The pericentriolar recycling endosome plays a key role in Vpu-mediated enhancement of HIV-1 particle release

The HIV-1 accessory gene product Vpu is required for efficient viral particle release from infected human cells. The mechanism by which Vpu enhances particle assembly or release is not yet defined. Here, we identify an intracellular site that is critical for Vpu-mediated enhancement of particle release. Vpu was found to co-localize with markers for the pericentriolar recycling endosome. Expression of dominant negative mutants of Rab11a and myosin Vb that disrupt protein sorting through the recycling endosome abrogated the ability of Vpu to augment particle release. Remarkably, the effects of blocking recycling endosome function on HIV particle release were demonstrable only in human cell lines known to be responsive to Vpu, while no effect on particle release was seen in African green monkey cells. Inhibition of recycling endosome function in human cells also blocked the ability of HIV-2 envelope to enhance particle release. These studies indicate that Vpu and HIV-2 envelope glycoprotein enhance particle release via a common mechanism that requires the activity of the pericentriolar recycling endosome.     

Identification of an intracellular trafficking and assembly pathway for HIV-1 gag

Retroviral Gag proteins are membrane-bound polyproteins that are necessary and sufficient for virus-like particle (VLP) formation. It is not known how Gag traffics through the cell or how the site of particle production is determined. Here we use two techniques, biarsenical/tetracysteine (TC) labeling and release from a cycloheximide block, to follow the trafficking of newly synthesized HIV-1 Gag. Gag first appears diffusely distributed in the cytosol, accumulates in perinuclear clusters, passes transiently through a multivesicular body (MVB)-like compartment, and then travels to the plasma membrane (PM). Sequential passage of Gag through these temporal intermediates was confirmed by live cell imaging. Induction of a transient rise in cytoplasmic calcium increased the amounts of Gag, Gag assembly intermediates and VLPs in MVBs, and resulted in a dramatic increase in VLP release. These results define an intracellular trafficking pathway for HIV-1 Gag that uses perinuclear compartments and the MVB as trafficking intermediates. We propose that the regulation of Gag association with MVB-like compartments regulates the site of HIV-1 budding and particle formation.     

Plectin regulates the signaling and trafficking of the HIV-1 co-receptor CXCR4 and plays a role in HIV-1 infection

The CXC chemokine CXCL12 and its cognate receptor CXCR4 play an important role in inflammation, human immunodeficiency virus (HIV) infection and cancer metastasis. The signal transduction and intracellular trafficking of CXCR4 are involved in these functions, but the underlying mechanisms remain incompletely understood. In the present study, we demonstrated that the CXCR4 formed a complex with the cytolinker protein plectin in a ligand-dependent manner in HEK293 cells stably expressing CXCR4. The glutathione-S-transferase (GST)-CXCR4 C-terminal fusion proteins co-precipitated with the full-length and the N-terminal fragments of plectin isoform 1 but not with the N-terminal deletion mutants of plectin isoform 1, thereby suggesting an interaction between the N-terminus of plectin and the C-terminus of CXCR4. This interaction was confirmed by confocal microscopic reconstructions showing co-distribution of these two proteins in the internal vesicles after ligand-induced internalization of CXCR4 in HEK293 cells stably expressing CXCR4. Knockdown of plectin with RNA interference (RNAi) significantly inhibited ligand-dependent CXCR4 internalization and attenuated CXCR4-mediated intracellular calcium mobilization and activation of extracellular signal regulated kinase 1/2 (ERK1/2). CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 and of Jurkat T cells was inhibited by the plectin RNAi. Moreover, CXCR4 tropic HIV-1 infection in MAGI (HeLa-CD4-LTR-Gal) cells was inhibited by the RNAi of plectin. Thus, plectin appears to interact with CXCR4 and plays an important role in CXCR4 signaling and trafficking and HIV-1 infection.     

