JW - HIV Flashcards
Describe the life cycle of HIV-1 (7)
1) Mature virion enters via fusion with the plasma membrane
2) Capsid is released in to the cytoplasm of the host cell
3) Capsid dissassembles itself and releases RNA
4) Reverse transcriptase acts on RNA and allows for integration it into the host DNA
5) Viral RNA is transcribed and put back into translation to make Gag protein
6) Transported via host mechanism to plasma membrane and forms immature virions outside the cell via budding
7) Process is repeated as immature virion → mature viron via maturation
Assembly & maturation of HIV-1
(2)
- Assembly of unprocessed GAG into the immature virion (non-infectious)
- HIV protease cleaves Gag into domains MA, CA, NC causing maturation of the virus particle (infectious)
What are the main components and functions of the Gag protein?
GAG is a modular protein
MA - Self-trimer
NCA - Self hexamer
CCA - Self dimer
NC - RNA
What are 2 features of the Matrix (MA) region?
- Forms trimers
- Associates with the membrane via myristoyl modification
What is seen in the NMR structure of Matrix (MA)? (3)
- Overlay of 20 structures
- In the helical core all structures overlay well, but N- and C-termini show large variations
- No constraints means structure is not defined (see N- and C-termini above)
What does comparison of X-ray and NMR structures of MA determine?
Final Structure
- Difference around Ser72 because in the crystal MA forms trimers (MAA,MAB,MAC) and the region around Ser72 is in the trimer interface
What are 3 general features of the differences between solution and crystal structures?
1) Disordered regions of proteins do no diffract and hence x-ray structure not defined (e.g. here N and C-termini)
2) Core structures agree well between x-ray and solution state
3) Loop regions may be different
- because of packing interactions in crystal ( eg ~Ser72)
- because in solution these regions are flexible (Ile19-Gln28)
What 2 observations are made about the capsid region based on its structure?
- After cleavage of matrix domain (mature form of capsid) N-terminal loop of capsid (yellow) folds back on itself as shown here.
- In immature GAG (when matrix and capsid are covalently linked) the N-terminus of capsid is extended and forms a flexible linkage with the matrix domain
What binds viral RNA
Nucleocapsid (NC)
Describe the interdomain linkage regions?
MA to NCA: flexible linker
NCA to CCA: flexible linker
SP1 region: under investigation:
- some measurements suggest single helical structure
- others suggest flexible
SP2 and p6: flexible regions (with recognition sites for host proteins)
What is used to determine the shape of full length GAG protein>
X-ray scattering
Immature viral particle vs Mature viral particle and the interfaces in each
“Immature” viral particle
- Overall shape is spherical
interfaces:
MA trimer; NCA hexamer, CCA dimer
NCA hexamers are only partially ordered
“Mature” viral particle (capsid only)
* Overall shape is a cone (fullerene cage)
Interfaces:
NCA hexamer (some pentamers & some heptamers)
CCA dimers
Is viral assembly homogenous?
Viral Cores obtained from virally infected cells have variable morphology
- Hence HIV assembly is not homogeneous
Can we interfere with assembly? (2)
Inhibition of assembly would render virus non-infectious.
- Target common assembly interface in both mature and immature viral particles
- CCA domain good candidate
Forms dimers in mature and immature viral particles
What is the action of Capsid Assembly Inhibitor (CAI)?
Shown to inhibit HIV-1 assembly in vitro (two step inhibitor):
- Immature, spherical particles
- Mature, tubular particles
