36. Viral Pathogenes: Classification, Biology, Diseases I Flashcards
List the parts that can make up the different kinds of viral genomes.
Single-stranded RNA (ssRNA)
Double-stranded RNA (dsRNA)
Single-stranded DNA (ssDNA)
Double-stranded RNA (dsDNA)
Double-stranded genomes have complementary base pairing
RNA genomes can be linear and segmented i.e. more than one RNA per capsid
DNA genomes can be linear or circular.
What is the central dogma?
It is the directional relationship describing the flow of information from DNA to RNA to proteins.
Describe the structure of the mature HIV-1 particle
The outer envelope of HIV consists of a lipid bilayer with protruding Env spikes (heterotrimers of SU3TM3).
Inside the envelope lie shells of Gag proteins. In the immature particle, Gag itself forms a single shell.
MA associates with the membrane CA forms the conical 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).
Retroviruses synthesise 3 polyproteins. List them.
Gag; group specific antigen; viral core proteins; MA (matrix), CA (capsid), NC (nucleocapsid)
Pol; viral enzymes; protease (PR), reverse transcriptase (RT) and integrase (IN)
Env; envelope glycoprotein; gp120 SU (surface); gp41 TM (transmembrane)
List some HIV-1 regulatory/accessory proteins and their functions
- 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 (?)
What are the ‘stages’ of the retroviral replication cycle?
- Entry
- Reverse transcription
- Integration
- Gene expression
- Assembly and release
How do retroviruses (specifically HIV-1) know which cells to infect?
Env-receptor interactions between the virus and a certain cell are specific, so the virus knows which cells it needs to infiltrate.
HIV-1 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.
Describe the entry of the HIV-1 virus
HIV-1 entry requires two membrane proteins: CD4 and a 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, which is thought to be for fusion peptide insertion. There is then the formation of the 6-helix bundle for membrane fusion.
Describe how the HIV-1 virus enters the nuclear space.
The uncoating step, uncoating the capsid and you will still have the RNA content covered by enzymes and nucleocapsid proteins.
It travels down the microtubules in a directional manner to get to the nucleus. This is called intracellular trafficking. Whilst moving from the membrane to the nucleus, the virus replicates it’s DNA.
There are multiple degenerate nuclear entry pathways of HIV-1. There are molecules of capsin that come in with the virus, and help select what microtubules are used, and what destination on the nuclear membrane the virus actually takes.
The capsin directs the virus through the cytoplasm, getting it to the NPC or the nuclear pore complex. The virus uses this to gain entry into the nuclear space.
At the NPC, it reacts with the Nup proteins, which direct the genome into its next path and into the nucleus.
Describe reverse transcriptase (RT)
While travelling to the nucleus, we think that the virus converts all its RNA to DNA. This is because when it encounters the nucleus, it will encounter all the DNA within the nucleus, and as such it is just easier for it to interact with the DNA. This conversion is brought bout by reverse transcriptase, or RT.
RT is heterodimer of p66 and p51 subunits.
The catalytic properties are in the p66 subunit, while p51 serves a structural role and lacks an RNAse H domain.
RT displays three distinct enzymatic activities:
- RNA-dependent DNA polymerase
- RNAse H (cleaves RNA from RNA/DNA hybrid)
- DNA-dependent DNA polymerase
What are the basic steps of reverse transcription?
- We have an RNA structure (all scrunched up, forming extrinsic structures)
- The enzyme RT recognises this specificity of the structure of RNA and binds to it.
- RNA pol moves down it and make the RNA primer
- The RNA structure then transfers to the other end of the genome.
- This means you will produce even more RNA.
- There is also a DNA primer that is produced from the RNA.
- This DNA primer is then used to produce DNA, so the DNA copies back n itself.
- Thus, you produce the DNA copy of an RNA genome.
How is the HIV DNA genome (provirus) integrated into the host chromosome?
The target sequences find each other, and then the integrase protein (brought by the virus) allows the integration of the viral genome into the DNA.
How does viral integrase work?
The integrase enzymes have DNA break repair pathways.
The viral DNA is looped around the target DNA, which brings the termini (TTA sequences) into physical contact with the viral genome.
The integrase then breaks open the DNA and insert new viral DNA inside, eventually forming one linear sequence of DNA that can be copied.
How is HIV-1 integrase targetted to the cell chromatin?
LEDGF/P75 binds HIV-1 integrase facilities its targeting to chromatin.
As the virus addresses the nuclear membrane, the LEDGF protein allows the integrase protein to recognise the specific target sequences within host cell DNA in the nucleus.
LEDGF is not a viral protein, the HIV picks it up along the way and utilises it to reach specific chromatin sequences.
How is the virus preferentially transcribed?
The virus encodes the Tat protein, which binds to the TAR element on the genome.
We think the Tat-TAR complex is preferentially bringing the RNA polymerase to viral genome.