Role of myristylation in HIV-1 Gag assembly

Assembly of the human immunodeficiency virus type 1 (HIV-1) first occurs on the plasma membrane of host cells where binding is driven by strong electrostatic interactions between the N-terminal matrix (MA) domain of the structural precursor polyprotein, Gag, and the membrane. MA is also myristylated, but the exact role this modification plays is not clear. In this study, we compared the protein oligomerization and membrane binding properties of Myr(+) and Myr(-) Gag(MA) expressed in COS-1 cells. Sedimentation studies in solution showed that both the myristylated Gag precursor and the mature MA product were detected in larger complexes than their unmyristylated counterparts, and the myristylated MA protein bound liposomes with approximately 3-fold greater affinity than unmyristylated MA. Aromatic residues near the N-terminal region of the MA protein were more accessible to chymotrypsin in the unmyristylated form and, consistent with this, an epitope in the N-terminal region was more exposed. Moreover, the cyclophilin binding site in the CA domain downstream of MA was more accessible in the unmyristylated Gag protein, while the Tsg101 binding site in the C-terminal region was equally available in the unmyristylated and myristylated Gag proteins. Taken together, our results suggest that myristylation promotes assembly by inducing conformational changes and facilitating MA multimerization. This observation offers a novel role for myristylation.     

Interactions of HIV-1 Gag with assembly cofactors

HIV-1 Gag is the only protein required for retroviral particle assembly. There is evidence suggesting that phosphatidylinositol phosphate and nucleic acid are essential for viruslike particle assembly. To elucidate structural foundations of interactions of HIV-1 Gag with the assembly cofactors PI(4,5)P2 and RNA, we employed mass spectrometric protein footprinting. In particular, the NHS-biotin modification approach was used to identify the lysine residues that are exposed to the solvent in free Gag and are protected from biotinylation by direct protein-ligand or protein-protein contacts in Gag complexes with PI(4,5)P2 and/or RNA. Of 21 surface lysines readily modified in free Gag, only K30 and K32, located in the matrix domain, were strongly protected in the Gag-PI(4,5)P2 complex. Nucleic acid also protected these lysines, but only at significantly higher concentrations. In contrast, nucleic acids and not PI(4,5)P2 exhibited strong protection of two nucleocapsid domain residues: K391 and K424. In addition, K314, located in the capsid domain, was specifically protected only in the presence of both PI(4,5)P2 and nucleic acid. We suggest that concerted binding of PI(4,5)P2 and nucleic acid to the matrix and nucleocapsid domains, respectively, promotes protein-protein interactions involving capsid domains. These protein-protein interactions must be involved in virus particle assembly.     

Role of HIV-1 Gag domains in viral assembly

After entry of the human immunodeficiency virus type 1 (HIV-1) into T cells and the subsequent synthesis of viral products, viral proteins and RNA must somehow find each other in the host cells and assemble on the plasma membrane to form the budding viral particle. In this general review of HIV-1 assembly, we present a brief overview of the HIV life cycle and then discuss assembly of the HIV Gag polyprotein on RNA and membrane substrates from a biochemical perspective. The role of the domains of Gag in targeting to the plasma membrane and the role of the cellular host protein cyclophilin are also reviewed.     

Role of HIV-1 Gag domains in viral assembly

After entry of the human immunodeficiency virus type 1 (HIV-1) into T cells and the subsequent synthesis of viral products, viral proteins and RNA must somehow find each other in the host cells and assemble on the plasma membrane to form the budding viral particle. In this general review of HIV-1 assembly, we present a brief overview of the HIV life cycle and then discuss assembly of the HIV Gag polyprotein on RNA and membrane substrates from a biochemical perspective. The role of the domains of Gag in targeting to the plasma membrane and the role of the cellular host protein cyclophilin are also reviewed.     

