TS3: Viruses

Question 1

Define: virion

Answer 1

An independent viral particle released from the cell

Question 2

Define: defective particles

Answer 2

An empty virus shell without the genetic material inside

Question 3

Define: zoonosis

Answer 3

Any disease or infection naturally transmissible from animals to humans

Question 4

Define: viral tropism

Answer 4

The ability of a virus to infect and replicate within specific types of cells or tissues in the body.

Different viruses have different preferences, and this is determined by the specific viral proteins that interact with receptors on the surface of host cells.

Question 5

How can the genome size of RNA viruses make them harder to target than DNA viruses?

Answer 5

RNA viruses tend to have much smaller genomes, and so the mutations have greater impact and each generation will likely be completely different to the ‘master strain’.

This makes them incredibly hard to target.

Question 6

What are the 6 strategies that viruses can use to make multiple proteins from one gene? Give an example of a virus that uses each of these.

Answer 6

1. mRNA splicing (HIV)
2. Read-through of stop codons (Rous sarcoma virus)
3. Multiple start sites (HBV)
4. Overlapping genes (HBV)
5. Polyprotein cleavage (Zika virus)
6. Ribosomal frameshifting (HCV)

Question 7

What’s the difference between an RNA virus and a retrovirus?

Answer 7

RNA viruses are viruses that use RNA as their genetic material instead of DNA. RNA viruses can be further classified into different groups based on their genome structure, replication strategies, and mode of transmission.

Retroviruses, on the other hand, are a specific type of RNA virus that have a unique replication strategy that involves reverse transcription. Retroviruses use an RNA genome to produce a DNA intermediate that is then integrated into the host genome, where it can persist for the lifetime of the infected cell.

Question 8

Compare the following for RNA to DNA viruses:
- Genome size
- Stability
- Polymerase fidelity

Answer 8

For all of these, RNA viruses are smaller/weaker compared to DNA viruses.

Question 9

What is the typical clinical course of HIV infection? What are the symptoms at each stage?

Answer 9

Acute infection - virus replicates and the immune system responds, giving flu-like symptoms.

Clinical latency - can last several years or even decades where the virus replicates at low levels, gradually weakening the immune system. Many don’t experience symptoms at this stage.

AIDS - severe immune deficiency and development of opportunistic infections and cancers. Symptoms include weight loss, persistent fever and frequent infections.

Question 10

How does HIV replicate within host cells?

Answer 10

1. RTase converts its RNA genome into DNA
2. Rnase H degrades the RNA template
3. DNA is transcribed to form ds-DNA
4. Integration of the ds-DNA using integrase
5. ds-DNA is transcribed into mRNA
6. mRNA can be spliced, or left alone to encode Gag, RT and genomic RNAs
7. Genomic RNAs bud off from the host cell
8. Viral protease cleavage and maturation

Question 11

What are the genes encoded by the HIV genome? What are each of their roles?

Answer 11

HIV contains two pieces of single-stranded RNA.

1. 5’ cap
2. Gag (form the capsid of the virus)
3. Pro (protease that processes polyprotein precursors)
4. Pol (reverse transcriptase, RNase H and integrase)
5. Env (envelope proteins)

Question 12

How does HIV enter host cells?

Answer 12

1. HIV binds the CD4 receptor via its gp120 glycoprotein trimer
2. gp120 conformational change
3. CCR5 binding site is now exposed
4. Binds CCR5 co-receptor
5. Triggers membrane fusion and insertion of the HIV capsid

Question 13

What treatment has been used to cure 3 people of HIV?

Answer 13

The Berlin patient was the first person to be cured of HIV. He has leukemia and had a bone marrow transplant from a donor with a rare genetic mutation called CCR5-delta32. This mutations confers resistance to HIV. Following the bone marrow transplant, the patients HIV levels became undetectable.

The other two individuals - the London patients - have had similar treatments and successes.

Question 14

Give a detailed explanation of the process of reverse transcription of HIV.

Answer 14

1. Primer tRNA anneals to the PBS sequence in the gRNA
2. tRNA is extended to form a DNA copy of the 5’ end of the genomic RNA
3. RNase removes the RNA template
4. FIRST JUMP: DNA hybridizes to remaining RNA at the 3’ end
5. DNA (-) strand is extended and completed. RNA is removed.
6. (+) strand DNA primes at the PPT and synthesizes to the 3’ end.
7. RNase H degrades tRNA and PPT
8. SECOND JUMP: (+) strand DNA binds the PBS at the 3’ end of the (-) strand.
9. Both strands extended and completed to give ds-DNA

Question 15

How do retroviruses integrate their DNA into the host genome? What machinery is involved?

