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HSCI 338 (2)
Lecture 1

HSCI 338 Lecture 1: hsci 338 notes lecture 1-6

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Simon Fraser University
Health Sciences
HSCI 338
Nienke Van Houten

Quantification Plaque Assay Drug is effective Formation of plaques at 10^-2, dosage of drug not high enough to kill the virus, or it may be high amount of virus at 10^-3 for mutants or drug resistance Midterm 1h 50min Short answers maybe some mc Interpret assays Includes up to chapter 6 -- does not inlcude chapter 7 Office hours tuesday 12-1:30pm before midterm Methods for identifying receptors not covered Entry and coating What are the similarities between gp120 and HA1? - Explain the genome structure? - How is end-replication problem solved? Homework: Write an exam question - one of the question will be on the exam. Comparison between viruses compare/contrast, list silimarlities Define the end replciation problem ---- Oct 7th 2016 Next week: submit a paragraph - Why the experiment was done - Brief explanation of the method - Brief explanation of the results - Summary statement - In lay terms - For figure 2A and 2B In relation to class 6 and 7 virus - Choose retrovirus or hepadenovirus genome replication - paraphrase the replication cycle. You can use point from, draw labeled diagram, include in your response a description of template jump and how dna synthesis primed - Define differential splicing and consider why it is important for viral genome expression RNA dependent RNA polymerases ---> replicases - Eukaryotic cells do not have these dsRNA - conservative replication - Ds RNA template ( + and - strand) - Separate, neg. Strand becomes template - More + strands are synthesized Ss + RNA - Direct translation to proteins - Ss + strand makes dsRNA - dsRNA separates and makes “antisense genome strand” Make a similar drawing for negative strand r Ss - RNA - Makes dsRNA intermediate - Makes more + RNA - Translate to protein MOI = multiplicity of infection You purify some viral particles but they are not infectious in a plaque assay - They have to co-infect with an infectious virus Run a gel electrophoresis - Size difference of initial genome and the defective particle by running a gel - PCR, making cDNA with RNA by reverse. Transcriptase --- Oct 14th 2016 Midterm 2: ch7, 8, 9, 10, 11, 12 - Genome replication: 8 and 9 - Genome expression: 10 and 11 - Assembly: 12 Homework: - Find peer review primary literature article - Bring two review questions for the midterm - Superficial immunology review - draw flow chart Chapter 7 - List general features shared by RNA viruses (section 7.1) - Unable to proofread, more mutations - Must replicate via dsRNA intermediate aka “antigenome strand” - Then uses antigenome strand as template to make genome - Formation of replication intermediate, which consits of partially ssRNA and partially dsRNA - Describe characteristics of Baltimore class 3,4 & 5 viruses and identify whether enzymes are brought in at infection or synthesized upon entry in relation to genome. - Class 3: - Uses polymerase contained within the virion, brought in at infection, - Replicates in cytoplasm - Class 4: - Translated immediately to make RNA polymerase and protein - Class 5: - Complementary sequences at the terminals - Carries RNA polymerase into the virion - Explain why RNA genomes are more prone to increased mutation than DNA viruses - Lack of proofreading - Discuss why all RNA viruses require a dsRNA intermediate during genome replication - Viruses that replicate via RNA intermediates need an RNA-dependent RNA-polymerase to replicate their RNA - Explain 2 ways how DI particles are generated and consider how they might be identified in a laboratory setting - DI viruses are mutants where the genomes have large deletions, leaving little bit left of the infectious genome - Unable to reproduce without infectious virus present - panhandle/stems from H-bonds - Deletion occurs when the polymerase falls off and reattaches at a different point in the genome or to newly synthesized incomplete strand - - describe the significance of reverse genetics as a research tool for RNA viruses and provide overview of how it works Reverse genetics: How it works: - Problem: RNA cannot be manipulated by restriction enzymes or mutagenesis - Solution: make RNA virus using DNA copy of RNA genome - By cloning cDNA using reverse transcriptase from RNA genome - Why: - Introduce specific mutations in the DNA form - Generate virus in cells - Provide evidence for functions of genome sequences and viral proteins, etc - Compare and contrast synthesis of Baltimore class 3,4,& 5 virus genomes MOI - muliplicity of infection DI - defective interfering particle, - Often looks like the infectious particle Describe characteristics of Baltimore class 3,4 & 5 viruses and identify whether enzymes are brought in at