tail contracts and injects the genome through cell wall and membranes (like a syringe) ○ 3.) Synthesis ■ The phage DNA is transcribed and translated into proteins by host cell machinery ■ Early proteins are translated within minutes from the viral DNA, these prevent host gene expression ● Nucleases degrade host DNA ● Protein modify host RNA polymerase so that they can't make any more transcript for themselves ■ Late proteins: structural proteins that are produced towards the end of the cycle ● Capsomeres = capsid, tail proteins, tail fibers ○ 4.) Assembly ■ Some components spontaneously assemble, others require protein scaffolds ■ Multi-step sequence: ● 1.) Head (capsid) is formed and packed with DNA) ● 2.) The tail is formed and attached to the head ● 3.) Tail spikes/fibers are attached ○ 5.) Release ■ Endolysin: breaks down the cell wall on the particular host cell ■ Burst size: how many virions are made before it releases Temperate phages - have 2 options: ● Can produce a lytic infection ● Can produce a lysogenic infection ○ Lysogenic infection: incorporate DNA into host cell genome ○ Prophage: phage DNA that incorporates into host chromosome ○ Prophage is replicated along with host DNA during binary fission ● The decision between lysogenic or lytic appears to be random however, metabolic state of the host cell influences the decision ○ If the cell is growing slowly because of limited nutrients, a ○

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lysogenic infection is more likely The number of host cells also makes an impact on the decision ■ If there is a low host cell population, lysogenic is better ■ Wait for the population of bacteria to grow before you kill all of them, because they need them to survive

Phage begins with lytic infection (sense # of other phases) ■ Lysogenic cycle is inhibited at the first encounter of a phage with a bacterial population ■ aimR and aimP are expressed immediately upon infection ● aimR protein activates AimX expression ○ aimX is an inhibitor to lysogenic gene expression ○ This results in a lytic cycle ● At the same time, AimP is expressed, translated, and secreted ○ This gene make a communication peptide called arbitrium ● The arbitrium peptide accumulates in the environment ● The arbitrium is internalized into the host cell by transporter (OPP) ● Arbitrium molecules bind to the aimR activator. AimR cannot activate the expression of AimX, leading to lysogeny **This process allows for viruses to coordinate their attack ○ At the begging of the infection, it makes sense for viruses to quickly replicate (lytic cycle) - large bacteria population ○ If they don't switch strategies, there won't be any hosts left for future generations of viruses to infect ○ At some point, the viruses need to switch strategies and become dormants so that the bacterial population can recover Lambda phage as an example of temperate phages ○ The molecule can either direct a lytic infection or integrate into the E. coli chromosome ○ Phage enzyme integrase inserts viral DNA at a specific site, now the integrated phage DNA is called a prophage. ■ The prophage replicates in the host chromosome ■ It can be escised by phage-encoded enzymes

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This process is call induction, whist results in lytic infection ■ Phage repressor protein prevents excision, maintains lysogenic state ○ If DNA is damaged, SOS repair system turns on and activates protease ○ Protease destroys phage repressor that is responsible for keeping prophage in chromosome and lytic infection ■ This allows prophage to be excised and enter lytic cycle ** if the DNA is in the chromosome it is a lysogenic infection **if the DNA is not in the chromosome it will enter the lytic cycle ●

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Lysogen (the infected cell) is morphologically identical to an uninfected cell, but other aspects may change ○ Immunity to superinfection: lysogens protected against infection by same (another) phage ■ Phage repressor binds to operator of incoming (new) phage DNA ■ Prevents expression of genes that direct lytic infection ■ Once there is a prophage atready in the bacteria, another type of phage can not infect that same cell ○ Lysogenic conversion: prophage changes phenotype of lysogen ■ Often toxins are encoded by genes on prophage ■ Only strains carrying prophage produce the toxins ■ The bacteria carries genes that the bacteria can use ** bacteria can be pathogenic when they are infected by a temperate phage

Filamentous Phages ● Do NOT cause lytic infections ○ The host cells are not killed but the host cells grow slower while virions are produced ● M13 phage as an example ○ Attaches to F pilus of E. coli ○ Single stranded DNA genome enters cytoplasm ■ ssDNA enters host and is replicated, then transcribed ■ ssDNA becomes dsDNA and is called replicative form (RF) ■ (-) strand used as template for synthesis of mRNA

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and as template for DNA replication ■ (+) strand packaged into new virions M13 phage produces capsomeres and those are inserted into the cytoplasmic membrane Other viral proteins form pores that span from cytoplasmic membrane to the outer membrane As phage DNA is excreted through pores, capsomeres coat the DNA, and form the nucleocapsid - this process is called extrusion This all occurs as the cell is dividing so progeny of host cell are also infected (carrier cells)

