scholarly journals Retention of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein in the Endoplasmic Reticulum Does Not Redirect Virus Assembly from the Plasma Membrane

1998 ◽  
Vol 72 (9) ◽  
pp. 7523-7531 ◽  
Author(s):  
Karl Salzwedel ◽  
John T. West ◽  
Mark J. Mulligan ◽  
Eric Hunter
Keyword(s):  
Human Immunodeficiency Virus ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Envelope Glycoprotein ◽  
Virus Assembly ◽  
Immunofluorescent Staining ◽  
Immunodeficiency Virus

ABSTRACT The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) has been shown to redirect the site of virus assembly in polarized epithelial cells. To test whether localization of the glycoprotein exclusively to the endoplasmic reticulum (ER) could redirect virus assembly to that organelle in nonpolarized cells, an ER -retrieval signal was engineered into an epitope-tagged variant of Env. The epitope tag, attached to the C terminus of Env, did not affect the normal maturation and transport of the glycoprotein or the incorporation of Env into virions. The epitope-tagged Env was also capable of mediating syncytium formation and virus entry with a similar efficiency to that of wild-type Env. When the epitope was modified to contain a consensus K(X)KXX ER retrieval signal, however, the glycoprotein was no longer proteolytically processed into its surface and transmembrane subunits and Env could not be detected at the cell surface by biotinylation. Endoglycosidase H analysis revealed that the modified Env was not transported to the Golgi apparatus. Immunofluorescent staining patterns were also consistent with the exclusion of Env from the Golgi. As expected, cells expressing the modified Env failed to form syncytia with CD4+ permissive cells. Despite this tight localization of Env to the ER, when the modified Env was expressed in the context of virus, virions continued to be produced efficiently from the plasma membrane of transfected cells. However, these virions contained no detectable glycoprotein and were noninfectious. Electron microscopy revealed virus budding from the plasma membrane of these cells, but no virus was seen assembling at the ER membrane and no assembled virions were found within the cell. These results suggest that the accumulation of Env in an intracellular compartment is not sufficient to redirect the assembly of HIV Gag in nonpolarized cells.

scholarly journals Opposing Effects of Human Immunodeficiency Virus Type 1 Matrix Mutations Support a Myristyl Switch Model of Gag Membrane Targeting

1999 ◽  
Vol 73 (4) ◽  
pp. 2604-2612 ◽  
Author(s):  
Jean-Christophe Paillart ◽  
Heinrich G. Göttlinger
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Virus Assembly ◽  
Membrane Binding ◽  
Single Amino Acid ◽  
Immunodeficiency Virus ◽  
N Terminus

ABSTRACT Targeting of the human immunodeficiency virus type 1 (HIV-1) Gag precursor Pr55 gag to the plasma membrane, the site of virus assembly, is primarily mediated by the N-terminal matrix (MA) domain. N-myristylation of MA is essential for the stable association of Pr55 gag with membranes and for virus assembly. We now show that single amino acid substitutions near the N terminus of MA can dramatically impair assembly without compromising myristylation. Subcellular fractionation demonstrated that Gag membrane binding was compromised to a similar extent as in the absence of the myristyl acceptor site, indicating that the myristyl group was not available for membrane insertion. Remarkably, the effects of the N-terminal modifications could be completely suppressed by second-site mutations in the globular core of MA. The compensatory mutations enhanced Gag membrane binding and increased viral particle yields above wild-type levels, consistent with an increase in the exposure of the myristyl group. Our results support a model in which the compact globular core of MA sequesters the myristyl group to prevent aberrant binding to intracellular membranes, while the N terminus is critical to allow the controlled exposure of the myristyl group for insertion into the plasma membrane.

scholarly journals Vpu and Tsg101 Regulate Intracellular Targeting of the Human Immunodeficiency Virus Type 1 Core Protein Precursor Pr55gag

2006 ◽  
Vol 80 (8) ◽  
pp. 3765-3772 ◽  
Author(s):  
Kirsi Harila ◽  
Ian Prior ◽  
Mathilda Sjöberg ◽  
Antti Salminen ◽  
Jorma Hinkula ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Core Protein ◽  
Virus Assembly ◽  
Host Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Assembly of human immunodeficiency virus type 1 (HIV-1) is directed by the viral core protein Pr55 gag . Depending on the cell type, Pr55 gag accumulates either at the plasma membrane or on late endosomes/multivesicular bodies. Intracellular localization of Pr55 gag determines the site of virus assembly, but molecular mechanisms that define cell surface or endosomal targeting of Pr55 gag are poorly characterized. We have analyzed targeting of newly synthesized Pr55 gag in HeLa H1 cells by pulse-chase studies and subcellular fractionations. Our results indicated that Pr55 gag was inserted into the plasma membrane and, when coexpressed with the viral accessory protein Vpu, Pr55 gag remained at the plasma membrane and virions assembled at this site. In contrast, Pr55 gag expressed in the absence of Vpu was initially inserted into the plasma membrane, but subsequently endocytosed, and virus assembly was partially shifted to internal membranes. This endocytosis of Pr55 gag required the host protein Tsg101. These results identified a previously unknown role for Vpu and Tsg101 as regulators for the endocytic uptake of Pr55 gag and suggested that the site of HIV-1 assembly is determined by factors that regulate the endocytosis of Pr55 gag .

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