Viral Pathogens I

Question 1

1. Which two viruses can both cause respiratory diseases but vary in their structure and genomes?

Answer 1

• > Adenoviruses

- >Influenza

Question 2

2. What are the 4 main configurations of the viral genome configuration?

Answer 2

Single-stranded RNA (ssRNA)

Double-stranded RNA (dsRNA)

Single-stranded DNA (ssDNA)

Double-stranded RNA (dsDNA).

Question 3

3. What are some features of double stranded genomes?

Answer 3

•Double-stranded genomes have complementary base pairing and can be linear or circular

Question 4

4. What are some features of RNA genomes?

Answer 4

• RNA genomes can be linear and segmented i.e. more than one RNA per capsid

Question 5

5. Are genes encoded in a positive or negative sense?

Answer 5

• Genomes can be encode information (genes) in positive or negative sense; 5’-3’ or 3’-5’ respectively.

Question 6

6. What is the central dogma?

Answer 6

describes the flow of genetic information in cells from DNA replication, transcription (RNA) and translation (proteins)

Question 7

7. How do viruses use the central dogma?

Answer 7

Reverse transcription is the transfer of information from RNA to DNA (the reverse of normal transcription). This is known to occur in the case of retroviruses,

RNA replication is the copying of one RNA to another. Many viruses replicate this way. The enzymes that copy RNA to new RNA, called RNA-dependent RNA polymerases,

Question 8

8. What is the baltimore classification?

Answer 8

Viruses grouped according to their type of genome (DNA/RNA/ss/ds) and method of replication.

Question 9

9. Explain the structure of of a mature HIV-1 particle ?

Answer 9

• The outer envelope of HIV consists of a lipid bilayer with protruding enveloped glycoprotein spikes (heterotrimers of SU3TM3) called Gag proteins. Gag proteins form a single shell in immature particles.
• Matrix proteins (MA) associates with the membrane capsid (CA) forms the conical (cone shaped) capsid and a nuclear capsid (NC) coats the viral RNA genome.
• The core contains two genomic RNA strands (plus strand), tRNALys3, and ~50 copies of each viral enzyme (PR, RT, and IN).

Question 10

10. HIV synthesise three polyproteins, what are these?

Answer 10

1. Gag (group specific antigen): viral core proteins; MA (matrix), CA (capsid), NC (nucleocapsid)
2. Pol: viral enzymes e.g. protease (PR), reverse transcriptase (RT) and integrase (IN).
3. Env (envelope glycoprotein): gp120 SU (surface) and gp41 TM (transmembrane).

Question 11

11. List some important regulatory /Accessory proteins of HIV-1 and their functions?

Answer 11

o Tat - potent activator of viral transcription
o Rev - mediates unspliced RNA nuclear export
o Vif - critical regulator of virus infectivity
o Nef - immune modulator, T-cell activation, virus spread (?)
o Vpu - immune modulator, virus release
o Vpr - cell cycle, virus nuclear import (?)

Question 12

12. HIV has bimodal organisation, what does this mean?

Answer 12

HIV RNA undergoes reverse transcription into DNA and vice versa

Question 13

13. How does HIV have entry into the host cell?

Answer 13

• The HIV-1 envelope receptor consists of a trimer of glycoprotein (gp41 and gp120) peptide subunits and is covered with glycans.
• HIV entry at cell surface is triggered by conformational changes in the Env receptor, this contrasts with the pH-dependent entry of adenovirus, influenza virus, etc.
• HIV-1 entry requires two membrane proteins: CD4 and co-receptors chemokine receptor (CCR5/CXCR4).
• HIV-1 is specific for CD4 expressing cells (helper T cells and macrophages), the loss of these cells results in immunodeficiency (& AIDS).
• 6 helix bundle structure: Fusion of viral and cellular plasma membrane using the 2 trimer envelope structures.

Question 14

14. Once HIV is inside the host cell , how can it move and what happens?

Answer 14

• As the virus moves across the cytoplasm using the microtubules (provides directionality for intracellular trafficking), it replicates its genome.
• The virus capsid is removed, it is then uncoated and reverse transcription occurs to replicate the genome.

Question 15

15. How does the HIV know which ENTRY pathway to take?

Answer 15

• Capsid molecules select/determine which intracellular pathway will be taken and what part of the nuclear membrane the destination of this is.
• The virus uses the nuclear pore complex to enter the nucleus. Here the interactions with specific Nup proteins directs the next pathway that will be used.

Question 16

16. Describe the structure of Reverse transciptase?

Answer 16

2. Reverse Transcriptase (heterodimer of p66 and p51 subunits):
   • p66 subunit provides the catalytic properties, p51 serves structural role and lacks RNAse H domain.

Question 17

17. What three distinct enzymatic activities does reverse transcriptase play?

Answer 17

o RNA-dependent DNA polymerase.
o RNAse H (cleaves RNA from RNA/DNA hybrid).
o DNA-dependent DNA polymerase.