★ Roles of bacteriophages in horizontal gene transfer ○ Phages can accidently transfers bacterial DNA from one bacteria (donor) to another (recipient) ■ This is called transduction, there are two types ● Generalized transduction: any part of genome can be transferred from infected cell to another ● Specialized transduction: a certain part of the genome transferred ★ Generalized transduction ○ Results from packaging error during phage assembly ○ Lytic and temperate phages degrade host chromosome via nuclease ○ Host DNA fragments mistakenly packaged into phage head ■ These are called generalized transducing particles ■ After release, it can bind to a new host, and inject DNA ■ DNA may integrate via homologous recombination, replacing host DNA ■ Any gene from donor cell can be transferred ★ Specialized Transduction ○ Excision mistake during induction (transition from lysogenic to lytic) of a temperate phage ○ Excised DNA incorporated into phage heads ■ Defective transducing particles are released ○ Bacterial DNA may integrate via homologous recombination ○ Only bacterial genes adjacent to prophage is transferred ★ Preventing phage attachment ○ Alter or cover specific receptors on cell surface ■ Bacteria have capsules, slime layers, biofilms ■ Staphylococcus aureus produces protein A, which covers phage preceptors on its cell wall ● Protein A binds to Fc region of antibodies, preventing phagocytosis ○ Restriction modification systems ■ Requires two enzymes

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1. Restriction enzymes: recognize short nucleotide sequences (the phage DNA) and cut them up at a specific site. ○ Bacteria have hundreds of varieties, each recognizing different sequences ● 2. Modification enzymes ○ Methylate bacterial DNA so not attacked by their own restriction enzymes ■ Enzymes may accidentally methlate phage DNA, this causes infection CRISPR system ■ Cells that survive phage infections insert pieces of phage DNA (spacer DNA) into the region of DNA called CRISPR ■ This provides a record of the infection ■ CRISPR region is transcribed, cut into small pieces call crRNAs (guide RNA) ■ crRNAs bind to Cas proteins ■ When injected phage DNA binds to CAS-crRNA complex it is triggered for destruction ■ When another phage tried to infect the bacteria, it knows how to fight it

★ Methods used to study bacteriophages ○ Viruses multiply only inside living cells ■ Must cultivate suitable host cells to grow viruses. ■ Plaque assays are used to quantify phage particles in samples ● Only works for lytic phages ● Zones of clearing from infected cells lysing, called plaques. ● Counting phage forming units (PFU) yields titer ● Very difficult with temperate and filamentous phages ★ Animal viruses ○ Five-step infection cycle ■ Knowledge of proteins are involved in infection allows researchers to develop antiviral medications ○ Step 1. Attachment ■ Viral spikes bind to receptors on the host cell's surface ● The spikes are usually glycoproteins on cytoplasmic membrane ● Specific host cell receptors are required for attachment, this limits the host range of the virus ○ Some have a narrow host range ■ Ex. influenza virus (respiratory tract of humans) ○ Broad host range ■ Rabies virus (nerve cells of many different animals) ○ Step 2. Penetration and uncoating (big difference between phages and animal viruses)

The entire virus enters into the cells Penetration through fusion or endocytosis ● Only enveloped viruses enter via fusion ● All non enveloped virions enter by triggering endocytosis ○ Attach to receptors that are used for endocytosis ○ If enveloped, the viral envelope fused with the endosome membrane, leaving a free nucleocapsid ○ If nakes, the nucleocapsid is released in the cytoplasm one it is in the endosome Uncoating is the separation of nucleic acid from the protein coat ■ Uncoating occurs through virus-specific complex processes triggered by virus-host-cell interactions **Then the nucleic acid can enter the nucleus via nuclear pores to replicate Step 3. Synthesis (production of new viral particles) requires two interrelated events ■ A) expression of viral genes to produce viral structural and catalytic proteins (make the proteins that the virus need to replicate itself) ■ B) synthesis of multiple copies of genome, it replicates itself ■ ■

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Replication of DNA viruses ● Usually occurs in the host cells nucleus ● Prefer to use host DNA polymerase ● ●

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dsDNA replication follows the central dogma of molecular biology ssDNA replication requires a dsDNA intermediate ○ (-) strand used to make mRNA ○ (+) new copies of viral genome

Replication of RNA viruses ● Replicate in the cytoplasm ● Have to make their own enzyme to replicate itself, called replicase ○ Replicase is DNA dependent RNA polymerase ■ This is unique because normal RNA polymerase can only synthesize from DNA, which is DNA dependent RNA polymerase. ● ss (+) RNA functions as mRNA and immediately binds to host ribosomes ○ First, replicase translate from mRNA using host ribosome ○ Then, replicase replicates viral genome ● ss (-) RNA ○ Has to carry replicase in the nucleocapsid to synthesize (+) strand ■ This is because the host cell does not have a

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polymerase to make ss (-) RNA to mRNA dsRNA ○ Also carry replicase in the nucleocapsid ○ Uses both (-) and (+)

