Gaurav D. Gaiha

Ragon Institute of MGH, MIT and Harvard, Charlestown, Maryland, United States

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Publications (21)100.42 Total impact

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    [Show abstract] [Hide abstract] ABSTRACT: Decreased HIV-specific CD8(+) T cell proliferation is a hallmark of chronic infection, but the mechanisms of decline are unclear. We analyzed gene expression profiles from antigen-stimulated HIV-specific CD8(+) T cells from patients with controlled and uncontrolled infection and identified caspase-8 as a correlate of dysfunctional CD8(+) T cell proliferation. Caspase-8 activity was upregulated in HIV-specific CD8(+) T cells from progressors and correlated positively with disease progression and programmed cell death-1 (PD-1) expression, but negatively with proliferation. In addition, progressor cells displayed a decreased ability to upregulate membrane-associated caspase-8 activity and increased necrotic cell death following antigenic stimulation, implicating the programmed cell death pathway necroptosis. In vitro necroptosis blockade rescued HIV-specific CD8(+) T cell proliferation in progressors, as did silencing of necroptosis mediator RIPK3. Thus, chronic stimulation leading to upregulated caspase-8 activity contributes to dysfunctional HIV-specific CD8(+) T cell proliferation through activation of necroptosis and increased cell death. Copyright © 2014 Elsevier Inc. All rights reserved.
    Full-text · Article · Dec 2014 · Immunity
  • [Show abstract] [Hide abstract] ABSTRACT: RNAi screens have implicated hundreds of host proteins as HIV-1 dependency factors (HDFs). While informative, these early studies overlap poorly due to false positives and false negatives. To ameliorate these issues, we combined information from the existing HDF screens together with new screens performed with multiple orthologous RNAi reagents (MORR). In addition to being traditionally validated, the MORR screens and the historical HDF screens were quantitatively integrated by the adaptation of an established analysis program, RIGER, for the collective interpretation of each gene's phenotypic significance. False positives were addressed by the removal of poorly expressed candidates through gene expression filtering, as well as with GESS, which identifies off-target effects. This workflow produced a quantitatively integrated network of genes that modulate HIV-1 replication. We further investigated the roles of GOLGI49, SEC13, and COG in HIV-1 replication. Collectively, the MORR-RIGER method minimized the caveats of RNAi screening and improved our understanding of HIV-1-host cell interactions.
    No preview · Article · Oct 2014 · Cell Reports
  • Gaurav D Gaiha · Abraham L Brass
    [Show abstract] [Hide abstract] ABSTRACT: A cytosolic protein that senses fragments of HIV-1 DNA triggers the death of uninfected CD4 T cells.
    No preview · Article · Jan 2014 · Science
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    Dataset: Figure S1
    [Show abstract] [Hide abstract] ABSTRACT: Characterization of iMDDCs by flow cytometry. The iMDDCs were initially analysed in FACS dot plot showing size (FSC-H) and granularity (SSC-H) characteristics. A gate (R1) was used to select for immature DCs and these were further analysed for surface markers for cell type and differentiation using FITC labeled antibodies for: CD11c, CD83 and MHC II (HLA-DR). iMDDCs were positive for the DC-specific markers CD11c and HLA-DR, but not for the mature marker CD83. The dot plot and histograms are representative of the FACS screening performed before iMDDCs were used for experimentation. (TIF)
    Full-text · Dataset · Mar 2013
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    [Show abstract] [Hide abstract] ABSTRACT: Surfactant Protein D (SP-D) is an oligomerized C-type lectin molecule with immunomodulatory properties and involvement in lung surfactant homeostasis in the respiratory tract. SP-D binds to the enveloped viruses, influenza A virus and respiratory syncytial virus and inhibits their replication in vitro and in vivo. SP-D has been shown to bind to HIV via the HIV envelope protein gp120 and inhibit infectivity in vitro. Here we show that SP-D binds to different strains of HIV (BaL and IIIB) and the binding occurs at both pH 7.4 and 5.0 resembling physiological relevant pH values found in the body and the female urogenital tract, respectively. The binding of SP-D to HIV particles and gp120 was inhibited by the presence of several hexoses with mannose found to be the strongest inhibitor. Competition studies showed that soluble CD4 and CVN did not interfere with the interaction between SP-D and gp120. However, soluble recombinant DC-SIGN was shown to inhibit the binding between SP-D and gp120. SP-D agglutinated HIV and gp120 in a calcium dependent manner. SP-D inhibited the infectivity of HIV strains at both pH values of 7.4 and 5.0 in a concentration dependent manner. The inhibition of the infectivity was abolished by the presence of mannose. SP-D enhanced the binding of HIV to immature monocyte derived dendritic cells (iMDDCs) and was also found to enhance HIV capture and transfer to the T-cell like line PM1. These results suggest that SP-D can bind to and inhibit direct infection of T-cells by HIV but also enhance the transfer of infectious HIV particles from DCs to T-cells in vivo.
