Deficiency of Niemann-Pick Type C-1 Protein Impairs Release of Human Immunodeficiency Virus Type 1 and Results in Gag Accumulation in Late Endosomal/Lysosomal Compartments

Human immunodeficiency virus type 1 (HIV-1) relies on cholesterol-laden lipid raft membrane microdomains for entry into and egress out of susceptible cells. In the present study, we examine the need for intracellular cholesterol trafficking pathways with respect to HIV-1 biogenesis using Niemann-Pick type C-1 (NPC1)-deficient (NPCD) cells, wherein these pathways are severely compromised, causing massive accumulation of cholesterol in late endosomal/lysosomal (LE/L) compartments. We have found that induction of an NPC disease-like phenotype through treatment of various cell types with the commonly used hydrophobic amine drug U18666A resulted in profound suppression of HIV-1 release. Further, NPCD Epstein-Barr virus-transformed B lymphocytes and fibroblasts from patients with NPC disease infected with a CD4-independent strain of HIV-1 or transfected with an HIV-1 proviral clone, respectively, replicated HIV-1 poorly compared to normal cells. Infection of the NPCD fibroblasts with a vesicular stomatitis virus G-pseudotyped strain of HIV-1 produced similar results, suggesting a postentry block to HIV-1 replication in these cells. Examination of these cells using confocal microscopy showed an accumulation and stabilization of Gag in LE/L compartments. Additionally, normal HIV-1 production could be restored in NPCD cells upon expression of a functional NPC1 protein, and overexpression of NPC1 increased HIV-1 release. Taken together, our findings demonstrate that intact intracellular cholesterol trafficking pathways mediated by NPC1 are needed for efficient HIV-1 production.Human immunodeficiency virus type 1 (HIV-1) is a complex retrovirus highly dependent upon a myriad of cellular mechanisms for successful virus replication. Cholesterol plays a pivotal role throughout the HIV-1 life cycle (23, 40, 41, 64). HIV-1 entry, assembly, and budding processes occur at cholesterol-enriched membrane microdomains known as lipid rafts, and depletion of cellular cholesterol markedly and specifically reduces HIV-1 particle production. Virion-associated cholesterol is required for fusion and subsequent infection of susceptible cells (41), and cholesterol-sequestering drugs, such as β-cyclodextrin, render the virus incompetent for cell entry (4, 25, 57). Therefore, intracellular cholesterol trafficking pathways that allow nascent HIV-1 particles to acquire lipids appear critical for virus replication.Recent evidence supports a critical role for cholesterol trafficking and homeostasis in viral replication, showing that the HIV-1 accessory protein Nef increases synthesis and transport of cholesterol to both lipid rafts and progeny virions and induces multiple genes involved in cholesterol synthesis (80, 88). More recent studies have revealed that binding of Nef to the ATP-binding cassette transporter A1 (ABCA1) leads to impairment of ABCA1-dependent cholesterol efflux and an accumulation of lipids within the cell (51).Mammalian cells acquire cholesterol primarily from endocytosed low-density lipoproteins (LDL). The Niemann-Pick type C-1 (NPC1) protein is well known for its role in intracellular trafficking of LDL-derived free unesterified cholesterol. Dysfunctional NPC1 activity leads to development of NPC disease, a rare, autosomal recessive, neurodegenerative disorder characterized by the massive accumulation of cholesterol and glycosphingolipids in late endosomal/lysosomal (LE/L) compartments (61). In normal cells, endocytosed LDLs are delivered to the LE/Ls, where they are hydrolyzed and free cholesterol is released. Homeostasis is achieved when cholesterol is then rapidly transported out of the LE/Ls to the plasma membrane and endoplasmic reticulum (ER) (17, 19, 42, 73, 85), or first to the trans-Golgi (TG) network (TGN) and then to the ER (76). In NPC1-deficient (NPCD) cells, the cholesterol does not exit the endocytic pathway, resulting in its accumulation within LE/L structures.In 95% of NPC patients, the disease is caused by mutations in the NPC1 gene, while the remaining 5% harbor mutations in the NPC2 gene (50, 72, 79). One of the