Medical Microbiology: Viral Pathogens: Classification, Biology, Diseases - I Flashcards
1
Q
What are the different types of viral genome structure?
A
- Single-stranded RNA (ssRNA)
- Double-stranded RNA (dsRNA)
- Single-stranded DNA (ssDNA)
- Double-stranded RNA (dsDNA)
2
Q
Describe some features of the different types of viral genome structure
A
- Double-stranded genomes have complementary base pairing
- RNA genomes can be linear and segmented
- DNA genomes can be linear or circular
3
Q
How can RNA genomes encode information (genes)?
A
- RNA genomes can encode onformation in a postive sense (5’-3’) or in a negative sense (3’-5’)
4
Q
Explain how viruses use the central dogma
A
- Viruses can use reverse transcriptase enzyme to produce viral DNA from their + sense viral RNA
- Once viral DNA intergrated into host DNA it can be transcribed into more + sense RNA
- sense RNA, along with host DNA, can then be translated to produce viral proteins
- Viruses can also use RNA dependent RNA polymerase to replicate + sense RNA into -sense RNA and vice versa
5
Q
What is the Baltimore classification?
A
- A virus classification system that groups viruses into families, depending on their type of genome and their method of replication
6
Q
Describe the structure of a mature HIV-1 particle
A
- The outer envelope of HIV consists of a lipid bilayer with protruding Env spikes (heterotrimers of SU3TM3)
- Inside the envelope lie shells of Gag protein - In the immature particle, Gag itself forms a single shell
- Capsid of HIV has 3 main components:
- Matrix protein (MA) which associates with the membrane
- Capsid protein (CA) which forms the conical capsid
- Nucleocapsid (NC) which coats the viral RNA genome
- The core contains two genomic RNA strands (plus strand), tRNALys3, and around 50 copies of each viral enzyme:
- Protease (PR), Reverse transcriptase (RT), and Integrase (IN)
7
Q
Describe the genome organisation of the HIV-1 retrovirus
A
- Retroviruses such as HIV contain genes that encode for 3 polyproteins:
- Gag (group specific antigen) - synthesises the matrix, capsid and nucleocapsid
- Pol (polymerase) - synthesises protease, reverse transcriptase and intergrase enzymes
- Env - synthesises envelope glycoprotein, gp120 SU (surface); gp41 TM (transmembrane)
8
Q
What are some of the regulatory/accessory proteins encoded by the HIV genome?
A
- 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
9
Q
Describe the structure of HIV RNA
A
- Contains repeat untis upstream of both 5’ and 3’ untranslated regions
- Contains regulatory RNA elements:
- Rev responsive element (RRE)
- Trans-activation response element (TAR)
10
Q
Explain how a HIV-1 particle enters a cell
A
- HIV-1 consists of a trimer of gp41 and gp120 envelope glycoprotein subunits
- HIV-1 samples membrane looking for suitable host cell - cell that contains both CD4 and chemokine receptor (CCR5/CXCR4)
- gp120 glycoprotein binds to CD4 receptor present on T-cell membrane
- This binding then promotes further binding of the gp120 glycoprotein to a coreceptor also prsent on the T-cell membrane (CCR5/CXCR4)
- This co-receptor binding causes conformational change in shape of gp120 which allows gp41 to insert its hyrophobic terminus into the T-cell membrane
- gp41 then folds in on itself which draws te HIV-1 membrane to the T-cell membrane
- This eventually leads to fusion of the 2 membranes resulting in the formation of the 6-Helix bundle
- Fusion of 2 membranes allows HIV-1 to enter cell
11
Q
Once HIV-1 enters host cell it has to travel from the plasma membrane to the nucleus of host cell, as it travels it performs a variery of different processes. What are these processes?
A
- Uncoating step - loses capsid so instead you have RNA genome covered by viral enzymes surronded by nucleocapsid proteins
- Reverse transcription - HIV-1 RNA is reverse transcribed to form HIV-1 DNA
- Incorportaion of cellular proteins
- Nuclear entry - HIV-1 moves through nuclear membrane into nucleus of nucleus of host cell
12
Q
How does HIV-1 move through the host cell into the nucleus?
A
- Moves via intracellular trafficking - Uses microtubules formed via the microtubule organising complex (MTOC) to move specifically from the plasma membrane of cell to the nuclear membrane
- There’s a variety of mictrotubule pathways that lead to different areas of cell
- Because of this there are molecules on capsid that travel with HIV-1 which help select which microtubule pathyways are used and the destination of HIV-1
- Ideal microtubule pathway is to get HIV-1 to nuclear pore complex (NPC) on nuclear membrane
13
Q
What happens once HIV-1 reaches the Nuclear pore complex (NPC)?
A
- At NPC HIV-1 interacts with Nup358 and Nup153 which directs the HIV-1 genome into the nucleus
14
Q
What is reverse transcriptase and what is its structure?
