Virology study guide

Today my virology class is taking a midterm examination. To prepare for this in-class test, I provided the students with a study guide highlighting what I believe to be the main concepts that they should learn from each lecture.  Once all the students have taken the exam, I’ll post it here for everyone else to take. To prepare for that day, you might want to start by reviewing the first half of the course, which covered the infectious cycle.

Virology W3310 is held in Hamilton Hall (image), an original McKim, Mead, and White building.

Lecture 1 €“ What is a virus?

• Viruses were originally discovered because of their size, relative to known bacterial cells. Tobacco mosaic virus was called a filterable infectious agent by this criterion. Why is size not a good defining feature for viruses? What is a better definition?
• Are viruses living? Why or why not?
• What is the three part strategy followed by every virus that makes it distinct from all other forms of self-replicating matter?
• It is said that viruses violate the cell theory (cells only arise from preexisting cells).  To which phase of a virus life cycle (the single step growth curve) does this statement refer?  What is the explanation for this phase of the growth curve?
• A viral genome must make mRNA: TRUE OR FALSE?  Does it ever encode the ability to decode that mRNA?  What does that last question mean?
• How do viruses achieve their remarkable adaptability with such small genomes?
• What are the four key features used in classifying viruses?  Are these the best features? Could one do better?
• What does (+) and (-) strand RNA mean?
• What is the Baltimore system? What does it tell us?

Lecture 2 €“ The infectious cycle

• What is a susceptible cell? A resistant cell? A permissive cell? Which describe the only cell that can take up a virus particle and replicate it?
• Before the advent of cell culture, how were viruses propagated?
• What is the difference between primary cells, cell lines, and diploid cell strains?
• Name one famous immortal cell line.
• What is cytopathic effect? Name two examples.
• How does a plaque assay work? How many viruses are needed to form a plaque?
• Are all virus particles infectious?  How would we know?  What is the term used by virologists to define this phenomenon?
• What are the individual phases of the one step growth cycle, and what occurs during each?
• How does a viral growth differ from bacterial growth?
• What is MOI?
• Do you believe this? At an MOI of 1, a significant number of cells will see no virions and a similar number will get one virion. Some (a smaller number) will get 2, 3, or more.  How do you know?
• The numbers can be calculated using the Poisson distribution.  Try your hand: fill out the following table. Remember:

P(k)=e^-mm^k/k!

P(k) is the fraction of cells infected by k virus particles, m is the moi.

Also  P₀ = e^-m

P₀= probability of a cell not being infected

• In addition to the plaque assay, what other ways are used to measure virus particles? Describe at least one.
• What€™s the big deal about green fluorescent protein? Who is Marty Chalfie anyway?
• What€™s the problem with Koch€™s postulates these days?

Lecture 3 €“ Genomes and genetics

• What information is or is not encoded in a viral genome?
• The genome inside a virion is always a nucleic acid. If you could get it into a cell, would it start the replication cycle? YES, NO, MAYBE?
• What are the only viral genome structures that have been identified?  There are less than 8 and more than 6.
• Can you imagine a viral genome in a virion that never actually replicates?
• Pick one of the algorithms for a virus replication strategy from chapter 3 and explain it.
• What can viruses with segmented genomes do that other viruses with unimolecular genomes cannot?
• Try to explain the ambisense strategy. Will this concept work for a DNA virus?
• What is wild-type?
• What is the name of the process by which viral DNA is introduced into cells? What about non-viral DNA?
• There is an infectious DNA clone for at least one member of nearly every virus family. Why is this so important?

Lecture 4: Structure of viruses

• What are the functions of virion proteins?
• Racaniello said capsids are metastable.  What was he talking about?
• He also said virions can be active containers. What does that mean?
• Know the difference between capsid, nucleocapsid, envelope, and virion
• How big are viruses compared with an alpha helix and a ribosome?
• What are the two rules of symmetry that instruct how virions self assemble?
• Helical virions often have precise lengths. How can this be?
• Helical virions always have the nucleic acid inside the helical structure.  How can this be?
• An icosahedron has three characteristics (these are invariable). What are they?
• What does the T number describe?
• How is the simplest icosahedral virion constructed?
• If capsid proteins are not larger than 20-60 kDa, how do you make larger virions?
• What is quasiequivalence?
• What is a complex virion?  Why would evolution come up with such structures?
• What is the function of viral envelope proteins?
• What is the difference between a structured and an unstructured envelope?

