Tryptophan-based motifs in the LLP3 region of the HIV-1 envelope glycoprotein cytoplasmic tail direct trafficking to the endosomal recycling compartment and mediate particle incorporation.

IMPORTANCE: The HIV-1 envelope glycoprotein (Env) is an essential component of the virus and has an exceedingly long cytoplasmic tail (CT). Previous studies have suggested that trafficking signals in the CT interact with host factors to regulate the incorporation of Env into particles. One particular area of interest is termed lentiviral lytic peptide 3 (LLP3), as small deletions in this region have been shown to disrupt Env incorporation. In this study, we identify a small region within LLP3 that regulates how Env associates with cellular recycling compartments. Mutants that reduced or eliminated Env from the recycling compartment also reduced Env incorporation into particles. These findings emphasize the importance of two tryptophan motifs in LLP3 for the incorporation of Env into particles and provide additional support for the idea that the CT interacts with host recycling pathways to determine particle incorporation.

KIF16B Mediates Anterograde Transport and Modulates Lysosomal Degradation of the HIV-1 Envelope Glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) is incorporated into virions at the site of particle assembly on the plasma membrane (PM). The route taken by Env to reach the site of assembly and particle incorporation remains incompletely understood. Following initial delivery to the PM through the secretory pathway, Env is rapidly endocytosed, suggesting that recycling is required for particle incorporation. Endosomes marked by the small GTPase Rab14 have been previously shown to play a role in Env trafficking. Here, we examined the role of KIF16B, the molecular motor protein that directs outward movement of Rab14-dependent cargo, in Env trafficking. Env colocalized extensively with KIF16B+ endosomes at the cellular periphery, while expression of a motor-deficient mutant of KIF16B redistributed Env to a perinuclear location. The half-life of Env labeled at the cell surface was markedly reduced in the absence of KIF16B, while a normal half-life was restored through inhibition of lysosomal degradation. In the absence of KIF16B, Env expression on the surface of cells was reduced, leading to a reduction in Env incorporation into particles and a corresponding reduction in particle infectivity. HIV-1 replication in KIF16B knockout cells was substantially reduced compared to that in wild-type cells. These results indicated that KIF16B regulates an outward sorting step involved in Env trafficking, thereby limiting lysosomal degradation and enhancing particle incorporation. IMPORTANCE The HIV-1 envelope glycoprotein is an essential component of HIV-1 particles. The cellular pathways that contribute to incorporation of envelope into particles are not fully understood. Here, we have identified KIF16B, a motor protein that directs movement from internal compartments toward the plasma membrane, as a host factor that prevents envelope degradation and enhances particle incorporation. This is the first host motor protein identified that contributes to HIV-1 envelope incorporation and replication.

A Bivalent, Spherical Virus-Like Particle Vaccine Enhances Breadth of Immune Responses against Pathogenic Ebola Viruses in Rhesus Macaques.

The 2013-2016 Ebola outbreak in West Africa led to accelerated efforts to develop vaccines against these highly virulent viruses. A live, recombinant vesicular stomatitis virus-based vaccine has been deployed in outbreak settings and appears highly effective. Vaccines based on replication-deficient adenovirus vectors either alone or in combination with a multivalent modified vaccinia Ankara (MVA) Ebola vaccine also appear promising and are progressing in clinical evaluation. However, the ability of current live vector-based approaches to protect against multiple pathogenic species of Ebola is not yet established, and eliciting durable responses may require additional booster vaccinations. Here, we report the development of a bivalent, spherical Ebola virus-like particle (VLP) vaccine that incorporates glycoproteins (GPs) from Zaire Ebola virus (EBOV) and Sudan Ebola virus (SUDV) and is designed to extend the breadth of immunity beyond EBOV. Immunization of rabbits with bivalent Ebola VLPs produced antibodies that neutralized all four pathogenic species of Ebola viruses and elicited antibody-dependent cell-mediated cytotoxicity (ADCC) responses against EBOV and SUDV. Vaccination of rhesus macaques with bivalent VLPs generated strong humoral immune responses, including high titers of binding, as well as neutralizing antibodies and ADCC responses. VLP vaccination led to a significant increase in the frequency of Ebola GP-specific CD4 and CD8 T cell responses. These results demonstrate that a novel bivalent Ebola VLP vaccine elicits strong humoral and cellular immune responses against pathogenic Ebola viruses and support further evaluation of this approach as a potential addition to Ebola vaccine development efforts.IMPORTANCE Ebola outbreaks result in significant morbidity and mortality in affected countries. Although several leading candidate Ebola vaccines have been developed and advanced in clinical testing, additional vaccine candidates may be needed to provide protection against different Ebola species and to extend the durability of protection. A novel approach demonstrated here is to express two genetically diverse glycoproteins on a spherical core, generating a vaccine that can broaden immune responses against known pathogenic Ebola viruses. This approach provides a new method to broaden and potentially extend protective immune responses against Ebola viruses.

HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C560-649) and examined the consequences on Env trafficking and incorporation into particles. FIP1C560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane.IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes trapped inside the cell within the endosomal recycling compartment. Intracellular trapping resulted in a loss of envelope protein on released particles and a corresponding loss of infectivity. Mutations of specific trafficking motifs in the envelope protein tail prevented its trapping in the recycling compartment. These results establish that trafficking to the endosomal recycling compartment is an essential step in HIV envelope protein particle incorporation.

Siglec-1 initiates formation of the virus-containing compartment and enhances macrophage-to-T cell transmission of HIV-1.

HIV-1 particles assemble and bud from the plasma membrane of infected T lymphocytes. Infected macrophages, in contrast, accumulate particles within an apparent intracellular compartment known as the virus-containing compartment or VCC. Many aspects of the formation and function of the VCC remain unclear. Here we demonstrate that VCC formation does not actually require infection of the macrophage, but can be reproduced through the exogenous addition of non-infectious virus-like particles or infectious virions to macrophage cultures. Particles were captured by Siglec-1, a prominent cell surface lectin that attaches to gangliosides on the lipid envelope of the virus. VCCs formed within infected macrophages were readily targeted by the addition of ganglioside-containing virus-like particles to the extracellular media. Depletion of Siglec-1 from the macrophage or depletion of gangliosides from viral particles prevented particle uptake into the VCC and resulted in substantial reductions of VCC volume. Furthermore, Siglec-1-mediated virion capture and subsequent VCC formation was required for efficient trans-infection of autologous T cells. Our results help to define the nature of this intracellular compartment, arguing that it is a compartment formed by particle uptake from the periphery, and that this compartment can readily transmit virus to target T lymphocytes. Inhibiting or eliminating the VCC may be an important component of strategies to reduce HIV transmission and to eradicate HIV reservoirs.

A tyrosine-based motif in the HIV-1 envelope glycoprotein tail mediates cell-type- and Rab11-FIP1C-dependent incorporation into virions.

Lentiviruses such as HIV-1 encode envelope glycoproteins (Env) with long cytoplasmic tails (CTs) that include motifs mediating interactions with host-cell-trafficking factors. We demonstrated recently that Rab11-family interacting protein 1C (FIP1C) is required for CT-dependent incorporation of Env into HIV-1 particles. Here, we used viruses bearing targeted substitutions within CT to map the FIP1C-dependent incorporation of Env. We identified YW795 as a critical motif mediating cell-type-dependent Env incorporation. Disruption of YW795 reproduced the cell-type-dependent particle incorporation of Env that had previously been observed with large truncations of CT. A revertant virus bearing a single amino acid change near the C terminus of CT restored wild-type levels of Env incorporation, Gag-Env colocalization on the plasma membrane, and viral replication. These findings highlight the importance of YW795 in the cell-type-dependent incorporation of Env and support a model of HIV assembly in which FIP1C/RCP mediates Env trafficking to the particle assembly site.

A Putative Cyclin-binding Motif in Human SAMHD1 Contributes to Protein Phosphorylation, Localization, and Stability.

