Virology Lab Quiz 3

Labs 11-14

What is forward genetics?

Known phenotype with unknown genetic driver, sequence to determine genetic differences, identify underlying gen or genetic drivers

What is reverse genetics?

Known gene or genetic difference, introduce genetic changes, determine phenotype caused by genetic change

What is needed to undergo viral reverse genetics?

• easily manipulable viral DNA

• cells capable of expressing all the components needed to generate virus particles (virions)

  • must be able to get the DNA in (transfectable)

What are the characteristics of HIV

• Positive-sense RNA genome → gag, pol, env

• Retrovirus

  • replicates through a DNA intermediate

• Enveloped with conical capsid containing two copies of the viral genome.

• Infects T cells, macrophages, dendritic cells

• Causes chronic infection in humans due to genome integration

What is the HIV-1 Replication Cycle?

1. Attachment and entry

2. RT

3. Integration

4. Gene expression

5. Assembly and release

6. Maturation

HIV-1 plasmid - pNL4-3

Mutations in RT(D186N), IN (D116N), and Env (insertion) that make any

released particles completely non-infectious

These non-infectious particles are called virus-like particles (VLPs)

Methods for Transfection

Physical methods → Electroporation, Microinjection

Chemical Methods → Cationic lipids or polymers or Ca2+ ions

We used polyethylenimine (PEI)

HIV-1 Assembly, Release, and Maturation

VLP assembly and release are driven by the Gag polyprotein which must:

• Associate with the plasma membrane.

• Assemble into a spherical, hexametric protein lattice.

• Recruit cellular factors to drive membrane scission and release

Gag must be cleaved by a protease for the morphological transform into a mature VLP

What are the four parts of HIV-1 Gag?

Matrix (MA): Facilitates Gag binding to the inner leaflet of the plasma membrane. This targets Gag to sites of virus-like particle assembly.

Capsid (CA): Facilitates the Gag-Gag interactions required for assembly. Spacer peptide 1 (SP1) also participates in these interactions.

Nucleocapsid (NC): Facilitates Gag-RNA interactions required for assembly and genome packaging.

p6: Recruits endosomal sorting complexes required for transport (ESCRT) machinery that drives the release of VLPs from the plasma membrane.

What does a SDS-PAGE do?

Method to separate protein species based on size, by using electrical charge. Creates a mass-to-charge ratio that is the same for every protein, allowing the protein to migrate through the gel based solely on size.

Purpose of membrane blocking?

We need to incubate the membrane in something with lots of protein that will

“fill-in” all of the potential non-specific binding sites with protein that won’t

be detected.

What is the full western blot process?

Why is so important to quantify the the cellular p24 (i.e. why can’t we

just compare how much p24 is found in the released VLPs?)

Factors unrelated to the efficiency with which our mutants assemble

and release may affect the number of VLPs that we quantify.

Examples?

1. Differences in transfection efficiency between mutant plasmids.

2. Differences between cell viability between wells.

3. Certain mutants may affect the expression/stability of Gag in

cells.