Lecture 5 €“ Attachment and entry

• Virions attach to cells.  What are the steps? Why is it sequential? Why is it dynamic? What steps are reversible?
• What are the functions of cell receptors for viruses? Why must viruses attach to receptors to enter cells?
• How does an icosahedral virion attach to cell receptors? An enveloped virion?
• Why are two receptors sometimes required for entry into a cell?
• How does a virion deliver the genome payload? A naked capsid, an enveloped virion must be different. Yes, No, Maybe?
• Why must membrane fusion be regulated? How is it regulated?
• How does the viral genome behave after the virion uncoats?  Obviously the different genome types end up in different places inside the cell. How could this happen?  Diffusion plays a role, yes or no?
• Are all viral nucleic acids released from the capsid/nucleocapsid during entry?
• What is the role of the cellular cytoskeleton in virus entry?
• Most DNA viral genomes must enter the nucleus. How is this done?

Lecture 6 €“ RNA-directed RNA synthesis

• What are the two requirements for copying RNA viral genomes?
• Why must (-) strand viral RNA be coated with protein in the virion? Why not (+) strand viral genomes? Are there exceptions?
• What structural features are common to all nucleic acid polymerases?
• All RNA viruses encode their own replication system. True or False?  Why?
• It is amazing that RNA viral genomes can replicate. Do you agree with this statement? Why or why not?
• If you discovered a previously unknown RNA virus, what would be the best guess about its genome topology: linear, circular, double stranded, single stranded?  Why?
• How do viral RNA genomes work? Compare +, -, and ds RNA genomes
• How do they encode information? Compare +, -, and ds RNA genomes
• How is this information used to operate an infectious cycle? What must happen first when one of these RNA genomes enters a cell?  Or, put another way, where€™s the polymerase?
• In the (€“) sense RNA genomes, the (+) strands and mRNA are the same. True or False? Why or why not?
• Consider a (+) sense ssRNA genome. What is the difference between the viral mRNA and the (-) strand complement of the genome?
• What is the primer for picornavirus replication?
• Compare the monopartite strategy with the segmented genome strategy for minus strand ssRNA genomes.
• When the genome of a negative sense ssRNA virus is purified and introduced into cells that are permissive to the original intact virus, what will happen?  Why?  If you did this same experiment with a plus-sense ssRNA virus, what would happen?
• How is poly(A) added to mRNAs of RNA viruses?
• Influenza virus will NOT GROW in a cell from which the nucleus has been removed. Why is that? It is an RNA virus.
• What is cap snatching and why is it necessary for influenza replication?
• Influenza virus replicates in the nucleus of cells and therefore does not require a viral polymerase to replicate its genome.  True or False? Why?
• Why is viral RNA synthesis a source of diversity?
• What is RNA editing? What good is it?

Lecture 7 €“ Reverse transcription and integration

• Remember I asked you before about the possibility of a viral genome that never replicates?  That would be the retrovirus genomes. What is their remarkable strategy?
• What are the unique viral enzymes that are required to make more retroviral genomes? What do they do? What is the origin of the name of the enzyme?
• What does this statement mean?  Retroviral genomes are diploid.  What good is this property?
• What is a provirus?  How many proviral copies are produced after a retrovirus infection?
• Take a shot at explaining how reverse transcription works. For example, what are the primers for the polymerase? What are the three kinds of biochemical reactions catalyzed by reverse transcriptase?
• What are the functions of RNAse H and integrase during retroviral replication?
• Which enzyme produces viral mRNAs in a cell infected with a retrovirus? Where is the promoter for mRNA synthesis?
• If you expose polio virions and retroviral virions to UV radiation, the amazing fact is that retroviral virions are orders of magnitude more resistant to UV. Why is this so?  It has nothing to do with the fact that retrovirus virions are enveloped!
• Both hepadnaviruses and retroviruses use reverse transcriptase to replicate, yet the retroviral virion has a (+) ssRNA inside while the hepadnaviral virion has a gapped dsDNA molecule inside. What€™s that all about?
• Does the hepadnavirus genome encode an RNAse H? An integrase? Why or why not?
• Someone suggested that hepadnaviruses evolved from an ancestral retrovirus. What do you think of this idea?
• Half of your DNA is made of mobile genetic elements. What does this mean?