    Full-text · Article · Mar 2013 · PLoS ONE
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    [Show abstract] [Hide abstract] ABSTRACT: HIV-1 depends on many host factors for propagation. Other host factors, however, antagonize HIV-1 and may have profound effects on viral activation. Curing HIV-1 requires the reduction of latent viral reservoirs that remain in the face of antiretroviral therapy. Using orthologous genetic screens, we identified bromodomain containing 4 (BRD4) as a negative regulator of HIV-1 replication. Antagonism of BRD4, via RNA interference or with a small molecule inhibitor, JQ1, both increased proviral transcriptional elongation and alleviated HIV-1 latency in cell-line models. In multiple instances, JQ1, when used in combination with the NF-κB activators Prostratin or PHA, enhanced the in vitro reactivation of latent HIV-1 in primary T cells. These data are consistent with a model wherein BRD4 competes with the virus for HIV-1 dependency factors (HDFs) and suggests that combinatorial therapies that activate HDFs and antagonize HIV-1 competitive factors may be useful for curing HIV-1 infection.
    Full-text · Article · Oct 2012 · Cell Reports
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    G Gaiha · K Mckim · M Woods · M Lichterfeld · A Brass · B Wallker
    Full-text · Article · Oct 2012 · Retrovirology
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    G Gaiha · M Anahtar · E Rossin · B Walker
    Full-text · Article · Sep 2012 · Retrovirology
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    Dataset: Figure S2
    [Show abstract] [Hide abstract] ABSTRACT: IFITM3 arrests influenza A virus in acidic cytosolic inclusions preventing vRNP nuclear translocation. A) Normal diploid human fibroblasts (WI-38 cells) were stably transduced with retroviruses containing IFITM3 (WI-38 IFITM3) or (B) a non-targeting control shRNA (WI-38 shScramble). Cells were incubated with PR8 on ice, and then warm media containing LTRed (red) was added at time zero. Cells were fixed at 150 min. p.i. and stained for NP (green) and DNA, then analyzed by confocal microscopy. Image analysis software was used to define each cell's cytosolic (white lines) and nuclear peripheries (blue lines, based on DIC images and DNA staining, respectively). Images are representative of four independent experiments. (Scale bar: 12 µM). C) Quantitation of nuclear vRNP particles. The number of vRNP particles per nucleus of the WI-38 cell lines (with or without IFN treatment) at the indicated time points are shown. Values represent the mean +/− the SD of three independent experiments. D) Percent colocalization of vRNPs and LTRed compartments in WI-38 shScramble, shIFITM3 or IFITM3 expressing cells at the indicated times p.i. Values represent the mean +/− the SD of three independent experiments. E) Western blot of lysates from WI-38 cells probed with the indicated antibodies. shIFITM3-3 is referred to as shIFITM3 in the preceding figures and was selected for use based on its superior knockdown of the target protein. (PDF)
    Preview · Dataset · Oct 2011
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    Dataset: Figure S7
    [Show abstract] [Hide abstract] ABSTRACT: Fusion of viral pseudoparticles expressing HA envelope subtypes, but not a MLV envelope, is decreased by IFITM3. IFITM3 inhibits the replication of infectious H5N1 virus. A) MDCK cells stably transduced with IFITM3 or empty vector were incubated with pseudoparticles expressing N1 and HA subtypes (H1N1pp, H3N1pp, or H5N1pp). Cells were then fixed and assayed for cleavage of CCF2 using flow cytometry. These results are representative of three independent experiments. B) Chicken embryonic fibroblasts (ChEF) cells stably expressing the empty vector control or IFITM3 were incubated with pseudoparticles expressing N1 and either of the two avian influenza A viral HA subtypes, H5 or H7, as in (A). These data are representative of three independent experiments. C) ChEF cells stably transduced with the empty vector control or overexpressing IFITM3, were infected with WSN/33 for 12 h then stained for HA protein (red) and DNA (blue). Average percent infection is given for three independent experiments +/− SD. 4× magnification. D) MDCK-Vector or MDCK-IFITM3 cells were incubated with pseudoparticles expressing the amphotropic MLV envelope protein (MLVpp) and then assayed for cleavage of CCF2 using flow cytometry. These results are representative of two independent experiments. E) Infectivity of HA-expressing pseudoparticles is decreased by IFITM3. MDCK-Vector or MDCK-IFITM3 cell lines were infected with the indicated pseudoparticles for 48 h. Cells were then immunostained for expression of HIV-1 p24 protein expressed from the integrated lentiviral genomes. Percent infection is provided. These results are representative of three independent experiments. 