most frequently found and extensively characterized NPC1 mutations is the I1061T mutation (37, 38, 86). This mutation results in misfolding of the NPC1 protein, leading to its degradation and causing an 85% decrease in cellular NPC1 expression (20). Cells with such low levels of functional NPC1 maintain only 38% of normal sphingomyelinase activity and have impaired cholesterol esterification and trafficking.NPC1 is a large, multispanning protein that resides in the limiting membrane of the LE and binds cholesterol via its N-terminal domain (31). While the complete physiological function of NPC1 is still unclear, NPC1 does share homology with the resistance-nodulation-division family of prokaryotic permeases and may function as a transmembrane efflux pump to transport cargos in LEs (9, 75). Other studies suggest that NPC1 might also function in vesicle-mediated pathways for cargo transportation from LEs to other intracellular sites (21, 33). Recent studies by Infante et al. have propelled forward our understanding of how NPC1 works together with NPC2, also known to bind cholesterol, to support cholesterol efflux from the LE (32). Their findings provide a basis for either of two possible models, with respect to cholesterol trafficking: (i) NPC1 binds cholesterol found within the LE and mediates either direct export or transfer to NPC2 for delivery to a cholesterol efflux transporter, such as ABCA1; or (ii) NPC2 is the first to bind cholesterol and then mediate its delivery to NPC1 for direct export or transfer to ABCA1. These recent findings underscore the highly critical role of these proteins in maintaining intracellular cholesterol homeostasis.In addition to its role in sterol trafficking, some studies suggest that the NPC pathway may be directly involved in trafficking multiple proteins from LE/L compartments. LEs act as sorting stations to deliver endocytosed molecules to L''s for degradation, while at the same time retrieving other classes of proteins and lipids for transport back to nondegradative compartments (3, 14, 15, 28, 63, 69, 78). LE compartments also serve as sorting stations for HIV-1 viral proteins and represent a major site for HIV-1 assembly and budding (7, 12, 16, 22, 24, 57, 59).The endosomal trafficking defects observed in NPCD cells extend to proteins such as IGF2/MPR, NPC1, and annexin II, all of which utilize the endosomal recycling pathway (42, 74). Electron microscopy studies have shown that within the LEs of NPCD cells these proteins are trapped in the cholesterol-enriched membrane-bound vesicular structures (47). Cholesterol and glycosphingolipid accumulation within NPCD cells appears to disrupt Rab9 GTPase function in LE-to-TGN transport, trapping Rab9-associated proteins, such as vimentin, Tip47, and the mannose-6-phosphate receptor in LEs (18, 83). Overexpression of Rab7 and Rab9 GTPases can reverse the cholesterol accumulation phenotype caused by NPCD (8, 84). These observations suggest that NPC1, directly or indirectly, plays a role in protein export from LEs. It is unknown whether NPC1 is involved in the export of HIV-1 proteins from LEs; however, the Rab9 GTPase-mediated pathway is known to be required for HIV-1 replication (53). This strongly suggests that HIV assembly will be hindered when the NPC pathway is disrupted.Given the function of NPC1 in mediating intracellular cholesterol trafficking within the LE and given the need of HIV-1 for cholesterol, NPC1 involvement in HIV-1 biogenesis is highly likely. In the present study, using cells treated with U18666A or NPCD cells, we show that impaired NPC1 function results in profound suppression of HIV-1 replication. Further, our findings demonstrate that the NPC1 protein is essential for proper trafficking of the HIV-1 Gag protein during the late stages of assembly and budding. It appears that in NPCD cells, in which cholesterol and cellular proteins accumulate in LE/L compartments, the viral Gag protein fails to traffic properly and accumulates within these compartments, resulting in decreased particle production. Our findings not only reinforce the dependence of HIV-1 on cholesterol homeostasis but also support a role for NPC1 in HIV-1 viral protein trafficking and particle release from infected cells.