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1.
PLoS One ; 14(11): e0225582, 2019.
Article in English | MEDLINE | ID: mdl-31770409

ABSTRACT

The biodistribution of AAVHSC7, AAVHSC15, and AAVHSC17 following systemic delivery was assessed in cynomolgus macaques (Macaca fascicularis). Animals received a single intravenous (IV) injection of a self-complementary AAVHSC-enhanced green fluorescent protein (eGFP) vector and tissues were harvested at two weeks post-dose for anti-eGFP immunohistochemistry and vector genome analyses. IV delivery of AAVHSC vectors produced widespread distribution of eGFP staining in glial cells throughout the central nervous system, with the highest levels seen in the pons and lateral geniculate nuclei (LGN). eGFP-positive neurons were also observed throughout the central and peripheral nervous systems for all three AAVHSC vectors including brain, spinal cord, and dorsal root ganglia (DRG) with staining evident in neuronal cell bodies, axons and dendritic arborizations. Co-labeling of sections from brain, spinal cord, and DRG with anti-eGFP antibodies and cell-specific markers confirmed eGFP-staining in neurons and glia, including protoplasmic and fibrous astrocytes and oligodendrocytes. For all capsids tested, 50 to 70% of glial cells (S100-ß+) and on average 8% of neurons (NeuroTrace+) in the LGN were positive for eGFP expression. In the DRG, 45 to 62% of neurons and 8 to 12% of satellite cells were eGFP-positive for the capsids tested. eGFP staining was also observed in peripheral tissues with abundant staining in hepatocytes, skeletal- and cardio-myocytes and in acinar cells of the pancreas. Biodistribution of AAVHSC vector genomes in the central and peripheral organs generally correlated with eGFP staining and were highest in the liver for all AAVHSC vectors tested. These data demonstrate that AAVHSCs have broad tissue tropism and cross the blood-nerve and blood-brain-barriers following systemic delivery in nonhuman primates, making them suitable gene editing or gene transfer vectors for therapeutic application in human genetic diseases.


Subject(s)
Blood-Brain Barrier/metabolism , Central Nervous System/metabolism , Dependovirus/genetics , Genetic Vectors/metabolism , Administration, Intravenous , Animals , Ganglia, Spinal/metabolism , Genetic Therapy/methods , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/immunology , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Immunohistochemistry , Macaca , Neuroglia/metabolism , Neurons/metabolism , Tissue Distribution
2.
Cell Rep ; 22(9): 2493-2503, 2018 02 27.
Article in English | MEDLINE | ID: mdl-29490283

ABSTRACT

Accessory proteins of lentiviruses, such as HIV-1, target cellular restriction factors to enhance viral replication. Systematic analyses of proteins that are targeted for degradation by HIV-1 accessory proteins may provide a better understanding of viral immune evasion strategies. Here, we describe a high-throughput platform developed to study cellular protein stability in a highly parallelized matrix format. We used this approach to identify cellular targets of the HIV-1 accessory protein Vpu through arrayed coexpression with 433 interferon-stimulated genes, followed by differential fluorescent labeling and automated image analysis. Among the previously unreported Vpu targets identified by this approach, we find that the E2 ligase mediating ISG15 conjugation, UBE2L6, and the transmembrane protein PLP2 are targeted by Vpu during HIV-1 infection to facilitate late-stage replication. This study provides a framework for the systematic and high-throughput evaluation of protein stability and establishes a more comprehensive portrait of cellular Vpu targets.


