Formation of virus assembly intermediate complexes in the cytoplasm by wild-type and assembly-defective mutant human immunodeficiency virus type 1 and their association with membranes
Ym. Lee et al., Formation of virus assembly intermediate complexes in the cytoplasm by wild-type and assembly-defective mutant human immunodeficiency virus type 1 and their association with membranes, J VIROLOGY, 73(7), 1999, pp. 5654-5662
We have previously identified two distinct forms of putative viral assembly
intermediate complexes, a detergent-resistant complex (DRC) and a detergen
t-sensitive complex (DSC), in human immunodeficiency virus type 1 (HIV-1)-i
nfected CD4(+) T cells (Y. M. Lee and X. F. Yu, Virology 243:78-93, 1998).
In the present study, the intracellular localization of these two viral ass
embly intermediate complexes was investigated by use of a newly developed m
ethod of subcellular fractionation, In wild-type HN-l-infected H9 cells, th
e DRC fractionated with the soluble cytoplasmic fraction, whereas the DSC w
as associated with the membrane fraction. The DRC was also detected in the
cytoplasmic fraction in H9 cells expressing HIV-1 Myr- mutant Gag. However,
Little of the unmyristylated Gag and Gag-Pol proteins was found in the mem
brane fraction, Furthermore, HIV-1 Gag proteins synthesized in vitro in a r
abbit reticulocyte lysate system in the absence of exogenous lipid membrane
were able to assemble into a viral Gag complex similar to that of the DRC
identified in infected H9 cells. The density of the viral Gag complex was n
ot altered by treatment with the nonionic detergent Triton X-100, suggestin
g a lack of association of this complex with endogenous lipid. Formation of
the DRC was not significantly affected by mutations in assembly domains M
and L of the Gag protein but was drastically inhibited by a mutation in the
assembly I domain. Purified DRC could be disrupted by high-salt treatment,
suggesting electrostatic interactions are important for stabilizing the DR
C. The Gag precursor proteins in the DRC were more sensitive to trypsin dig
estion than those in the DSC. These findings suggest that HIV-1 Gag and Gag
-Pol precursors assemble into DRC in the cytoplasm, a process which require
s the protein-protein Interaction domain (I) in NCp7; subsequently, the DRC
is transported to the plasma membrane through a process mediated by the M
domain of the matrix protein. It appears that during this process, a confor
mational change might occur in the DRC either before or after its associati
on with the plasma membrane, and this change is followed by the detection o
f virus budding structure at the plasma membrane.