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RNA viruses have replicase, and replicase does not have proofreading ability ○ This is an advantage because it will cause genetic change, antigenic drift. So our bodies can't recognize the virus. ● Segmented RNA viruses have genes that are encoded by 2 or more nucleic acid strands ○ Antigenic shift can occur if the host cell is infected by two or more segmented viruses at the same time. ○ May cause a pandemic **Drift = lack of proofreading in replicase ** Shift = two or more different strains infect the same cell, big changes in the segment of virus **recently discovered, coronavirus and larger RNA viruses have proofreading exonucleases. This is good because then it wont keep coming back ●

Replication of reverse-transcribing viruses (retroviruses) ○ ss (+) RNA genome ○ Lack replicase gene, but carries reverse transcriptase in virion: makes ssDNA from RNA (RNA dependant DNA polymerase) ○ Complementary strand synthesized by host cell DNA polymerase ○ dsDNA can integrate into host cell chromosome via integrase and forms provirus ○ Can direct productive infection or remain latent ■ Productive = flu-like symptoms ■ Latent = can't get rid of it **RNA to make DNA

The HIV life cycle: 1.) Binds to receptors on a CD4 cell 2.) Fuses with the membrane 3.) HIV RNA, reverse transcriptase a.) DNA is made 4.) Integration: intergrase inserts into a chromosome in our DNA 5.) Replication: Then, transcripts can be made. 6.) Assembly: new HIV proteins and HIV RNA move to the surface and assemble into

immature HIV 7.) Budding: the immature HIV pushes itself out of the cell

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Step 4: Assembly ■ Non-enveloped viruses are completely assembled in the cytoplasm ■ Enveloped viruses are completed as they are released from the cell via budding, take the host cell cytoplasmic membrane with it.

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Step 5: release ■ Most enveloped viruses via budding ■ Viral protein spikes insert host cell membrane, matrix proteins accumulates ■ Non-enveloped viruses released when host cell dies, many trigger apoptosis

★ Influenza Virus - segmented ss(-) virus ○ 1.) Haemagglutinin (spike) attaches to sialic acid (host receptor) ○ 2.) Induces endocytosis ○ 3.) viral envelope fuses w endosome, releasing nucleocapsid ○ 4.) Capsid uncoats and segmented ssRNA (-) is released ○ 5.) Replicase makes ssRNA (+) ○ 6.) ssRNA (+) enters nucleus and is replicated and mRNA is made ○ 7.) ssRNA (-) and mature mRNA leave nucleus and enter cytoplasm for translation ○ 8.) Viral proteins assemble around membrane and bud off ○ 9.) Neuraminidase (enzyme) on surface prevents attachment to the same cell ■ Neuraminidase breaks the sialic acid off the surface of the cell ■ Neuraminidase inhibitors - Tamiflu, binds to and prevents function of neuraminidase. So they just stay on that cell, cant leave. ★ Vaccines **the vaccines: professionals guess which strain is going to attack that year and put in in the vaccine to prepare our bodies to fight it off. **Vaccinating people against a disease they’re never going to get is a risky proposition: -We don't know how the body would respond to a ton of flu vaccines - the patient might also develop a strong immune response to an insignificant strain **new vaccines each year is caused by antigenic shift

★ Antiviral drugs ○ Viruses use the metabolic machinery of their hosts, which limits many of the potential points of attack

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Current drugs inhibit virus-specific enzymes and life cycle processes

★ Mechanism of action of antiviral medications ○ Prevent viral entry ■ Maraviroc block co-receptor for CCR5. prevents attachment ■ Enfuvirtide - blocks protein spike that prevents fusion ○ Interfere with viral uncoating ■ Amantadine ● Block influenza A viruses from uncoating, blocks M-protein functions ○ Interferes with nucleic acid synthesis ■ Nucleoside analogs: structure similar to nucleosides ● Nucleotide analogs are formed by being phosphorylated ● Acyclovir is used to treat herpesvirus. ● Analog is added instead of a nucleotide, so it can stop replication ■ Polymerase inhibitors ● Inhibits replicase ■ Reverse transcriptase inhibitors ● Inhibits reverse transcriptase. ○ Preventing genome integration ■ Raltegravir ● Inhibits HIV-encoded enzyme integrase ○ Prevent assembly and release of viral particles ■ Protease inhibitors are virus specific ■ Neuraminidase inhibitors - tamiflu ● Bind so that nothing else can infect that cell (prevents the rebinding of influenza to budding cell) ★ Categories of Animal virus infections ○ Acute: ■ Rapid onset ■ Short duration ■ Result of productive infections (a large amount of viruses are produced) ■ Analogous to the lytic phage ○ Persistent: ■ Continue for years or lifetime ■ May or may not have symptoms ■ There are two types ● Chronic - continuous production of low levels or virus particles ○ HIV, gives you initial effects and also long term ○ Analogous to filamentous phage ○ Host cell may survive with slow release or viral particles ○ Or the virus lyses but only impact a few cells at any given time

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Latent - viral genome (provirus) remains silent in host cell and can reactivate ○ Analogous to the temperate phage ○ Initial infection is followed by symptomless period, the reactivation ○ The viral genome remains silent/latent with a host c...