infection or synthesized upon entry in relation to genome. Class 3 virus (dsRNA) - reovirus - Semi conservative, complementary strands are displaced into separate progeny - Conservative replication - Positive strand used as template, 100% radiolabelled neg. Strand hybridized 1. Infection 2. Proteins in reovirus particles are removed by protease digestion during uncoating 3. Subviral particle is form, found in cytoplasm 4. Nucleic acid found in cytoplasm outside subviral particle is mRNA which is generated by transcription Class 4 viruses (+ve ssRNA) *can be directly translated* E.g. picornavirus, coronavirus, rubella, help c - Genome replication starts at 3’ polyA tail - poly A binding protein - role in mRNA translation, binds to polyA tail - Poly C binding protein - binds near 5’ end cloverleaf structure - Picornavirus 3CD protein - a polymerase precursor that binds genome through cellular proteins, is activated by proteolytic cleavage 1. +ve sense strand is used to generate -ve strand, with VPg at the 5’ end 2. Polymerase moves along and many initiation start a. These copies are templates - Treatment with RNase creases psuedo dsRNA, same length as viral genome and with nicks in the newly synthesized strand of RNA Class 5 virsues (-ve ssRNA genome) - Two types: single molecule genome or segmented genomes - Monoegavirales are non segmented - Measles, rabies, ebola - Influenza virus - RNA synthesis can only occur with RNA-dependent RNA polymerase that pre-existed in the virus particle - - Replication of non-segmented virus: Vesicular stomatitis virus (VSV) - ssRNA genome always found associated with three virus proteins in helical complex - Nucleoprotein (NP) most abundant - Phosphoprotein (P) - Large protein (L) - Catalytic component of the replication complex - Called the nucleocapsid complex… produces mRNA - Requires continuous protein synthesis; addition of inhibitors stops the replication - Viral mRNA and protein synthesis must happen before replication starts - -ve strand is replicated to +ve strand, then used as template - Treatment with RNase generates a ds Replicative Form RNA similar to picorna RI - Replication of RNA genome of segmented class 5: - NP interacts directly with genome RNA - +ve strand RNA is initiated from the beginning - Absence of cap structure on the synthesized RNA - Synthesis of RNA does not stop in response to transcription termination - NP acts to prevent termination at polyadenylation signal Chapter 8 - Diagram the lifecycle of a typical retrovirus 1. Attachment 2. Entry 3. Reverse transcription (RNA to DNA) 4. Integration 5. Transcription 6. Translation 7. Assembly 8. Budding - Define “provirus” and identify differences between genome RNA and proviral DNA - Provirus = Genetic material incorporated into genome of host - Pinpoint templates for genome replication and transcription - U5, R, U3 - Identify part of RT enzyme and their function - P66 - RNaseH domain - p51 - Explain the importance of RT discovery as a tool for molecular biology - Allows production of cDNA from mRNA - Describe the steps of reverse transcription and genome replication for retroviruses - Explain “template jumping” and list where it occurs for retrovirus and hepadnaviruses. - Retroviral: 1. Base pairing of (-)DNA to RNA 3’ end 2. Base pairing between -DNA and +DNA 3’ end - Explain role of RNase in Class 6 and 7 genome replication - Degrades RNA template after synthesis of -DNA - Compare and contrast RT by retroviruses and hepadnaviruses --- Class 6 - retroviruses 1) Synthesis of -DNA strand from +RNA template a) -ve DNA initated at tb with tRNA 2) Degradation of RNA template by RNaseH 3) Synthesis of + DNA strand to form ds DNA a) +ve strand DNA initiated from poly P - Contain 2 identical ssRNA genomes (diploid) - Used as vehicle for gene therapy 1. Binding 2. Fusion 3. Penetration 4. Reverse Transcription 5. Transport 6. Integration 1. tRNA acts as primer ​by base pairing to genome to synthesize (-)DNA, positioned next to U5, initiation happens 2. Synthesis extends to the 5’ end of the template, making a RNA:DNA hybrid 3. A domain on the RT enzyme called RNase H degrades 5’ end of RNA which removes U5 and R region 4. The newly freed DNA sequence is now available to base pair with R sequence from the 3’ end of the same RNA molecule, called the first jump 5. cDNA is extended using RNA as template and viral RNA is degraded by RNase H along the way 6. Once the strand is completed, second strand synthesis is initiated from the viral RNA 7. Poly P primes (+)DNA synthesis, using -DNA as template 8. Base pairing between (+)DNA and (-)DNA, called the 2nd jump 9. Synthesis of both strands makes a dsDNA a) Entry b) Reverse transcription c) Transport, integrate d) Transcription e) RNA export f) Packaging g) Assembly, exit Reverse transcription 1. Reverse transcription - synthesi
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