Biology Reference
In-Depth Information
the b-subunit of the AP-2 adaptor protein complex. The Nef-GFP protein was
also found in the perinuclear region of the cell that was hypothesized to be the
Golgi apparatus. Colocalization of Nef-GFP with components of the clathrin
coat in the CD4
ÿ
®broblast cell line indicated that colocalization does not re-
quire expression of the CD4 molecule. Analysis of a panel of chimeric mole-
cules containing mutant Nef moieties demonstrated that the N-terminal mem-
brane targeting signal cooperates with additional element(s) in the disordered
loops in the Nef molecule to colocalize the Nef protein with AP-2 adaptor
complexes at the cell margin. This localization of Nef-GFP correlated with, but
was not su½cient for, down-regulation of surface CD4. In T cells coexpressing
CD4 and Nef-GFP, CD4 at the cell surface is redistributed into a discrete pat-
tern that colocalized with that of Nef-GFP. This observation indicates that Nef
may interact with a component of the AP-2-containing coat at this location.
Welker and colleagues have shown that myristoylation and an N-terminal
cluster of basic amino acids were required for virion incorporation and plasma
membrane targeting of Nef (Welker et al., 1998). Fusion of the N-terminal
domain of Nef alone to GFP led to membrane targeting and virion incor-
poration of the label protein, thus proving the high targeting potency of the
sequence.
Human thioesterase II was demonstrated to bind to a site on HIV-1 Nef
critical for CD4 down-regulation (Cohen et al., 2000). Both HIV-1 and SIV
Nef-GFP fusion proteins were found to redistribute from cytoplasm and
plasma membrane into peroxisomes in the presence of the thioesterase II, thus
con®rming high-a½nity interaction between Nef and the enzyme.
A Nef-GFP fusion protein was used to follow microinjected cells in studies
of the interaction of Nef with p21-activated kinase 1 ( Fackler et al., 2000). The
authors have used the fact that Nef fused to GFP retains full biological activity.
Gag proteins
The Gag proteins of HIV-1 are necessary and su½cient for the assembly of
virus-like particles. The roles played by HIV-1 Gag proteins during the life
cycle are complex and involve not only assembly but also virion maturation
after particle release and early steps in virus replication. GFP fusion to the C-
terminus of full-length or truncated Gag proteins was used for studying the
interaction of the Pr55
Gag
molecule with the plasma membrane (Sandefur et al.,
1998). This interaction is an essential step in the viral life cycle. The study was
undertaken to identify a region outside of MA that determines e½cient plasma
membrane interaction of Gag polyprotein. Application of GFP tagging allowed
simultaneous and rapid subcellular localization by ¯uorescence microscopy and
quanti®cation of proteins in subcellular fractions. The authors have used a
human codon-optimized form of GFP and a vaccinia virus-T7 expression sys-
tem because of it ability to readily produce Gag retrovirus-like particles. The
study proved that membrane-targeting/binding function within MA by itself
was insu½cient to direct GFP to the plasma membrane. Extension of the Gag
