Viral Pathogens: Classification, Biology and Disease I Flashcards
Viruses which cause respiratory disease
Adenovirus
Influenza virus
Structure of Viral Genome
Different viruses have many different structures of genomes
Central Dogma
theory that states that, in cells, information only flows from DNA to RNA to proteins via transcription and translation
How does the virus use the Central Dogma?
If the virus has DNA genome, it can use the first part of central Dogma and simply use DNA polymerase enzymes to replicate that DNA.
If virus wishes to make RNA from the DNA genome, it can follow through the central Dogma using RNA polymerase and ribosomes, transcription and translation to make the proteins that it wishes to make.
Viruses with +sense and -sense RNA genomes and how they make proteins
Viruses with a +sense RNA genome can use RNA polymerase to replicate this + sense genome in the 5 prime to 3 prime end and from that +sense you can produce proteins.
Viruses with a –sense RNA genome are coded in the 3-prime end to 5 prime end. This –sense RNA is translated into +sense by RNA dependent RNA polymerase which reads this 3 prime to 5 prime end directionality, produces a +sense RNA from it, from which you can make proteins.
Classification of viruses
Viruses are grouped into genome classifications
Structure of HIV
- Core contains two single RNA strands and enzymes (protease, reverse transcriptase, integrase)
- Protein capsid encapsulates RNA
- Matrix protein associated with capsid to form nucleocapsid which coats the two RNA strands
- Lipid bilayer enveloped with glycoprotein spikes (Gp120)/envelope spikes
Main HIV genes
3 polyproteins (genes):
Gag:
-group specific antigen; encodes viral core proteins, matrix, capsid, nucleocapsid
Pol:
-encodes viral enzymes, protease, reverse transcriptase, integrase
Env:
-forms envelope glycoprotein; gp120 SU (surface); gp41 TM (transmembrane)
HIV Regulatory Genes
· 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
HIV Entry
Envelope glycoprotein binds CD4 receptor on T cell/macrophage
This opens interaction with chemokine co-receptors CCR5 and CxCR4, resulting in a 6-helix bundle formation, meshing the membrane of the virus and the cellular plasma membrane
HIV tropism
HIV is tropic for CD4 expressing cells such as T-helper cells and macrophages
Loss of T-helper cells and macrophages results in immunodeficiency → AIDS
HIV Reverse Transcription
On the way from the plasma membrane to the nucleus:
Uncoating
- capsid is lost
- reverse transcription of RNA into DNA in cytoplasm by reverse transcriptase
Structure of reverse transcriptase
Heterodimer of p66 and p51 subunits with three distinct enzymatic activities:
- RNA dependent DNA polymerase
- RNAse H
- DNA dependent DNA polymerase
Steps of reverse transcription
1) Host cell tRNA binds to the HIV RNA and acts as a primer
2) Reverse transcriptase adds DNA nucleotides onto the 3’ end of the primer
3) RNAse H domain on reverse transcriptase cleaves RNA from this RNA/DNA hybrid structure
4) tRNA primer then “jumps” to the 3’ end of the viral RNA strand to hybridise the newly synthesised DNA to the viral RNA
5) The complementary DNA is further extended and the majority of the RNA is degraded by RNAse H domain
6) Same thing happens with the other RNA strand, resulting in a complete double stranded DNA copy of original viral RNA genome
What happens after reverse transcription of HIV in the cytoplasm?
Intracellular Trafficking
-virus travels down the microtubule to the microtubule organising complex (MTOC) which directs the virus to the NPC complex
The virus interacts with Nup proteins (Nup335 & Nup153) at the NPC complex which directs the HIV DNA genome into the nucleus so it can be integrated into the host’s chromosomes
How is the HIV DNA genome integrated into the host’s chromosomes?
Host cell’s chromosomes contains a target sequence which is complementary to specific sequences at the end of the HIV genome
Integrase enzyme loops the viral DNA around the host’s target DNA, bringing them into physical contact. Integrase then breaks the DNA one strand at a time and anneals the viral DNA to the cellular DNA, resulting in one linear bit of DNA (provirus).
What binds to integrase and facilitates targeting specific sequences within host cell chromatin?
LEDGF/p75 protein
What happens after the provirus is produced?
Virus wants to replicate itself
- transcription factors bind HIV promoter
- RNA polymerase II recruited for transcription
- viral Tat protein binds TAR element within the genome, and the Tat-TAR interaction enhances the elongation of RNA polymerase II
- full length RNA is transcribed & different mRNAs are produced for protein production in the cytoplasm (through splicing of different mRNAs)
How are the synthesised mRNAs exported out of the nucleus?
Virus produces Rev protein which interacts with RRE on unspliced and singly spliced viral RNA to mediate nuclear export.
Crm1 cellular protein binds to the RRE-Rev interaction and dictates movement out of the nucleus and which way the RNA will travel into the cytoplasm, where the mRNAs are translated to produce essential viral proteins (Crm1 Dependent Pathway)
What happens after nuclear export of RNAs?
Spliced mRNAs are used to produce essential viral proteins
Unspliced viral RNA form ‘kissing loops’ and interact with each other, allowing for the packaging of the two genomes
Virus then assembles at the membrane
How does the virus assemble at the membrane?
Myristolytation of Gag and Gag-pol polyproteins:
-feature added to end of polyproteins which allows proteins to interact with membrane
How are Gag and Gag pol proteins produced?
Gag is a polyprotein which normally encodes for matrix, capsid, nucleocapsid and p6 proteins.
Gag-pol polyprotein is produced by -1 ribosomal framshifting induced by a ‘slippery’ sequence, which encodes protease, reverse transcriptase and integrase proteins.
How does HIV bud out of the cell?
Cellular ESCRT protein Tsg101 is recruited to the assembly site and binds HIV PT(S)AP motif of the Gag p6 protein.
As the virus is pushed out, the viral protease cleaves the Gag and Gag-pol polyproteins to produce the individual proteins which make the capsid, thus producing the mature virion.
