Viral Pathogens I

Question 1

Define a virus

Answer 1

An infective agent which consists of a nucleic acid in a protein coat, too small to be seen by a microscope and is not able to multiply without a host

Question 2

List the different types of genomes that can consist in a virus

Answer 2

Viruses can have

• Single stranded RNA or DNA
  
  • ssDNA/ ssRNA
• Double stranded RNA or DNA
  
  • dsRNA/ dsDNA

Double stranded genomes have complemetary base pairing.

RNA genomes can be linear and segmented (more than one RNA per capsid)

DNA genomes can be linear or circular

Question 3

What is the central dogma?

Answer 3

It is the directional relationship describing the flow of information from DNA to RNA to proteins

Question 4

Describe the structure of the mature HIV-1 particle

Answer 4

• Outer envelope of HIV consists of a lipid bilayer with protuding Env spikes
  
  • Gp 120 Receptor Ligand, Gp41 transmembrane domain
    Heterotrimers of SU₃TM₃
• Inside envelope shell lie Gag proteins
  
  • In the immature particle, Gag itself will form a single shell
• MA (matrix) associates with the outer membrane
• CA forms the caspid
• Inside the capsid
  
  • two RNA strands with are encapsulated via a nucelocapsid
• The capsid also contains reverse transcriptase, integrase and protease

Question 5

What are the three polyproteins synthesised by the retrovirus?

Answer 5

1. Gag (Group specific antigen)
   
   • Viral core proteins = Matrix, capsid and nucleocapsid
2. Pol (polymerase)
   
   • Protease (PR), reverse transcriptase (RT) and integrase (IN)
3. Env (envelope)
   
   • gp120 SU (surface); gp41 TM (transmembrane)

Question 6

List some HIV-1 regulatory/ accessory proteins and their functions

Answer 6

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

Some of these proteins can shut down apoptosis in permissive cells and permit viral replication

Question 7

What are the stages of the retroviral replication cycle

Answer 7

1. Entry
2. Reverse transcription
3. Integration
4. Gene expression
5. Assembly and release

Question 8

Describe what the HIV-1 Env structure

Answer 8

• HIV Env consists of a trimer of gp41 and gp120 peptide subunits and is covered with glycans
• gp41 and gp120 interact and stick out of the surface of the membrane
  
  • Gp120 is the receptor ligand which allows for primary attachment to CD4+ cells on T-cells, macrophages, and dendritic cells
  • Gp41 is the transmembrane domain + helps the virus enter cells

Question 9

Describe how the HIV-1 virus will enter the host cell

Answer 9

• HIV-1 requires two membrane proteins → CD4 and chemokine receptor (CCR5/CXCR4)
• The Env first samples the membrane and comes across a CD4 protein and binds to it
• This is then followed by CoR binding (chemokine receptors)
  
  • This allows for formation of the 6-Helix bundle formation
  • This then allows for membrane fusion and the virus to enter the cell

Question 10

Define viral tropism

Answer 10

Viral tropism refers to the cell types a virus infects

HIV virus will infect CD4+ T cells

Question 11

Describe how HIV-1 reaches the nucleus

Answer 11

• Uncoating step (lose capsid) where genetic component covered by nucleocapsid and surrounding enzymes (RT, IN, PR) enter the cells
• It will travel down the microtubules in a directional manner and get to the nucleus
  
  • Not using intracellular trafficking could mean replication takes years
• It also undergoes reverse transcription so the RNA is converted to DNA
• There are multiple degenerate pathways of HIV-1
  
  • Capsin comes in with the virus and helps select what microtubules are used and what destination on the nuclear membrane the virus will take
  • Capsin directs the virus through the cytoplasm to the NPC (nuclear pore complex)
    
    • The virus uses this to gain entry to the nuclear space
  • At the NPC it reacts with Nup proteins = directing the genome into its next path and into the nucleus

Question 12

Describe reverse transcriptase

Answer 12

Reverse transcription occurs before the virus enters the nucleus, this is because conversion of RNA → DNA allows for easier interaction with host cell DNA in the nucleus

Its located in the capsin protein

Reverse transcriptase is a heterotrimer of p66 and p51 subunits

The catalytic properties are in the p66 subunit, while 51 serves the structural role and lacks an RNAase domain

Question 13

What is the difference between the p51 and p66 subunit in reverse transcriptase?

Answer 13

• The catalytic properties are in the p66 domain
• p51 = structural role and lacks an RNAse H domain

Question 14

What are the three enzymatic activities reverse transcriptase displays?

