Viral pathogens: classification, biology, diseases

Question 1

What is the central dogma?

Answer 1

Genome from which you produce viral proteins

Viral proteins required for virus replication and production of new viruses

Protein production follows central dogma

• DNA polymerase replicates DNA
• RNA pol transcribes RNA from DNA
• Ribosome produces protein from RNA template

Question 2

Describe the structure of HIV

Answer 2

Genome has 2 copies of RNA, covered by protein and membrane

glycoproteins stick out as spikes

viral enzymes within virus - integrase, protease, reverse transcriptase

Outer envelope consists of a lipid bilayer with protruding Env spikes (heterotrimers of SU3TM3) (surface subunit and transmembrane subunit)

Inside envelope there is shells of Gag proteins. In immature particle, Gag itself forms a single shell.

• Gag composed of three different particles, Matrix (Ma), Capsid (CA) and nuclear capsid (NC)

MA associates with the membrane CA forms the conical capsid. NC coats the viral RNA genome

Core of the capsid composed of Gag contains two genomic RNA strands (plus strand), tRNA Lys3 and 50 copies of each viral enzyme (PR, RT and IN)

Question 3

What 3 polyproteins are synthesised to produce proteins?

Answer 3

Gag; group specific antigen; produces viral core proteins MA (matrix), CA (Capsid) and NC (nucleocapsid)

Pol; viral enzymes; produces protease (PR), reverse transcriptase (RT) and integrase (IN)

Env; envelope glycoprotein; produced by gp120 SU (surface); gp41 TM (transmembrane)

Question 4

How does HIV-1 virus enter the cell?

Answer 4

HIV envelope has trimer of gp41 and gp120

entry needs membrane proteins CD4 and chemokine receptor CCR5/CXCR4

CD4 recognises structure on surface subunit of HIV envelope - the native trimer

gp120 binds CD4, causes confirmational change in envelope protein - open and away from native confirmation

open confirmation uncovers transmembrane subunits, HIV recognises co-receptor

co-receptor interacts with surface subunits, stimulates transmembrane region to go into cell membrane

trying to insert transmembrane domain into lipid bilayer, causes 6-helix bundle formation and membrane fusion - pushes membrane apart

Question 5

What does HIV being tropic for CD4 mean?

Answer 5

If the cell doesn’t express CD4, virus can’t get into cell

if cell doesn’t express CD4 and co receptor then envelope protein can’t function - tropism

Question 6

What occurs in the early phase of HIV-1 infection once the virus has entered the cell?

Answer 6

Virus moves to a site of replication, area in which replication of the genome is most favoured

HIV wants to replicate in cell nucleus, because this is where we already find RNA and DNA replication within the cell, therefore can utilise those factors to replicate its own genome

However, distance between plasma membrane and nuclear membrane is great distance, takes virus time to travel

Once virus has fused and deposited the core containing genome into the cell it utilises the cellular microtubule network to move the core containing the genome to the nuclear membrane

Question 7

What does mutations in capsid dictate in HIV replication?

Answer 7

Capsid mtuations in HIV dictate how capsid containing genome gets to the nuclear pore capsid

or which NPC (nuclear pore complex) is used to gain access to the nucleus

Question 8

What enzyme does the virus use once it gets into the nucleus?

Answer 8

Once the virus has got to the nucleus, it utilises reverse transcription.

Transcription is process of forming RNA from DNA – goes DNA to RNA

Reverse transcription is of DNA from RNA – RNA to DNA

Viral RNA in capsid, within capsid reverse transcriptase enzyme mediates the production of DNA from RNA.

Question 9

The HIV DNA genome (provirus) is integrated into the host chromosomes

Answer 9

RNA is made from cellular DNA, which is a good template for making RNA.

Virus uses this to its advantage, integrates using viral integrase enzyme.

Viral linear DNA has specific sequences at ends. Integrase enzyme recognises these sequences at the termini of the viral DNA genome.

Integrase binds the viral DNA and binds the cellular DNA.

Cuts the cellular DNA and pastes the viral DNA into the cut.

Requires contortion retroviral DNA and HIV viral bent around, brings into close proximity to cellular DNA.

Integrase cuts cellular DNA and repairs the cut so that the cellular DNA is bound to viral DNA.