The Phe105 loop of Alix Bro1 domain plays a key role in HIV-1 release

Alix and cellular paralogs HD-PTP and Brox contain N-terminal Bro1 domains that bind ESCRT-III CHMP4. In contrast to HD-PTP and Brox, expression of the Bro1 domain of Alix alleviates HIV-1 release defects that result from interrupted access to ESCRT. In an attempt to elucidate this functional discrepancy, we solved the crystal structures of the Bro1 domains of HD-PTP and Brox. They revealed typical boomerang folds they share with the Bro1 Alix domain. However, they each contain unique structural features that may be relevant to their specific function(s). In particular, phenylalanine residue in position 105 (Phe105) of Alix belongs to a long loop that is unique to its Bro1 domain. Concurrently, mutation of Phe105 and surrounding residues at the tip of the loop compromise the function of Alix in HIV-1 budding without affecting its interactions with Gag or CHMP4. These studies identify a new functional determinant in the Bro1 domain of Alix.     

Role of the major homology region in assembly of HIV-1 Gag

The major homology region (MHR) is a highly conserved sequence in the gag gene of all retroviruses, including HIV-1. Its role in assembly is unknown, but deletion of the motif significantly impairs membrane binding and viral particle formation. To begin characterizing this defect, we have determined the contribution of this region to the energetics of the assembly process. Intrinsic fluorescence studies were conducted to determine the change in free energy associated with membrane and RNA binding using tRNA and large unilamellar vesicles of 1-palmitoyl-2-oleoylphosphatidylserine as models. For the wild-type protein, the change in free energy was within RT [600 cal/(mol.K)] whether Gag binds first to RNA or to the membrane. Thus, the initial binding of Gag can be to either substrate, but in vivo conditions favor initial association to RNA presumably due to its higher local concentration. After establishing the pattern of assembly, we compared the binding energy of Gag(WT) versus the deletion mutant, Gag(Delta)(MHR). Gag(WT) bound to membranes with a 2-fold higher affinity than Gag(Delta)(MHR), and the binding to RNA was similar for the two proteins. Gag prebound to RNA or to membrane exhibited approximately 2-4-fold greater binding affinity than Gag(Delta)(MHR) for binding the membrane or RNA, respectively. Most importantly, the mutant was significantly impaired in its ability to self-associate on RNA or on membrane surfaces. This key role of the MHR in promoting productive protein-protein interactions was also seen in altered amounts of cleavage products and the lack of membrane-bound, RNA-containing replication intermediates in infected cells. These results suggest that Gag first binds to RNA and then assembles into a multimeric complex with a large membrane-binding face that facilitates subsequent membrane binding. Deletion of the MHR disrupts the protein-protein interactions required to complete this process.     

ADP-ribosylation factor 1 of Arabidopsis plays a critical role in intracellular trafficking and maintenance of endoplasmic reticulum morphology in Arabidopsis

ADP-ribosylation factors (Arf), a family of small GTP-binding proteins, play important roles in intracellular trafficking in animal and yeast cells. Here, we investigated the roles of two Arf homologs, Arf1 and Arf3 of Arabidopsis, in intracellular trafficking in plant cells. We generated dominant negative mutant forms of Arf 1 and Arf3 and examined their effect on trafficking of reporter proteins in protoplasts. Arf1[T31N] inhibited trafficking of H(+)-ATPase:green fluorescent protein (GFP) and sialyltransferase (ST):GFP to the plasma membrane and the Golgi apparatus. In addition, Arf1[T31N] caused relocalization of the Golgi reporter protein ST:GFP to the endoplasmic reticulum (ER). In protoplasts expressing Arf1[T31N], ST:red fluorescent protein remained in the ER, whereas H(+)-ATPase:GFP was mistargeted to another organelle. Also, expression of Arf1[T31N] in protoplasts resulted in profound changes in the morphology of the ER. The treatment of protoplasts with brefeldin A had exactly the same effect as Arf1[T31N] on various intracellular trafficking pathways. In contrast, Arf3[T31N] did not affect trafficking of any of these reporter proteins. Inhibition experiments using mutants with various domains swapped between Arf1 and Arf3 revealed that the N-terminal domain is interchangeable for trafficking inhibition. However, in addition to the T31N mutation, motifs in domains II, III, and IV of Arf1 were necessary for inhibition of trafficking of H(+)-ATPase:GFP. Together, these results strongly suggest that Arf1 plays a role in the intracellular trafficking of cargo proteins in Arabidopsis, and that Arf1 functions through a brefeldin A-sensitive factor.     