SAMHD1 (sterile α motif and HD domain-containing protein 1) is a mammalian protein that regulates intracellular dNTP levels through its hydrolysis of dNTPs. SAMHD1 functions as an important retroviral restriction factor through a mechanism relying on its dNTPase activity. We and others have reported that human SAMHD1 interacts with the cell cycle regulatory proteins cyclin A, CDK1, and CDK2, which mediates phosphorylation of SAMHD1 at threonine 592, a post-translational modification that has been implicated in abrogating SAMHD1 restriction function and ability to form stable tetramers. Utilizing co-immunoprecipitation and co-localization approaches, we show that endogenous SAMHD1 is able to interact with the cyclin A-CDK1-CDK2 complexin monocytic THP-1 cells and primary monocyte-derived macrophages. Sequence analysis of SAMHD1 identifies a putative cyclin-binding motif found in many cyclin-CDK complex substrates. Using a mutagenesis-based approach, we demonstrate that the conserved residues in the putative cyclin-binding motif are important for protein expression, protein half-life, and optimal phosphorylation of SAMHD1 at Thr592 Furthermore, we observed that SAMHD1 mutants of the cyclin-binding motif mislocalized to a nuclear compartment and had reduced ability to interact with cyclin A-CDK complexes and to form the tetramer. These findings help define the mechanisms by which SAMHD1 is phosphorylated and suggest the contribution of cyclin binding to SAMHD1 expression and stability in dividing cells.

Three-Dimensional Structural Characterization of HIV-1 Tethered to Human Cells.

UNLABELLED: Tetherin (BST2, CD317, or HM1.24) is a host cellular restriction factor that prevents the release of enveloped viruses by mechanically linking virions to the plasma membrane. The precise arrangement of tetherin molecules at the plasma membrane site of HIV-1 assembly, budding, and restriction is not well understood. To gain insight into the biophysical mechanism underlying tetherin-mediated restriction of HIV-1, we utilized cryo-electron tomography (cryo-ET) to directly visualize HIV-1 virus-like particles (VLPs) and virions tethered to human cells in three dimensions (3D). Rod-like densities that we refer to as tethers were seen connecting HIV-1 virions to each other and to the plasma membrane. Native immunogold labeling showed tetherin molecules located on HIV-1 VLPs and virions in positions similar to those of the densities observed by cryo-ET. The location of the tethers with respect to the ordered immature Gag lattice or mature conical core was random. However, tethers were not uniformly distributed on the viral membrane but rather formed clusters at sites of contact with the cell or other virions. Chains of tethered HIV-1 virions often were arranged in a linear fashion, primarily as single chains and, to a lesser degree, as branched chains. Distance measurements support the extended tetherin model, in which the coiled-coil ectodomains are oriented perpendicular with respect to the viral and plasma membranes. IMPORTANCE: Tetherin is a cellular factor that restricts HIV-1 release by directly cross-linking the virus to the host cell plasma membrane. We used cryo-electron tomography to visualize HIV-1 tethered to human cells in 3D. We determined that tetherin-restricted HIV-1 virions were physically connected to each other or to the plasma membrane by filamentous tethers that resembled rods ∼15 nm in length, which is consistent with the extended tetherin model. In addition, we found the position of the tethers to be arbitrary relative to the ordered immature Gag lattice or the mature conical cores. However, when present as multiple copies, the tethers clustered at the interface between virions. Tethered HIV-1 virions were arranged in a linear fashion, with the majority as single chains. This study advances our understanding of tetherin-mediated HIV-1 restriction by defining the spatial arrangement and orientation of tetherin molecules at sites of HIV-1 restriction.

Platelet geometry sensing spatially regulates α-granule secretion to enable matrix self-deposition.