Question 18

18. What is the basic steps of reverse transcription?

Answer 18

• The polymerase recognises and binds to the RNA using the RNA primer.
• An RNA molecule is produced.
• The polymerase and primer structure transfers to the other side of the molecule.
• Another RNA molecule is produced.
• Then a DNA primer is produced using the RNA primer, this then used to produce more DNA which can then be copied back.

Question 19

19. How does the HIV dna Genome integrate into the host chromosome?

Answer 19

• The host cell genome has target sequences that have specific complimentary sequences at the ends of the viral genome. The target and viral sequences are brought together.
• The linear viral DNA is bound to integrase protein that travels to the nucleus.
• It recognises the target sequences and flips to integrate the viral DNA into the host cell chromatin.

Question 20

20. How does the viral integrase enzyme bind both the host and viral DNA?

Answer 20

• The host cell contains DNA break repair enzymes that cleave specific sequences and insert new DNA into them.
• Integrase copies this mechanism to integrate the viral genome.

Question 21

21.What are the basic Basic steps of retroviral integration?

Answer 21

• Integrase loops the viral DNA around the target DNA to bring their terminals into close contact/proximity.
• It then uses diodone cations to break the DNA open and anneal the viral DNA into the cellular DNA, one strand at a time.

Question 22

22.Why does LEDGF/P75 bind to HIV-1 integrase

Answer 22

facilitates targeting to chromatin:
• The virus binds to the LEDGF protein to be brought in through the nuclear membrane.
• It also allows integrase to recognise specific sequences on the host cell genome.

Question 23

23. How does the viral DNA become expressed?

Answer 23

•The viral DNA has evolved so that it can be recognised and bind to many transcription factors to result in transcription.

Question 24

24.What happens when TAR Rna bind to the Tat protein?

Answer 24

The TAR RNA binds to Tat protein to enhance elongation of RNA pol II:
• The virus encodes a Tat protein that binds to the viral TAR element.
• This Tat-TAR interaction preferentially brings the RNA polymerase to the viral genome. The virus then utilises the polymerase to transcribe its genome.

Question 25

•Viruses need to transcribe one full length of the viral genome but also specific proteins , how does it do this?

Answer 25

by utilising splicing

Question 26

26. Explain the Nuclear export of unspliced retrovirus RNA:

Answer 26

Nuclear export of unspliced retrovirus RNA:
• Full length transcript is spliced to remove the 3 polyproteins.
HIV-1 Rev protein mediates nuclear export of unspliced and singly spliced viral RNA:
• The cell doesn’t have mechanism for preferentially taking RNA out of the nucleus, the virus produces a protein to utilises protein interactions to preferentially move them out.
• The virus produces Rev protein which binds to RRE, these interactions export the RNA out through the nuclear pore.

Question 27

27. Why is HIV Rev is essential for the nuclear export of intron-containing viral mRNAs:

Answer 27

• Normally, intron containing RNA (e.g. viral RNA) cannot leave the cell nucleus until they are spliced out.
• This is why Rev is important for its export.

Question 28

28. How does HIV-1 Rev protein interacts with Crm1 and the RRE RNA:

Answer 28

Crm1 binds to the RRE interactions to dictate which exit pathway out of the nucleus will be taken.

Question 29

29. Explain the 5. Assembly and release: The unspliced HIV-1 RNA is the mRNA for Gag and Gag-Pol proteins.

Answer 29

• The virus is assembled at the plasma membrane because it has a smaller distance to travel.
• The 2 viral RNA genomes are surrounded by viral proteins. A kissing loop structure is formed due to interactions between the 2 viral molecules, this allows the dimerisation and package of the 2 unspliced viral DNA. Gag and Gag-Pol proteins assemble viral particles.
• Normally, the ribosome reads the RNA to translate the Gag protein. When the ribosome encounters a ‘slippery sequence’, it slips and mis-reads the RNA due to a -1 frame-shift resulting in the formation of the Gag-pol protein.
• Myristoylation (form of lipidation where myristic acid is added to the polyprotein) of glycines in the MA domain of Gag allows association with the plasma membrane.
• HIV-1 PT(S)AP motif is required for virus budding and mediates binding of the host Tsg101 protein.
o Virus utilises the normal cellular exit machinery (Tsg101) by binding P6 to it.

Question 30

30. Why is ESCRT Machinery is hijacked by HIV to perform membrane abscission during viral release:

Answer 30

• During cell division, this machinery is used in cytokinesis to break apart the 2 daughter cell.
• The virus hijacks it to push its own variant out of the cell (rather than 2 daughter cells).

Question 31

31. Explain how Protease releases the individual proteins from Gag and Gag-Pol polyproteins

Answer 31

• Viral surface polyproteins are cleaved (by viral protease) to produce individual proteins that make up the capsid.
• Gag processing (cleavage of immature polyproteins by protease) generates mature virions.
• The envelope glycoproteins produce the 2 glycoprotein spikes. These are already at the membrane, they are taken up and pushed up through the membrane to be incorporated into the membrane.