4× magnification. F) A549 cells were stably transduced with retroviruses containing IFITM3 or empty viral vector alone, then infected with A/Vietnam/1203/04 (H5N1) influenza A virus (VN/04). After 12 h, the cells were fixed and stained for viral NP expression (green) and for DNA (blue). Values given are percentage infected cells and are representative of two independent experiments. 4× magnification. G) Western blot of lysates from A549-IFITM3 or A549-Vector cell lines probed with the indicated antibodies. H) A549 cell lines were infected with increasing amounts of H5N1 VN/04. Twelve hours after infection the cells were immunostained for NP expression and scored for infection status. Values are representative of two independent experiments. (PDF)
    Preview · Dataset · Oct 2011
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    Dataset: Figure S10
    [Show abstract] [Hide abstract] ABSTRACT: IFN treatment both expands Rab7- and IFITM3-containing structures, and increases the size and number of acidified organelles. A) Confocal images of WI-38 cells treated with buffer, IFN-α, or IFN-γ, and then immunostained for either IFITM3 (endogenous, red) or Rab7 (endogenous, green), and DNA (blue). Arrows denote larger structures staining for Rab7 and IFITM3 that were seen predominantly with IFN-γ treatment. (Scale bar: 20 µM). B) A549 cells treated with either buffer or IFN-γ, then incubated with LTRed before fixation and DNA staining (blue) followed by confocal imaging. Images in this figure are representative of three independent experiments. (PDF)
    Preview · Dataset · Oct 2011
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    Dataset: Figure S9
    [Show abstract] [Hide abstract] ABSTRACT: A549 cells depleted of IFITM3 show increased susceptibility to influenza A virus infection. MxA is expressed and is IFN-inducible in A549 and WI-38 cells. A) A549 cells, stably transduced with retroviruses expressing IFITM3, a negative control shRNA against firefly luciferase (shLuc), or one of three shRNAs against IFITM3 (1, 2 or 3), were treated with buffer, IFN-α or IFN-γ for 24 h, then challenged with WSN/33. After 12 h of infection, the cells were fixed and immunostained for HA and stained for DNA. IF images were captured and the percentage of infected cells determined based on HA staining. Values represent the average of three independent experiments +/−SD. B) Western lysates of A549 cells from (A) probed with the indicated antibodies. Western lysates of (C) A549 cells or (D) WI-38 cells, treated with buffer, IFN-α or -γ, then probed with the indicated antibodies. E) Confocal images of WI-38 cells treated with buffer or IFN-α, then fixed, permeabilized and immunostained for either IFITM3 (endogenous, red), or MxA (endogenous, green), and for DNA (blue, scale bar: 20 µM). (PDF)
    Preview · Dataset · Oct 2011
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    Dataset: Figure S4
    [Show abstract] [Hide abstract] ABSTRACT: IFITM3 overexpression halts H3N2 influenza A virus in acidic cytosolic inclusions prior to vRNP nuclear translocation. MDCK cells stably expressing (A) the empty vector control or (B) IFITM3 were incubated with Aichi H3N2 virus on ice, and then warm media was added at time zero along with LTRed (red). Cells were then fixed at the indicated times p.i. and stained for NP (green), and DNA (blue lines denote nuclear periphery), then imaged by confocal microscopy. Images are representative of three independent experiments. (Scale bar: 20 µm). B) Quantitation of nuclear vRNP particles. The number of vRNP particles per nucleus of the MDCK cell lines at the indicated time points are shown. Values represent the mean +/− the SD of three independent experiments. C) Percent colocalization of vRNP and LTRed compartments in MDCK-Vector and MDCK-IFITM3 cell lines at the indicated times p.i. (PDF)