Answer 14

• RNA-dependant DNA polymerase
• RNAse H (cleaves RNA from RNA/DNA hybrid)
• DNA-dependant DNA polymerase

Important to note that reverse transcriptase contains both polymerases RNA and DNA

Question 15

Describe the basic steps of reverse transcription

Answer 15

1. Reverse transcriptase recognises the specificity of the structure of RNA and binds to it
2. RNA polymerase moves down it and makes the RNA primer
3. The RNA structure then transfers to the other end of the genome
4. This means you produce even more RNA
5. There is also a DNA primer that it produced from the RNA
6. This DNA primer is used to produce DNA, so the DNA copies back on itself

Question 16

Describe how the HIV DNA genome is integrated into the host chromosome.

What is formed following integration?

Answer 16

• Integrase will recognise the target sequences on the viral genome and flip it into the host cell DNA
  
  • Integrase is brought by the virus and allows for integration of viral DNA into host cell DNA

This forms a provirus = Virus genome that is integrated into the DNA of a host cell

Question 17

How does the viral integrase enzyme work?

Answer 17

• Viral integrase enzyme binds both host DNA + viral DNA
• It will bring the target sequences from the viral genome in physical contact with the target host genome
• Integrase enzymes have DNA break repair pathways
• Once they come into physical contact integrases use divalent cations to break open the host cell DNA and insert viral DNA inside forming a linear sequence that can be copies

Question 18

What is the role of LEDGF/P75?

Answer 18

• LEDGF/P75 will bind to HIV-1 integrase
• It is picked up in the cytoplasm (not a viral protein)
• It allows entry into nucleus via nuclear pore
• It will facilitate targetting to chromatin and allows integrase to recognise specific target sequences in the host cell DNA

Question 19

How is the virus preferentially transcribed?

Answer 19

• Viruses have evolved to posses the abillity to bind all transcription factors
• The TAT protein will bind to the Tar element in the viral genome
• The Tat-TAR complex is preferentially bringing RNA polymerase to the viral genome

Question 20

How does the HIV-1 provirus create different mRNAs to produce the viral proteins?

Answer 20

• Once the primary transcript has been formed it can produce genome and gag/pol mRNA
• SPLICING of the mRNA will allow for env mRNA to be produced

Question 21

How does transcribed mRNAs get out of the nucleus to make new viruses?

Answer 21

• The HIV-1 Rev protein mediates nuclear export of unspliced and singly spliced viral RNA
• The cell doesnt have a mechanism to preferentially take out RNA from the nucleus
• The virus will produce a protein (in this case Rev) which will interact with a viral structure (in this case RRE)
• The Rev-RRE complex will interact with the Crm1 protein
• Crm1 = determines which way out RNA leaves and where it goes
• ! Rev is also important for nuclear export of intron containing viral mRNAs!

Question 22

Where does virus assembly occur?

Answer 22

It occurs on the plasma membrane → So it has a smaller distance to travel

Question 23

How do the viral RNA copies come together?

Answer 23

• Dimerisation of unspliced viral RNA allows for packing of two genomes
• Dimerisation occurs due to SLI motifs
• These motifs will interact with each other making a kissing loop complex between 2 genomic structures

Question 24

How are viral proteins (Gag and Pol) generated?

Answer 24

• Gag-pol proteins are generated by 1 ribosomal framshifting induced by a ‘slippery’ sequence and a RNA hairpin structure
• When the ribosome is normally translates the RNA molecule you get the Gag polyprotein
  
  • matrix (MA), capsid (CA), nuclear capsule (NC)
• If the ribosome slips you will get a different open reading frame = Pol polyprotein produced
  
  • protease (PR), reverse transcriptase and integrase (IN) components

Question 25

How does the Gag polyprotein know how to get to the plasma membrane?

Answer 25

Myristoylation of glycines in the MA domain of Gag mediates its association with the plasma membrane

Question 26

How does budding of the plasma membrane occur?

Answer 26

• The HIV-1 PT(S) AP motif is required for virus budding and mediates binding of the host Tsg101 protein
• P6 end of the polyprotein binds machinery
• The amino acids PT(S)AP and YPLTSL are present in any protein which requires machinery to be pushed out of the cell via budding
• ESCRT machinery is hijacked by HIV to perform membrane abscission during viral release

Question 27

How will we get the final viral variant?

Answer 27

• Polyproteins at the membrane will use protease to cleave them and produce individual proteins which make up the capsid
• Protease will release individual proteins from Gag and Gag-Pol polyproteins

Question 28

What is the difference between immature and mature virions?

Answer 28

• Immature
  
  • Very ordered rays of the polyproteins surrounded by the membrane which has been taken out as the virion buds out of the cell
• Mature
  
  • The viral protease has digested the immature polyproteins to produce individual proteins