Viral Pathogens: Classification, Biology, Diseases I

Question 1

it is very difficult to segregate viruses into…

Answer 1

into 1 paticular group of biology
-this is because they have a range of genome structures

for example, adenoviruses and influenza viruses differ greatly in their structure, pathogenesis and microbiology despite both causing significant respiratory disease in humans

Question 2

there are many different types of viral genome - give some examples:

Answer 2

Single-stranded RNA (ssRNA)

Double-stranded RNA (dsRNA)

Single-stranded DNA (ssDNA)

Double-stranded RNA (dsDNA)

all of these viral genomes are encapsulated in different ways

Question 3

what 2 shapes can DNA genomes be?

Answer 3

linear or circular

Question 4

double stranded genomes (dsDNA and dsRNA) have what characteristic?

Answer 4

double-stranded genomes have complementary base pairing

Question 5

what 2 shapes can RNA genomes be?

Answer 5

linear or segmented i.e. more than one RNA per capsid

Question 6

explain the Baltimore Classification

Answer 6

viruses being grouped together not necessarily by disease, but by the way that their genome is put together

Question 7

describe the structure of a mature HIV-1 particle

Answer 7

outer envelope of HIV consists of a lipid bilayer with protruding Env spikes (mediate entry into the cell). Inside the envelope are shells of Gag proteins

the matrix protein (MA) associates with the membrane capsid protein (CA), forming the conical capsid

Nucleocapsid (NC) coats the viral RNA genome, bridging the gap between the genome and the capsid.

The core contains two genomic RNA (+) strands, tRNALys3, and 50 copies of each viral enzyme (PR, RT, and IN).

Question 8

describe the genome organisation of retroviruses

Answer 8

retroviruses synthesise 3 poly proteins from the RNA:
1. GAG
-group specific antigen, viral core proteins
(MA, CA and NC)

2. POL
   - viral enzymes (protease (PR), integrase (IN) and reverse transcriptase)
3. ENV
   - envelope glycoprotein (gp120 SU (surface) gp41 TM (transmembrane))

ALSO there are also large areas of open reading frames, encoding regulatory/accessory proteins

Question 9

name some HIV-1 Regulatory / Accessory Proteins

Answer 9

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

needs this as its entering the human body, which has the most complex immune system

Question 10

when HIV comes into the cell what happens with its RNA?

Answer 10

RNA reverse transcribed to DNA

From DNA, you can get transcription of the genome but also transcription that produces all the proteins products.

Question 11

The retroviral replication cycle - HIV ENTRY

Answer 11

ENV glycoproteins consists of gp41 (attached to viral membrane) trimer and gp120 (big globular head) peptide subunits and is covered with glycans. These 2 interact, stick out from the protein surface, and are able to recognise cellular receptors

Interaction of envelope with receptors on host cell is specific and helps the virus get into the cell it wants.

Question 12

in the case of HIV, what is the host cell receptor the envelope glycoprotein interacts with?

Answer 12

interacts with CD4 in a protein protein interaction

CD4 is a molecule specific for human helper T cells and macrophages

In order to make sure its the correct TCR, HIV also has co receptors (ccr5 and cxcr4) which bind

formation of a 6 helix bundle formation - this is the structure of the envelope meshing the the viral and cellular membranes together. The membrane fusion allows the virus to enter the cell.

Question 13

now the virus has entered the cell, where does it go?

Answer 13

the virus has to get from cytoplasm to nucleus, and as its travelling it is also replicating its genome and converting it from RNA to DNA – uncoating step where you lose the capsid, (RNA genome is covered by enzymes and nuclear capsid protein)

moves down the microtubling organising complex - intracellular trafficking - have to use microtubules otherwise it would take AGES for the virus to get to the nucleus

capsid molecules that come in with the virus help select what microtubules are used and what destination on the nuclear membrane the virus will take - there are various degenerate pathways, but the virus only wants to select the one that will get it to the NPC complex

capsid molecule directs virus through the cytoplasm and gets it to the NPC (nuclear pore complex) – this is what the virus uses to gain entry into the nuclear space.