Although the biology of platelet adhesion on subendothelial matrix after vascular injury is well characterized, how the matrix biophysical properties affect platelet physiology is unknown. Here we demonstrate that geometric orientation of the matrix itself regulates platelet α-granule secretion, a key component of platelet activation. Using protein microcontact printing, we show that platelets spread beyond the geometric constraints of fibrinogen or collagen micropatterns with <5-µm features. Interestingly, α-granule exocytosis and deposition of the α-granule contents such as fibrinogen and fibronectin were primarily observed in those areas of platelet extension beyond the matrix protein micropatterns. This enables platelets to "self-deposit" additional matrix, provide more cellular membrane to extend spreading, and reinforce platelet-platelet connections. Mechanistically, this phenomenon is mediated by actin polymerization, Rac1 activation, and αIIbß3 integrin redistribution and activation, and is attenuated in gray platelet syndrome platelets, which lack α-granules, and Wiskott-Aldrich syndrome platelets, which have cytoskeletal defects. Overall, these studies demonstrate how platelets transduce geometric cues of the underlying matrix geometry into intracellular signals to extend spreading, which endows platelets spatial flexibility when spreading onto small sites of exposed subendothelium.

ROCK1 and LIM kinase modulate retrovirus particle release and cell-cell transmission events.

UNLABELLED: The assembly and release of retroviruses from the host cells require dynamic interactions between viral structural proteins and a variety of cellular factors. It has been long speculated that the actin cytoskeleton is involved in retrovirus production, and actin and actin-related proteins are enriched in HIV-1 virions. However, the specific role of actin in retrovirus assembly and release remains unknown. Here we identified LIM kinase 1 (LIMK1) as a cellular factor regulating HIV-1 and Mason-Pfizer monkey virus (M-PMV) particle release. Depletion of LIMK1 reduced not only particle output but also virus cell-cell transmission and was rescued by LIMK1 replenishment. Depletion of the upstream LIMK1 regulator ROCK1 inhibited particle release, as did a competitive peptide inhibitor of LIMK1 activity that prevented cofilin phosphorylation. Disruption of either ROCK1 or LIMK1 led to enhanced particle accumulation on the plasma membrane as revealed by total internal reflection fluorescence microscopy (TIRFM). Electron microscopy demonstrated a block to particle release, with clusters of fully mature particles on the surface of the cells. Our studies support a model in which ROCK1- and LIMK1-regulated phosphorylation of cofilin and subsequent local disruption of dynamic actin turnover play a role in retrovirus release from host cells and in cell-cell transmission events. IMPORTANCE: Viruses often interact with the cellular cytoskeletal machinery in order to deliver their components to the site of assembly and budding. This study indicates that a key regulator of actin dynamics at the plasma membrane, LIM kinase, is important for the release of viral particles for HIV as well as for particle release by a distantly related retrovirus, Mason-Pfizer monkey virus. Moreover, disruption of LIM kinase greatly diminished the spread of HIV from cell to cell. These findings suggest that LIM kinase and its dynamic modulation of the actin cytoskeleton in the cell may be an important host factor for the production, release, and transmission of retroviruses.

Rab11-FIP1C and Rab14 direct plasma membrane sorting and particle incorporation of the HIV-1 envelope glycoprotein complex.

The incorporation of the envelope glycoprotein complex (Env) onto the developing particle is a crucial step in the HIV-1 lifecycle. The long cytoplasmic tail (CT) of Env is required for the incorporation of Env onto HIV particles in T cells and macrophages. Here we identify the Rab11a-FIP1C/RCP protein as an essential cofactor for HIV-1 Env incorporation onto particles in relevant human cells. Depletion of FIP1C reduced Env incorporation in a cytoplasmic tail-dependent manner, and was rescued by replenishment of FIP1C. FIP1C was redistributed out of the endosomal recycling complex to the plasma membrane by wild type Env protein but not by CT-truncated Env. Rab14 was required for HIV-1 Env incorporation, and FIP1C mutants incapable of binding Rab14 failed to rescue Env incorporation. Expression of FIP1C and Rab14 led to an enhancement of Env incorporation, indicating that these trafficking factors are normally limiting for CT-dependent Env incorporation onto particles. These findings support a model for HIV-1 Env incorporation in which specific targeting to the particle assembly microdomain on the plasma membrane is mediated by FIP1C and Rab14.

The tetherin/BST-2 coiled-coil ectodomain mediates plasma membrane microdomain localization and restriction of particle release.