    Preview · Dataset · Oct 2011
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    Dataset: Figure S3
    [Show abstract] [Hide abstract] ABSTRACT: A549 cells overexpressing IFITM3 inhibit vRNP nuclear entry. A549 cells overexpressing the empty vector control (A) or IFITM3 (B) were incubated with PR8 on ice (moi 500). At time zero warm media was added along with LTRed (red). At the indicated times, cells were processed and stained for NP (green) and DNA (blue lines represent the nuclear periphery based on staining), then imaged using a confocal microscope. (Scale bar: 20 µM). These images are representative of three independent experiments. C) Whole cell lysates of A549 cells used in (A) and (B) treated with either buffer, IFN-α or IFN-γ, were subjected to immunoblotting using the indicated antibodies. (PDF)
    Preview · Dataset · Oct 2011
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    Dataset: Figure S6
    [Show abstract] [Hide abstract] ABSTRACT: Ifitm3 expression rescues IFN-γ-mediated inhibition of vRNP nuclear translocation in IfitmDel−/− MEFs. A) IfitmDel−/− MEFs stably overexpressing Ifitm3 (IfitmDel−/−Ifitm3), were left untreated (left panels, Buffer), or treated (right panels) with IFN-γ. The following day cells were incubated on ice with PR8 (moi 500). Cells were next incubated in warm media containing LTRed (0 min.). Cells were then fixed at the indicated times p.i., immunostained with anti-NP antibodies (green) and imaged by confocal microscopy. Image analysis software was used to define the nuclear boundaries (blue lines). Images are representative of three independent experiments. (Scale bar 12 γM). B) Percent colocalization of vRNP and LTRed compartments in the indicated MEF cell lines, with or without IFN-γ treatment, are shown for the indicated times p.i. C) Quantitation of nuclear vRNP particles. The number of vRNP particles per nucleus of the MEF cell lines, with or without IFN-γ treatment, at the indicated time points are shown. Values represent the mean +/− the SD of three independent experiments. D) Western blot of whole cell lysates from the indicated MEFs probed with anti-mouse Ifitm3 and using GAPDH as a loading control. (PDF)
    Preview · Dataset · Oct 2011
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    Dataset: Figure S1
    [Show abstract] [Hide abstract] ABSTRACT: IFITM3 overexpression does not alter the surface levels of (α-2,3) or (α-2,6) sialylated proteins. A549 cells stably transduced with IFITM3 or the empty vector were incubated with biotinylated Maackia Amurensis lectin II (MAL) to detect (α-2,3) sialic acid linkages, followed by streptavidin-PE-Cy7, as well as FITC-conjugated Sambucus Nigra lectin (SNA) to detect (α-2,6) sialic acid linkages. A) The percentage of IFITM3 or vector cells staining positive for both sialic acid linkages (upper right hand quadrant), compared to unstained controls. B) IFITM3 overexpressing and vector cells are compared with regard to each sialic acid linkage in the double-stained populations. (PDF)
    Preview · Dataset · Oct 2011
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    Dataset: Figure S8
    [Show abstract] [Hide abstract] ABSTRACT: IFITM3 is required for IFN's inhibition of HA-mediated fusion. A) HeLa cells were stably transduced with retroviruses containing either IFITM3, a shRNA against IFITM3 (shIFITM3), or a non-targeting control shRNA (shScr). Cells were left untreated (left panels), or treated with IFN-γ (right panels), then exposed for 2 h to H1N1pps (WSN/33) containing BLAM-Vpr. After incubation with the pseudoparticles, the cells were fixed and assayed for cleavage of CCF2 by flow cytometry. These results are representative of three independent experiments. B) The indicated HeLa cell lines were treated with IFN-γ for 24 h then infected with increasing amounts of WSN/33. After 12 h of infection the cells were stained for HA expression. These results are representative of three independent experiments. C) Western blot of the indicated HeLa cell line lysates probed with the indicated antibodies. (PDF)
    Preview · Dataset · Oct 2011
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    Dataset: Figure S5
