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

被引:58
作者
Lee, YM [1 ]
Liu, BD [1 ]
Yu, XF [1 ]
机构
[1] Johns Hopkins Univ, Sch Hyg & Publ Hlth, Dept Mol Microbiol & Immunol, Baltimore, MD 21205 USA
关键词
D O I
10.1128/JVI.73.7.5654-5662.1999
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
We have previously identified two distinct forms of putative viral assembly intermediate complexes, a detergent-resistant complex (DRC) and a detergent-sensitive complex (DSC), in human immunodeficiency virus type 1 (HIV-1)-infected 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 assembly intermediate complexes was investigated by use of a newly developed method of subcellular fractionation, In wild-type HN-l-infected H9 cells, the DRC fractionated with the soluble cytoplasmic fraction, whereas the DSC was 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 membrane fraction, Furthermore, HIV-1 Gag proteins synthesized in vitro in a rabbit 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 not altered by treatment with the nonionic detergent Triton X-100, suggesting 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 DRC. The Gag precursor proteins in the DRC were more sensitive to trypsin digestion 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 requires 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 conformational change might occur in the DRC either before or after its association with the plasma membrane, and this change is followed by the detection of virus budding structure at the plasma membrane.
引用
收藏
页码:5654 / 5662
页数:9
相关论文
共 46 条
[1]   FUNCTIONAL CHIMERAS OF THE ROUS-SARCOMA VIRUS AND HUMAN-IMMUNODEFICIENCY-VIRUS GAG PROTEINS [J].
BENNETT, RP ;
NELLE, TD ;
WILLS, JW .
JOURNAL OF VIROLOGY, 1993, 67 (11) :6487-6498
[2]   Importance of basic residues in the nucleocapsid sequence for retrovirus Gag assembly and complementation rescue [J].
Bowzard, JB ;
Bennett, RP ;
Krisina, NK ;
Ernst, SM ;
Rein, A ;
Wills, JW .
JOURNAL OF VIROLOGY, 1998, 72 (11) :9034-9044
[3]   MYRISTOYLATION-DEPENDENT REPLICATION AND ASSEMBLY OF HUMAN IMMUNODEFICIENCY VIRUS-1 [J].
BRYANT, M ;
RATNER, L .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (02) :523-527
[4]   In vitro assembly of virus-like particles with Rous sarcoma virus Gag deletion mutants: Identification of the p10 domain as a morphological determinant in the formation of spherical particles [J].
Campbell, S ;
Vogt, VM .
JOURNAL OF VIROLOGY, 1997, 71 (06) :4425-4435
[5]   SELF-ASSEMBLY IN-VITRO OF PURIFIED CA-NC PROTEINS FROM ROUS-SARCOMA VIRUS AND HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 [J].
CAMPBELL, S ;
VOGT, VM .
JOURNAL OF VIROLOGY, 1995, 69 (10) :6487-6497
[6]   The role of nucleocapsid of HIV-1 in virus assembly [J].
Dawson, L ;
Yu, XF .
VIROLOGY, 1998, 251 (01) :141-157
[7]   FOLDING, INTERACTION WITH GRP78-BIP, ASSEMBLY, AND TRANSPORT OF THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 ENVELOPE PROTEIN [J].
EARL, PL ;
MOSS, B ;
DOMS, RW .
JOURNAL OF VIROLOGY, 1991, 65 (04) :2047-2055
[8]   ELECTROPHORETIC ANALYSIS OF MAJOR POLYPEPTIDES OF HUMAN ERYTHROCYTE MEMBRANE [J].
FAIRBANKS, G ;
STECK, TL ;
WALLACH, DFH .
BIOCHEMISTRY, 1971, 10 (13) :2606-+
[9]   SINGLE AMINO-ACID CHANGES IN THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 MATRIX PROTEIN BLOCK VIRUS PARTICLE-PRODUCTION [J].
FREED, EO ;
ORENSTEIN, JM ;
BUCKLERWHITE, AJ ;
MARTIN, MA .
JOURNAL OF VIROLOGY, 1994, 68 (08) :5311-5320
[10]   ROLE OF THE C-TERMINUS GAG PROTEIN IN HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 VIRION ASSEMBLY AND MATURATION [J].
FU, XY ;
MATSUDA, Z ;
YU, QC ;
LEE, TH ;
ESSEX, M .
JOURNAL OF GENERAL VIROLOGY, 1995, 76 :3171-3179