Vpr-GFP Virion Particle Identifies HIV-Infected Targets and Preserves HIV-1Vpr Function in Macrophages and T-Cells

Muthumani, Karuppiah; Montaner, Luis J.; Ayyavoo, Velpandi; Weiner, D.B.
March 2000
DNA & Cell Biology;Mar2000, Vol. 19 Issue 3, p179
Academic Journal
Human immunodeficiency virus type 1 (HIV-1) is known for its ability to infect immune cells, including Tcells and macrophages. The 96-amino acid Vpr, a virion-associated protein, is essential for viral replication in monocytes/macrophages and increases viral replication in primary and established T-cell lines. The Vpr protein regulates a number of host cellular events, including proliferation, differentiation, apoptosis, cytokine production, and NF-kappaB-mediated transcription. Most of these functions have been analyzed using either endogenous Vpr protein or cells transfected with a Vpr expression plasmid. We developed a lentiviral vector complemented with a Vpr expression plasmid that results in viral particles packaged with Vpr protein. To facilitate identification of the target cells infected with the particles containing Vpr, we fused green fluorescent protein (GFP) with the Vpr open reading frame and analyzed the biology of this novel particle. Vpr itself is expressed as a 14-kDa protein; however, in vitro translation of the pVpr-GFP plasmid resulted in the expression of 39-kDa fusion protein. The fusion molecule exhibited the same activity in arresting the cell cycle in G[sub 2] as does the wildtype Vpr molecule. Subcellular localization of Vpr and Vpr-GFP by immunofluoresence in human and murine cell lines indicated that Vpr by itself or with the reporter GFP showed a perinuclear staining pattern. Replication kinetics showed no significant difference between Vpr-GFP and native complemented pseudovirus replication in a single-round infectivity assay. A flow cytometry analysis of peripheral blood lymphocytes and macrophages infected with Vpr-GFP-packaged virions and selected by GFP showed 56.7% infectivity for lymphocytes and 84.6% infectivity for macrophages. Additional analysis of CD24 (HSA)-positive cells showed infection of CD4 + cells, macrophages, and, importantly, dendritic cells. This system will allow us to identify specific cell populations including antigen-presenting cells, and allow quantitative analysis of the precise effect of Vpr on both target and bystander cells in vitro as well as in vivo.


Related Articles

  • Global Dynamics of an HIV Infection Model with Two Classes of Target Cells and Distributed Delays. Elaiw, A. M. // Discrete Dynamics in Nature & Society;2012, Special section p1 

    We investigate the global dynamics of an HIV infection model with two classes of target cells and multiple distributed intracellular delays. The model is a 5-dimensional nonlinear delay ODEs that describes the interaction of the HIV with two classes of target cells, CD4+ T cells and macrophages....

  • Small molecules that inhibit Vif-induced degradation of APOBEC3G. Masashi Matsui; Keisuke Shindo; Taisuke Izumi; Katsuhiro Io; Masanobu Shinohara; Jun Komano; Masayuki Kobayashi; Norimitsu Kadowaki; Harris, Reuben S.; Takaori-Kondo, Akifumi // Virology Journal;2014, Vol. 11 Issue 1, p1 

    Background HIV-1 Vif is essential for virus replication in natural target cells such as T cells and macrophages. Vif recruits a ubiquitin ligase to degrade restrictive APOBEC3 proteins. APOBEC3G is one of the most potent retroviral restriction factors targeted by Vif and, as such, the...

  • Role of Vpr in HIV-1 Nuclear Import: Therapeutic Implications. Aida, Yoko; Matsuda, Go // Current HIV Research;Mar2009, Vol. 7 Issue 2, p136 

    The replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells, such as terminally differentiated macrophages, critically depends on the import of the viral pre-integration complex (PIC) into the nucleus. Vpr, one of the accessory gene products of HIV-1, plays a key...