Compartmentalization directs assembly of the signal recognition particle

Many ribonucleoprotein complexes assemble stepwise in distinct cellular compartments, a process that usually involves bidirectional transport of both RNA and proteins between the nucleus and cytoplasm. The biological rationale for such complex transport steps in RNP assembly is obscure. One important example is the eukaryotic signal recognition particle (SRP), a cytoplasmic RNP consisting of one RNA and six proteins. Prior in vivo studies support an "SRP54-late" assembly model in which all SRP proteins, except SRP54, are imported from the cytoplasm to the nucleus to bind SRP RNA. This partially assembled complex is then exported to the cytoplasm where SRP54 binds and forms the SRP holocomplex. Here we show that native SRP assembly requires segregated and ordered binding by its protein components. A native ternary complex forms in vitro when SRP19 binds the SRP RNA prior to binding by SRP54, which approximates the eukaryotic cellular pathway. In contrast, the presence of SRP54 disrupts native assembly of SRP19, such that two RNA-binding loops in SRP19 misfold. These results imply that SRP54 must be sequestered during early SRP assembly steps, as apparently occurs in vivo, for proper assembly of the SRP to occur. Our findings emphasize that spatial compartmentalization provides an additional level of regulation that prevents competition among components and can function to promote native assembly of the eukaryotic SRP.     

Phospholipase D1 plays a key role in TNF-alpha signaling

The primary characteristic features of any inflammatory or infectious lesions are immune cell infiltration, cellular proliferation, and the generation of proinflammatory mediators. TNF-alpha is a potent proinflammatory and immuno-regulatory cytokine. Decades of research have been focused on the physiological/pathophysiological events triggered by TNF-alpha. However, the signaling network initiated by TNF-alpha in human leukocytes is still poorly understood. In this study, we report that TNF-alpha activates phospholipase D1 (PLD1), in a dose-dependent manner, and PLD1 is required for the activation of sphingosine kinase and cytosolic calcium signals. PLD1 is also required for NFkappaB and ERK1/2 activation in human monocytic cells. Using antisense oligonucleotides to reduce specifically the expression of PLD isozymes showed PLD1, but not PLD2, to be coupled to TNF-alpha signaling and that PLD1 is required to mediate receptor activation of sphingosine kinase and calcium transients. In addition, the coupling of TNF-alpha to activation of the phosphorylation of ERK1/2 and the activation of NFkappaB were inhibited by pretreating cells with antisense to PLD1, but not to PLD2; thus, demonstrating a specific requirement for PLD1. Furthermore, use of antisense oligonucleotides to reduce expression of PLD1 or PLD2 demonstrated that PLD1 is required for TNF-alpha-induced production of several important cytokines, such as IL-1beta, IL-5, IL-6, and IL-13, in human monocytes. These studies demonstrate the critical role of PLD1 in the intracellular signaling cascades initiated by TNF-alpha and its functional role for coordinating the signals to inflammatory responses.     

Requirement for microtubule integrity in the SOCS1-mediated intracellular dynamics of HIV-1 Gag

Suppressor of cytokine signaling 1 (SOCS1) is a recently identified host factor that positively regulates the intracellular trafficking and stability of HIV-1 Gag. We here examine the molecular mechanism by which SOCS1 regulates intercellular Gag trafficking and virus particle production. We find that SOCS1 colocalizes with Gag along the microtubule network and promotes microtubule stability. SOCS1 also increases the amount of Gag associated with microtubules. Both nocodazole treatment and the expression of the microtubule-destabilizing protein, stathmin, inhibit the enhancement of HIV-1 particle production by SOCS1. SOCS1 facilitates Gag ubiquitination and the co-expression of a dominant-negative ubiquitin significantly inhibits the association of Gag with microtubules. We thus propose that the microtubule network plays a role in SOCS1-mediated HIV-1 Gag transport and virus particle formation.

