One Protein to Rule them All: Modulation of Cell Surface Receptors and Molecules by HIV Nef

The HIV-1, HIV-2 and SIV Nef protein are known to modulate the expression of several cell surface receptors and molecules to escape the immune system, to alter T cell activation, to enhance viral replication, infectivity and transmission and overall to ensure the optimal environment for infection outcome. Consistent and continuous efforts have been made over the years to characterize the modulation of expression of each of these molecules, in the hope that a better understanding of these processes essential for HIV infection and/or pathogenesis will eventually highlight new therapeutic targets. In this article we provide an extensive review of the knowledge gained so far on this important and evolving topic

Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function

Peer reviewe

Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-induced CD4 down-regulation

Background: Down-modulation of the CD4 receptor is one of the hallmarks of HIV-1 infection and it is believed to confer a selective replicative advantage to the virus in vivo. This process is mainly mediated by three viral proteins: Env, Vpu and Nef. To date, the mechanisms that lead to CD4 depletion from the surface of infected cells during HIV-1 infection are still only partially characterized. In this study, we sought to identify and characterize cellular host factors in HIV-1-induced CD4 down-modulation. Results: To identify host factors involved in CD4 down-regulation, we used a whole genome-targeting shRNA lentiviral library in HeLa CD4+ cells expressing Nef as an inducer of CD4 down-modulation. We identified 55 genes, mainly encoding for proteins involved in various steps of clathrin-mediated endocytosis. For confirmation and further selection of the hits we performed several rounds of validation, using individual shRNA lentiviral vectors with a different target sequence for gene knock-down in HIV-1-infected T cells. By this stringent validation set-up, we could demonstrate that the knock-down of DNM3 (dynamin 3), SNX22 (sorting nexin 22), ATP6AP1 (ATPase, H+ Transporting, Lysosomal Accessory Protein 1), HRBL (HIV-Rev binding protein Like), IDH3G (Isocitrate dehydrogenase), HSP90B1 (Heat shock protein 90 kDa beta member 1) and EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15) significantly increases CD4 levels in HIV-infected SupT1 T cells compared to the non-targeting shRNA control. Moreover, EPS15, DNM3, IDH3G and ATP6AP1 knock-down significantly decreases HIV-1 replication in T cells. Conclusions: We identified seven genes as cellular co-factors for HIV-1-mediated CD4 down-regulation in T cells. The knock-down of four out of seven of these genes also significantly reduces HIV-1 replication in T cells. Next to a role in HIV-mediated CD4 down-regulation, these genes might however affect HIV-1 replication in another way. Our findings give insights in the HIV-1-mediated CD4 down-regulation at the level of the plasma membrane and early endosomes and identify four possible new HIV-1 replication co-factors

Un progetto di restituzione digitale degli Statuti Comunali Ferraresi del 1173. Testo e traduzione del fr. 7 Franceschini e di un frammento inedito

Il contributo analizza, discute e traduce i frammenti noti deglli antichi statuti comunali epigrafici di Ferrara del 1173 e un frammento inedito ricostruito dopo una recente campagna di acquisizione. Nel contributo sono anche referite le tecniche digitali che hanno consentito una più efficace comprensione del testo e le prospettive per una loro più ampia comunicazione pubblic

Efficient, Vpx independent shRNA transduction of monocyte derived dendritic cells as a tool for studying HIV transmission to primary T-cells

Background: Dendritic cells present at mucosal sites are considered initial targets for HIV transmission. Due to SAMHD1 expression, they are highly resistant to HIV infection themselves, but act as shuttles for viral transmission to T cells. Knock-down of HIV host cellular partners is a powerful method in HIV research. At present, lentiviral shRNA delivery to Monocyte Derived Dendritic Cells (MDDC) requires the use of virus-like particles carrying the Vpx protein in order to alleviate SAMHD1 mediated restriction. Such an approach results in the desired high transduction efficiency, but simultaneously abrogates resistance to HIV thus allowing for viral replication which might obscure the actual outcomes of MDDC-HIV interactions. We present a method for efficient shRNA transduction of MDDC for subsequent studies of HIV transmission to CD4+ T-cells, without the need for Vpx. Methods: MDDC were obtained by culturing primary monocytes for 6 days in presence of lL-4 and GM-CSF. At day 1 after monocyte isolation, cells were transduced with shRNA encoding lentiviral vectors (Sigma MISSION®) at MOI of 1 (titration on HEK293T cell line) by addition of polybrene and using spinoculation. Transduction efficiency was determined by measuring the fraction of eGFP expression (encoded by the lentiviral vector) by flow cytometry. At day 5 post transduction, cells were exposed to NL4.3 HIV virus harboring a marker gene. Twenty four hours later the cells were extensively washed with culture medium to remove unbound virus and autologous, primary CD4+ T cells were added at 1:2 ratio. Infection of T-cells in MDDC-T-cell cocultures was subsequently monitored by flow cytometry to detect marker gene expressing cells in low scatter and/or CD3+ population. Results: Efficient transduction of MDDC (≈95%) as judged by eGFP expression was obtained. This high efficiency avoids puromycin selection and therefore simplifies the protocol while preventing possible side effects. Notably, the transduced MDDC did not show a marked increase in CD80, CD83 and CD86 expression levels. Transduced MDDC are able to capture and transmit the virus to T cells. We have observed a positive correlation between shRNA mediated downregulation of certain MDDC surface markers and transmission of HIV to CD4+ T cells as compared to cells transduced with a control vector encoding a scrambled shRNA sequence not known to target any human gene. Conclusions: We have established a method for lentiviral encoded shRNA transductions allowing for studies of MDDC cellular factors involved in HIV transmission to CD4+ T cells without the need to alleviate SAMHD1 induced restriction. Transduced MDDC remain immature as judged by expression of CD80, CD83 and CD86 cell surface receptors. Preliminary results provide validation of the approach by positively correlating downregulation of MDDC surface markers with trans-infection of CD4+ T cells