  • Acute Human Immunodeficiency Virus Replication Causes a Rapid and Persistent Impairment of V 9V 2 T Cells in Chronically Infected Patients Undergoing Structured Treatment Interruption. Martini, Federica; Poccia, Fabrizio; Goletti, Delia; Carrara, Stefania; Vincenti, Donatella; D'Offizi, Gianpiero; Agrati, Chiara; Ippolito, Giuseppe; Montesano, Carla // Journal of Infectious Diseases;9/15/2002, Vol. 186 Issue 6, p847 

    T cells expressing V-γ9Vδ2 display lytic and proliferative responses against human immunodeficiency virus (HIV)-infected cells and release antiviral soluble factors. Chronic HIVpositive patients have deep changes in the composition and function of the circulating γδ T cell pool that...

  • HIV-1 Vpr Induces Interferon-Stimulated Genes in Human Monocyte-Derived Macrophages. Zahoor, Muhammad Atif; Xue, Guangai; Sato, Hirotaka; Murakami, Tomoyuki; Takeshima, Shin-nosuke; Aida, Yoko // PLoS ONE;Aug2014, Vol. 9 Issue 8, p1 

    Macrophages act as reservoirs of human immunodeficiency virus type 1 (HIV-1) and play an important role in its transmission to other cells. HIV-1 Vpr is a multi-functional protein involved in HIV-1 replication and pathogenesis; however, its exact role in HIV-1-infected human macrophages remains...

  • The Replication of Human Immunodeficiency Virus Type 1 in Macrophages Is Enhanced after Phagocytosis of Apoptotic Cells. Lima, Rosangela G.; Van Weyenbergh, Johan; Saraiva, Elvira M.B.; Barral-Netto, Manoel; Galvao-Castro, Bernardo; Bou-Habib, Dumith Chequer // Journal of Infectious Diseases;6/1/2002, Vol. 185 Issue 11, p1561 

    Clearance of apoptotic cells increases macrophage secretion of antiinflammatory mediators and might modulate viral replication in human immunodeficiency virus (HIV) type 1--infected macrophages. To study this, primary macrophages were infected with HIV-1 and exposed to apoptotic cells. It was...

  • How HIV-1 Takes Advantage of the Cytoskeleton during Replication and Cell-to-Cell Transmission. Lehmann, Martin; Nikolic, Damjan S.; Piguet, Vincent // Viruses (1999-4915);Sep2011, Vol. 3 Issue 9, p1757 

    Human immunodeficiency virus 1 (HIV-1) infects T cells, macrophages and dendritic cells and can manipulate their cytoskeleton structures at multiple steps during its replication cycle. Based on pharmacological and genetic targeting of cytoskeleton modulators, new imaging approaches and primary...

  • Antiviral Profile of HIV Inhibitors in Macrophages: Implications for Therapy. Perno, C.-F.; Balestra, E.; Francesconi, M.; Abdelahad, D.; Caliò, R.; Balzarini, J.; Aquaro, S. // Current Topics in Medicinal Chemistry;May2004, Vol. 4 Issue 9, p1009 

    Macrophages (M / M) are identified as the second cellular target of HIV and a crucial virus reservoir. M / M are persistently infected cells and not susceptible to the HIV cytophatic effects typical of infected CD4+ T-lymphocytes. HIV replication in M / M is a crucial pathogenetic event during...

  • Selective killing of HIV-1-positive macrophages and T cells by the Rev-dependent lentivirus carrying anthrolysin O from Bacillus anthracis. Young, Jessica; Zhongwei Tang; Quan Yu; Dongyang Yu; Yuntao Wu // Retrovirology;2008, Vol. 5, Special section p1 

    Background: The ability of Human Immunodeficiency Virus (HIV) to persist in the body has proven to be a long-standing challenge to virus eradication. Current antiretroviral therapy cannot selectively destroy infected cells; it only halts active viral replication. With therapeutic cessation or...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics