Viral Pathogens: Classification, Biology, Diseases I

Question 1

What is a virus?

Answer 1

An infective agent that typically consists of a nucleic acid molecule in a protein coat, is too small to be seen by light microscopy, and is able to multiply only within the living cells of a host.

Question 2

What are the essential structural features of a virus?

Answer 2

On image

Question 3

Describe the structure of viral genomes

Answer 3

On image

Question 4

What is the central dogma?

How do viruses use the central dogma?

Answer 4

DNA replication - using DNA polymerase

Transcription - DNA to RNA using RNA polymerase

Translation - RNA to protein using ribosome

Reverse transcription using reverse transcriptase

Production of viral RNA USING RNA dependant RNA polymerase

Question 5

What is the The Baltimore Classification?

Answer 5

• We group viruses into their genome classifications
• Some have DNA, some have RNA
• We have different nucleic acids, and all these nucleic acids can be transcribed into RNA, and the protein which makes new viruses itself

Question 6

Describe the structure of HIV-1

What does the virus encode?

Answer 6

• Two RNA strands encapsulated by a protein capsid
• A lipid bilayer
• Protein envelope glycoproteins on the top which mediate entry into the cell
• This structure is what we want to get out of the cell once the virus replicates.
• The virus encodes some enzymes -> integrase, reverse transcriptase and protease

Question 7

How does the structure of the HIV-1 virus interact with the genome?

Answer 7

• Nucleic acid surrounded by capsid, surrounded by lipid bilayer, surrounded by envelope glycoprotein
• To make up the capsid, there is a matrix protein which forms around the capsid. Capsid protein is the main component of the capsid.
• Nucleocapsid interacts with the RNA genome and bridges the interaction between the genome and the capsid as the virion forms
• Nucleocapsid binds to particular copies of this RNA genome

Question 8

What 3 polyproteins do retroviruses synthesise?

Answer 8

• 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)

Question 9

What does HIV-1 do with the DNA from cells?

Answer 9

• RNA comes into the cell and is reverse transcribed
• Reverse transcription gives you the DNA version and from DNA you can get transcription of the genome back into the HIV RNA but also transcription that produces all the extra proteins.

Question 10

What stages make up the viral replication cycle?

Answer 10

• Entry
• Reverse transcription
• Integration
• Gene expression
• Assembly and release

Question 11

How does HIV-1 enter the body and cells?

How does it identify specific T-cells?

Answer 11

HIV’s envelope glycoprotein interacts with specific receptors on the host cell membrane.

ENV polyprotein consists of trimer of peptide subunits- Gp120 globular head and Gp41 tail. Stick out from cell surface.

Cellular receptor for HIV is CD4- specific to human T cells.

Native trimer (HIV) binds to CD4 binding site and, to identify specific T cell, coreceptor will also bind to chemokine receptor (CCR5 or CXCR4).

6 helix bundle will fuse the two membranes together to allow the virus to enter.

Question 12

What proteins does HIV-1 require for entry into cells?

Answer 12

HIV-1 entry requires two membrane proteins: CD4 and a chemokine receptor (CCR5/CXCR4)
HIV-1 is therefore tropic for CD4 expressing cells such as helper T cells and macrophages; the loss of which results in immunodeficiency (& AIDS).

Question 13

Describe the early phases of HIV-1 infection

Answer 13

• The virus has to traverse the distance from the plasma membrane to the nuclear membrane.
• As it is traversing, it duplicates its genome which is advantageous.
• There is an uncoating step  you lose the capsid, but you still have the RNA genome covered by the enzymes and the nucleocapsid protein
• Moving down the microtubule allows for directionality.

Virus must travel from membrane to the nucleosome whilst replicating genome at same time.
Uncoating- lose capsid. Now RNA is covered by enzymes and nucleocapsid protein.
Travels down microtubule to microtubule organising complex (MTOC), allowing directionality.
Known as intracellular trafficking.
Would take years to travel by diffusion.
Molecules of capsid in virus help select the correct microtubule and destination on nuclear membrane to enter by- nuclear pore complex (NPC).
At NPC, interacts with Nup proteins that directs the virus into the nuclear space.

Question 14

What helps the virus to get into the nucleus of cells?

Answer 14

• There are molecules of capsid that come in with the virus and help select what microtubules are used and what destination on the nuclear membrane the virus takes.
• NPC (Nuclear pore complex) is the complex that the virus uses to gain entry into the nucleus  at the NPC, the virus interacts with Nup proteins and that directs the genome into its next path into the nucleus

Question 15

Describe the structure of Reverse Transcriptase

What are the subunits of RT?

What are the 3 enzymatic activities of RT?

What is the virus doing when it is moving?