Lecture 8 €“ Translation

• What is the function of the 5€™-cap on mRNAs?
• How is translation from an IRES different from cap-dependent translation? Why are there IRESes?
• Does poliovirus require cap-dependent translation for expression of its viral gene products?
• Does initiation of translation always begin with a methionine?
• How do viruses get around the ‘one mRNA, one protein€™ limitation of the eukaryotic translation apparatus?
• What is leaky in the leaky scanning model?
• What is an eIF2a kinase and what does it do? How do viruses get around it?
• How does cleavage of eIF4G interfere with translation? Why does this occur in virus infected cells?

Lecture 9 €“ Genome replication of DNA viruses

• What are the different topologies of DNA viral genomes? How are the DNAs replicated?
• Where in the cell do DNA viruses replicate?
• Why is viral DNA replication always delayed after infection?
• What are the two possible outcomes of viral DNA replication?
• What are the minimal requirements for DNA replication?
• Do DNA viral genomes always encode their own DNA polymerase?  If not, where does the polymerase come from?
• What viral proteins are involved in DNA replication?
• What is a viral origin of replication? How do they work?
• What are the two basic modes of DNA replication?
• What is the function of polyomavirus T antigen?
• Do DNA genomes exist that do not require a DNA polymerase to copy them?
• What is bidirectional replication? Does it happen on all DNA viral genomes?
• Synthesis of the leading DNA strand is easy. Why is synthesis of the lagging strand difficult?
• What events occur after a polyomavirus DNA genome is replicated?
• Explain how the hepadnaviral DNA genome can replicate. That genome as it comes from the virion is a mess!
• Silverstein said that ‘adenoviral DNA replication is semiconservative and assymetric€™. What does he mean by that statement?
• All DNA viruses must replicate in the nucleus. True or False?
• What is a primer? What is the function?  What kinds of primers are there?
• There is one DNA virus family that encodes its own entire replication and transcription system. Which one?  Why does it do this?
• How might a herpes simplex virus genome generate four isomers?
• What happens to genes encoded within the repeat regions of herpes virus DNA when the inversions occur?

Lecture 10 €“ Transcription and RNA processing

• What is transcription? What are the generic steps in this process?
• What happens to RNA transcripts?
• What are the different possible fates of viral mRNAs?
• What are the steps in the transcription of pre-mRNA?
• Which control elements regulate mRNA synthesis?
• What are the steps in initiation of mRNA synthesis?
• Which virus was instrumental in the discovery of splicing? What is the purpose of splicing?
• How does an enhancer work?
• Host or viral proteins may regulate transcription. How do they function?
• Describe positive autoregulatory and cascade regulation.
• What is a transcriptional cascade and why is it necessary?
• A primary transcript does not become a mRNA until it is exported. True or False.
• HIV infected cells export unspliced and incompletely spliced mRNA molecules from the nucleus to the cytoplasm. Mammalian cells, however, do not normally export incompletely spliced mRNA molecules. How do viruses overcome this obstacle?
• What€™s a Charon and can you get to hell if you don€™t know?

Lecture 11 €“ Assembly

• True or false: the overall virion assembly process must be irreversible.
• Why must viral proteins achieve high concentrations in the cell? How is this achieved?
• How do viral structural proteins go to the ‘right place€™ in the cell?
• True or false: the virion structure is at an energy minimum.  Why or why not?  Certainly, it must be very stable because it has to survive in the environment outside the infected host.
• What features or motifs are found on integral membrane proteins that are necessary for them to function in the plasma membrane?
• How do virion components achieve high concentrations for efficient assembly?
• What is a sub-assembly? Provide an example.
• Distinguish between assisted assembly and self-assembly.
• We distinguished between sequential and concerted assembly. How do these processes differ?
• How does the viral nucleic acid get packaged into a virion? How is the viral genome distinguished from other nucleic acids in the cell?
• Egress of a retrovirus and a herpesvirus are remarkably different even though they both are enveloped viruses. Where does the envelope for each virus originate?
• How does an L domain participate in viral budding?
• Most viruses leave cells by one of which two general mechanisms?
• What is the advantage of cell-cell spread over release of particles?
• Can you imagine targeted egress or release of virions from infected cells? That is, virions emerge from a specific place and not all over a cell.  What advantage would this process provide for infection of an epithelial cell or a neuron?