Answer 15

Integrase attacks the ends of the viral genes, creating 3’OH whilst the host DNA is simultaneously being attacked to form blunt ends, typically in hot spots. Cellular repair enzymes resolve this joining reaction and form the integrated provirus.

Question 16

What are the functions of each HIV accessory genes?

Answer 16

Vpr: (viral protein R) facilitates viral replication in non-dividing cells by transported subviral particles across the nuclear membrane.

Tat: (trans-activator of transcription) potent transcriptional activator that binds in the LTR promoter.

Rev: (regulator of expression of virion proteins) essential for nuclear export of unspliced and partially spliced mRNA.

Nef/Vif/Vpu: enhance production and release of infectious viral particles

Question 17

How are some people resistant to HIV? What is thought to have caused this?

Answer 17

A mutation in CCR5 prevents HIV from binding properly. This is a fairly new mutation, thought to be caused by positive selection from smallpox as this also uses CCR5 to enter cells.

Question 18

Describe the structure of the hepatitis B virus.

Answer 18

A small, partially double-stranded genome within a protein capsid, surrounded by an outer envelope.

Question 19

How does HBV enter and replicate within the host cell?

Answer 19

1. Core particle is uncoated to release the partially ds-DNA genome
2. Within the nucleus, the genome is converted into cccDNA (covalently closed circular)
3. cccDNA remains in the nucleus and is transcribed by cellular machinery (RNA)
4. Viral transcripts are used as templates for the synthesis of new viral genomes by the viral polymerase (reverse transcription to DNA)
5. The DNA is used to form the genome of new viral core particles that bud off from the infected hepatocyte and are released into the bloodstream.

Question 20

What type of cells do HIV and HBV infect?

Answer 20

HIV: CD4+ T cells
HBV: hepatocytes

Question 21

What is cccDNA? Why is it important for HBV?

Answer 21

Covalently-closed circular DNA - a stable mini-chromosome that is present in the nucleus of infected hepatocytes.

It serves as a long-term reservoir for the viral genome, and is responsible for the persistence of chronic HBV infection.

Question 22

What is pgRNA? What is its role in the HBV life cycle?

Answer 22

pregenomic RNA - intermediate molecule that links the transcription of the viral genome from the cccDNA to the synthesis of new viral DNA genomes and the assembly of new viral particles.

Question 23

Where do HBV virions bud from?

Answer 23

The ER

Question 24

Why might a reverse transcriptase drug not target HBV as well as HIV?

Answer 24

HIV is a retrovirus and so it relies on RT to integrates its genome into the host genome.

HBV is a hepadnavirus, and so by the time RT is used, it will have already infected the cell nucleus.

Question 25

Give a detailed explanation of reverse transcription in HBV.

Answer 25

1. RT-RNase H binds to the e sequence on the RNA (5’ end). This signal is present at bond the 5’ and 3’ end, but only the 5’ signal functions for encapsidation.
2. Core is recruited and the capsid assembles. In the absence of RNase H, core assembles into capsids that packages DNA randomly.
3. In the capsid, first-strand synthesis is initiated by using the -OH of a tyrosine in RNase H as the primer. 4 nucleotides are added using the RNA template before the first jump to the 3’ end.
4. DNA synthesis of the first strand continues until the 5’ end of the RNA is reached.
5. RNase H degrades the RNA, but leaves the 5’ RNA cap. This cap is transferred to the 5’ of the new DNA as a primer for second strand synthesis.
6. The new DNA begins to cyclize and the cap at the 5’ end forms a ‘bridge’ that connects the 5’ end of the cDNA to the 3’ end.

Question 26

Give the steps of a generic viral replication cycle.

Answer 26

1. Attachment
2. Entry and uncoating
3. Synthesis of viral proteins and nucleic acids
4. Synthesis of viral structural proteins
5. Maturation/assembly
6. Release of new virions

Question 27

What makes a good antiviral drug?