Tetherin/BST-2 forms a proteinaceous tether that restricts the release of a number of enveloped viruses following viral budding. Tetherin is an unusual membrane glycoprotein with two membrane anchors and an extended coiled-coil ectodomain. The ectodomain itself forms an imperfect coil that may undergo conformational shifts to accommodate membrane dynamics during the budding process. The coiled-coil ectodomain is required for restriction, but precisely how it contributes to the restriction of particle release remains under investigation. In this study, mutagenesis of the ectodomain was used to further define the role of the coiled-coil ectodomain in restriction. Scanning mutagenesis throughout much of the ectodomain failed to disrupt the ability of tetherin to restrict HIV particle release, indicating a high degree of plasticity. Targeted N- and C-terminal substitutions disrupting the coiled coil led to both a loss of restriction and an alteration of subcellular distribution. Two ectodomain mutants deficient in restriction were endocytosed inefficiently, and the levels of these mutants on the cell surface were significantly enhanced. An ectodomain mutant with four targeted serine substitutions (4S) failed to cluster in membrane microdomains, was deficient in restriction of particle release, and exhibited an increase in lateral mobility on the membrane. These results suggest that the tetherin ectodomain contributes to microdomain localization and to constrained lateral mobility. We propose that focal clustering of tetherin via ectodomain interactions plays a role in restriction of particle release.

Tryptophan-based motifs in the LLP3 Region of the HIV-1 envelope glycoprotein cytoplasmic tail direct trafficking to the endosomal recycling compartment and mediate particle incorporation.

The HIV-1 envelope glycoprotein complex (Env) is incorporated into developing particles at the plasma membrane (PM). The cytoplasmic tail (CT) of Env is known to play an essential role in particle incorporation, while the exact mechanisms underlying this function of the CT remain uncertain. Upon reaching the PM, trafficking signals in the CT interact with host cell endocytic machinery, directing Env into endosomal compartments within the cell. Prior studies have suggested that Env must traffic through the endosomal recycling compartment (ERC) in order for Env to return to the plasma membrane (PM) site of particle assembly. Expression of a truncated form of the ERC-resident trafficking adaptor Rab11-Family Interacting Proteins C (FIP1C) resulted in CT-dependent sequestration of Env in the condensed ERC, preventing recycling of Env to the PM. In this work, the motifs within the CT responsible for ERC localization of Env were systematically mapped. A small deletion encompassing the N-terminal portion of LLP3 eliminated ERC localization. Site-directed mutagenesis identified two tryptophan-based motifs (WE 790-791 and WW 796-797 ) within the N-terminus of LLP3 that were essential for ERC localization of Env. Mutant viruses bearing substitutions in these motifs were deficient in Env incorporation, with a corresponding loss of particle infectivity and a significant defect in replication in a spreading infection assay. These results identify two tryptophan-based motifs at the N-terminal portion of LLP3 that mediate ERC localization and Env incorporation, providing additional supporting evidence for the importance of cellular recycling pathways in HIV-1 particle assembly. IMPORTANCE: The HIV-1 envelope glycoprotein (Env) is an essential component of the virus, and has an exceedingly long cytoplasmic tail (CT). Previous studies have suggested that trafficking signals in the CT interact with host factors to regulate the incorporation of Env into particles. One particular area of interest is termed lentiviral lytic peptide 3 (LLP3), as small deletions in this region have been shown to disrupt Env incorporation. In this study, we identify a small region within LLP3 that regulates how Env associates with cellular recycling compartments. Mutants that reduced or eliminated Env from the recycling compartment also reduced Env incorporation into particles. These findings emphasize the importance of two tryptophan motifs in LLP3 to the incorporation of Env into particles, and provide additional support for the idea that the CT interacts with host recycling pathways to determine particle incorporation.

Incorporation of the HIV-1 envelope glycoprotein into viral particles is regulated by the tubular recycling endosome in a cell type-specific manner.

The HIV-1 envelope glycoprotein (Env) is incorporated into particles during assembly on the plasma membrane (PM). Env initially reaches the PM through the secretory pathway, after which it is rapidly endocytosed via an AP-2- and clathrin-dependent mechanism. Here we show that endocytosed cell surface Env enters the tubular recycling endosome compartment (TRE). Trafficking to the TRE was dependent upon motifs within the CT previously implicated in Env recycling and particle incorporation. Depletion of TRE components MICAL-L1 or EHD1 led to defects in Env incorporation, particle infectivity, and viral replication. Remarkably, defects were limited to cell types defined as nonpermissive for incorporation of CT-deleted Env, including monocyte-derived macrophages, and not observed in 293T, HeLa, or MT-4 cells. This work identifies the TRE as an essential component of Env trafficking and particle incorporation, and provides evidence that the cell type-dependent incorporation of Env is defined by interactions with components of the TRE.

Development and optimization of a Zika virus antibody-dependent cell-mediated cytotoxicity (ADCC) assay.

Zika virus (ZIKV) has become a global public health issue due to its teratogenicity and ability to cause Guillain-Barré syndrome in adults. Although anti-ZIKV envelope protein neutralizing antibodies correlate with protection, the non-neutralizing function of ZIKV antibodies including antibody-dependent cell-mediated cytotoxicity (ADCC) is incompletely understood. To study the role of ADCC antibodies during ZIKV infections, we generated a stably transfected, dual-reporter target cell line with inducible expression of a chimeric ZIKV prM-E protein on the cell surface as the target cell for the assay. By using this assay, nine of ten serum samples from ZIKV-infected patients had >20% ADCC killing of target cells, whereas none of the 12 healthy control sera had >10% ADCC killing. We also observed a time-dependent ADCC response in 2 patients with Zika. This demonstrates that this assay can detect ZIKV ADCC with high sensitivity and specificity, which could be useful for measurement of ADCC responses to ZIKV infection or vaccination.

The ability of SAMHD1 to block HIV-1 but not SIV requires expression of MxB.

SAMHD1 is a human restriction factor known to prevent infection of macrophages, resting CD4+ T cells, and dendritic cells by HIV-1. To test the contribution of MxB to the ability of SAMHD1 to block HIV-1 infection, we created human THP-1 cell lines that were knocked out for expression of MxB, SAMHD1, or both. Interestingly, MxB depletion renders SAMHD1 ineffective against HIV-1 but not SIVmac. We observed similar results in human primary macrophages that were knockdown for the expression of MxB. To understand how MxB assists SAMHD1 restriction of HIV-1, we examined direct interaction between SAMHD1 and MxB in pull-down experiments. In addition, we investigated several properties of SAMHD1 in the absence of MxB expression, including subcellular localization, phosphorylation of the SAMHD1 residue T592, and dNTPs levels. These experiments showed that SAMHD1 restriction of HIV-1 requires expression of MxB.

Myristoylation is required for human immunodeficiency virus type 1 Gag-Gag multimerization in mammalian cells.

The Gag protein of human immunodeficiency virus type 1 directs the virion assembly process. Gag proteins must extensively multimerize during the formation of the spherical immature virion shell. In vitro, virus-like particles can be generated from Gag proteins that lack the N-terminal myristic acid modification or the nucleocapsid (NC) protein. The precise requirements for Gag-Gag multimerization under conditions present in mammalian cells, however, have not been fully elucidated. In this study, a Gag-Gag multimerization assay measuring fluorescence resonance energy transfer was employed to define the Gag domains that are essential for homomultimerization. Three essential components were identified: protein-protein interactions contributed by residues within both the N- and C-terminal domains of capsid (CA), basic residues in NC, and the presence of myristic acid. The requirement of myristic acid for multimerization was reproduced using the heterologous myristoylation sequence from v-src. Only when a leucine zipper dimerization motif was placed in the position of NC was a nonmyristoylated Gag protein able to multimerize. These results support a three-component model for Gag-Gag multimerization that includes membrane interactions mediated by the myristoylated N terminus of Gag, protein-protein interactions between CA domains, and NC-RNA interactions.

Targeted Elimination of Tumorigenic Human Pluripotent Stem Cells Using Suicide-Inducing Virus-like Particles.