A
- Reverse transcriptase is a viral enzyme used to convert RNA into DNA
- Structure:
- Bound to RNA of retrovirus
- Heterodimer of p66 and p51 subunit
- p66 subunit: Catalytic properties
- p51 subunit: Serves structural role and lacks RNAse H domain
15
Q
What enzymatic activity does reverse transcriptase have?
A
- RNA-dependent DNA polymerase
- RNAse H (cleaves RNA from RNA/DNA hybrid)
- DNA-dependent DNA polymerase
16
Q
Describe the basci steps of reverse transcription
A
- RNA dependent RNA polymerase recognises and binds to specific RNA structures within HIV-1 RNA and binds to it
- RNA polymerase makes RNA primer using HIV-1 RNA as template
- RNA primer then transfers to other end of genome
- RNA primer is then used to produce more RNA at end of genome RNA moved to - this results in 2 RNA structures being produced
- DNA polymerase actovity of reverse transcriptase takes over and produces DNA primer from RNA
- DNA primer then used to produce more DNA which eventually leads to complete DNA copy of RNA genome being produced
17
Q
Explain how viral integrase is able to integrate the HIV-1 DNA genome into the DNA of the host cell
A
- Integrase loops viral DNA around the target DNA which brings viral TTAA sequences, on ends of viral DNA, in physical contact with target DNA
- Integrase then uses chelation to break open target DNA and then, one strand at at time, anneal each target DNA strand to a HIV-1 DNA strand
- This results in the formation of one linear strand of double stranded DNA called a provirus
18
Q
How does HIV-1 get through the nuclear membrane?
A
- LEDGF/P75 binds HIV-1 integrase which allows the HIV-1 to get through the nuclear membrane
- This also allows integrase to recognise specific target sequences within the host cell DNA
19
Q
How is viral transcription of HIV-1 within the host cell genome controlled?
A
- Controlled by binding of a no. of transcription factors to the HIV-1 DNA
- Once transcription factor binds it recruits RNA polymerase which leads to transcription
- HIV-1 promoter contains binding sites for transcription factors that are present in T-lymphocytes
20
Q
How does HIV-1 ensure that it is preferentially transcribed over other host cell genes?
A
- HIV-1 encodes a TAT protein
- TAT protein then binds to TAR element to produce a TAT-TAR complex
- TAT-TAR complex preferentially brings RNA polymerase to the HIV-1 genome
21
Q
What are the different products of transcription of the HIV-1 genome?
A
- Entire HIV-1 DNA genome can be transcribed which forms Genomic RNA, Gag-Pol mRNA and pre-mRNA (full-length transcript)
- To produce the regulatory/accessory proteins of HIV-1 the mRNA has to be spliced
-
Singly spliced mRNAs produce:
- Vif
- Vpr
- Vpu
- Env
-
Mulitply spliced mRNAs produce:
- Tat
- Rev
- Nef
22
Q
How are all the viral mRNA transcripts produced by the granscription of viral DNA exported from the nucleus?
A
- Rev protein produced from HIV-1 interacts with the RRE of the full length HIV-1 transcript
- This interaction allows for the recruitment of other cellular proteins which help preferentially export the viral mRNAs out of the nucleus
26
Q
How are the 2 RNA genomes able to interact with each other during assembly of a new HIV-1 virus?
A
- Unspliced HIV-1 RNA is the mRNA for Gag and Gag-Pol proteins
- Kissing loops either in the SL 1 or SL 4 domain of the unspliced viral RNA interact with each other to form a kissing-loop complex
28
Q
How do you differentiate between producing Gag and producing Pol polyproteins from the translation of HIV-1 long genomic RNA?
A
- If ribosome starts at one end of genomic RNA then Gag polyprotein is produced
- This includes:
- Matrix
- Capsid
- Nucleocapsid
- p6 proteins
- If however the ribosome “slips” into a different reading frame then the Pol polyprotein is produced as well as the Gag protein
- This includes:
- Protease
- Reverse transcriptase
- Integrase proteins
- All the Gag proteins
29
Q
Once produced how do the polyproteins travel to the plasma membrane?
A
- Gag and Pol polyproteins undergo a process of Myristoylation
- This is the addition of mystric acid to glycine residues on the end of Gag and Pol polyproteins which allows them to interact with the plasma membrane
30
Q
How does the newly assembled HIV-1 virus bud out of the cell?
A
- HIV-1 PT(S)AP motif present with p6 domain of Gag polyprotein is required for virus budding as it binds to host Tsg101 protein
- ESCRT machinery is then hijacked by HIV which causes it to perform membrane abscission during viral release
31
Q
What occurs to the HIV-1 particle once it has budded out of the cell in order for a mature HIV-1 particle to be produced?
A
- As HIV-1 buds of plasma membrane viral proteases cleave the Gag and Pol polyproteins in order to produce the individual proteins which either form the capsid or bind viral RNA, if they are viral enzymes