Subject(s)
HIV-1/metabolism , Human Immunodeficiency Virus Proteins/metabolism , Proteolysis , Viral Regulatory and Accessory Proteins/metabolism , Antiviral Agents/metabolism , Down-Regulation , HEK293 Cells , HeLa Cells , Humans , Interferons/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Stability , Reproducibility of Results , Virion/metabolism
3.
J Virol ; 89(19): 9781-90, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26178989

ABSTRACT

UNLABELLED: The accessory HIV protein Vpu inhibits a number of cellular pathways that trigger host innate restriction mechanisms. HIV Vpu-mediated degradation of tetherin allows efficient particle release and hampers the activation of the NF-κB pathway thereby limiting the expression of proinflammatory genes. In addition, Vpu reduces cell surface expression of several cellular molecules such as newly synthesized CD4. However, the role of HIV Vpu in regulating the type 1 interferon response to viral infection by degradation of the interferon regulatory factor 3 (IRF3) has been subject of conflicting reports. We therefore systematically investigated the expression of IRF3 in primary CD4(+) T cells and macrophages infected with HIV at different time points. In addition, we also tested the ability of Vpu to interfere with innate immune signaling pathways such as the NF-κB and the IRF3 pathways. We report here that HIV Vpu failed to degrade IRF3 in infected primary cells. Moreover, we observed that HIV NL4.3 Vpu had no effect on IRF3-dependent gene expression in reporter assays. On the other hand, HIV NL4.3 Vpu downmodulated NF-κB-dependent transcription. Mutation of two serines (positions 52 and 56) involved in the binding of NL4.3 Vpu to the ßTrCP ubiquitin ligase abolishes its ability to inhibit NF-κB activity. Taken together, these results suggest that HIV Vpu regulates antiviral innate response in primary human cells by acting specifically on the NF-κB pathway. IMPORTANCE: HIV Vpu plays a pivotal role in enhancing HIV infection by counteraction of Tetherin. However, Vpu also regulates host response to HIV infection by hampering the type 1 interferon response. The molecular mechanism by which Vpu inhibits the interferon response is still controversial. Here we report that Vpu affects interferon expression by inhibiting NF-κB activity without affecting IRF3 levels or activity. These data suggest that Vpu facilitates HIV infection by regulating NF-κB transcription to levels sufficient for viral transcription while limiting cellular responses to infection.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Gene Expression Regulation/immunology , HIV-1/immunology , Human Immunodeficiency Virus Proteins/metabolism , NF-kappa B/metabolism , Signal Transduction/immunology , Viral Regulatory and Accessory Proteins/metabolism , CD4-Positive T-Lymphocytes/virology , DNA Primers/genetics , Flow Cytometry , Green Fluorescent Proteins/metabolism , HEK293 Cells , HIV-1/metabolism , Humans , Immunoblotting , Interferon Regulatory Factor-3/metabolism , Luciferases , Plasmids/genetics
4.
Cell ; 161(6): 1293-1305, 2015 Jun 04.
Article in English | MEDLINE | ID: mdl-26046437

ABSTRACT

Dendritic cells (DCs) play a critical role in the immune response to viral infection through the facilitation of cell-intrinsic antiviral activity and the activation of adaptive immunity. HIV-1 infection of DCs triggers an IRF3-dependent innate immune response, which requires the activity of cyclic GAMP synthase (cGAS). We report the results of a targeted RNAi screen utilizing primary human monocyte-derived DCs (MDDCs) to identify immune regulators that directly interface with HIV-1-encoded features to initiate this innate response. Polyglutamine binding protein 1 (PQBP1) emerged as a strong candidate through this analysis. We found that PQBP1 directly binds to reverse-transcribed HIV-1 DNA and interacts with cGAS to initiate an IRF3-dependent innate response. MDDCs derived from Renpenning syndrome patients, who harbor mutations in the PQBP1 locus, possess a severely attenuated innate immune response to HIV-1 challenge, underscoring the role of PQBP1 as a proximal innate sensor of a HIV-1 infection.


Subject(s)
Carrier Proteins/metabolism , HIV-1/immunology , Immunity, Innate , Nuclear Proteins/metabolism , Nucleotidyltransferases/metabolism , Base Sequence , Cell Line , Cerebral Palsy/immunology , DNA, Viral/genetics , DNA-Binding Proteins , HIV-1/physiology , Humans , Mental Retardation, X-Linked/immunology , Molecular Sequence Data
5.
PLoS One ; 9(5): e96687, 2014.
Article in English | MEDLINE | ID: mdl-24817247