    [Show abstract] [Hide abstract] ABSTRACT: vRNPs are retained in LAMP1-containing organelles in cells overexpressing IFITM3. A) MDCK-Vector or IFITM3 cells stably expressing a LAMP1-red fluorescence protein (LAMP1-RFP) were challenged with PR8 as in Fig. S4. Cells were immunostained for NP (green), stained for DNA, and then imaged confocally along with the collection of LAMP1-RFP fluorescence (orange). Images are representative of three independent experiments. Blue lines represent the nuclear margins based on DNA staining. (Scale bar: 20 µM). B) Quantitation of nuclear vRNP particles. The number of vRNP particles present per nucleus of the MDCK cell lines at the indicated time points are shown. Values represent the mean +/− the SD of three independent experiments. C) Percent colocalization of vRNP particles and LAMP1-RFP-containing compartments in MDCK-Vector and MDCK-IFITM3 cell lines at the indicated times p.i. Values represent the mean +/− the SD of three independent experiments. (PDF)
    Preview · Dataset · Oct 2011
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    [Show abstract] [Hide abstract] ABSTRACT: To replicate, viruses must gain access to the host cell's resources. Interferon (IFN) regulates the actions of a large complement of interferon effector genes (IEGs) that prevent viral replication. The interferon inducible transmembrane protein family members, IFITM1, 2 and 3, are IEGs required for inhibition of influenza A virus, dengue virus, and West Nile virus replication in vitro. Here we report that IFN prevents emergence of viral genomes from the endosomal pathway, and that IFITM3 is both necessary and sufficient for this function. Notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by IFN, and IFITM3 was required and sufficient for this action. We further demonstrate that IFN expands Rab7 and LAMP1-containing structures, and that IFITM3 overexpression is sufficient for this phenotype. Moreover, IFITM3 partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. Collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to IFITM3's actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. Multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by IFN. Therefore these findings are likely to have significance for the intrinsic immune system's neutralization of a diverse array of threats.
    Full-text · Article · Oct 2011 · PLoS Pathogens
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    [Show abstract] [Hide abstract] ABSTRACT: Type 1 interferons (IFNs) induce the expression of the tripartite interaction motif (TRIM) family of E3 ligases, but the contribution of these antiviral factors to HIV pathogenesis is not completely understood. We hypothesized that the increased expression of select type 1 IFN and TRIM isoforms is associated with a significantly lower likelihood of HIV-1 acquisition and viral control during primary HIV-1 infection. We measured IFN-α, IFN-β, myxovirus resistance protein A (MxA), human TRIM5α (huTRIM5α), and TRIM22 mRNA levels in peripheral blood mononuclear cells (PBMCs) of high-risk, HIV-1-uninfected participants and HIV-1-positive study participants. Samples were available for 32 uninfected subjects and 28 infected persons, all within 1 year of infection. HIV-1-positive participants had higher levels of IFN-β (P = 0.0005), MxA (P = 0.007), and TRIM22 (P = 0.01) and lower levels of huTRIM5α (P < 0.001) than did HIV-1-negative participants. TRIM22 but not huTRIM5α correlated positively with type 1 IFN (IFN-α, IFN-β, and MxA) (all P < 0.0001). In a multivariate model, increased MxA expression showed a significant positive association with viral load (P = 0.0418). Furthermore, TRIM22 but not huTRIM5α, IFN-α, IFN-β, or MxA showed a negative correlation with plasma viral load (P = 0.0307) and a positive correlation with CD4+ T-cell counts (P = 0.0281). In vitro studies revealed that HIV infection induced TRIM22 expression in PBMCs obtained from HIV-negative donors. Stable TRIM22 knockdown resulted in increased HIV-1 particle release and replication in Jurkat reporter cells. Collectively, these data suggest concordance between type 1 IFN and TRIM22 but not huTRIM5α expression in PBMCs and that TRIM22 likely acts as an antiviral effector in vivo.
    Full-text · Article · Oct 2010 · Journal of Virology