Integrase promotes a repair process, making sure the cuts are resolved.

Question 10

What is the pre-integration complex, what does it do?

Answer 10

LEDGF/P75 and TRN-SR2, pre-integration complex.

Recongnises the cellular DNA and guides the viral DNA to it.

Question 11

Virus replication is about making RNA

How does HIV make its RNA?

Answer 11

Double stranded DNA integrated into cellular DNA

Virus uses all the cellular processes in the nucleus

Recruits cellular proteins to the viral genome required for mRNA transcription

Promoter and enhancer regions of the viral genome can be seen, very extreme terminus of viral DNA genome

In red are two major transcriptional factors that the virus can promote the binding of on its promoter enhancer region

These enhance and promote transcription from the viral genome

Question 12

Now that transcription from viral genome has began, how do we ensure that we get preferrential transcription from viral geome?

Via 2 ways

Answer 12

1.

viral Tat protein produced from viral genome, viral RNA binding protein, binds specifically to viral RNA and enhances production of RNA

HIV ensures it gets preferential treatment by ensuring that its RNA are preferably produced

Occurs during positive feedback mechanism

2.

Virally encoded process that prioritises viral RNA over cellular RNA:

• Have primary transcript or genome transcripts that can be spliced
• And unspliced viral transcripts
• Virus therefore produces protein called Rev
• Rev protein forms a positive feedback loop
• Rev protein gets produced, comes back into nucleus and binds to genomic DNA specifically through RRE region
• Binding of Rev to RRE promotes the movement of viral RNA out of the nucleus
• Virally encoded protein goes back into nucleus, finds viral RNA and promotes the nuclear export of RNA over the nuclear export of cellular DNA

Question 13

What is HIV rev essential for?

Answer 13

Essential for nuclear export of intro-containing viral mRNAs

Rev protein binds RRE element

Also binds cellular protein Crm1

Crm1 interacts with nuclear pore, therefore promoting export of viral RNA over export of cellular DNA

Question 14

Where does HIV coordinate the produce of its viral proteins in the cell?

Answer 14

Towards the cell surface

Viral genomic DNA exported from nucleus to cytoplasm

production of viral envelope glycoproteins through golgi apparatus, moving to cell surface

mature protein goes onto infect cell

Question 15

What is dimerisation? What does it allow?

Answer 15

Coming together of two copies of the viral RNA.

Dimerisation of unspliced viral RNA promotes its movement using cellular factors to the plasma membrane from cytoplasm.

And subsequent packaging / incorporation into novel capsids that are made in the plasma membrane

Question 16

Viral protein is produced two ways

Answer 16

Can either have ribosome reads through the viral RNA, producing polyprotein, which is the fusion of all the proteins – matrix, capsid, nuclear capsid and P6

Or you can read through of your transcript, ribosomal frameshifting.

Ribosome comes to a particular site in your RNA and then jumps or skips to the next codon

Next codon encompasses all information required for the production of a second polyprotein

Ribosome reads through, makes nuclear capsid, instead of producing P6 it produces polyprotein that contains viral enzymes – protease, reverse transcriptase and integrase (PR, RT, IN)

Question 17

How is the polyprotein brough to the cell surface?

Answer 17

Post translational modification, or the addition of new moiety to the viral protein

e.g an Myr / Myristic site at the amino terminus of polyprotein

Myristylation post translational modification promotes association of the protein with the plasma membrane

Goes to the plasma membrane and sticks there.

Question 18

What role does the Tsg101 protein play?

Answer 18

Polyprotein transferred using Tsg101 to cell surface and myristylation sticks it to the cell surface at plasma membrane

Question 19

Now everything is at the plasma membrane, what is formed before viral release?

Answer 19

ESCRT machinery is hijacked by HIV to form abscission before viral release

Abscission is the organisation of all the proteins and RNA’s into a new capsid, pushes capsid into the extracellular space

Question 20

How is mature virions generated?

Answer 20

Via gag processing.

Immature capsid, had all the polyproteins yet to be cleaved being pushed out by ESCRT complex during abscission

mature capsid, when all the polyproteins have been cleaved to the constituent part and now form a recognisable core around the two copies of the RNA viral genome