Question 14

what is Reverse Transcriptase>

Answer 14

A viral enzyme bound to RNA in the capsid, converts RNA to DNA whilst virus is travelling to the nucleus

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:

1. RNA-dependent DNA polymerase
2. RNAse H (cleaves RNA from RNA/DNA hybrid - don’t want these intermediates)
3. DNA-dependent DNA polymerase

Question 15

Basic steps of reverse transcription

Answer 15

RNA with RNA structures is is recognised by RT, which binds to it

RNA primer formed, which then gets transferred to the other end of the genome (don’t know why)

So, there are 2 RNA structures, and then DNA polymerase forms a DNA primer from the RNA - DNA copy of the RNA genome is produced.

Question 16

so the RT produces viral DNA - what happens to it?

Answer 16

The HIV DNA genome (provirus) is integrated into the host chromosomes via INTEGRASE

There is a target sequence within the host cell chromatin, and specific sequences at the end of the HIV genome (TTAA’s at both sides).

The LEDGF protein brings the virus (containing integrase and DNA copy of the viral genome) through the membrane and allows integrase to recognise the specific target sequences within the host cell DNA.

Target sequences find each other. Integrase loops viral DNA around the target DNA. Now they’re in physical contact, the integrase has the ability to break open the DNA 1 strand at a time and anneal the viral DNA to the cellular DNA. Flips the integrated form of DNA genome into the cell.

Nothing deleted from the host cell genome, just insertion of viral genome.

Question 17

is LEDGF a viral protein

Answer 17

no

Question 18

HIV-1 promoter contains what?

Answer 18

binding sites for transcription factors that are present in T-lymphocytes - the virus become preferentially transcribed

The virus has evolved to be recognised by a number of different transcription factors

Question 19

How does the virus allow itself to be preferentially transcribed?

Answer 19

Tat-Tar interaction - The TAR RNA binds the Tat protein

The virus utilizes the cellular RNA polymerase to get transcription from the genome. So, the virus encodes the Tat protein which binds to the Tar element within the genome.

The Tat-Tar interaction preferentially brings the RNA polymerase to the viral genome - high affinity site for RNA polymerase.

Binding of Tat protein to TAR RNA enhances elongation of RNA pol II

Question 20

what does the HIV-1 provirus generate?

Answer 20

different mRNAs for the viral proteins
-this is done through RNA splicing

provirus

primary transcript (genome and GAG/POL mRNA)
spliced ENV mRNA

polyproteins are produced, and splicing produces the env polyprotein

Question 21

how does viral RNA leave the nucleus?

Answer 21

Rev-rre interaction

The virus produces a protein (rev) that interacts with a particular viral structure (rre) and utilises the Rev-rre interaction to bring other cellular proteins along and preferentially get the RNAs out of the cell via the nuclear pore

To get RNA out of the nucleus into the cell you need specific proteins - Crm 1 is the cellular protein that binds to the rev-rre interaction and dictates the interaction of the viral genome getting out via the nuclear pore

Question 22

Dimerisation of the unspliced viral RNA allows?

Answer 22

packing of two genomes

RNA elements within the RNA interact
-kissing loop formed

Question 23

Gag-pol protein is generated by what?

Answer 23

ribosomal frameshifting induced by a ‘slippery’ sequence and an RNA hairpin structure

ribosome misreads the slippery RNA sequence and so you get different protein

Question 24

what is the mRNA for Gag and Gag-Pol proteins

Answer 24

the unspliced HIV-1 RNA

Question 25

where are viruses assembled and why?

Answer 25

viruses are assembled at the plasma membrane – this is because if the virus buds out of the plasma membrane it has less distance to travel as opposed to if it was assembled in the cytoplasm.

Question 26

Myristoylation - what is it and what is its role?

Answer 26

Myristoylation of Glycines in the MA domain of Gag mediates association with the plasma membrane

Long acidic chains that stick into the lipid bilayer

Question 27

explain how the virus is pushed out of the cell

Answer 27

Tsg101 protein - involved in pushing the virus through the membrane out of the cell

ESCRT machinery is hijacked - usually used in cytokenesis where it pushes 2 daughter cells away from each other, but here it pushes its own varion out of the cell.

Question 28

As the virus is pushed out, what happens?

Answer 28

the viral protease chews up the polyproteins into individual proteins that make up the capsid
-release of the individual proteins from
Gag and Gag-Pol polyproteins

Question 29

Gag processing generates ?

Answer 29

mature virions