ABSTRACT

Recent proteomic and genetic studies have aimed to identify a complete network of interactions between HIV and human proteins and genes. This HIV-human interaction network provides invaluable information as to how HIV exploits the host machinery and can be used as a starting point for further functional analyses. We integrated this network with complementary datasets of protein function and interaction to nominate human protein complexes with likely roles in viral infection. Based on our approach we identified a global map of 40 HIV-human protein complexes with putative roles in HIV infection, some of which are involved in DNA replication and repair, transcription, translation, and cytoskeletal regulation. Targeted RNAi screens were used to validate several proteins and complexes for functional impact on viral infection. Thus, our HIV-human protein complex map provides a significant resource of potential HIV-host interactions for further study.


Subject(s)
HIV Infections/metabolism , HIV-1/metabolism , Human Immunodeficiency Virus Proteins/metabolism , Protein Interaction Maps , HEK293 Cells , HIV Infections/genetics , HIV Infections/virology , HIV-1/genetics , HIV-1/physiology , Host-Pathogen Interactions , Human Immunodeficiency Virus Proteins/genetics , Humans , Jurkat Cells , Protein Binding , Protein Interaction Mapping/methods , Proteomics/methods , RNA Interference , Signal Transduction
6.
Retrovirology ; 10: 23, 2013 Feb 25.
Article in English | MEDLINE | ID: mdl-23442224

ABSTRACT

BACKGROUND: Type I interferon (IFN) treatment of some cells, including dendritic cells, macrophages and monocytic THP-1 cells, restricts HIV-1 infection and prevents viral cDNA accumulation. Sterile alpha motif and HD domain protein 1 (SAMHD1), a dGTP-regulated deoxynucleotide triphosphohydrolase, reduces HIV-1 infectivity in myeloid cells, likely by limiting dNTPs available for reverse transcription, and has been described as IFNα-inducible. Myeloid cell infection by HIV-1 is enhanced by HIV-2/SIVSM Vpx, which promotes SAMHD1 degradation, or by exogenous deoxyribonucleoside (dN) addition. FINDINGS: SAMHD1 expression was not substantially influenced by IFNα treatment of monocyte-derived macrophages or THP-1 cells. The contributions of SAMHD1 to the inhibition of HIV-1 infectivity by IFNα were assessed through the provision of Vpx, exogenous dN addition, or via RNAi-mediated SAMHD1 knock-down. Both Vpx and dN efficiently restored infection in IFNα-treated macrophages, albeit not to the levels seen with these treatments in the absence of IFNα. Similarly using differentiated THP-1 cells, the addition of Vpx or dNs, or SAMHD1 knock-down, also stimulated infection, but failing to match the levels observed without IFNα. Neither Vpx addition nor SAMHD1 knock-down reversed the IFNα-induced blocks to HIV-1 infection seen in dividing U87-MG or THP-1 cells. Therefore, altered SAMHD1 expression or function cannot account for the IFNα-induced restriction to HIV-1 infection seen in many cells and cell lines. CONCLUSION: IFNα establishes an anti-HIV-1 phenotype in many cell types, and appears to accomplish this without potentiating SAMHD1 function. We conclude that additional IFNα-induced suppressors of the early stages of HIV-1 infection await identification.


Subject(s)
HIV-1/immunology , Interferon-alpha/immunology , Macrophages/immunology , Macrophages/virology , Monomeric GTP-Binding Proteins/metabolism , Cell Line , Gene Knockdown Techniques , Humans , Monomeric GTP-Binding Proteins/genetics , Nucleotides/metabolism , SAM Domain and HD Domain-Containing Protein 1 , Viral Regulatory and Accessory Proteins/metabolism
7.
Retrovirology ; 9: 33, 2012 Jun 22.
Article in English | MEDLINE | ID: mdl-22534017

ABSTRACT

BACKGROUND: HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. RESULTS: To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3). We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6), an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. CONCLUSIONS: Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of exocyst involvement in polarized targeting for intercellular transfer of viral proteins and viruses.


Subject(s)
Vesicular Transport Proteins/metabolism , nef Gene Products, Human Immunodeficiency Virus/metabolism , Cell Line , Humans , Models, Biological , Mutant Proteins/metabolism , Protein Binding , Protein Interaction Mapping , Protein Interaction Maps , Proteomics , nef Gene Products, Human Immunodeficiency Virus/genetics , p21-Activated Kinases/metabolism
8.
Retrovirology ; 8: 64, 2011 Aug 05.
Article in English | MEDLINE | ID: mdl-21819585

ABSTRACT

BACKGROUND: The HIV-1 accessory protein Nef is an important determinant of lentiviral pathogenicity that contributes to disease progression by enhancing viral replication and other poorly understood mechanisms. Nef mediates diverse functions including downmodulation of cell surface CD4 and MHC Class I, enhancement of viral infectivity, and enhancement of T cell activation. Nef interacts with a multiprotein signaling complex that includes Src family kinases, Vav1, CDC42, and activated PAK2 (p21-activated kinase 2). Although previous studies have attempted to identify a biological role for the Nef-PAK2 signaling complex, the importance of this complex and its constituent proteins in Nef function remains unclear. RESULTS: Here, we show that Nef mutants defective for PAK2-association, but functional for CD4 and MHC Class I downmodulation and infectivity enhancement, are also defective for the ability to enhance viral replication in primary T cells that are infected and subsequently activated by sub-maximal stimuli (1 µg/ml PHA-P). In contrast, these Nef mutants had little or no effect on HIV-1 replication in T cells activated by stronger stimuli (2 µg/ml PHA-P or anti-CD3/CD28-coated beads). Viruses bearing wild-type Nefs, but not Nef mutants defective for PAK2 association, enhanced NFAT and IL2 receptor promoter activity in Jurkat cells. Moreover, expression of wild-type Nefs, but not mutant Nefs defective for PAK2 association, was sufficient to enhance responsiveness of primary CD4 and CD8 T cells to activating stimuli in Nef-expressing and bystander cells. siRNA knockdown of PAK2 in Jurkat cells reduced NFAT activation induced by anti-CD3/CD28 stimulation both in the presence and absence of Nef, and expression of a PAK2 dominant mutant inhibited Nef-mediated enhancement of CD25 expression. CONCLUSION: Nef-mediated enhancement of cellular activation and viral replication in primary T cells is dependent on PAK2 and on the strength of the activating stimuli, and correlates with the ability of Nef to associate with PAK2. PAK2 is likely to play a role in Nef-mediated enhancement of viral replication and immune activation in vivo.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , HIV Infections/enzymology , HIV-1/physiology , Virus Replication , nef Gene Products, Human Immunodeficiency Virus/metabolism , p21-Activated Kinases/metabolism , CD4-Positive T-Lymphocytes/virology , Cell Line , Cells, Cultured , HIV Infections/genetics , HIV Infections/immunology , HIV Infections/virology , HIV-1/genetics , HIV-1/immunology , Humans , Lymphocyte Activation , Protein Binding , nef Gene Products, Human Immunodeficiency Virus/genetics , nef Gene Products, Human Immunodeficiency Virus/immunology , p21-Activated Kinases/genetics , p21-Activated Kinases/immunology
9.
AIDS Res Hum Retroviruses ; 26(4): 495-500, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20377428

ABSTRACT

Human immunodeficiency virus type 1 (HIV) infection of the central nervous system frequently causes HIV-associated neurocognitive disorders (HAND). The role of HIV Nef and other accessory proteins in HAND pathogenesis is unclear. To determine whether HIV nef undergoes adaptive selection in brain, we cloned 100 nef sequences (n = 30 brain and n = 70 lymphoid) from four patients with AIDS and HIV-associated dementia (HAD). Normalized nonsynonymous substitutions were more frequent at the divergence of lymphoid and brain sequences, indicating stronger adaptive selection in brain compared to lymphoid tissue. Brain-specific nonsynonymous substitutions were found within an NH(3)-terminal CTL epitope, the PACS1 binding motif, or positions predicted to be important for activation of the myeloid-restricted Src family tyrosine kinase Hck. These results suggest that adaptive selection of HIV nef in brain may reflect altered requirements for efficient replication in macrophages and brain-specific immune selection pressures.