Answer 15

• RT is a heterodimer of p66 and p51 subunits.
• Catalytic properties are in p66 subunit, p51 serves structural role and lacks RNAse H domain
• RT displays three distinct enzymatic activities:
• RNA-dependent DNA polymerase
• RNAse H (cleaves RNA from RNA/DNA hybrid) 3. DNA-dependent DNA polymerase
• While the virus is moving, it is converting its RNA into DNA
• This is principally caused by reverse transcriptase  it binds to RNA in the capsid  reverse transcriptase is an RNA dependent polymerase
• It has an RNAse H domain
• Then there is a DNA-dependent polymerase

Question 16

Describe the basic steps of RT

Answer 16

• At the top there is RNA with its specific structures  reverse transcriptase finds the specificity in the RNA structures and binds to it
• You then make an RNA primer
• The RNA primer then transfers to the other end of the genome and then you produce even more RNA
• We know have two RNA structures and from that, the DNA polymerase aspect of reverse transcriptase takes over and produces a DNA primer
• The DNA primer is then used to produce more DNA
• Important to realise that HIV virus reverse transcriptase has a DNA polymerase and an RNA polymerase

Question 17

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

Answer 17

• The virus integrates within the host cell genome
• The viral DNA is pasted into the host cell chromatin
• In the purple and red areas, there is a target sequence within the host cell chromatin and there are specific sequences at the end of the HIV genome
• The two target sequences find each other and the integrase protein helps the sequence to integrate into the host DNA
• Nothing is deleted from the host cell genome, only something is added

Question 18

What enzyme binds both host and viral DNA?

Answer 18

• Integrase loops the blue viral DNA around the target DNA and brings the terminal sequences (TTA) into physical contact with the viral genome

Question 19

What does intergrase do?

Answer 19

The integrase uses bivalent cations to break open the DNA and one strand at a time anneal the viral DNA to the cellular DNA

Question 20

What facilitates targeting of HIV-1 to chromatin? (INTERGRATION)

Answer 20

• LEDGF protein
• As the viruses addreses the nuclear membrane and the pore, LEDGF is bound by the virus and this brings it not only through the membrane but also allows the integrase protein to recognise specific target sequences within your host cell DNA.

Question 21

Describe the process of intergrase

Answer 21

 Uses mechanism similar to DNA repair pathway.
 DNA break repair enzymes break apart mutated DNA to allow repair in normal cell.
 Integrase brings the genomes in physical contact and then breaks open the host DNA
then binding the termini sequences of viral DNA, strand by strand.
 This process results in a provirus.

Question 22

Describe the process of transcription

How does the virus then allow itself to be preferentially transcribed?

Answer 22

• Many transcription factors can recognise the viral DNA to enable transcription in
• many different cells, etc. To ensure its replication.
• Preferential transcription.
• Virus encodes TAT protein bind TAR element in RNA genome. TAT-TAR preferentially
• brings RNA pol to the viral DNA- high affinity.
• Transcription all along genome to produce genomic RNA.
• Splicing can be used to translate the sequence into proteins.
• Singly spliced or multiply spliced.
• The virus also encodes TAT protein
• TAT binds to TAR in the genome  this interaction preferentially brings the RNA polymerase to the viral genome
• The virus uses the cellular RNA polymerase to get transcription from the genome.

Question 23

Describe the process of nuclear export

Answer 23

• REV protein produced from provirus interacts with RRE element of viral RNA.
• Utilises interaction to bring other cellular proteins to preferentially export viral RNA (both spliced and unspliced).
• Viral mRNA has introns- cell does not allow RNA containing introns without splicing so REV essential for export.

Question 24

What is the Crm1 dependant pathway?

Answer 24

Crm1 protein binds REV-RRE and dictates interaction of nuclear pore and viral RNA, as well as transport direction.

Question 25

Describe the assembly at the plasma membrane

Answer 25

• At plasma membrane so less distance to travel to bud off the cell.
• 2 copies of RNA genome and contains many intrinsic structures.
• Kissing loops of RNA can interact with each other.
• SL1 and SL4 domains.

Question 26

What are polyproteins?

Answer 26

• Translation of mRNA at ribosome.
• Ribosomal frameshift of -1 produces gag-pol protein induced by ‘slippery sequence’
• and RNA hairpin structure.
• Post-translational modification- myristoylation (acidic molecule- myristic acid- added
• to end of polyprotein and allows for binding to membrane).
• Polyproteins surround RNA genome.

Question 27

How does HIV-1 exit from the cell?

Answer 27

• Protein p6- binds Tsg101 host protein.
• Part of ESCRT machinery usually used in processes, such as cytokinesis.
• Virus hijacks the machinery to push virion from cell.
• A virion escapes cell, polyproteins are cleaved by protease to make up the capsid,
• releasing individual gag and gag-pol polyproteins.
• Gag processing matures the virions.