Structured summary

MINT-7014185: Gag (uniprotkb:P05888) and SOCS1 (uniprotkb:O15524) colocalize (MI:0403) by cosedimentation (MI:0027)MINT-7014239: Cullin 2 (uniprotkb:Q13617) physically interacts (MI:0218) with RelA (uniprotkb:Q04206), RBX1 (uniprotkb:P62877), SOCS1 (uniprotkb:O15524), elongin B (uniprotkb:Q15369) and elongin C (uniprotkb:Q15370) by pull-down (MI:0096)MINT-7014046: gag (uniprotkb:P05888), SOCS1 (uniprotkb:O15524) and tubulin alpha (uniprotkb:Q13748) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7014269: tubulin alpha (uniprotkb:Q13748) physically interacts (MI:0218) with Gag (uniprotkb:P05888) by anti tag coimmunoprecipitation (MI:0007)MINT-7014036: tubulin alpha (uniprotkb:Q13748) and SOCS1 (uniprotkb:O15524) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7014201: Cullin 2 (uniprotkb:Q13617) physically interacts (MI:0218) with RBX1 (uniprotkb:P62877), SOCS1 (uniprotkb:O15524), elongin B (uniprotkb:Q15369) and elongin C (uniprotkb:Q15370) by pull-down (MI:0096)MINT-7014257: Gag (uniprotkb:P05888) physically interacts (MI:0218) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7014221: Cullin 2 (uniprotkb:Q13617) physically interacts (MI:0218) with Gag (uniprotkb:P05888), elongin C (uniprotkb:Q15370), elongin B (uniprotkb:Q15369), SOCS1 (uniprotkb:O15524) and RBX1 (uniprotkb:P62877) by pull-down (MI:0096)     

Trypanosoma brucei ARF1 plays a central role in endocytosis and golgi-lysosome trafficking

The ADP ribosylation factor (Arf)1 orthologue in the divergent eukaryote Trypanosoma brucei (Tb) shares characteristics with both Arf1 and Arf6 and has a vital role in intracellular protein trafficking. TbARF1 is Golgi localized in trypanosomes but associates with the plasma membrane when expressed in human cells. Depletion of TbARF1 by RNA interference causes a major decrease in endocytosis, which correlates with Rab5 dissociation from early endosomes. Although the Golgi remains intact, parasites display enlarged flagellar pockets and intracellular flagella. An increase in active GTP-bound TbARF1 in bloodstream parasites is rapidly lethal, correlating with a defect in Golgi-to-lysosome transport. We conclude that the essential Golgi-localizing T. brucei ARF1 has a primary role in the maintenance of both post-Golgi transport and endocytosis and that it is significantly divergent from other characterized ARFs.     

The clathrin adaptor complex AP-1 binds HIV-1 and MLV Gag and facilitates their budding

Retroviral assembly is driven by Gag, and nascent viral particles escape cells by recruiting the machinery that forms intralumenal vesicles of multivesicular bodies. In this study, we show that the clathrin adaptor complex AP-1 is involved in retroviral release. The absence of AP-1mu obtained by genetic knock-out or by RNA interference reduces budding of murine leukemia virus (MLV) and HIV-1, leading to a delay of viral propagation in cell culture. In contrast, overexpression of AP-1mu enhances release of HIV-1 Gag. We show that the AP-1 complex facilitates retroviral budding through a direct interaction between the matrix and AP-1mu. Less MLV Gag is found associated with late endosomes in cells lacking AP-1, and our results suggest that AP-1 and AP-3 could function on the same pathway that leads to Gag release. In addition, we find that AP-1 interacts with Tsg101 and Nedd4.1, two cellular proteins known to be involved in HIV-1 and MLV budding. We propose that AP-1 promotes Gag release by transporting it to intracellular sites of active budding, and/or by facilitating its interactions with other cellular partners.     

