Medical Microbiology: Viral Pathogens: Classification, Biology, Diseases - I

Question 1

What are the different types of viral genome structure?

Answer 1

• Single-stranded RNA (ssRNA)
• Double-stranded RNA (dsRNA)
• Single-stranded DNA (ssDNA)
• Double-stranded RNA (dsDNA)

Question 2

Describe some features of the different types of viral genome structure

Answer 2

• Double-stranded genomes have complementary base pairing
• RNA genomes can be linear and segmented
• DNA genomes can be linear or circular

Question 3

How can RNA genomes encode information (genes)?

Answer 3

• RNA genomes can encode onformation in a postive sense (5’-3’) or in a negative sense (3’-5’)

Question 4

Explain how viruses use the central dogma

Answer 4

• 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

Question 5

What is the Baltimore classification?

Answer 5

• A virus classification system that groups viruses into families, depending on their type of genome and their method of replication

Question 6

Describe the structure of a mature HIV-1 particle

Answer 6

• 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), tRNA^Lys3, and around 50 copies of each viral enzyme:
  • Protease (PR), Reverse transcriptase (RT), and Integrase (IN)

Question 7

Describe the genome organisation of the HIV-1 retrovirus

Answer 7

• 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)

Question 8

What are some of the regulatory/accessory proteins encoded by the HIV genome?

Answer 8

• 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

Question 9

Describe the structure of HIV RNA

Answer 9

• Contains repeat untis upstream of both 5’ and 3’ untranslated regions
• Contains regulatory RNA elements:
  
  • Rev responsive element (RRE)
  • Trans-activation response element (TAR)

Question 10

Explain how a HIV-1 particle enters a cell

Answer 10

• 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)

1. gp120 glycoprotein binds to CD4 receptor present on T-cell membrane
2. This binding then promotes further binding of the gp120 glycoprotein to a coreceptor also prsent on the T-cell membrane (CCR5/CXCR4)
3. This co-receptor binding causes conformational change in shape of gp120 which allows gp41 to insert its hyrophobic terminus into the T-cell membrane
4. gp41 then folds in on itself which draws te HIV-1 membrane to the T-cell membrane
5. This eventually leads to fusion of the 2 membranes resulting in the formation of the 6-Helix bundle
6. Fusion of 2 membranes allows HIV-1 to enter cell

Question 11

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?

Answer 11

• 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

Question 12

How does HIV-1 move through the host cell into the nucleus?

Answer 12

• 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

Question 13

What happens once HIV-1 reaches the Nuclear pore complex (NPC)?

Answer 13

• At NPC HIV-1 interacts with Nup358 and Nup153 which directs the HIV-1 genome into the nucleus

Question 14

What is reverse transcriptase and what is its structure?

Answer 14

• 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

Question 15

What enzymatic activity does reverse transcriptase have?

Answer 15

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

Question 16

Describe the basci steps of reverse transcription

Answer 16

1. RNA dependent RNA polymerase recognises and binds to specific RNA structures within HIV-1 RNA and binds to it
2. RNA polymerase makes RNA primer using HIV-1 RNA as template
3. RNA primer then transfers to other end of genome
4. RNA primer is then used to produce more RNA at end of genome RNA moved to - this results in 2 RNA structures being produced
5. DNA polymerase actovity of reverse transcriptase takes over and produces DNA primer from RNA
6. DNA primer then used to produce more DNA which eventually leads to complete DNA copy of RNA genome being produced

Question 17

Explain how viral integrase is able to integrate the HIV-1 DNA genome into the DNA of the host cell

Answer 17

1. Integrase loops viral DNA around the target DNA which brings viral TTAA sequences, on ends of viral DNA, in physical contact with target DNA
2. 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
3. This results in the formation of one linear strand of double stranded DNA called a provirus

Question 18

How does HIV-1 get through the nuclear membrane?

Answer 18

• 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

Question 19

How is viral transcription of HIV-1 within the host cell genome controlled?

Answer 19

• 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

Question 20

How does HIV-1 ensure that it is preferentially transcribed over other host cell genes?

Answer 20

• 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

Question 21

What are the different products of transcription of the HIV-1 genome?

Answer 21

• 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

Question 22

How are all the viral mRNA transcripts produced by the granscription of viral DNA exported from the nucleus?

Answer 22

• 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

Question 23

How is Long genomic RNA of virus able to be exported out of the nucleus if it has introns?

Answer 23

• Long genomic RNA (full length transcript) has introns so normally wouldn’t be allowed to be exported out of nucleus
• Long genomic RNA ablr o be exported via binding of Rev protein to RRE
• This pathway is called a Crm1-dependent pathway
• This is because Crm1 binds to Rev-RRE complex and directs the viral mRNA out of the nuclear pore into the cytoplasm

Question 24

Why does the assembly of a new virus occur at the plasma membrane of the host cell?

Answer 24

• If newly assembled virus buds out of plasma membrane it has less distance to travel

Question 25

Briefly describe how a new HIV-1 virus is assembled

Answer 25

* HIV-1 virus assembled at plasma membrane * Interaction between 2 RNA genomes * These 2 RNA genomes are then surrounded by proteins and released from the plasma membrane

Question 26

How are the 2 RNA genomes able to interact with each other during assembly of a new HIV-1 virus?

Answer 26

* 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

Question 27

What types of proteins are produced from the 3 different polyproteins present within the HIV-1 genome?

Answer 27

* **Gag polyprotein** - produces structural proteins * **Pol polyprotein** - produces viral enzymes * **Env polyprotein** - produces glycoproteins

Question 28

How do you differentiate between producing Gag and producing Pol polyproteins from the translation of HIV-1 long genomic RNA?

Answer 28

* 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**

Question 29

Once produced how do the polyproteins travel to the plasma membrane?

Answer 29

* 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

Question 30

How does the newly assembled HIV-1 virus bud out of the cell?

Answer 30

* 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

Question 31

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?

Answer 31

* 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

Question 32

How are the envelope Glycoproteins added to the HIV-1 virus as it's assembled?

Answer 32

* As newly assembled HIV-1 buds off plasma membrane gp120 and gp41, which are already at membrane, get pushed through along with rest of HIV-1 and get incorporated into the newly assembled HIV-1