Sensitization to prodrugs via transgenic expression of suicide genes is a leading strategy for the selective elimination of potentially tumorigenic human pluripotent stem cells (hPSCs) in regenerative medicine, but transgenic modification poses safety risks such as deleterious mutagenesis. We describe here an alternative method of delivering suicide-inducing molecules explicitly to hPSCs using virus-like particles (VLPs) and demonstrate its use in eliminating undifferentiated hPSCs in vitro. VLPs were engineered from Qß bacteriophage capsids to contain enhanced green fluorescent protein (EGFP) or cytosine deaminase (CD) and to simultaneously display multiple IgG-binding ZZ domains. After labeling with antibodies against the hPSC-specific surface glycan SSEA-5, EGFP-containing particles were shown to specifically bind undifferentiated cells in culture, and CD-containing particles were able to eliminate undifferentiated hPSCs with virtually no cytotoxicity to differentiated cells upon treatment with the prodrug 5-fluorocytosine.

HIV-1 decreases Nrf2/ARE activity and phagocytic function in alveolar macrophages.

Respiratory complications occur frequently in individuals living with human immunodeficiency-1 virus (HIV) infection, and there is evidence that HIV-related oxidative stress impairs alveolar macrophage immune function. We hypothesized that nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a master transcription factor that activates the antioxidant response element (ARE) and regulates antioxidant defenses, has an important role in alveolar macrophage (AMs) immune dysfunction in individuals with HIV infections. To test that hypothesis, we analyzed human monocyte-derived macrophages (MDMs) that were either infected with HIV-1 or were exposed to the HIV-related proteins gp120 and Tat ex vivo and determined that either stress affected the expression of Nrf2 and the Nrf2-ARE-dependent genes for NAD(P)H dehydrogenase, quinone 1 (NQO1) and glutamate-cysteine ligase, catalytic subunit (GCLC). We then determined that the expression of Nrf2, NQO1, and GCLC was significantly decreased in primary AMs isolated from HIV-1 transgenic rats. In parallel, treating a rat macrophage cell line (NR8383 cells) with the HIV-related proteins gp120 or Tat similarly decreased the gene and protein expression of Nrf2, NQO1, and GCLC. Further, phagocytic function was decreased in both human MDMs infected with HIV-1 and primary AMs from HIV-1 transgenic rats. Importantly, treating HIV-1-infected human MDMs or AMs from HIV-1 transgenic rats with sulforaphane (SFN, an Nrf2 activator) significantly improved their phagocytic function. The salutary effects of SFN were abrogated by silencing RNA to Nrf2 in wild-type rat macrophages. Our findings demonstrate that HIV-1 infection and exposure to HIV-1-related proteins inhibit Nrf2-ARE activity in the AMs and impair their phagocytic function. Treatments targeted at increasing Nrf2-ARE activity could, therefore, enhance lung innate immunity in people living with HIV-1.

An siRNA screen of membrane trafficking genes highlights pathways common to HIV-1 and M-PMV virus assembly and release.

The assembly and release of retroviruses from the host cells requires a coordinated series of interactions between viral structural proteins and cellular trafficking pathways. Although a number of cellular factors involved in retrovirus assembly have been identified, it is likely that retroviruses utilize additional trafficking factors to expedite their assembly and budding that have not yet been defined. We performed a screen using an siRNA library targeting host membrane trafficking genes in order to identify new host factors that contribute to retrovirus assembly or release. We utilized two retroviruses that follow very distinct assembly pathways, HIV-1 and Mason-Pfizer monkey virus (M-PMV) in order to identify host pathways that are generally applicable in retrovirus assembly versus those that are unique to HIV or M-PMV. Here we report the identification of 24 host proteins identified in the screen and subsequently validated in follow-up experiments as contributors to the assembly or release of both viruses. In addition to identifying a number of previously unsuspected individual trafficking factors, we noted multiple hits among proteins involved in modulation of the actin cytoskeleton, clathrin-mediated transport pathways, and phosphoinositide metabolism. Our study shows that distant genera of retroviruses share a number of common interaction strategies with host cell trafficking machinery, and identifies new cellular factors involved in the late stages of retroviral replication.