New host factors and pathways involved in CD4 downregulation in HIV-1 infected cells

Background: Downregulation of the CD4 receptor is one of the hallmarks of HIV infection. The virus has evolved redundant mechanisms to remove the receptor from the cell surface and accelerate its degradation, mainly mediated by three viral proteins: Vpu, Env and Nef. We were interested in the discovery of pathways and human proteins involved in the process, which eventually could represent new drug targets. Materials and methods: A genome-wide short-hairpin RNA (shRNA) screening using a SBI shRNA lentiviral interference delivery system library compatible with the GeneChip® Human Genome U133 Plus 2.0 Array (Affymetrix) was performed in HeLa CD4+ cells expressing the Nef protein introduced by retroviral transduction. CD4 surface levels were measured by flow cytometry. The read-out in the screen showed the rescue of the CD4-high phenotype despite Nef expression. shRNA sequences enriched in the CD4-high cells compared to the CD4-low cells were identified and filtered via pathway analysis. For the confirmation and further selection of the hits two cell lines in different conditions were used: 1. HeLa CD4+ cells expressing Nef after retroviral transduction (similar to previous screening effort). 2. SupT1 lymphocytic cells infected with replication competent HIV-1 encoding a GFP reporter. 3. SupT1 cells expressing Nef or Vpu after retroviral transduction. In all three experimental set-ups, the cells were selectively knocked-down for each of the hits individually after transduction with a different set of shRNA encoding lentiviral vectors (Mission Consortium, Sigma Aldrich) prior to HIV-1 infection or retroviral transduction. Results: The genome-wide screen with the SBI library was repeated 4 times to obtain a final list of 75 genes as a first selection of possible new host co-factors in CD4 downregulation by Nef. Of these, 22 proteins were confirmed independently with individual Mission consortium vectors in the same cell line. Eight proteins contributed to CD4 downregulation in HIV-1 infected SupT1 cells. The host factors identified show differential effect on CD4 surface levels in SupT1 cells expressing either HIV-1 Vpu or Nef proteins individually, that together determine CD4 levels on infected cells. These proteins are mainly involved in endosomal and trans Golgi network (TGN) trafficking. Conclusion: Several host proteins involved in endosomal and TGN trafficking differentially affect Nef or Vpu mediated CD4 down-regulation in HIV1-1 infected cells

New host factors and pathways involved in CD4 downregulation in HIV-1 infected cells

Background: Downregulation of the CD4 receptor is one of the hallmarks of HIV infection. The virus has evolved redundant mechanisms to remove the receptor from the cell surface and accelerate its degradation, mainly mediated by three viral proteins: Vpu, Env and Nef. We were interested in the discovery of pathways and human proteins involved in the process, which eventually could represent new drug targets. Materials and methods: A genome-wide short-hairpin RNA (shRNA) screening using a SBI shRNA lentiviral interference delivery system library compatible with the GeneChip® Human Genome U133 Plus 2.0 Array (Affymetrix) was performed in HeLa CD4+ cells expressing the Nef protein introduced by retroviral transduction. CD4 surface levels were measured by flow cytometry. The read-out in the screen showed the rescue of the CD4-high phenotype despite Nef expression. shRNA sequences enriched in the CD4-high cells compared to the CD4-low cells were identified and filtered via pathway analysis. For the confirmation and further selection of the hits two cell lines in different conditions were used: 1. HeLa CD4+ cells expressing Nef after retroviral transduction (similar to previous screening effort). 2. SupT1 lymphocytic cells infected with replication competent HIV-1 encoding a GFP reporter. 3. SupT1 cells expressing Nef or Vpu after retroviral transduction. In all three experimental set-ups, the cells were selectively knocked-down for each of the hits individually after transduction with a different set of shRNA encoding lentiviral vectors (Mission Consortium, Sigma Aldrich) prior to HIV-1 infection or retroviral transduction. Results: The genome-wide screen with the SBI library was repeated 4 times to obtain a final list of 75 genes as a first selection of possible new host co-factors in CD4 downregulation by Nef. Of these, 22 proteins were confirmed independently with individual Mission consortium vectors in the same cell line. Eight proteins contributed to CD4 downregulation in HIV-1 infected SupT1 cells. The host factors identified show differential effect on CD4 surface levels in SupT1 cells expressing either HIV-1 Vpu or Nef proteins individually, that together determine CD4 levels on infected cells. These proteins are mainly involved in endosomal and trans Golgi network (TGN) trafficking. Conclusion: Several host proteins involved in endosomal and TGN trafficking differentially affect Nef or Vpu mediated CD4 down-regulation in HIV1-1 infected cells