Lecture 17 Retroviruses

For the final

Reverse transcription

Process where reverse transcriptase synthesizes DNA from RNA

Allows retroviruses to integrate their RNA into host’s DNA

Reverse transcriptase

The enzyme that converts RNA into DNA

Needed to convert RNA to cDNA

Heterodimer

RdRp

Why was the discovery of reverse transcription significant?

Proved that genetic information could flow the other way (RNA → DNA)

Allows us to understand retroviruses (HIV)

Can treat diseases like AIDS

Cancer research

Where do retrovirus buds form?

Plasma membrane

What is the shape of an HIV capsid?

Highly unusual conical shape

A cone

List the order of the components in the RNA genome

1) 5’ cap

2) R

3) U5, U3

4) PBS

5) Psi packaging signal

6) DLS

7) 3’ PolyA tail

Cap and PolyA tail in RNA genome

Provide stability

Similar to mRNA

R

Repeats

Aid in reverse transcription

U5 & U3

Unique regions at each end of the RNA genome

PBS

Primer binding site in RNA

Psi packaging signal

Required for gRNA incorporation into virions

DLS

Dimerization site in RNA genomes

What kind of genome do retroviruses have?

Two identical copies of ssRNA

What are the unique features of a retrovirus virion?

• Diploid genome (2 copies of (+)ssRNA)

• Reverse transcriptase

• Integrated provirus

• Host-derived envelope

• Gag protein structures

• Specific RNA sequences (R, U3, U5)

What does reverse transcriptase convert retrovirus RNA into?

cDNA

Where is the viral ribonucleoprotein complex in retroviruses?

Inside the capsid

What are differences in simple and complex retroviruses?

Simple: only have gag, pol, and env genes & rely on host cell’s machinery

Complex: Contains gag, pol, and env genes AND others; use trans-acting factors & cis-acting elements for regulation (i.e. splicing)

Components of simple retroviruses

gag

pol

env

Example of a simple retrovirus

Murine leukemia virus (MLV)

Components of complex retroviruses

gag

pol

env

Additional genes that are regulatory or accessory

What is the polarity of a retrovirus genome?

Positive sense

Two (+)ssRNAs

Nucleocapsids in retroviruses

Needed to facilitate cDNA synthesis

Integrase in retroviruses

Needed to insert cDNA into host genome

What is an example of a complex retrovirus?

Human immunodeficiency virus (HIV)

gag

Group-specific antigen

Forms viral core (matrix, capsid, and nucleocapsid)

Crucial for structure & assembly

What are the proteins in a retrovirus viral core?

1) Matrix (MA)

2) Capsid (CA)

3) Nucleocapsid (NC)

pol

Encodes the essential enzymes (RT, protease, and integrase)

Protease

Cleaves polyproteins into functional units

env

Produces surface and transmembrane glycoproteins

Forms the spikes that bind to host cell receptors/enable entry

Viral oncogenes (v-onc)

When expressed as Gag-Onc fusion proteins, they drive uncontrolled cell growth & cancer

Typical retrovirus replication cycle (using HIV as an example)

1) Fusion

2) Transport to nucleus

3) Reverse transcription

4) Nuclear import

5) Capsid uncoating

6) Integration

7) Transcription

8 ) Nuclear export

9) Translation

10) Assembly & exit

Receptor(s) of HIV

CD4 protein

Initial attachment point

Where is CD4 protein found in immune cells?

Cell surface

Coreceptor(s) of HIV

CCR5 or CXCR4

Facilitates fusion of cell & viral membranes

How does reverse transcriptase cleave RNA?

RNAse H

What does the PBS in RT’s substrate pair to?

tRNA-Lys3

What kinds of cells can simple retroviruses infect?

Dividing cells

What kinds of cells can complex retroviruses infect?

Non-dividing, differentiated cells

Macrophages

HIV latency

Stage of chronic, asymptomatic infection

Virus remains dormant inside the body w/o causing immediate symptoms

Fusion

Cellular receptors facilitate retrovirus entry into the cell via merging viral & cell membranes

Uses receptors (CD4) and coreceptors

What happens during transport to the nucleus?

Viral core is delivered into the cytoplasm

Host transport proteins

FEZ1

Host proteins involved in capsid protection & disassembly

1) Cyclophilin A

2) Pin1

3) PDZD8

What type of polymerase is found in retroviruses?

RNA-dependent DNA polymerase (RdDP)

What is the key determinant for lentivirus nuclear import?

The capsid

How does HIV uncoating occur?

Currently unknown

What is the proposed mechanism for uncoating retrovirus capsids?

Since vDNA is too long, it supposedly breaks down the capsid

How does integrase integrate cDNA into the host’s DNA?

Integrase has a 3’ end processing, 5’ strand transfer activities, and gap repair functions

Duplicates bases of repeated flanking sequences

T/F: Retrovirus vDNA integration can be reversed

False

T/F: It’s impossible to distinguish b/w integrated vDNA from host DNA

True

ART suppression

Viral replication is inhibited

HIV levels in blood drop to undetectable levels

What is TAT in HIV?

Responsive element

Secondary RNA structure at start of mRNAs

Transactivates transcription by acting on HTLV LTR

LTRs

Long terminal repeats

Pair of identical DNA sequences that flank retroviruses

Contain promoter & enhancer sequences needed for integration & transcription

Structure of LTRs

1) U3

2) R

3) U5

U3

Unique 3’ end

Contains most of regulatory elements (promoter & enhancer)

U5

Contains sequences from 5’ end of genome

What is the main function of REV/REX?

Regulate splicing of viral mRNAs

Check nuclear mRNA export before they are fully spliced

How is Gag-Pol translated?

1) Gag translated as long precursor protein

2) 5% of Gags made as Gag/Pol precursor

3) Translational read through (in MLV)

4) Fram shift (in HIV)

Long precursor proteins

Large proteins that are cleaved to make viruses and their parts (capsids/nucleocapsids, enzymes)

How does retrovirus assembly rely on polyproteins?

Gag-polyproteins direct budding of immature viral particles from cell membrane, forming the core, packaging RNA, and recruiting other viral components

Why do you need to use cocktails to treat HIV patients?

They attack the virus at multiple points and make it harder for HIV to become drug resistant

A form of ART (antiretroviral therapy)

Why is it difficult to develop an HIV vaccine?

1) It mutates rapidly, making it easy to evade host’s immune system

2) It can hide its DNA within the host’s, since retroviruses integrate their DNA within the host’s