The membrane-spanning domain of gp41 plays a critical role in intracellular trafficking of the HIV envelope protein

Background

Each of the pathogenic human retroviruses (HIV-1/2 and HTLV-1) has a nonhuman primate counterpart, and the presence of these retroviruses in humans results from interspecies transmission. The passage of another simian retrovirus, simian foamy virus (SFV), from apes or monkeys to humans has been reported. Mandrillus sphinx, a monkey species living in central Africa, is naturally infected with SFV. We evaluated the natural history of the virus in a free-ranging colony of mandrills and investigated possible transmission of mandrill SFV to humans.

Results

We studied 84 semi-free-ranging captive mandrills at the Primate Centre of the Centre International de Recherches Médicales de Franceville (Gabon) and 15 wild mandrills caught in various areas of the country. The presence of SFV was also evaluated in 20 people who worked closely with mandrills and other nonhuman primates. SFV infection was determined by specific serological (Western blot) and molecular (nested PCR of the integrase region in the polymerase gene) assays. Seropositivity for SFV was found in 70/84 (83%) captive and 9/15 (60%) wild-caught mandrills and in 2/20 (10%) humans. The 425-bp SFV integrase fragment was detected in peripheral blood DNA from 53 captive and 8 wild-caught mandrills and in two personnel. Sequence and phylogenetic studies demonstrated the presence of two distinct strains of mandrill SFV, one clade including SFVs from mandrills living in the northern part of Gabon and the second consisting of SFV from animals living in the south. One man who had been bitten 10 years earlier by a mandrill and another bitten 22 years earlier by a macaque were found to be SFV infected, both at the Primate Centre. The second man had a sequence close to SFVmac sequences. Comparative sequence analysis of the virus from the first man and from the mandrill showed nearly identical sequences, indicating genetic stability of SFV over time.

Conclusion

Our results show a high prevalence of SFV infection in a semi-free-ranging colony of mandrills, with the presence of two different strains. We also showed transmission of SFV from a mandrill and a macaque to humans.     

Sts1 plays a key role in targeting proteasomes to the nucleus

The evidence that nuclear proteins can be degraded by cytosolic proteasomes has received considerable experimental support. However, the presence of proteasome subunits in the nucleus also suggests that protein degradation could occur within this organelle. We determined that Sts1 can target proteasomes to the nucleus and facilitate the degradation of a nuclear protein. Specific sts1 mutants showed reduced nuclear proteasomes at the nonpermissive temperature. In contrast, high expression of Sts1 increased the levels of nuclear proteasomes. Sts1 targets proteasomes to the nucleus by interacting with Srp1, a nuclear import factor that binds nuclear localization signals. Deletion of the NLS in Sts1 prevented its interaction with Srp1 and caused proteasome mislocalization. In agreement with this observation, a mutation in Srp1 that weakened its interaction with Sts1 also reduced nuclear targeting of proteasomes. We reported that Sts1 could suppress growth and proteolytic defects of rad23Δ rpn10Δ. We show here that Sts1 suppresses a previously undetected proteasome localization defect in this mutant. Taken together, these findings explain the suppression of rad23Δ rpn10Δ by Sts1 and suggest that the degradation of nuclear substrates requires efficient proteasome localization.     

Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers

Assembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions. We show that Gag diffuses as a monomer on the subsecond timescale with severely reduced mobility. Reduction of mobility is associated with basic residues in its nucleocapsid (NC) domain, whereas capsid (CA) and matrix (MA) domains do not contribute significantly. Strikingly, another diffusive Gag species was observed on the seconds timescale that oligomerized in a concentration-dependent manner. Both NC- and CA-mediated interactions strongly assist this process. Our results reveal potential nucleation steps of cytosolic Gag fractions before membrane-assisted Gag assembly.