CD63 is not required for production of infectious human immunodeficiency virus type 1 in human macrophages.

During the assembly of human immunodeficiency virus type 1 (HIV-1) particles, the tetraspanin CD63 can be incorporated into the viral membrane. Indeed, cell surface tetraspanin microdomains that include CD63 have been proposed as sites for virus release. In addition, antibodies against CD63 can inhibit HIV infection of macrophages. In this cell type, HIV assembles into intracellularly sequestered plasma membrane domains that contain several other tetraspanins, including CD81, CD9, and CD53. CD63 is recruited to this domain following HIV infection. Together, these observations suggest that CD63 may have some function in the assembly of infectious virus particles and/or the infectivity of assembled virions. Here we have used RNA interference to knock down CD63 expression in monocyte-derived primary macrophages. We show that in the absence of CD63, HIV assembly is quantitatively comparable to that seen in CD63-expressing macrophages and that virus assembly occurs on compartments positive for CD81, CD9, and CD53. Moreover, the infectivity of macrophage-derived virus is unaffected by the loss of CD63. Together, our results indicate that at least in tissue culture, CD63 expression is not required for either the production or the infectivity of HIV-1.

In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53.

In macrophages, HIV-1 has been shown to bud into intracellular structures that contain the late endosome marker CD63. We show that these organelles are not endosomes, but an internally sequestered plasma membrane domain. Using immunofluorescence microscopy and immunoelectron microscopy, we find that HIV-1 buds into a compartment that contains the tetraspanins CD81, CD9, and CD53. On uninfected macrophages, these proteins are seen at the cell surface and in intracellular vacuole-like structures with a complex content of vesicles and interconnected membranes that lack endosome markers, including CD63. Significantly, these structures are accessible to small tracers (horseradish peroxidase or ruthenium red) applied to cells at 4 degrees C, indicating that they are connected to the cell surface. HIV assembles on, and accumulates within, these intracellular compartments. Furthermore, CD63 is recruited to the virus-containing structures and incorporated into virions. These results indicate that, in macrophages, HIV-1 exploits a previously undescribed intracellular plasma membrane domain to assemble infectious particles.

HIV interaction with endosomes in macrophages and dendritic cells.

The human immunodeficiency virus type 1 (HIV-1) is an enveloped retrovirus that undergoes assembly at specific sites in infected cells. In macrophages, at least, this assembly occurs primarily on a subset of endocytic organelles that contain some of the markers found in late endosomes or multivesicular bodies (MVBs), in particular CD63. The budding of virus particles into endosomes has many features in common with the formation of exosomes and some limited biochemical comparison of HIV-1 particles produced from macrophages with exosomes suggests that the two have similar cellular origins. Here we show that macrophages infected with HIV contain large intracellular pools of infectious virus that can be released by homogenisation of intact cells. Immunoprecipitation experiments indicate this virus has a similar complement of cellular membrane proteins to viruses that can be recovered from the extracellular medium, further suggesting that viruses released from macrophages initially bud into endosomal organelles and are then released by fusion of these organelles with the plasma membrane.

Phosphatidylinositol 4-kinasebeta is critical for functional association of rab11 with the Golgi complex.

Phosphatidylinositol 4-kinasebeta (PI4Kbeta) plays an essential role in maintaining the structural integrity of the Golgi complex. In a search for PI4Kbeta-interacting proteins, we found that PI4Kbeta specifically interacts with the GTP-bound form of the small GTPase rab11. The PI4Kbeta-rab11 interaction is of functional significance because inhibition of rab11 binding to PI4Kbeta abolished the localization of rab11 to the Golgi complex and significantly inhibited transport of vesicular stomatitis virus G protein from the Golgi complex to the plasma membrane. We propose that a novel function of PI4Kbeta is to act as a docking protein for rab11 in the Golgi complex, which is important for biosynthetic membrane transport from the Golgi complex to the plasma membrane.

Regulation of membrane transport by rab GTPases.

Membrane flow through the cell is a highly dynamic process in which intracellular compartments communicate via tubulo-vesicular structures shuttling cargo molecules to their destinations. Transport carriers are formed at a donor compartment and navigate through the cytoplasm to the target organelle, on which they subsequently dock and fuse. Many of these events are regulated by the cooperative action of monomeric rab GTPases and their effector proteins. Research in recent years resulted in the identification of many rab effectors, providing first glimpses how the GTPase switch of individual rab proteins is utilized in discrete transport steps.

Rabaptin-5alpha/rabaptin-4 serves as a linker between rab4 and gamma(1)-adaptin in membrane recycling from endosomes.

Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin-5alpha and its putative partner gamma(1)-adaptin in membrane recycling. We found that rabaptin-5alpha forms a ternary complex with the gamma(1)-sigma(1) subcomplex of AP-1, via a direct interaction with the gamma(1)-subunit. The binding site for gamma(1)-adaptin is in the hinge region of rabaptin-5alpha, which is distinct from rab4- and rab5-binding domains. Endogenous or ectopically expressed gamma(1)- adaptin localized to both the trans-Golgi network and endosomes. Co-expressed rabaptin-5alpha and gamma(1)-adaptin, however, co-localized in a rab4-dependent manner on recycling endosomes. Transfection of rabaptin-5alpha caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4-GTP acts as a scaffold for a rabaptin-5alpha- gamma(1)-adaptin complex on recycling endosomes and that interactions between rab4, rabaptin-5alpha and gamma(1)-adaptin regulate membrane recycling.