Lecture 5

Question 1

coronavirus genome

Answer 1

• linear
• single stranded positive sense RNA
• largest known RNA genome
• since it is a positive strand RNA genome, it can start translating proteins directly from the genome, therefore the genome itself is infectious
• order of genes is highly conserved
• ends have 5’ terminal cap and 3’ poly(A) tail, just like mRNA, can be used to directly make proteins

Question 2

what family is coronavirus in?

Answer 2

Coronaviridae

Question 3

What are the 3 genera of coronaviruses?

Answer 3

1. alphacoronavirus
2. betacoronavirus (causative agents for 3 deadly outbreaks in humans)
3. gammacoronavirus

Question 4

what factors are increasing zoonosis emergence?

Answer 4

• deforestation and other land changes
• illegal and poorly regulated wildlife trade
• climate change
• intensified agriculture and livestock production
• antimicrobial resistance

Question 5

History of coronavirus infection in humans

Answer 5

• had not been observed in humans before 2002-03
• huge number of coronavriuses have been found in bats
• Bats are the most common natural reservoir and do not fall ill
• wide variety of intermediary animal hosts pose a risk of transfer to humans

Question 6

coronaviruses structure

Answer 6

• spherical enveloped parcels studded with trimeric spikes
• spikes form the corona or the crown
• mostly cause respiratory illnesses in humans
• in rare cases can cause gastroenteritis (inflammation of the GI tract) and encephalitis (inflammation of the brain)
• cause veterinary diseases such as feline peritonitis (inflammation of abdominal linings), infectious bronchitis (inflammation of bronchi), murine hepatitis, and others

Question 7

pros and cons of virus-specific antiviral strategies

Answer 7

Pros
- proven efficacy
- easier design, 1 viral target
- safe compared to other strategies
Cons
- narrow application
- low barrier to drug resistance development
- long development time

Question 7

pros and cons of host-targeted broad spectrum antiviral strategies

Answer 7

Pros
- host proteins are broadly required by viruses
- demonstrated antiviral effect (ability of a substance, compound, or immune response to inhibit the replication or spread of a virus within a host organism)
- higher barrier to drug resistance development
Cons
- not selective
- potential for toxicity

Question 8

pros and cons of virus-targeted broad-spectrum antiviral strategies

Answer 8

Pros
- less potential for toxicity
- potential for repurposing
Cons
- more complex design
- limited examples

Question 8

Ways to prevent virus entry into host cell

Answer 8

1. Intercept virus before it reaches the cell using neutralizing antibodies
   - basis for vaccines
   - best strategy
2. Flood extracellular space with truncated, soluble version of the receptor proteins, prevents the binding of the virion to receptors on the cell surface
3. Use molecules that bind to the receptor which occupy the receptor sites or induces receptor internalization
4. interfere with cellular processes needed for internalization/penetration
5. inhibit uncoating of capsids
6. inhibit membrane fusion using peptides designed to bind conformational intermediates of viral fusion proteins

Question 9

How to interfere with cellular processes needed for internalization/penetation

Answer 9

• can prevent endosome acidification by using lysosomotropic agents, which are basic compounds that elevate the pH
• this causes H+ to leak out and raises the overall pH
• can also use carboxylic ionophores that bind to monovalent ions and increase membrane permeability, that gets rid of the ion gradient across the membrane
• specific inhibitors can also be used

Question 10

What is a limitation in antiviral strategies

Answer 10

can give strong side effects because viruses use a lot of cell machinery to carry out their replication cycles, have to be careful not to inhibit processes that normally happen in the cell

Question 11

Steps in the uncoating of adenovirus

Answer 11

• binds to the cell receptor via interaction of the fiber protein with its receptor
• this also leads to interaction with integrin, that leads to internalization by endocytosis
• acidification of the endosome causes destabilization of the capsid and release of viral proteins
• the viral proteins lyse the endosomal membrane
• this releases the viral particle into the cytoplasm
• it is transported along microtubules and docks on to the nuclear pore complex
• uncoating occurs and the genome enters the nucleus

Question 12

why do many viruses use the nucleus as site of replication

Answer 12

• take advantage of cellular machinery for transcription
• can also establish latency in the nucleus
• can integrate their genome into the host genome

Question 13

Strategies for the import of Viral Genomes into the Nucleus

Answer 13

1. Interact with nuclear targeting receptors such as importins that bind to nuclear localization signals in proteins and is then imported through the nuclear pore complex
2. Wait until the cell divides and make use of the dissociation of the nuclear membrane during mitosis for the genome to enter
3. Partial disassembly in cytoplasm and entry though nuclear pore via importins
4. Disassembly at nuclear pore for larger capsids
5. Transport of intact virions through the nuclear pore

Question 14

Intracellular transport

Answer 14

• take advantage of cellular transport systems
• transport in endocytic vesicles o as free nucleocapsids
• transported along microtubles towards microtuble organizing centre using molecular motors like dynein and dynactin
• some can also use actin cytoskeleton by polymerizing and de-polymerizing actin
• small viruses can also freely diffuse

Question 15

How do non-enveloped viruses penetrate the membrane

Answer 15

• membrane lysis or pore formation
• can be caused by major conformational changes in response to receptor binding or low pH in endosomes
• can also penetrate witout any lysis or rupture of host proteins
• can be pH independent but still depend on endocytosis
• capsid wall loosens in response to conformational changes triggered by binding to receptors
• hydrophobic proteins in the capsid can also create membrane channels

Question 16

General process of membrane fusion for enveloped viruses

Answer 16

1. Exposure to low pH in endosome exposes the fusion peptides and they insert into outer bilayer leaflet
2. Membranes do not fuse yet as distance between them is too great, further conformational change needed
3. Molecules of the virus cluster together and each trimer folds back on itself forcing the 2 membranes closer together
4. The two bilayers fuse
5. Narrow channel is formed between the internal cavities of the virus and the cytosol of the host cell

Question 17

fusion peptides

Answer 17

• part of type 1 transmembrane proteins
• most of the mass is external to viral membrane, sticks out of the virus
• can form oligomers (molecules that consist of a few repeating unit)

Question 18

Class 1 fusion proteins

Answer 18

• mostly alpha helical
• tend to form trimers
• start perpendicular to the envelope surface
• when triggered, bends to form hairpin conformation that brings the membranes closer together
• the fusion proteins also come together and form a 6-helix bundle

Question 19

Class 3 fusion proteins

Answer 19

• combination of alpha helices and beta sheets
• consist of 2 subunits: fusion subunit and receptor binding subunit
• functions similar to class 1 and 2
• fold out like an umbrella when the conditions change
• can attach to other membranes
• folds into a hairpin which brings membranes closer together so they can fuse

Question 19

Class 2 fusion proteins

Answer 19

• mostly beta sheets
• tend to form dimers
• horizontal arrangement in native form
• rearrange after pH triggering before the final trimer is formed
• fusion involves rearrangement of protein subunit but only minor conformational changes at tertiary levels
• extends to contact other membrane then hairpin folding bring membranes together for fusion