Answer 27

• Active against WT and mutants without allowing for drug resistance emergence
• Minimal adverse effects
• Good pharmacokinetics
• Long elimination half life
• Easy to synthesize and formulate

Question 28

How do nucleoside analogues function as an antiviral drug? What type of inhibitor are they? What is their general structure?

Answer 28

A nucleoside is a unit of a nucleotide base with a pentose (RNA) or deoxypentose (DNA) sugar. 3 phosphates must then be added by a kinase for them to become active.

Analogues of these can incorporate into macromolecules and stop DNA or RNA synthesis via obligate chain termination. Hence, they’re competitive inhibitors.

Most nucleoside analogues are some spacer sugar that has a hydroxymethyl group and a nitrogenous base attached.

Question 29

What variations are there of nucleoside analogue antivirals? Give an example and explain how it’s specific to infected cells.

Answer 29

Modifications are usually only on the sugar, not the base as this can easily change the hydrogen bonding chemistry.

Acyclic nucleoside analogues have their rings removed. e.g., Acyclovir which is an analogue of deoxyguanosine.

In uninfected cells, the drug cannot be phosphorylated for activation. In infected cells, the virus encodes its own kinase.

Question 30

What are the potential problems with nucleoside analogues?

Answer 30

1. Delivery
2. Escape mutants
3. Toxicity
4. Metabolic instability

Question 31

How do HIV protease inhibitors work?

Answer 31

They bind to the active site of the HIV protease enzyme, preventing it from cleaving the precursor polypeptide and producing the mature viral proteins that are necessary for the production of new viral particles.

Question 32

State the steps involved in developing a protease inhibitor.

Answer 32

1. Identify the protease cleavage site.
2. Build a peptide substrate
3. Build a peptidic inhibitor that modifies the cleavage site
4. Modify the inhibitor to reduce the peptidic nature
5. Solve the structure of the enzyme/inhibitor complex
6. Improve the fit of the inhibitor

Question 33

What are interferons? Why are they tightly regulated? How are they used in antiviral immunity pathways?

Answer 33

Proteins released by cells in response to viral entry. They have short half-lives thanks to destabilization sequences as they can be damaging to the host.

When a viral infection is detected by host cells, they produce and release IFNs that bind to neighbouring cells and induce activation of antiviral pathways.

Question 34

What is the Covid Moonshot? How did the project work?

Answer 34

A collaborative project with the goal of developing an un-patented oral antiviral drug against SARS-CoV2.

Scientists identified a protease (Mpro) from the virus and crystallized it. Fragment screening was used to identify compounds that bound irreversibly to the protease. Crowdsourcing was then used to identify unique compound designs. A supercomputer accelerated the speed of this.

Question 35

What drugs are needed to prepare for future pandemics? Why is it important that we prepare, rather than wait for the next pandemic?

Answer 35

Directly acting antivirals - specifically target the virus
Immunomodulatory drugs - modify the negative effects of an overreacting immune system
Host-targeting antivirals - inhibits human proteins that the virus uses

There will never be time to develop a specific DAA from scratch, so we must keep developing drugs against viruses inbetween pandemics to reduce vulnerabilities.

Question 36

What do all of the global viruses have in common? How can this be used in treatments against these viruses?

Answer 36

All viruses use common host-cell glycosylation machinery. The human host is known in advance so we can optimize drugs against this system before the next virus emerges.

e.g., inhibition of the glucosidases that impact viral glycoprotein folding in the ER

Question 37

What happens when an interferon binds a neighbouring cell?

Answer 37

The receptors dimerize, autophosphorylate and recruit STAT proteins. These also become phosphorylated and dimerize before entering the nucleus to upregulate interferon-induced genes. These genes aim to make a hostile environment for incoming viruses, as well as genes to synthesize more MHC molecules for upregulation of antigen presentation.

Question 38

What’s the difference between a vaccine and an antiviral drug?

Answer 38

Unlike a vaccine, which increases immunity, an antiviral drug treats someone who’s already sick by attacking the virus itself.

Question 39

Why is Mpro a good target for all coronavirus drug treatments?

Answer 39

It’s present in other coronaviruses and doesn’t mutate easily so variants are less likely.

Question 40

What therapeutic uses do iminosugars have against viruses?

Answer 40

Iminosugars mimic the structures of carbohydrates. They can therefore interfere with the processing of viral glycoproteins that are vital for viral entry and release.