Subject(s)
AIDS Dementia Complex/virology , Brain/virology , Evolution, Molecular , HIV-1/physiology , Lymphocytes/virology , nef Gene Products, Human Immunodeficiency Virus/genetics , Amino Acid Sequence , Amino Acid Substitution , Genetic Variation , Humans , Molecular Sequence Data , Protein Structure, Tertiary/genetics , Selection, Genetic , Virus Replication , nef Gene Products, Human Immunodeficiency Virus/chemistry
10.
Retrovirology ; 5: 42, 2008 May 29.
Article in English | MEDLINE | ID: mdl-18510766

ABSTRACT

AIDS-associated, CCR5-tropic (R5) HIV-1 clones, isolated from a patient that never developed CXCR4-tropic HIV-1, replicate to a greater extent and cause greater cytopathic effects than R5 HIV-1 clones isolated before the onset of AIDS. Previously, we showed that HIV-1 Env substantially contributed to the enhanced replication of an AIDS clone. In order to determine if Nef makes a similar contribution, we cloned and phenotypically analyzed nef genes from a series of patient ACH142 derived R5 HIV-1 clones. The AIDS-associated Nef contains a series of residues found in Nef proteins from progressors 1. In contrast to other reports 123, this AIDS-associated Nef downmodulated MHC-I to a greater extent and CD4 less than pre-AIDS Nef proteins. Additionally, all Nef proteins enhanced infectivity similarly in a single round of replication. Combined with our previous study, these data show that evolution of the HIV-1 env gene, but not the nef gene, within patient ACH142 significantly contributed to the enhanced replication and cytopathic effects of the AIDS-associated R5 HIV-1 clone.


Subject(s)
HIV Infections/virology , HIV-1/physiology , Virus Replication , nef Gene Products, Human Immunodeficiency Virus/physiology , Amino Acid Sequence , CD4 Antigens/biosynthesis , Cloning, Molecular , Cytopathogenic Effect, Viral , Down-Regulation , HIV-1/genetics , HIV-1/growth & development , HIV-1/pathogenicity , Histocompatibility Antigens Class I/biosynthesis , Humans , Molecular Sequence Data , Sequence Alignment , nef Gene Products, Human Immunodeficiency Virus/genetics
11.
Virology ; 358(1): 23-38, 2007 Feb 05.
Article in English | MEDLINE | ID: mdl-16999983

ABSTRACT

Late stage AIDS associated CCR5 tropic HIV-1 clones (R5-AIDS HIV-1) exhibit greater cytopathic effects (CPE) than earlier isolates from the same patients. In this study, envelopes from a series of three biological clones derived from the same patient were evaluated as a cytopathic determinant of R5-AIDS HIV-1 for thymocytes. In a single round of replication in thymocytes, the AIDS associated clone mediated greater initiation of reverse transcription. This enhancement was not due to broadened coreceptor tropism, as all clones studied were exclusively R5 tropic. The full-length R5-AIDS env mediated greater infectivity than R5 pre-AIDS env when used to pseudotype a reporter virus. R5-AIDS env pseudotypes were more resistant to TAK-779 and showed more rapid infection kinetics but similar resistance to a CD4 blocking mAb. We conclude that the enhanced thymic replication and CPE shown by the R5-AIDS clone is due to enhanced efficiency of Env-mediated entry via CCR5.


Subject(s)
Cytopathogenic Effect, Viral , HIV Envelope Protein gp120/physiology , HIV Envelope Protein gp41/physiology , HIV-1/genetics , HIV-1/pathogenicity , Virus Replication/physiology , Amides/pharmacology , Anti-HIV Agents/pharmacology , Antibodies, Monoclonal/immunology , CCR5 Receptor Antagonists , Cell Line , Cells, Cultured , Genes, env , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp41/genetics , Humans , Mutation , Organ Culture Techniques , Quaternary Ammonium Compounds/pharmacology , Receptors, CCR5/physiology , Reverse Transcription , Thymus Gland/virology , Viral Load
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